The efficacy at which an administration of the gold and silver NPs inhibits mouse Lewis lung carcinoma (LLC) growth in vivo was compared. The data suggest that in vivo antitumor activity of the studied NPs strongly depends on gold and silver interaction arising from their ordered topological distribution. NPs with Ag core covered by Au shell were the most effective among the NPs tested towards LLC tumor growth and metastasizing inhibition. Our data show that among the NPs tested in this study, AgcoreAushell NPs may serve as a suitable anticancerous prototype.Nanoparticles (NPs) of noble metals, namely gold and silver, remain promising anticancer agents capable of enhancing current surgery- and chemotherapeutic-based approaches in cancer treatment. Bimetallic AgAu composition can be used as a more effective agent due to the synergetic effect. Among the physicochemical parameters affecting gold and silver nanoparticle biological activity, a primary concern relates to their size, shape, composition, charge, etc. However, the impact of metal components/composition as well as metal topological distribution within NPs is incompletely characterized and remains to be further elucidated and clarified. In the present work, we tested a series of colloidal solutions of AgAu NPs of alloy and core-shell type for an antitumor activity depending on metal molar ratios  and topological distribution of gold and silver within NPs (Au The data, revealed in the present study, emphasize that both parameters, metal molar ratio as well as metal topological distribution within bimetallic gold and silver nanoparticles, are critical features of NPs and can affect their antitumor activity. Among the NPs tested in this study, AgcoreAushell NPs may serve as the most suitable prototype for the development of an anticarcinomatous agent.Rapid advances in nanomedicine have generated an increasing number of potential diagnostic and therapeutic applications of nanoparticles in recent years , 2. Amon3 and HAuCl4, Merck, Germany) with amino acid tryptophan . The components interacted in a molar ratio Trp:M = 2:1. The concentration of metal in the resulting solution was C(M) = 10-4M followed by concentrating it to 10−3 M. Bimetallic NPs of alloy type were obtained via simultaneous reduction of metal ions using initial metal molar ratios of Ag:Au = 3:1, 1:1, and 1:3, indicated as AgAu(3:1), AgAu(1:1), and AgAu(1:3) correspondingly. For the preparation of NPs of core-shell type with different topology, metal ions were reduced sequentially using the molar ratio Ag:Au = 1:1 and were indicated as AucoreAgshell and AgcoreAushell, For all colloids, the initial solutions of tryptophan were adjusted to pH = 10. Thus, amino acid was used in an anionic state that exists in alkaline medium. The pH control of the medium was performed using a pH meter I-160MI. As a working electrode, a glass electrode was used. Silver chloride electrode served as a reference one. Metals were injected into boiling solution of Trp followed by continuous heating of the reaction mixture.Colloidal solutions of bimetallic silver and gold NPs were obtained via chemical reduction of silver nitrate and tetrachlorauratic acid  in 1-cm quartz cells.The size and morphology of nanoparticles were characterized with a transmission electron microscope (TEM) JEM-1230  with an accelerating voltage of 80 kV. One microliter of colloid was placed on carbon-coated copper grids and dried at room temperature. For the calculation of particle size distribution, the program ImageJ was used.2 setup.Scanning electron microscopy (SEM) was performed using a Tescan Mira 3 LMU microscope with an accelerating voltage of 5–20 kV. Energy-dispersive X-ray (EDX) spectra were taken using a built-in Oxford X-max 80 mmThe particle size distribution function was studied by a laser correlation spectrometer Zetasizer Nano S  equipped with a correlator (multicomputing correlator type 7032 ce) by a method based on the scattering of light on any micro-objects. The information signal from the random movement of nanoparticles was analyzed by multichannel spectrum analyzer and colorimeters; 1 ml of studied suspension was placed in a cylindrical optical glass cell with a diameter of 10 mm, which was located in a thermostated sample holder of a laser correlation spectrometer. Registration and statistical processing of the scattered laser light at 173° from the suspension (helium-neon laser LGN-111 was used with power output of 25 mW and wavelength of 633 nm) was performed three times during 120 s at 25 °C. The resulting autocorrelation function was treated with standard computer programs PCS–Size mode v 1.61.6 cells/ml). Starting from day 5 after tumor cell inoculation , the animals were randomly divided into groups receiving daily an intra-peritoneal injection of 100 μL of the colloidal solution of NPs  at a dose of 500 μg/kg/day for 12 days. Control group of mice received an intra-peritoneal injection of 100 μL of saline. General morphological parameters  and survival rate were monitored daily.All the mice employed in our studies were congenic in the C57BL/6J genetic background. Male mice aged 12 weeks and weighing 18–20 g were used in the experiments. The animals were housed in cages containing a sterile paddy husk as bedding in a ventilated animal facility with a controlled temperature (21 ± 2 °C). All the mice were maintained on a laboratory complete diet with free access to water. As a model of malignant tumor growth, Lewis lung carcinoma (LLC) was employed. Tumor strain was kindly provided by the National Bank of Cell Lines from Human and Animal Tissues . LLC transplantation was performed by intra-muscular injection into a femoral muscle of 0.2 ml 10% cell suspension in saline  and sedimented at 1000×g for 20 min at 21 °C. Mononuclear cells were harvested from the interface and pelleted three times in phosphate-buffered saline (PBS) without Ca2+ and Mg2+ to remove platelets (first at 650×g then twice at 235×g).Twenty-four hours after the last NP injection (day 18 after tumor cells inoculation), the mice underwent euthanasia. At the time of sacrifice, mice were weighed, blood was taken from the inferior vena cava, and liver, lungs, and primary tumor were immediately removed. Blood serum was collected after centrifugation at 1500×V) was calculated by the formula V = 0.52d3, where d is the tumor diameter − ions are simultaneously reduced. Its maximum is located between the maxima inherent to the LSPR bands of individual metals  band absorption. Only one LSPR band is characteristic for bimetallic alloy NPs formed when Agals Fig. a and is average = 1  (Table An in vivo antitumor activity of the administered NPs was estimated based on the rate of primary tumor growth inhibition (TGI), lung metastasis growth inhibition (MGI), and metastasis inhibition (MI) indexes . An inhil) Table . Based ol) Table .Table 1TcoreAushell NPs, MI index was 19 and 13%, respectively, whereas the treatment of mice with AgAu(1:3) (−28%), AucoreAgshell (−22%), and AgAu(1:1) (−10%) was characterized even by stimulation of metastasizing  index was calculated based on a frequency and a number of metastatic nodes in mouse lungs. During the administration of AgAu3:1) and Ag and AgcocoreAushell-treated group). Conversely, the treatment with AgAu(3:1) NPs led to an accelerated metastasis growth in mice (MGI = −36%).Lung metastases were observed in mice of all the treatment groups. Lung metastasis growth inhibition index ranged from 4% (in AgAu(1:3)-treated group) to 27%  NP administration had no significant effect on serum LDH activity in mice with LLC. This was consistent given the lowest primary tumor growth inhibition rate in this treatment group (Table NPs administration to mice was accompanied with low serum lactate dehydrogenase (LDH) activity, an indicator of tumor growth activity and antitumor treatment effectiveness . For ther, AgAu3: NP adminup Table .Fig. 5BlOxidative stress within the cells is considered as one of the primary mechanisms underlying NP antitumor activity –20. SilvcoreAushell or AucoreAgshell) was the most sufficient in inducing protein carbonylation in LLC primary tumor tissue. Conversely, the use of NPs with different metal molar ratios (AgAu(1:1) and AgAu(3:1)) did not result in higher tumor protein carbonylation and did not affect or even cause elevation of primary tumor protein sulfhydryl groups , NPs that consisted of Ag core covered by Au shell were the most active towards LLC tumor growth and metastasizing inhibition and possessed the lowest toxicity. Thus, the studied biological activity was the highest in case of ordered topological distribution of metal compounds within AgcoreAushell NPs among the studied series of bimetallic nanoparticles with different metal content and topology. This was also confirmed by low serum lactate dehydrogenase activity compared to untreated mice with LLC and higher tumor tissue protein carbonylation. AgcoreAushell NPs showed low genotoxicity towards mouse lymphocytes as evidenced by DNA comet analysis and did not affect hepatic lipid and protein oxidation. Our data suggest that AgcoreAushell NPs used in this study may serve as a suitable prototype to develop anticarcinomatous agent and anticancer drug vehicle.Based on the parameters of primary tumor growth inhibition, lung metastasis growth inhibition, and metastasis inhibition index, the highest antitumor activity was attributed to Ag


We assessed the soil organic carbon (Corg) stocks, soil accretion rates  and soil Corg sequestration rates (g Corg m−2 yr−1) in 10 mangrove sites within four locations along the Saudi coast of the Central Red Sea. Soil Corg density and stock in Red Sea mangroves were among the lowest reported globally, with an average of 4 ± 0.3 mg Corg cm−3 and 43 ± 5 Mg Corg ha−1 (in 1 m-thick soils), respectively. Sequestration rates of Corg, estimated at 3 ± 1 and 15 ± 1 g Corg m−2 yr−1 for the long (millennia) and short (last century) temporal scales, respectively, were also relatively low compared to mangrove habitats from more humid bioregions. In contrast, the accretion rates of Central Red Sea mangroves soils were within the range reported for global mangrove forests. The relatively low Corg sink capacity of Red Sea mangroves could be due to the extreme environmental conditions such as low rainfall, nutrient limitation and high temperature, reducing the growth rates of the mangroves and increasing soil respiration rates.Mangroves forests of A However, these extend beyond the local communities to yield, through their role as a globally-relevant carbon sinks, climate change mitigation benefits to the global community5. Although mangroves occupy only 0.5% of the area of the global coastal ocean6 and represent only 0.7% of the tropical forests of the world7, they account for about 1% and 14% of the carbon sequestered annually by the world’s forests and the global ocean, respectively8.Mangrove forests supply important ecosystem services that support the livelihoods of coastal societies in the tropics and subtropicsorg) sequestration by mangrove forests is mainly due to their high net ecosystem production9, resulting in high loads of leaf litter and biomass to the soil, combined with their high capacity to trap and retain soil resulting in vertical accretion10 compared to other forests and coastal areas devoid of vegetation11. Further, high Corg preservation due to low oxygen availability13 combined with lack of