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Analysis of contents of heavy metals in wasteland soilThe test results show (Table 5) that the contents of Hg, Cd, As, Pb, Cr, Zn, Ni and Cu in the surface soil within Shigetai Coal Mine vary from 0.043 to 0.255, 0.44 to 2.23, 2.66 to 18.40, 11.80 to 42.80, 40.50 to 118.60, 18.90 to 70.10, 4.31 to 28.10, 4.96 to 46.25 mg/kg, respectively; the average contents of Hg, Cd, As, Pb, Cr, Zn, Ni and Cu are 0.128, 1.03, 4.73, 23.08, 76.22, 46.94, 16.11 and 12.10 mg/kg, respectively. The average contents of Hg, Cd, Pb and Cr in soil within the research area are 2.03, 1.36, 1.11 and 1.23 times of the soil background values in Shaanxi Province, respectively. The average contents of As, Zn and Cu are lower than the soil background value in Shaanxi Province, but the maximum contents of these three elements are 1.65, 1.01 and 2.16 times of the soil background values in Shaanxi Province, respectively. It is reported that the average concentration of lead in agricultural soil affected by coal mines is relatively high (433 mg kg−1)38. Lead is usually related to minerals in coal and occurs mainly in the form of sulfide such as PbS and PbSe39. In addition, aluminosilicate and carbonate also contain lead40. Chromium is a non-volatile element, which is related to aluminosilicate minerals41. In the mining process, chromium may be accumulated in coal, gangue or other tailings, and then enter the soil or water body through rain leaching42.Table 5 Statistics of contents of heavy metals in wasteland soil (n = 79).Full size tableThe coefficient of variation (CV) of Hg and Cd contents in soil within the research area is 0.050 and 0.37, respectively, with moderate variation, indicating that the content of these two heavy metals is less affected by the external factors; the coefficient of variation (CV) of As, Pb, Cr, Zn, Ni and Cu contents is 2.81, 7.46, 18.00, 13.51, 5.44 and 5.64, respectively, with strong variation (CV  > 0.50)43, indicating that the content of these eight heavy metals may be affected by some local pollution sources. The skewness coefficient (SK) ranges from − 3 to 3, and the larger its absolute value, the greater its skewness. When SK  > 0, it is positive skewness; when SK  More

Fauna, culture and chronology datasetsA geo-referenced dataset of chronometric dates covering the late MIS 3 (55–30 kyr cal bp) was compiled from the literature (dataset 1). The dataset included 363 radiocarbon, thermoluminescence, optically stimulated luminescence and uranium series dates obtained from 62 archaeological sites and seven palaeontological sites. These chronological determinations were obtained from ten palaeontological levels and 138 archaeological levels. The archaeological levels were culturally attributed to the Mousterian (n = 75), Châtelperronian (n = 6) and Aurignacian (n = 57) technocomplexes. A number of issues can potentially hamper the chronological assessment of Palaeolithic technocomplexes from radiocarbon dates, such as pretreatment protocols that do not remove sufficient contaminants or the quality of the bone collagen extracted. Moreover, discrepancies in cultural attributions or stratigraphic inconsistencies are commonly detected in Palaeolithic archaeology. Information regarding the quality of date determinations and cultural attribution or stratigraphic issues is provided in the Supplementary Information.Our dataset also included the presence of herbivore species recovered from each archaeo-palaeontological site (hereafter referred to as local faunal assemblages (LFAs)), their body masses and their chronology. The mean body mass of both sexes, for each species, was obtained from the PHYLACINE database53 and used in the macroecological modelling approach described below (see ‘Carrying capacity of herbivores’). For visual representation purposes, the herbivore species were grouped into four weight categories: small (500 kg). The chronology of the occurrence of each herbivore species was assumed to be the same as the dated archaeo-palaeontological layer where the species remains were recovered. Thus, to estimate the chronological range of each species in each region, all radiocarbon determinations were calibrated with the IntCal20 calibration curve54 and OxCAL4.2 software55. The BAMs were run to compute the upper and lower chronological boundaries at a CI of 95.4% of each LFA (see ‘Chronological assessment’ for more details). One of the purposes of the current study was to estimate the potential fluctuations in herbivore biomass during the stadial and interstadial periods of the late MIS 3. Accordingly, the time spans of the LFAs were classified into the discrete GS and GI phases provided by Rasmussen et al.51.Geographic settingsThe Iberian Peninsula locates at the southwestern edge of Europe (Fig. 1). It constitutes a large geographic area that exhibits a remarkable diversity of ecosystems, climates and landscapes. Both now and in the past, altitudinal, latitudinal and oceanic gradients affected the conformation of two biogeographical macroregions with different flora and fauna species pools: the Eurosiberian and Mediterranean regions13,46. In the north, along the Pyrenees and Cantabrian strip, the Eurosiberian region is characterized by oceanic influence and mild temperatures in the present day, whereas the Mediterranean region features drier summers and milder winters (Fig. 1). Between the Eurosiberian and Mediterranean regions, there is a transitional area termed Submediterranean or Supramediterranean. Lastly, the Mediterranean region is divided into two distinctive bioclimatic belts: (1) the Thermomediterranean region, located at lower latitudes, with high evapotranspiration rates and affected by its proximity to the coast; and (2) the Mesomediterranean region, with lower temperatures and wetter conditions (Fig. 1).Previous studies have shown that zoocoenosis and phytocenosis differed between these macroregions in the Pleistocene13,46. However, flora and fauna distributions changed during the stadial–interstadial cycles in the Iberian Peninsula, which suggests potential alterations in the boundaries of these biogeographical regions. The modelling approach used in this study to estimate the biomass of primary consumers is dependent on the reconstructed NPP and the herbivore guild structure in each biogeographical region. To test the suitability of the present-day biogeographical demarcations of the Iberian Peninsula during MIS 3, we assessed whether the temporal trends of NPP and the composition of each herbivore palaeocommunity differed between these biogeographical regions during the MUPT.Chouakria and Nagabhusan56 proposed a dissimilarity index to compare time series data by taking into consideration the proximity of values and the temporal correlation of the time series:$${rm{CORT}}(S_1,S_2) = frac{{mathop {sum}nolimits_{i = 1}^{p – 1} {left( {u_{left( {i + 1} right)} – u_i} right)} (v_{(i + 1)} – v_i)}}{{sqrt {mathop {sum}nolimits_{i = 1}^{p – 1} {(u_{(i + 1)} – u_i)^2} } sqrt {mathop {sum}nolimits_{i = 1}^{p – 1} {(v_{(i + 1)} – v)^2} } }}$$