fires in the aquatic environment where mangroves thrive3, results in high carbon storage in mangroves15.The intense organic carbon . Hence, we would expect mangrove forests in the Red Sea to have a relatively modest carbon sink capacity so that, despite the stability of the forests, their contribution to carbon sequestration would still be marginal. Yet, most estimates of carbon sequestration and stocks in mangrove forests have been, hitherto, derived from mangrove forests in the wet tropics8, and whether those growing in the arid topics contribute significantly to carbon sequestration remains an open question. However, a recent assessment reports a Corg sequestration for dwarf mangrove forests in the arid shores of Baja California of, on average, 1000 Mg Corg ha−1 in 1.5 m-thick soils  the stock of Corg contained within the top meter of the soil, and (b) the burial rate of Corg over short-term (last 100 years) and long-term (last millennia) periods. Further, we use stable carbon and nitrogen isotopes of the organic matter in the soil and putative sources  to estimate the potential contribution of different sources of Corg to mangrove soil carbon stocks.Here we assess the CP < 0.001), . Mangrove forests at Thuwal Island grow in the coarsest soil (80% particles between 0.125 and 1 mm) compared to the other study sites  compared to loamy sand in Petro Rabigh (32% of mud <0.063 mm) Table , where torg density and % Corg were generally low, ranging from 3 to 9 mg Corg cm−3 and 0.2% to 1.5% Corg , and high % Corg (0.2 to 17% Corg in the upper 25 cm), compared to the other locations  compared with those in other locations   was poor and involved considerable uncertainty  and 1/3 of Corg derived from aquatic photosynthesis . The 210Pb concentration profiles of 3 mangrove soil cores  showed evidence of mixing in the upper 2 to 7 cm  and down to 22, 12, 15 and 11 cm, in cores KMC, KME, RMF and TMF respectively. Supported 226Ra concentrations were similar among the 10 analyzed cores, averaging 10 ± 3 Bq kg−1, with KMC concentrations being significantly lower at 5.6 ± 0.6 Bq kg−1  was applied to these cores (below the mixed layer when present), obtaining SAR ranging from 0.7 ± 0.1 to 3.7 ± 0.7 mm yr−1 over the last 100 years. Due to the presence of mixing, these rates must be considered as upper limits, especially for KMC and RMF. The resulting SAR averaged 2.2 ± 0.6 mm yr−1 over the past 100 years .The  cm Fig. , and no g−1 Fig. . The CF:rs Table . The ranorg content at Khor Alkharar resulted in the highest Corg stock at this site, which was significantly higher compared to all other locations (Tables org sequestration rates averaged 15 ± 1 g Corg m−2 yr−1 (i.e. based on 210Pb dating) and varied three-fold, from the lowest rates at Petro Rabigh (9 ± 4 g Corg m−2 yr−1) to the highest rates at Khor Alkarar . These differences were driven mostly by the differences in Corg density, as soil accretion rates were rather uniform and did not differ significantly among locations. The long-term Corg sequestration rates did not differ among locations, averaging 3.5 ± 1.1 g Corg m−2 yr−1 (Table P > 0.05).The high Cs Tables  and 5. L26 who reported sandy sediments in Khor Alkharar Sea becoming coarser, with a higher gravel component, towards the beach, and sediment at Rabigh to be mainly composed of medium grain size sand. Although, the clay and silt content of mangrove soils at Khor Alkharar were relatively low compared to the other locations, site KME had considerably high content of mud 24% , hence the high content of fine sediments at surface layers together with high concentrations of Corg can explain the higher inventory of excess 210Pb found at this site compared to other locations.The sediment grain size we found was consistent with the findings of Gheith & Abou-ouforg density in Central Red Sea mangrove soils (0.0044 ± 0.00028 g Corg cm−3) is remarkably low compared to values reported from a global compilation . The Corg density in the mangrove soils of the Central Red Sea is 100 to 300-fold lower than in mangrove soils in wet temperate to subtropical climate at Rookery Bay, Florida  C−1) are close to the median global value of 2.8 mm yr−1 29 and half of the median value of 4.5 mm yr−1 reported for mangroves globally3, and within the range previously reported (0.1 to 21 mm yr−1 8). Moreover, the values obtained for the Red Sea mangroves using 210Pb chronologies are higher than those recently reported using comparable methods for mangroves in Moreton Bay and southeastern Australia , and lower than the average rate reported in Florida (2.7 mm y−1)32.In contrast, the recent soil accretion rates of Central Red Sea mangroves soil  is up to 4-fold higher than those based on 14C (1 mm yr−1), which encompass long-term (millennia) accretion, in areas such as Economic City, Petro Rabigh and Khor Alkharar. These results suggest an increase in SAR during the last decades, which is consistent with coastal development in the Saudi coast of the Central Red Sea, that experienced industrial and urban developments over the past decades. This is supported by the high value of 15N, that suggests an increase of nutrients from land runoff and coastal development as sewage and fertilizers are released to the sea. Moreover, decreasing δ13C values and increasing mud content (silt and clay) towards recent periods also support this hypothesis. However, the presence of mixing in top layers, decomposition of organic matter with ageing, compaction of soils during diagenesis, and intrinsic differences in 210Pb and 14C methodologies could also explain the higher SAR found in recent times35.The SAR obtained using the org density resulted in low carbon sequestration rates. The average soil Corg sequestration rates of 15 g Corg m−2 yr−1 for Central Red Sea mangroves soils is 10-fold lower than the average value of 163 g Corg m−2 yr−1 reported for mangroves globally29, and are in the low range of values previously reported (10 to 920 g C m−2 yr−1)8. The long-term  soil Corg sequestration rates in Central Red Sea mangroves (3.5 g Corg m−2 yr−1) was also well below the soil Corg sequestration rates derived from 14C chronologies reported for dwarf A. germinans forests in arid Baja California (256 g Corg m−2 yr−1)23 and Pohnpei Island, Micronesia dominated by Rhizophora apiculata, (93 g Corg m−2 yr−1)29, despite SARs at the Micronesia sites being comparable to those reported here for Red Sea mangroves, of 2 mm yr−1 36. Despite of the low long-term sequestration of carbon in Central Red Sea mangroves compared to other mangrove habitats, their capacity to sequester Corg is similar to that observed in tropical forest soils (2.3 to 2.5 g Corg m−2 yr−1)37.Whereas SAR are not particularly low for Central Red Sea mangrove forests, their remarkably low soil Corg stocks and Corg sequestration rates in Red Sea mangroves are most likely due to the oligotrophic nature and low allochthonous inputs to the Red Sea. The lack of rivers and the extremely arid conditions result in nutrient-limited mangrove growth19, reflected in low-biomass dwarfed trees, particularly within the study region in the Central Red Sea20. Moreover, the soils of Red Sea mangroves are mainly composed of biogenic coarse carbonates, which could also explain the relatively low Corg sequestration capacity of mangroves growing in unfavorable conditions for biomass production, soil accretion and preservation compared to mangrove habitats from temperate and sub-tropical habitats. The isotopic results showed that two-thirds of the soil Corg stocks originated from mangrove or tidal-marsh biomass, while one-third was derived from marine photosynthetic organisms. Previous studies demonstrated that terrigenous Corg inputs can contribute up to 30% of Corg stocks in mangrove soil associated with riverine ecosystems38, but the lack of rivers implies that there is no influx of riverine soil and organic matter in coastal areas and, therefore, Corg sequestration in Red Sea mangroves is limited to autochthonous production and fluxes from the ocean.The relatively low C39. Moreover, the seston carbon seems to be dominated by carbon derived from macrophytes as plankton-derived Corg is also relatively depleted in 13C 40.The lack of terrigenous inputs is also reflected in the heavy carbon isotopic signatures of suspended particulate matter (−11.3 ±1.6‰), indicative of sestonic organic matter of marine origin, as seston from river sources is characterized by lighter values ranging from −18.5 to −26.4‰2 capture by mangroves in the Red Sea would be extremely low due to the arid conditions of this region. Indeed, poor Corg preservation under coarse grained-soils, high hydraulic conductivity, and low moisture-holding capacity, could also result in low Corg storage, as previously demonstrated for mangroves from United Arab Emirates41. Considering the total mangrove area in the Red Sea (135 km2)18, the rates reported for Red Sea mangroves here represent about 2100 tons of carbon sequestered per year, which is a relatively low contribution. Whereas Red Sea mangroves remains a healthy and stable ecosystem in the Red Sea18, they offer a limited potential to support blue carbon strategies to mitigate CO2 emissions.We hypothesized that in contrast with other mangrove forests, COAvicennia marina at Thuwal Island, Economic City, Petro Rabigh and Khor Alkharar . Then, every second slice of whole cores and all subsamples from port cores were divided into two subsamples by quartering. One subsample was ground and analyzed for organic carbon (Corg), and stable carbon and nitrogen isotopic composition (δ13C and δ15N), and the other subsamples were used for soil grain-size, 14C and 210Pb analyses.PVC whole cores were cut lengthwise and cut into 1 cm-thick slices. Each slice from the whole cores and the subsamples from the port cores were weighed before and after oven drying at 60 °C until constant weight  to estimate dry bulk density  and the supernatant with acid residues was removed using a pipette, then washed in deionized water once, the residues were centrifuged again and the supernatant removed. The residual samples were re-dried (70 °C) and encapsulated for analysis using a Thermo Delta V Conflo III coupled to a Costech 4010 at the UH Hilo Analytical Laboratory, USA  samples. Organic carbon and Nitrogen isotope ratios are expressed as δ values in parts per thousand and relative to the Vienna Pee Dee Belemnite and atmospheric nitrogen using USGS 40 and USGS 41 standards, respectively. Analyses of replicates and reference materials were carried out to ensure reproducibility of the results.Samples were acid-rinsed to ensure complete removal of inorganic carbon  before CAvicennia marina macro-detritus , halophytes , seaweeds , seagrasses  and seston particulate organic matter (fraction retained on a 0.7 μm pore diameter filter) collected at the four study locations. The samples from living materials were milled, and encapsulated for elemental and isotopic analyses as described above. Living material containing carbonates in their tissues were acidified with 1 M HCl before analyses  digestion of <1 mm samples with 30% hydrogen peroxide at the Centro de Estudios Avanzados de Blanes, Spain. Grain size classification and texture were categorized following the Wentworth scale210Pb in the upper 20 to 30 cm of one to two cores per location were determined in the soil fraction <125 μm at the Universitat Autònoma de Barcelona (Spain) through the measurement of its granddaughter 210Po assuming radioactive equilibrium between both radionuclides49. Briefly, after addition of a known amount of 209Po as yield tracer, samples were acid digested in an analytical microwave, the polonium isotopes were auto-plated in silver discs and the concentration of 210Po quantified by alpha spectrometry. The concentrations of excess 210Pb used to obtain the age models were determined as the difference between total 210Pb and 226Ra (supported 210Pb). Concentrations of 226Ra were determined for selected samples along each core by low-background liquid scintillation counting method 50. These concentrations were found to be in agreement with the concentrations of total 210Pb at depth below the excess 210Pb horizons. Analyses of reagent blanks, replicates and a reference material  were carried out for both 210Pb and 226Ra to assess for any contamination and to ensure reproducibility of the results,  at two soil depths per core , at the AMS Direct Laboratory (USA). Samples consisted of pooled shells and bulk soil,  and aquatic  photosynthesis collected at the four study sites, using a 1% increment and 0.1 to 0.5‰ tolerance.The IsoSource software packageorg stocks per unit area (g Corg m−2) were estimated for 10 cm and 1 m soil thicknesses (i.e. cumulative mass). Where necessary (i.e. in 7 cores), we inferred Corg stocks below the limits of the reported data to 1 m, by extrapolating linearly the cumulative Corg stocks to 1 m.The soil Corg sequestration rates (expressed in g DW m−2 y−1) for the last century and the last millennia were estimated using 210Pb (CF:CS model)24, and 14C age models, respectively. Mean soil accretion rates  over the last 200 to 5000 years (i.e. based on 14C) were determined by calibrating the raw radiocarbon dates reported by the Laboratory using the R routine “Bacon” (Marine13 curve) for Bayesian chronology building52 and corrected for the marine reservoir effect 53. From the Bacon routine output, the mean age was used to produce an age-depth weighted regression model forced through 0 , using as weight the sum of the Euclidean distance of the minimum and maximum ages.Soil Corg were estimated by dividing the inventories in 100 cm-thick soil by the average soil accretion rate derived from 14C, whereas, the 210Pb-derived sequestration rate was calculated for 10 cm-thick soils by multiplying the soil accretion rate by the fraction of Corg accreted since 1900. For the cores that were not possibly dated, we assumed they supported the same accretion rates as the dated cores at the same location. The cores from Economic city could not be successfully dated with 210Pb (i. e. showed mixing) and we assumed that the soil accretion rate of that area is the average of the successfully dated cores in other locations.Sequestration rates of CStatistical analyses were carried out using JMP software, including descriptive statistics and ANOVA , and General Linear Mixed Models (GLMM)  to test for differences among forests, followed by Tukey HSD posthoc tests to assess pairwise differences among sites and soil depths.org, δ13C signatures and soil grain size fractions) were square-root transformed prior to analyses to homogenize their variances. Study sites  and soil depth nested within cores were treated as fixed factors, whereas replicate cores within sites was treated as random factor.Generalized Linear Mixed Models (GLMM) were used to take into account the potential non-independence of samples taken at different depths within the same core, since depth is a proxy for time in the cores. And given the spatial separation of cores within mangrove forests (hundreds of meters) we considered the cores themselves to be spatially independent. All response variables (bulk density, CSupplementary Materials


The reconstruction of pre-depositional cooking treatments used by prehistoric coastal populations for processing aquatic faunal resources is often difficult in archaeological shell midden assemblages. Besides limiting our knowledge of various social, cultural, economic and technological aspects of shell midden formation, unknown pre-depositional cooking techniques can also introduce large errors in palaeoclimate reconstructions as they can considerably alter the geochemical proxy signatures in calcareous skeletal structures such as bivalve shells or fish otoliths. Based on experimental and archaeological data, we show that carbonate clumped-isotope thermometry can be used to detect and reconstruct prehistoric processing methods in skeletal aragonite from archaeological shell midden assemblages. Given the temperature-dependent re-equilibration of clumped isotopes in aragonitic carbonates, this allows specific processing, cooking or trash dispersal strategies such as boiling, roasting, or burning to be differentiated. Besides permitting the detailed reconstruction of cultural or technological aspects of shell midden formation, this also allows erroneous palaeoclimate reconstructions to be avoided as all aragonitic shells subjected to pre-historic cooking methods show a clear alteration of their initial oxygen isotopic composition. The remains of molluscs (shells) and fish (otoliths) were often deposited in the vicinity of prehistoric settlements forming mounds known as shell middens. The composition, internal structure or the spatial and temporal distribution of these shell middens represent a valuable source of information about human dispersal, site-specific occupation pattern, subsistence strategies, associated dietary preferences or fishing and foraging seasonality4. Moreover, mollusc shells or fish otoliths derived from shell middens can provide proxy records such as oxygen isotopes which enable the reconstruction of highest-resolution  palaeoenvironmental conditions of ancient coastal environments7. Despite allowing the reconstruction of foraging seasonality4, past upwelling intensities10 or seasonal freshwater discharge patterns13, ontogenetic geochemical signatures of shell midden constituents have become an important tool for the reconstruction of ancient water temperature seasonality within coastal areas17. In particular shell midden deposits which cover time spans of up to several millennia allow the long-term reconstruction of environmental change with a sub-seasonal resolution18.Many prehistoric coastal populations have exploited the faunal resources of adjacent aquatic environments4, In particular molluscs, where the comparably small amount of edible flesh is protected by tightly closed valves or an operculum require sophisticated processing strategies in order to efficiently extract the small edible portion6. However, evidence for certain processing or cooking techniques such as pottery and tool fragments or blackened/burned stones are often sparse in shell midden deposits or allow a variety of potential interpretations regarding specific cooking or processing strategies. This is particularly true at transitory preparation sites near fishing or shellfish gathering grounds rather than close to prehistoric settlements.As subsistence was the primary reason for exploiting aquatic faunal resources for many prehistoric cultures, most shell midden constituents were most-likely subjected to certain processing or cooking techniques prior to deposition21. Hence, the reliable reconstruction of prehistoric processing methods is crucial in order to avoid erroneous palaeoclimate reconstructions based on geochemical proxies in shell midden constituents.While usually unknown processing methods limit our understanding of shell midden formation, processing or cooking methods can alter the geochemical proxy records of calcareous skeletal remains, potentially introducing large uncertainties into palaeoclimate reconstructions13C-18O bonds  in the carbonate molecule with decreasing carbonate formation temperature, independent of the isotopic composition of the precipitating fluid24 and therefore circumvents uncertainties in conventional oxygen-isotope thermometry which commonly derive from unknown ancient water chemistry25. In addition, clumped-isotope thermometry allows reconstructing the thermal histories of carbonate rocks as the relative abundance of 13C-18O bonds can be re-equilibrated during post-formational heating27. The rate and magnitude of 13C-18O bond re-ordering is a function of temperature and exposure time and can occur even in closed systems without any isotope exchange with ambient media28. While re-equilibration in calcite occurs over geological time scales, aragonite shows much higher re-ordering rates resetting the clumped-isotopic signature within minutes to hours29. Thus, clumped-isotope thermometry might represent an ideal tool to detect pre-depositional heating of aragonitic shell midden constituents and may allow certain processing or cooking practices to be differentiated.In this study, we tested the potential application of carbonate clumped-isotope thermometry on aragonitic mollusc shells and fish otoliths to reconstruct specific prehistoric cooking methods. Carbonate clumped-isotope thermometry is based on the preferential formation of Mercenaria campechiensis, Gmelin 1791) cultured in the Gulf of Mexico to a variety of prehistoric cooking methods. Our experiment comprised untreated shells , shells boiled in seawater (~100 °C), roasted over charcoal (174 ± 13 °C) and directly burned in charcoal (559 ± 95 °C). For all treatments, exposure times ranged from 15 minutes to 6 hours. Afterwards, we measured the bulk clumped-isotopic composition (Δ47), oxygen and carbon isotope values  as well as the potential conversion of the primary aragonite into secondary calcite.In order to test this hypothesis, we exposed the right valves of modern hard clams , radiocarbon dated to 5,020–5,320 cal. yrs. BP. These samples comprised four catfish otoliths (Carlarius spp.) and seven bivalve shells . We also analysed additional samples drilled from the hinge region of each bivalve shell, as well as high-resolution ontogenetic oxygen-isotope records of three exemplary shells.In addition, we measured bulk δDepending upon the cooking temperature, variable changes in shell mineralogy, visual appearance and outer shell structure occurred within the different cooking treatments Fig. . Boiling47). Re-ordering rates in aragonite were high enough to cause an immediate reduction in Δ47 where the magnitude is generally a function of cooking temperature and exposure time. The lowest amount of change occurred in the boiling treatment (~100 °C) where the reduction in the Δ47 values was not significantly different from the control shells. This agrees with the thermodynamic model for clumped-isotope re-ordering in aragonite at 100 °C29 which suggests that an exposure time of several hours to temperatures ~100 °C is not sufficient to cause a significant decrease in Δ47 values. In contrast, roasting and burning caused an immediate decrease in Δ47 to values ranging from 0.510 to 0.609‰, and 0.342 to 0.425‰, respectively. As shown in Fig. 47 values within the boiling and roasting treatments are in good agreement with thermodynamic models for the relevant temperatures over the time of exposure29.The exposure of bivalve shells to different cooking practices caused an alteration of their clumped-isotopic composition . Likewise, all mid-Holocene S. senilis shells were composed of 100% aragonite which is the initial mineralogy also found in modern shells31. From the mineralogical perspective, the archaeological samples can therefore be considered pristine. All mineralogical data are provided in Supplementary Tab. All mid-Holocene otoliths are mainly composed of aragonite (96–100%) which is in agreement with the initial mineralogy of modern 47-values (0.667 to 0.716‰) largely agree with predicted values based on modern water temperature observations32. In contrast, mid-Holocene bivalve shells show highly variable Δ47 values ranging from 0.601 to 0.716‰ which is considerably lower than expected for natural environments. Noteworthy, bulk samples of the shells show higher variations in Δ47 and tend towards lower Δ47 values (0.601 to 0.716‰) than corresponding samples from the hinge region of the same individuals (0.651 to 0.697‰). Subsequently, Δ47-based water temperature reconstructions using the bulk shell samples result in unreasonably high palaeo-water temperature estimates (23.9 to 51.0 °C). In contrast, the samples drilled from the hinge region of the same shells provide more realistic water temperature estimates for coastal intertidal environments (28.0 to 38.3 °C).Mid-Holocene otolith Δ18OCarbonate values are consistent amongst all otoliths and bivalve shells, ranging from −0.54 to +0.88‰ VPDB  which were also measured during the experiment  a sharp decrease in Δ47 during the initial heating of the primary aragonite  followed by a (2) partial conversion of aragonite into calcite causing a re-increase in Δ47 and (3) a second re-ordering phase of the secondary calcite resulting in a decrease in Δ47 values with considerably lower re-equilibration rates. This complex model of clumped-isotope re-ordering during the transition of aragonite into calcite is supported by our data because the measured trajectory of Δ47 alteration within the burning treatment exactly follows the pattern described by Piasecki38. Assuming a lower treatment temperature of only 386 °C increases the goodness of fit between the measured and predicted Δ47 values of the burning treatment  coals19. In contrast, Milano et al.20 showed only a clear decrease in the δ18OShell values of mollusc shells at temperatures of >300 °C. We suggest that the lower minimum temperature for alteration of oxygen isotopes in aragonitic skeletal structures found in this study as well as by Milano et al.20 compared to that of Andrus & Crowe19 is related to the direct exposure of the aragonitic mollusc shells to the cooking medium. Andrus & Crowe19 exposed the entire fish to the different cooking treatments with otoliths protected from direct heat inside the vestibular system. This hypothesis is supported by the general agreement between our data and those data of Milano et al.20 who also observed a similar decrease in the δ18O values of mollusc shells at temperatures between 300 and 700 °C. The experiment of Milano et al.20, however, did not include relevant roasting temperatures between 100 and 300 °C which most likely represent the temperature window where a decrease in the δ18OCarbonate values occurs without a conversion of primary aragonite into secondary calcite.The measured alterations of δ18OShell alteration between this study and Milano et al.20 at lower temperatures (<300 °C) indicates that experimental design plays a key-role in assessing the impact of prehistoric cooking practices on palaeoclimate proxy signatures of shell midden constituents. While controlled oven experiments are a suitable approach for studying the underlying processes of aragonite-calcite conversion and identifying isotope-exchange mechanisms, the realistic simulation of prehistoric cooking practices using coal/wood fire provides more reliable estimates regarding the absolute alteration of palaeoenvironmental proxies in shell midden constituents. The stronger alteration at lower temperatures found in this study is probably related to the presence of water in the boiling treatment, high-frequency temperature variability in the roasting and burning experiment, and higher CO2 and CO concentrations and fluxes compared to the closed oven setups of Milano et al.20. Such factors are likely to facilitate isotope-exchange reactions between the carbonate mineral and the ambient cooking medium and can thus explain the more extensive alteration found in this study with respect to δ18OCarbonate values at lower cooking temperatures. Together with temperature fluctuations of realistic roasting and burning experiments, such processes might also explain the observed non-linearity of δ18O alteration within the different treatments. However, experimentally determined alteration during prehistoric cooking will most likely also vary depending on the flame temperatures or different wood types used in the experiments.The different magnitudes of δ47 values of the mid-Holocene bivalve shells are considerably lower than expected for natural environments. While our experimental data suggests that these shells were heated prior to deposition, their pristine aragonitic mineralogy suggests that they did not experience temperatures higher than 300–400 °C. The overlap of the measured Δ47 values of the mid-Holocene shell with our experimental data obtained from the roasting treatment , which is also supported by our data , measured Δ47 values within individual shells (hinge versus bulk samples), as well as similar Δ47 values for the otoliths and bivalves excavated from the same shell midden. Thus, it appears unlikely to us that the exposure to increased sediment surface temperatures caused the observed trend in the clumped-isotopic signature of the mid-Holocene samples.An alternative explanation for the post-mortem alteration of clumped-isotopic signatures in the mid-Holocene bivalve shells might be the long-term sub-aerial exposure with increased soil temperatures as a consequence of high solar insolation in this sub-Saharan region. However, assuming a potential re-equilibration due to surface exposure throughout the Holocene, one would also expect different bivalve shells to exhibit the same degree of alteration, similar Δ18O-based water temperature records show a clear sinusoidal oscillation but with on average ~5 °C higher water temperatures during the mid-Holocene than observed today in this region  agree with the modern values of this region (13.6 °C)31, we presume that even if these shells were altered by pre-depositional heating, their internal δ18O-range might have been preserved and can still provide information about water temperature seasonality and, for example, the season of capture. However, future studies on the influence of prehistoric cooking on ontogenetic oxygen isotope records in mollusc shells are necessary to further examine the potential alteration of ontogenetic oxygen-isotope records.Although the clumped-isotopic signatures of the bivalve shells strongly suggests pre-depositional heating, the bulk oxygen isotopic composition provides slightly higher, but still reasonable, water temperature estimates for coastal regions of NW Africa during the mid-Holocene, potentially indicating only a minor degree of pre-depositional alteration. Similarly, the high-resolution ontogenetic δion Fig. 31. WhethThis study strongly suggests that pre-depositional heating of aragonitic shell midden constituents can cause considerable, but most likely unnoticeable, errors in palaeoclimate reconstructions. Depending on the cooking temperature, the alteration of oxygen isotopes in aragonitic shells is rather small (<0.9‰ ≈ <4.2 °C) and occurs even without a clear conversion of primary aragonite into secondary calcite, particularly at cooking temperatures <300 °C. This implies that such pre-depositional alterations are hardly differentiable from natural ancient water temperature variations and that conventional mineralogical analyses might fail to detect such pre-depositional heating. However, our data clearly show that clumped-isotope thermometry enables the reconstruction of the thermal history of aragonitic skeletal structures and therefore helps avoiding erroneous paleoclimate reconstructions introduced by their exposure to considerable heat prior to their deposition.While additional experiments are needed to fully understand the underlying thermodynamic mechanisms controlling isotope-exchange mechanisms, our data show that a careful assessment of sample preservation including clumped-isotope analysis is necessary in future studies to avoid erroneous paleoclimate reconstructions using aragonitic shell midden constituents. Considering the higher stability of calcite relative to aragonite, it is likely that calcitic mollusc shells may well retain their primary oxygen-isotope ratio during exposure to prehistoric cooking practises although this also needs to be tested in future studies.41 and information about the function of particular sites 42, the reconstruction of specific cooking methods may also help to test the validity of modern analogues of shell midden formation from ethnographic or ethnohistoric observations43. Detailed information about specific processing techniques potentially broaden our understanding of prehistoric trading activities as the use of processing methods which prevent spoiling was most likely a prerequisite for the long-term transportation of the edible portion of marine molluscs4. Moreover, potential seasonal differences in shellfish processing techniques can contribute to our understanding of fishing and foraging seasonality. For long-term shell midden assemblages deposited over several millennia, the reconstruction of specific processing techniques may enable tracing the technological advancement over time of shell midden accretion.As shown in this study, clumped-isotope thermometry represents a unique diagnostic tool for detecting but also differentiating between certain prehistoric cooking practices employing aragonitic shell midden constituents which can provide new insights into various aspects of shell midden formation. Despite evidence of human behaviour 13C values, potential alteration of 14C signatures and subsequent uncertainties in measured radiocarbon ages obtained from aragonitic shell midden constituents have to be expected. This hypothesis is supported by previous experiments showing that radiocarbon dates measured from food crusts on prehistoric pottery fragments can also be subjected to alteration during cooking by e.g. a reversed old-wood effect44. Detailed future studies are therefore needed to quantify the effect of pre-depositional heating on radiocarbon signatures in shell midden constituents.Given the apparent alteration of shell δM. campechiensis individuals were cultured by Southern Cross Sea Farms®, at Cedar-Key, FL, USA (N29° 08′ 32.09662″ W83° 00′ 19.66703″). All individuals had the same age (14 months), similar size (~3.52 ± 0.24 cm shell height) and were reared in the same water masses throughout ontogeny. All individuals were sampled on 30 April 2015 and shipped overnight to RSMAS and sacrificed on 01 May 2015 immediately after arrival. Shells were manually cleaned with tap water, dried at room temperature and stored at ~20 °C until the experiment.In total, 23 sub-adult ®Hickory) were used to simulate roasting and burning. The coal briquettes were continually replenished to keep the burning and roasting temperature as constant as possible throughout the entire experiment. For the roasting treatment, individual shells were placed into perforated aluminium cups approximately 15 cm above the coal fire. For the burning treatment, individual shells were placed into small aluminium cups preventing any loss of shell material during the experiment or contamination and placed directly on/in between the burning coals. The shells were not buried in burning coal. The temporal and spatial temperature variability was determined for both treatments recurrently by measuring the temperature at different spots using an Omega® HH806AU thermocouple thermometer equipped with an Uxcell® K-type (nickel-chromium/nickel) high-temperature thermocouple probe (100–1250 °C). The reported treatment temperatures represent average values with the 1σ standard deviations. The measured treatment temperatures were boiling in seawater at ~100 °C, roasting over charcoal at ~174 ± 13 °C (maximum temperature range 126–205 °C) and burning on charcoal at 559 ± 95 °C (maximum temperature range 386–721 °C), see Supplemental Fig. 45 at room temperature to remove the periostracum and other organic compounds and ground using a ceramic pestle and mortar. Powdered bulk samples were stored in sealed glass vials at room temperature.We used natural seawater from Virginia Key, FL, USA heated in a glass jar on a hotplate for the boiling treatment. Evaporation was compensated by adding periodically deionized water. A ~10 cm thick layer of commercial charcoal briquettes . Thick sections (~2 mm) were prepared along the direction of maximum growth and were sampled along the direction of growth in the outer shell layer using a NewWave/Merchantec Micromill system. Posterior halves of the shells and otoliths were ground using a ceramic pestle and mortar. Powdered samples were stored in sealed glass vials at room temperature until chemical analysis. The anterior halves of the otoliths were ground in a similar manner with ~50 mg aliquots used for mineralogical analyses and radiocarbon dating. Similarly, a piece of each shell was prepared for mineralogical analysis and radiocarbon dating by cutting a slice through the ventral margin of the anterior halves using a Dremel™ tool equipped with a diamond cutting wheel.Mid-Holocene specimens were excavated from a shell midden in the Ras el Sass area, Mauritania, NW Africa. For details of the study area and spatial distribution of shell midden deposits see Barusseau Shell mineralogy of the experimental shells was measured using a PANalytical X´Pert PRO diffractometer with a Cu-tube  at RSMAS, Miami, FL, USA. Measurements were done using a continuous scan from 23.0–35.0 2θ with a step size of 0.005 2θ and a measuring time of 0.1 seconds per step. Data collection and processing was done using the software X’Pert Data Collector and X’Pert HighScore Plus, respectively. Relative abundance of aragonite and calcite were determined using the relative peak areas of aragonite  peak (2θ = 26.1°) and low magnesium calcite  peak (2θ = 29.6°) against a calibrated standard. Archaeological XRD-samples were analysed with a Philips X´Pert Pro diffractometer equipped with a Cu-tube  at the University of Bremen, Germany. Measurements were done using a continuous scan from 3–85° 2θ with step sizes of 0.016° 2θ. We used the Philips software X´Pert HighScore™ for data processing. Abundance of aragonite and calcite was determined using the relative peak areas of aragonite  peak (2θ = 26.1°) and low magnesium calcite  peak (2θ = 29.6°) against a calibrated standard. Mineralogical data of the experimental and mid-Holocene samples are reported in the supplementary information  which is also supported by radiocarbon dates from this archaeological site. Raw as well as calibrated radiocarbon ages are reported in the supplementary information . Individual sample aliquots of ~8.0 mg powder were digested for 30 minutes at 90 °C in 3.5 ml ~105% phosphoric acid (H3PO4) using a modified “Fairbanks” device50 connected to a common acid bath. Resulting CO2 was then purified on the cryogenic vacuum extraction line.Clumped-isotope analysis of the powdered bulk samples was performed at the Stable Isotope Laboratory of the Rosenstiel School of Marine and Atmospheric Science (RSMAS), Miami, FL, United States, between April 17, 2015 and March 28, 2016 using the analytical procedure described in Murray 2 was measured at RSMAS using a dual-inlet MAT-253 gas source isotope ratio mass spectrometer . Samples were measured against an in-house working-gas gas standard. We analysed each sample at a signal intensity of 12 V on mass-44 over six acquisitions of 15 sample-standard measurements. Data reduction and normalisation for ∆47 and temperature calculations followed the methods of Affek and Eiler51 and Huntington et al.52. To check for potential contamination, samples were scrutinised based on their offset of δ48 and ∆4852. Precision of clumped-isotope analysis is reported using the average standard errors. The external precision of the instrument was monitored with repeated measurement of a Carrara marble standard. The average Carrara marble ∆47 value was found to be 0.4001 ± 0.0294‰ (n = 191) which is similar to reported average values from four different laboratories of 0.395‰24 even though a different sample of the Carrara marble standard was used.Purified CO13C and δ18O of the samples by means of masses 45/44 and 46/44, respectively using a method adapted from Craig53 modified for a multi-collector mass spectrometer. Shell and otolith δ13C and δ18O values were calibrated using NBS-19 and reported relative to Vienna Pee Dee Belemnite (VPDB). External precision of δ13C and δ18O analyses was better than ± 0.055 and ± 0.117‰, respectively. All Δ47 values defined as47, R46, R45 are abundance ratios of masses 47, 46 and 45 relative to the mass 4425, were calculated using the method described by Affek and Eiler51 and Huntington et al.52. Translation into the absolute reference frame (ARF) was accomplished using the method described by Dennis et al.24 with 1000, 50 and 25 °C water equilibrated gasses. All isotope data are reported in the supplementary information  for δ13C and δ18O and generally within the external precision of the analytical method. For Δ47, the suggested changes were more significant and dependent upon the δ13C value of the material being analysed. These differences increased from essentially no changes for a sample with a δ13C value close to 0‰ to 0.04‰ for a sample with a δ13C value of −40‰. We compared our method of calculating δ13C and δ18O values modified from Craig53 and found the difference between our method and that proposed by Brand et al.57 to show only a 0.03‰ difference for samples with a δ13C difference of −40‰. This decreases to less than 0.01‰ for δ13C values of samples analysed in this study. Hence, based on the range of δ13C values of the samples measured in this study we conclude that the artefacts noted by Schauer et al.54 and Daeron et al.55 do not significantly impact the results presented here. We have, nonetheless, processed all results presented herein using the revised parameters from Brand et al.55.Recent publications by Schauer 18O.Sub-samples drilled from the outer shell layer (~80–100 μg) were measured on a Finnigan MAT 251 gas isotope ratio mass spectrometer connected to a Kiel III automated carbonate preparation device at the stable isotope laboratory in the Center for Marine Environmental Sciences (MARUM), University of Bremen, Germany. The long-term standard deviation of the in-house standard (Solenhofen limestone) was better than 0.06‰ for δWe used a one-way analysis of variances (ANOVA) with a Bonferroni-corrected t-test as post hoc test. The assessment of equal variances and normal distributions was done using a Levene’s test and a Shapiro-Wilk test, respectively. In case of non-normal distributed data , we used a Kruskal-Wallis one way ANOVA on ranks and the Dunn’s method as post hoc test. The significant levels for all statistical test were set to α = 0.05. Results of the statistical analyses are reported in the supplementary information .Supplementary Information


Supplemental Digital Content is available in the text A genome-wide association study was conducted to identify expression quantitative trait loci (eQTL) for human telomerase.We tested the genetic associations of nucleotide variants with expression of the genes encoding human telomerase reverse transcriptase (hTERT) and telomerase RNA components (TERC) in lymphoblastoid cell lines derived from 373 Europeans.P < 5 × 10–8). One eQTL (rs17755753) was located in the intron 1 of the gene encoding R-spondin-3 (RSPO3), a well-known Wnt signaling regulator. Transcriptome-wide association analysis for these eQTLs revealed their additional associations with the expression of 29 genes (P < 4.75 × 10–6), including prickle planar cell polarity protein 2 (PRICKLE2) gene important for the Wnt signaling pathway. This concurs with previous studies in which significant expressional relationships between hTERT and some genes (β-catenin and Wnt-3a) in the Wnt signaling pathway have been observed.Our results revealed 6 eQTLs associated with hTERT (This study suggested 6 novel eQTLs for hTERT and the association of hTERT with the Wnt signaling pathway. Further studies are needed to understand their underlying mechanisms to improve our understanding of the role of hTERT in cancer. The telomerases consist of human telomerase reverse transcriptases (hTERT) that regulate telomerase activity and telomerase RNA components (TERC) that are used as template RNAs for lengthening telomeres.Telomeres play key roles in human genome stability through replenishing their G-rich sequences by telomerases. TERC are targeted by imetelstat sodium (GRN163L), an inhibitor of telomerase activity in cancer therapy. Nevertheless, genetic factors that regulate the variability of gene expression are not well understood. In particular, knowledge on genetic factors is limited to genetic variability in or near the genes encoding hTERT and TERC. Some nucleotide variants  were associated with telomere length in leukocytes.,6 Some intronic variants  of hTERT have been reported to have association with hTERT expression in prostate cancers. This study aimed to identify nucleotide variants associated with mRNA expression of hTERT and TERC through genome-wide analysis.It has been reported that mRNA expression level of hTERT is highly correlated with telomerase activity in tumor cells.22.1 were used to identify expression quantitative trait loci (eQTLs). Cell lines were derived from 373 Europeans of the following 4 populations: Utah residents with northern and western ancestry (n = 91), Finns (n = 95), British (n = 94), and Toscani (n = 93). We excluded Yoruba population from the project to avoid false positive associations produced by heterogeneous genetic background. Gene expression was calculated as the sum of reads per kilobase per million mapped reads (RPKM) for all transcripts of each gene in each individual. Their corresponding genotypic data were obtained from the 1000 Genomes Project (http://www.1000genomes.org/). Genotypes with minor allele frequency <5% or with missing rate of >5% were removed. After the quality control, genotypes of 5,851,914 SNPs were used for final analysis. Ethical approval was not necessary because we dealt with publically available data.Expression data of the genes encoding hTERT and TERC in lymphoblastoid cell lines generated from the Geuvadis RNA-sequencing project2.2–8. All the statistical analyses were conducted using PLINK. Linkage disequilibrium (LD) blocks at association signals were constructed using HaploView. The identified eQTLs were analyzed to determine if they were located in transcription factor binding sites using ChIP-seq data from the regulomeDB.Linear regression analysis was performed to discover eQTLs of hTERT and TERC. Multiple testing was employed with significance threshold value of 5 × 102.3–6 . Functional enrichment analysis was conducted with the identified genes to examine their functional relevance using the DAVID functional annotation tool.Further associations between 10,518 genes and SNPs identified to be associated with the expression of hTERT were examined. For transcriptome-wide association analysis, the significance threshold value was set at 4.75 × 103P < 5 × 10–8; Fig. P > 5 × 10–8; Fig. P = 1.50 × 10–10). Another intragenic association signal was located in intron 1 of R-spondin-3 (RSPO3) gene .Genome-wide association analysis revealed 37 eQTLs associated with mRNA expression of hTERT .Transcriptome-wide association analysis with the 6 eQTLs identified to have association with hTERT expression showed significant associations with mRNA expressions of additional 29 genes , but relative binding affinity remained unknown in these studies, especially by alleles. Further research on specific underlying mechanism on their interaction with binding sites is required to understand how gene expression is affected by alleles.One of the 6 association signals covered 26 nucleotide variants in strong linkage within LOC105370092. Some of these variants were corresponding to a transcription factor binding sites uncovered by ChIP-Seq , suggesting that these variants might regulate the expression of the uncharacterized ncRNA gene. Significant associations of these variants with the expression of CTNND2 were found ( Genes identified with eQTL of rs2636908 were enriched with cytoskeleton (Supplementary Table 2). Acute withdrawals of TERT in mouse have triggered a rapid change in the expression of genes with the functions in the cytoskeleton, suggesting that hTERT might be involved in cytoskeletal changes associated with the PCP pathway.Transcriptome-wide association analysis revealed that the 6 signals associated with hTERT expression were further associated with the expression of many other genes. The most significant association was observed between prickle planar cell polarity protein 2 (PRICKLE2) and rs2636908. Since the PRICKLE2 gene and this SNP were both located in chromosome 3, a regulatory element for expression of PRICKLE2 might include this SNP or other SNPs strongly linked to it. The complex of PRICKLE2 with Vangl2 can regulate the Wnt/planar cell polarity (PCP) pathway that changes cytoskeleton. The lymphoblastoid cell lines may be suitable for identifying eQTLs of telomerase genes since they demonstrated strong telomerase activity to be immortalized.–23 In EBV-infected B cell, expression of ectopic hTERT simultaneously increased with expression of basic leucine zipper ATF-like transcription factor (BATF) which maintains EBV latent status, and hTERT silenced by shRNA can induce B-cell death through transition into lytic cycle of EBV. Thus, lymphoblastoid cells transformed by EBV can be pertinent to hTERT expression studies. Also, we suspect that the Wnt signaling pathway can be instrumental in EBV-transformed lymphoblastoid cells. This is because β-catenins are expressed in lymphoblastoid cells, and moreover, EBV-transformed lymphoblastoid cells increased the expression of β-catenin more than EBV-negative cells.–28The current study dealt with expression of telomerase genes in lymphoblastoid cell lines transformed by Epstein–Barr virus (EBV).P > 5 × 10–8). However, many false negatives are suspected because we employed a conservative significance threshold. Some “suggestive” eQTLs for TERC are presented in Fig. –5 > P > 5 × 10–8).Any eQTLs for TERC, another important component of human telomerase, were not found in this study P > 5 × 0–8. HoweThe current GWAS identified 6 novel eQTLs for hTERT. Some of these eQTLs were involved in the Wnt signaling pathway critical to the production of tumors. Functional studies are needed in order to understand the underlying mechanisms of the Wnt signaling pathway influenced by hTERT. This will provide some evidence to unveil the reason why hTERT expression is associated with tumor.


We find that onsite electronic configurational entropy is sizable in all lanthanides, and reaches a maximum value of ≈4.7 kB per oxygen vacancy for Ce4+/Ce3+ reduction. This unique and large positive entropy source in ceria explains its excellent performance for high-temperature catalytic redox reactions such as water splitting. Our calculations also show that terbium dioxide has a high electronic entropy and thus could also be a potential candidate for solar thermochemical reactions.Previous studies have shown that a large solid-state entropy of reduction increases the thermodynamic efficiency of metal oxides, such as ceria, for two-step thermochemical water splitting cycles. In this context, the configurational entropy arising from oxygen off-stoichiometry in the oxide, has been the focus of most previous work. Here we report a different source of entropy, the onsite electronic configurational entropy, arising from coupling between orbital and spin angular momenta in lanthanide  f orbitals.Solid-state entropy of reduction increases the thermodynamic efficiency of ceria for two-step thermochemical water splitting. Here, the authors report a large and different source of entropy, the onsite electronic configurational entropy arising from coupling between orbital and spin angular momenta in  It is used in three-way exhaust automotive catalysts6, solid-state fuel cells10, two-step thermochemical water splitting cycles (TWSC)14, low-temperature water–gas shift reactions15, and several other industrial catalytic applications20. To a large extent, the performance of ceria in these applications depends strongly on its oxygen storage capacity and facile Ce4+/Ce3+ redox reaction.Ceria  temperature TL:Ceria is a highly promising redox active material for TWSCδL and δH are, respectively, the low-temperature  and high-temperature  oxygen non-stoichiometries21. Meredig and Wolverton22 examined the thermodynamics of these two-step reaction cycles and showed that a key thermodynamic quantity for increased efficiency is a large solid-state entropy of reduction 22 2CeO2 → Ce2O3 + 2 is the only one that shows 23:Sconf includes both electronic and ionic configurational entropy of ceria. The electronic part associated with electron or hole localization contributes to the total entropy in the same fashion as the ionic configurational entropy due to vacancies. Both configurational contributions can be determined accurately by Monte Carlo simulations based on a cluster expansion24. ΔSvib is the vibrational entropy of reduction and kB per oxygen atom25—hereafter, all the reported entropic contributions are per one oxygen vacancy, unless stated otherwise. By excluding the gas phase entropy, we have the solid-state entropy of reduction The entropy of reduction of Eq.  in the T2 by Bevan et al.26 and successively Panlener et al.23 showed that this quantity has a logarithmic dependence on the non-stoichiometry, δ, of CeOδ2−. They associated this behavior with the change in the configurational entropy (ΔSconf) with varying degrees of non-stoichiometry, while vibrational entropy was estimated to be negligible. Grieshammer et al.27 calculated the ΔSvib of defect formation in ceria at a fixed non-stoichiometry and found it to be ~ 2.5 kB, which is not negligible, but smaller than other entropic contributions. Gopal et al.28 did comprehensive calculations using Monte Carlo simulations based on the DFT-derived cluster expansion Hamiltonian. They calculated all configurational and vibrational entropic contributions + for several other lanthanide cations  that are stable in two valence states and for which reliable spectroscopic data are available (see ref. 29). We calculate the crystal field (CF) parameters of Ce3+ in the host fluorite CeO2 structure, where each cerium atom experiences a cubic crystal field from eight oxygen atoms. The results show that the electronic entropy of reduction of Ce4+ is the highest among f elements, even surpassing the calculated configurational entropy28 at large off-stoichiometry values, thereby explaining the unique entropic properties of the reduction of CeO2.In the present paper, we demonstrate that particularly in the case of lanthanides, a different type of electronic entropy should be considered. This source of electronic entropy, which becomes important for elements with partially filled L–S) coupling. In multi-electron atoms, L–S coupling between orbital and spin angular momenta (L–S) results in the creation of microstates, which are further split by CF interactions. As the interaction of the very contracted f-orbitals of the lanthanide ions with the CF is small30, it is reasonable to first treat the electronic energy levels of the lanthanide oxides by considering only free ion energies and then subsequently apply the crystal field. For f-orbitals of lanthanides, defining the electronic configuration by just number of valence electrons (4fn) is far less descriptive than the term symbol described by the L–S coupling scheme31. In this scheme, coupling of orbital and spin angular momentum results in S+12LJ term symbols in which 2S + 1 is the spin-multiplicity, L is the total orbital quantum number and J is the total angular momentum quantum number, ranging from |L + S| to |L − S| by steps of one. Indeed, Hund’s rules imply that the term with the largest value of multiplicity (2S + 1) is the most stable one. If several terms have the same multiplicity, then the term with the largest L is the most stable one. If the f shell is less than half-filled then the state with the lowest J has the lowest energy. On the other hand, if the f shell is more than half-filled then the state with the highest J has the lowest energy. The degeneracy of each J-multiplet is (2J + 1) and the total number of microstates (m) for a given term symbol S+12L is (2S + 1) × (2L + 1).Electronic configurational entropy J states to several subsets and breaks the spherical symmetry of the f-shell charge distribution, depending on the local symmetry of the ionic environment. Here, we used a fully ab-initio method, opposing crystal potential (OCP)32, to calculate the crystal field parameters of Ce3+ in the host fluorite CeO2 structure, where each cation is coordinated to eight oxygen atoms. This method is motivated by the above mentioned lowering of the spherical symmetry of the d/f charge distribution, e.g., to cubic symmetry in CeO2, due to crystal field interactions. OCP iteratively computes and subtracts CF interactions in order to effectively cancel them and recover spherical d/f charge distribution upon convergence. An on-site potential 32 for details). As previously discussed32, the goal of the constrained DFT calculations was to extract CF parameters rather than to introduce band gap and self-interaction corrections. Consequently the commonly adopted LDA + U approach was not necessary. As a benchmark, the CF parameters of PrO2 from constrained OCP calculations were in good agreement with our previous LDA + U calculations that directly compute the energy difference of different crystal field levels33.When a free ion is placed in a crystal, the CF further splits each of the degenerate f1 (Ce3+) energy-level splitting scheme in the presence of spin-orbit coupling (SOC) and calculated CF. Without CF, the f1 states split into 2F5/2 and 2F7/2 separated by approximately 0.28 eV29. The CF interaction further splits the six-fold degenerate 2F5/2 ground state into a four-fold degenerate Γ8 and two-fold degenerate Γ7 subsets, separated by 0.12 eV. Crystal field—calculated by OCP method for Ce3+—splits the eight-fold degenerate 2F7/2 state into states with energies 0.25, 0.32, 0.46 eV. These results are comparable with experimental and theoretical CF splittings of Ce3+ doped in CaF236 and YAG (Y3Al5O12)37 hosts.Figure Ei and degeneracy (gi) of each microstate, m different microstates can be calculated bypi) to the state with energy Ei is proportional to the Boltzmann factor byZ is the partition function defined byBy having the energy p) and the total number of microstates (m) that varies from 14 in f1/f13 to 66 in f5/f9 , a large fraction of microstates are accessible due to the increased thermal excitation, as seen in Fig. 3+ approaches the ideal limit of kB ln(m). As seen in Table f orbitals get occupied by a single electron—generating 14 microstates with ln (14) kB (≈2.6 kB) entropic contribution—the system gains a large entropy. For example f1 (Ce3+) is 2.34 kB and f4 (Nd2+) is 3.52 kB.Equations  and 6)  6) indicmentclass2pt{minimmentclass2pt{minimmentclass2pt{minim38 extracted the electronic entropy contribution of lanthanide ions (Ln3+) in lanthanide trihalides from absolute entropy data. Our calculated electronic entropies per ion at ≈300 K in units of kB compared with Myers et al.38 data  are the following: Ce3+, 1.79 (1.77); Pr3+, 2.19 (2.18), Nd3+, 2.30 (2.27); Eu3+, 1.13 (1.10); Tb3+, 2.56 (2.54). The calculated L–S coupling shows excellent agreement with previously reported data. Below, we will consider the effect of crystal field splitting on the calculation of Myers et al.fn) and after (fn+1) reduction is relevant, 4+ ions are reduced per oxygen vacancy. As discussed in the previous paragraph and shown in Table f orbital, in TWSC systems the electronic entropy of reduction reaches its maximum, when the entropy of the oxidized state is zero, 4+ → Ce3+, which undergoes an f0 → f1 redox reaction. Having the oxidized state f0 (1S) with zero onsite electronic entropy is a unique feature of ceria, resulting in a large kB per oxygen vacancy, which is a maximum for the reduction of any rare-earth cation. We assert that this unique entropic characteristic of the Ce4+/Ce3+ redox reaction helps facilitate the TWSC properties of CeO2. The second largest value of 4+ → Tb3+) with 2.30 kB per oxygen vacancy at 1900 K  states but with only one orbital degeneracy (L = 0), while Tb3+, has an orbital degeneracy of 7 (L = 3), providing extra entropy, see Table 4+ based materials promising candidates for TWSC applications, as Tb, like Ce, is stable in two valence states (Tb4+/Tb3+). This prediction agrees with a recent thermodynamic study that also suggested39 TbO2 as a potential candidate for TWSC applications.For TWSC applications, the absolute electronic entropy does not matter, only the entropy difference before kB = 4.16 kB per oxygen vacancy. On the other hand non-reduced Eu3+ has a large number of total degeneracy of 49 ), where n depends on the defect structure and here n = 3, is ≈10.4 kB. However, we note that a system with extensive ordering of oxygen vacancies43, such as ceria, will have short range order and hence the actual configurational entropy is non-ideal and smaller than the ideal solution model. For instance, the non-ideal ΔSconf + ΔSvib, calculated by Monte Carlo simulation based on a cluster expansion Hamiltonian, is about 5.9 kB28, less than half of the ideal ΔSconf. Our calculations show that the neglected electronic entropy (ΔSelec) is >4.7 kB, which is comparable to these other widely considered sources of entropy and can explain the ≈5 kB gap between the calculation and experiment. We note that as long as oxygen vacancy is compensated by two polarons Ce′  δ). Being independent of off-stoichiometry implies that at large δ the contribution from the electronic entropy surpasses that of the configurational entropy (which decreases with δ) and becomes the major entropy contribution. Using the calculated ΔSconf in ref. 28, we estimate that this crossover occurs at (δ ≈ 0.05).Finally, we compare entclass1pt{minima28 and the experimental data of Panlener et al.23 for small deviations from stoichiometry corresponding to δ < 0.03. At larger deviations, adding a constant onsite electronic entropy to the vibrational and configurational entropies from ref. δ values most of the polarons become bound to oxygen vacancies forming singly charged V3+ or neutral V3+ complexes44; the proximity of Ce3+ to an oxygen vacancy could slightly modify the electronic structure and hence reduce the electronic entropy associated with Ce3+, but as already discussed the overall effect of oxygen vacancy on the energy levels40 and electronic entropy is expected to be small. Furthermore, the experimental measurements of Panlener et al.23 found that the enthalpy of reduction is composition dependent even at very small δ; however, this finding has been challenged due to the large experimental uncertainty26. As the entropy is obtained from TΔS = ΔH − ΔG, the entropy values of Panlener et al. may be contaminated by contributions from the composition dependent contribution to ΔH. Indeed, the results of ref. 28 suggest that the entropy stays approximately constant for δ in the 0.05 to 0.15 range, while the data of Panlener et al.23 show a pronounced decrease in this range.Our results show that the electronic contribution to the entropy of reduction explains the gap between the results of the currently most detailed theoretical calculations of ref. 45. Unfortunately, the experimental data here are also contradictory. The data of Tuller and Nowick45 suggest that for small δ the spin degeneracy factor is one, which contradicts the Kramers theorem requiring that the ground state must be at least doubly degenerate. However, a later study by the same authors6 concluded that the agreement between the polaron model with spin degeneracy one and the experimental data for the Seebeck coefficient was poor, especially at low δ where impurities were thought to play an important role. On the theory side, the vibrational entropy of an isolated Ce3+ polaron has not been established accurately. Grieshammer et al.27 have calculated a very large value of about 7 kB for the entropy of polaron formation at zero pressure, but the largest contribution to this value is due to a volume contribution from the CeO2 host, which was treated in an approximate fashion. Such a large positive entropy is inconsistent with the available data on the Seebeck coefficients in the dilute limit45. Hence, thermoelectric measurements on pure, well-equilibrated samples of CeO2 and more accurate calculations of the vibrational entropy associated with free polarons are highly desirable.Measurements of the Seebeck coefficient provide another means of estimating the electronic entropy contribution in the dilute limit where all polarons are unbound3+ and Pr4+ and found that at temperatures above 1000 K, CF interactions affect the Selec by >3%. The results show that, in ceria, the magnitude of the entropy of reduction due to the commonly neglected onsite electronic entropy (ΔSelec) reaches a maximum of 4.7 kB per oxygen vacancy, which is twice as large as the vibrational entropy contribution and can be larger than the configurational entropy. This surprisingly large entropy is the result of the very unique electronic structure of cerium in ceria, where redox reactions change its electronic state from f0 to f1. These entropic properties, together with the excellent chemical stability and tolerance for large non-stoichiometry, put ceria in a unique position for two-step solar thermochemical CO2/H2O splitting cycles. In addition, we find that Tb (IV) based materials have the next highest electronic entropy, for Tb4+ → Tb3+ redox reactions. We therefore propose compounds containing Tb4+ should be experimentally investigated as promising candidates for TWSC applications.We calculated electronic entropies of different lanthanides in the presence of SOC and CF. We calculated CF splittings for Ce46 using the Perdew–Becke–Ernzerhof47 functional, projector augmented-wave potentials48, a 6 × 6 × 6 k-point mesh an energy cut-off of 520 eV and a convergence criterion of 10−10 eV/atom for the electronic structure to ensure satisfactory convergence of CF parameters. One electron was added to the neutral CeO2 cell in order to model Ce3+. Note that as previously discussed32, the goal of the constrained DFT calculations was only to extract CF parameters rather than to introduce band gap and self-interaction corrections.Constrained DFT calculations, required for the calculation of crystal field parameters using OCP method, were performed in the VASP packageThe data that support the findings of this study are available within the paper or from the corresponding author on request.Peer Review File


ZnO is a widely used metal-oxide semiconductor for photovoltaic application. In solar cell heterostructures they not only serve as a charge selective contact, but also act as electron acceptor. Although ZnO offers a suitable interface for exciton dissociation, charge separation efficiencies have stayed rather poor and conceptual differences to organic acceptors are rarely investigated. In this work, we employ Sn doping to ZnO nanowires in order to understand the role of defect and surface states in the charge separation process. Upon doping we are able to modify the metal-oxide work function and we show its direct correlation with the charge separation efficiency. For this purpose, we use the polymer poly(3-hexylthiophene) as donor and the squaraine dye SQ2 as interlayer. Interestingly, neither mobilities nor defects are prime performance limiting factor, but rather the density of available states around the conduction band is of crucial importance for hybrid interfaces. This work highlights crucial aspects to improve the charge generation process of metal-oxide based solar cells and reveals new strategies to improve the power conversion efficiency of hybrid solar cells. Besides high power conversion efficiencies due to an efficient charge generation process, it is essentially important to demonstrate devices with high ambient- and photostability made of low cost and nontoxic materials6. In the past, there have been numerous research efforts to improve the low performance of hybrid solar cells with respect to purely organic or inorganic devices7. Studies on the influence of crystal defects in the metal-oxides, different architectures, surface modifications, distinct recombination, and different organic/inorganic layers revealed performance limitations that are different to purely organic or inorganic devices16. Though none of these studies fully explained the rather low performance, it is generally invoked that the charge generation process at a polymer metal-oxide interface is not very efficient17. For this reason, it is fundamentally important to identify and address limiting factors to efficient charge separation and collection. Only recently, it has been shown that the charge generation process in hybrid solar cells is significantly enhanced if exciton separation happens at the metal-oxide-dye interface, while excitons from the polymer are resonantly transferred to the dye18. Additionally, trap and surface states on the metal-oxide surface are found to have a large impact on charge separation and recombination while the large binding energies of bound charge pairs at the organic/inorganic interface are a significant obstacle for exciton dissociation and charge separation23. Inorganic n-type metal-oxides such as ZnO, TiO2, and SnO2 are frequently used as electron transport layers and p-type conjugated polymers such as P3HT, PCPDTBT, and spiro-OMeTAD are used as hole transport layers28. Within this material combination it is important to note that typical hole mobilities in P3HT (10−6–10−4 cm2 V−1 s−1)30, PCPDTBT (10−4–10−3 cm2 V−1 s−1)31 and spiro-OMeTAD (10−4–10−3 cm2 V−1 s−1)32 are orders of magnitude lower than electron mobilities in bulk ZnO (200–300 cm2 V−1 s−1)35, ZnO nanorods (1–30 cm2 V−1 s−1)37, ZnO nanowires (20–220 cm2 V−1 s−1)38, single ZnO nanowires (1000 cm2 V−1 s−1)39, TiO2 (0.1–1 cm2 V−1 s−1)33, or SnO2 (100–250 cm2 V−1 s−1)40. This large discrepancy in mobilities raises the question on the influence of space charge formation and the impact on device performance.The concept of hybrid metal-oxide polymer solar cells is driven by the motivation of combining advantages of organic and inorganic solar cells42. By usage of a broad range of different dopants like Mg43, Sr44, Ca45, N46, Sn47 and Cs48 power conversion efficiencies have increased significantly. In general, improved open circuit voltages have been explained by an uplift in the conduction band or a decreased conductivity, while larger photocurrents are justified by rougher surfaces that enhance the interfacial area or by a reduced electron concentration and recombination at surface traps9. Although interfacial characteristics have been extensively investigated, little is known about the role of the metal-oxide bulk properties and its contribution towards interfacial charge separation. Herein, we investigate the effect of the bulk properties on the charge dynamics in hybrid solar cells using pristine and doped ZnO-P3HT as a model system. All cells are fabricated with a self-assembled monolayer (SAM) of the squaraine dye SQ2 acting as an interlayer between polymer and metal-oxide in order to not only track changes in surface characteristics but also to reduce the role of surface states. Interestingly, we find the influence of space-charges to be rather low. Our experiments show that a low work function of the metal-oxide favours a large driving force for exciton separation while intrinsic charges in the metal-oxide reduce the photocurrent generation.The impact of doping on metal-oxide properties and resulting consequences on solar cell performance have been studied extensively in the past−6 mbar.The hybrid solar cells fabricated in this work consist of an ITO/ZnO nanowire (NW)/SQ2/P3HT/Ag or ITO/Sn:ZnO NWs/SQ2/P3HT/Ag structure which is shown in Fig. 4+ doping of 0 mol%, 1 mol%, 2 mol% and 3 mol%. All samples were measured without any further cleaning treatment prior to their introduction into an ultra-high vacuum chamber. For XPS, the Mg Kα line at 1253.6 eV  is used in combination with an analyser (Leybold EA-11) with a pass energy of 100 eV, giving a full width at half maximum (FWHM) of 1.3 eV for the Au 4f7/2 peak. The analysed area was approximately a 2 × 5 mm2 rectangle, positioned near the geometric centre of each sample. XPS analysis was carried out at 0° take-off angle . In all XPS spectra, the binding energy (BE) of the predominant aliphatic contribution to the C 1 s peak at 284.8 eV was used as a measured BE reference. For the UPS measurements, the He I excitation line (21.2 eV) was used, and a negative bias of 12.23 V was applied to the specimen in order to separate the high binding energy cut-off from the analyser. The error in our UPS measurements is 0.1 eV. Since ZnO is a photosensitive material all UPS spectra were taken prior XPS measurements. Crystallographic properties were carried out by X-ray diffraction (XRD) via a Bruker AXS D8 Advance diffractometer and recorded at 2θ range 20–80° at a rate of 0.004 °/s with a Cu Kα source. A Keithley 2400 source meter was used for current density-voltage (J-V) and external quantum efficiency (EQE) measurements using a home-built LabVIEW program. The devices were illuminated with a LOT-Oriel LS0106 solar simulator equipped with an AM 1.5 G filter using a light intensity of 100 mWcm−2. For light intensity dependent J-V measurements a series of neutral density filters was used for attenuation of the incident light intensity. The cell area of 0.125 cm2 was defined through a shadow mask after calibrating with a Fraunhofer Institute certified Si reference solar cell using a KG5 filter. For EQE measurements the solar cells were illuminated with monochromatic light (LOT-Oriel Omni 150 monochromator in combination with a 150 W Xe lamp). The acquired absorption spectra were recorded using a Cary 5000 UV-VIS-NIR spectrometer equipped with an integrating sphere. For conductivity measurements a platinum-iridium (PtIr) (4:1) tip was used as top contact on Zn NW/ITO structures. In these measurements, samples are investigated in dry nitrogen and contacted to a Keithley 2401 serving as a voltage source and measurement unit for transient currents.The surface morphology and structural properties of nanowires were characterized using a scanning electron microscopy (SEM) (Zeiss Crossbeam 1540 SEM) at 6 keV accelerating voltage. Additionally, X-Ray and ultra-violet photoemission spectroscopy (XPS and UPS) measurements were carried out in order to characterize the chemical composition of their surfaces as well as the electronic structure of the valence band (VB), respectively. For this purpose, ZnO NWs on ITO substrates are used with nominal SnFigure 49. Upon Sn doping, we see a strong rise in JSC which results in an improvement of power conversion efficiency by more than 30%. In Fig. 18. We further track changes in surface properties by means of XPS and compare results to XRD and high resolution TEM (HRTEM) data. As shown in the Supplementary Information, XRD measurements reveal a loss in crystallinity upon Sn doping while XPS spectra are very similar and indicate only traces of SnOx on the surface with otherwise similar elemental composition. Note that Sn is not clearly visible on the XPS wide scan due to its very low concentration and distribution in the NWs. Indium of the substrate is not present either, which implies that the nanowires are closely packed, as it is confirmed by the electron microscopy observations.Table To investigate the electronic structure  of the pristine and doped ZnO, UPS measurements were performed Fig. . The mid51 and gains in intensity and contribution to the overall spectra as Sn doping increases. This enhancement of hybridization between the cation’s s-orbitals and the p-orbitals of neighbouring oxygen atoms in the Sn doped NW spectra is expected to contribute in overcoming the electron localization bottleneck and facilitating electron transfer. We suggest that Sn doping may favour orbital hybridization as a result of charge transfer between Sn and ZnO. Note that for the NW spectra, an upward binding energy shift is observed compared to thin film spectra, which is attributed to surface band bending, as well as an overall spectral broadening and an increase of the density of states of the hybridized metal and oxygen orbitals.The analysis of the full UPS spectrum reveals some additional interesting observations on the ZnO composition. In general, ZnO exhibits two characteristic peaks around 6 eV and 11 eV. The former can be attributed to 2p orbitals of oxygen from Zn-O bonds while the latter results from Zn 3d orbitals. In our NWs, both peaks are not very pronounced. In addition, the spectral shape shifts continuously towards higher binding energies upon doping and a broad peak at 8 eV becomes dominant. This signal has been correlated with a hybridization of Zn 4s-O 2p orbitals53.As our XRD results suggest the growth of the NWs is always in (0002) direction and is independent of doping concentration, we can conclude that crystal growth is still largely preserved upon doping. However, a strong hybridization of Zn 4 s - O 2p orbitals points to a broad density of energetically lower lying states. This has been proposed and demonstrated experimentally for a variety of metal-oxide based materialsOC as the CBM and the WF change upon doping, we do not observe such a significant dependence  by means of absorbance measurements  for all samples.The contact resistances were taken from the linear plot and are calculated as R(0%) ≈ 250 kΩ, R(1%) ≈ 17 MΩ, R(2%) ≈ 250 MΩ, and R(3%) ≈ 25 MΩ, respectively. Between 0 and 0.2 V, a linear regime in the Fowler-Nordheim plot Fig.  indicatec is the density of states in the conduction band, and A and B are constants containing parameters such as the effective mass, the static dielectric constant, and the energy level distribution of the traps61. The slope as well as the vertical axis intersection point are increased for 1 and 2 mol% doped samples and thus, the factor τNc is enhanced. For a 3 mol% doping concentration this factor is decreasing again. In general, a higher doping concentration should result in a larger number of mobile electrons in the conduction band (increased Nc) such that an increased slope points to an enhanced scattering time τ.The Poole-Frenkel Fig.  behaviouUpon such variation in the metal-oxide conductivity, improved solar cell performances could also be explained by a reduced influence of space-charge limitations. However, (as shown in the Supplementary Information) mobilities play only a minor role in performance enhancements since the space charge limited current region is unaffected and we observe a very efficient charge collection at short circuit condition for all devices while the FF is constant.18, showing that energy transfer is favoured compared to direct electron injection, this is a surprising result. It indicates the crucial role of a large density of states close to the conduction band while a large number of free electrons in the metal-oxide prevent excitons in the polymer from successful and efficient dissociation. As a result, photocurrent contributions by the polymer are reduced.In summary, we have investigated ZnO NW-P3HT hybrid solar cells as a model system where defects and surface properties of ZnO are varied via Sn-doping. Upon doping the WF increases while the photocurrent in solar cell devices is enhanced no matter which doping concentration is applied. Although dye absorption is reduced, photocurrent contributions from the polymer are improved. Based on previous workFurthermore, neither mobilities nor defect states close to the conduction band are primary performance limiting factors in hybrid solar cells. Instead, the energetic position of the WF is decisive whether electron injection is possible or hindered.Within this work we can show, that doping of the metal-oxide offers a promising strategy to tune the relative energetic position of the WF and therefore improve the charge separation efficiency for efficient hybrid solar cells. Our results reveal the potential of further device improvements if a deeper understanding on the role of metal-oxide properties is gained especially if the WF can be tuned without the need of dopants or reduction in crystallinity.Supplementary Information