(1)
where S1 and S2 are the time series of data, u and v represent the values of S1 and S2, respectively, and p is the length of values of each time series. CORT(S1, S2) belongs to the interval (−1,1). The value CORT(S1, S2) = 1 indicates that in any observed period (ti, ti+1), the values of the sequence S1 and those of S2 increase or decrease at the same rate, whereas CORT = −1 indicates that when S1 increases, S2 decreases or vice versa. Lastly, CORT(S1, S2) = 0 indicates that the observed trends in S1 are independent of those observed in S2. To complement this approach by considering not only the temporal correlation between each pair of time series but also the proximity between the raw values, these authors proposed an adaptive tuning function defined as follows:$$d{rm{CORT}}left( {S_1,S_2} right) = fleft({{rm{CORT}}left( {S_1,S_2} right)} right)times dleft( {S_1,S_2} right)$$
(2)
where$$fleft( x right) = frac{2}{{1 + exp left( {k,x} right)}},k ge 0$$
(3)
In this study, k was 2, meaning that the behaviour contribution was 76% and the contribution of the proximity between values was 24%57. Hence, f(x) modulates a conventional pairwise raw data distance (d(S1,S2)) according to the observed temporal correlation56. Consequently, dCORT adjusts the degree of similarity between each pair of observations according to the temporal correlation and the proximity between values. This function was used to compare the reconstructed NPP between biogeographical regions during MIS 3 in the Iberian Peninsula. However, two different biogeographical regions could have experienced similar evolutionary trends in their NPP, even though their biota composition was different. Therefore, this analysis was complemented with a JSI to assess whether the reconstructed herbivore species composition in each palaeocommunity differed among biogeographical regions during the late MIS 3. The JSI was based on presence–absence data and was calculated as follows:$${rm{JSI}} = frac{c}{{(a + b + c)}}$$
(4)
where c is the number of shared species in both regions and a and b are the numbers of species that were only present in one of the biogeographical regions. Therefore, the higher the value the more similar the palaeocommunities of both regions were.Chronological assessmentPivotal to any hypothesis of Neanderthal replacement patterns by AMHs is the chronology of that population turnover. To this end, we used three different approaches to provide greater confidence in the results: BAMs, the OLE model and SPD of archaeological assemblages. As detailed below, each of these approaches provides complementary information about the MUPT.First, we built a set of BAMs for the Mousterian, Châtelperronian and Aurignacian technocomplexes in each region during the MIS 3. As stated above, we compiled the available radiocarbon dates for Iberia between 55 and 30 kyr cal bp. However, not all dates or levels were included in the Bayesian chronology models. Radiocarbon determinations obtained from shell remains were incorporated in the dataset (dataset 1); however, the local variation of the reservoir age was unknown from 55 to 30 kyr bp. Because of uncertainties related to marine reservoir offsets, all BAMs that incorporated dates from marine shells were run twice: including and excluding these dates. All of the archaeological levels with cultural attribution issues or stratigraphic inconsistencies were excluded. The Supplementary Note provides a detailed description of the sites, levels and dates excluded and their justification. All BAMs were built for each technocomplex using the OxCAL4.2 software55 and IntCal20 calibration curve54.Bayesian chronology models were built for each archaeological and palaeontological level. Then, the dates associated with each technocomplex were grouped within a single phase to determine each culture’s regional appearance or disappearance. Our interest was not focused on the chronological duration of the Mousterian, Châtelperronian and Aurignacian cultures, but on the probability distribution function of the temporal boundaries of these cultures in each region. Thus, this chronological assessment aims to provide an updated chronological frame for Neanderthal replacement by AMHs in Iberia. For this reason, we did not differentiate between proto- and early Aurignacian cultures, since both are attributed to AMHs.In each BAM, we inserted into the same sequence the radiocarbon dates associated with a given technocomplex within a start and end boundary to bracket each culture, which allowed us to determine the probability distribution function for the beginning and end moment of each cultural phase6. The resolution of all models was set at 20 years. We used a t-type outlier model with an initial 5% probability for each determination, but when more than one radiocarbon date was obtained from the same bone remain, we used an s-type outlier model and the combine function. The thermoluminescence dating likelihoods were included in the models, together with their associated 1σ uncertainty ranges. When dates with low agreement ( More

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ChemicalsErbium (III) oxide nanoparticles (Er2O3-NPs) were purchased from Sigma-Aldrich Chemical Company (Saint Louis, USA) with pink appearance and product number (203,238). Powders of Er2O3-NPs with 99.9 trace metals basis were suspended in deionized distilled water to prepare the required concentrations and ultra-sonicated prior use.Cell lineHuman hepatocellular carcinoma (Hep-G2) cells were obtained from Nawah Scientific Inc., (Mokatam, Cairo Egypt). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL) and heat-inactivated fetal bovine serum (10) in humidified, 5% (v/v) CO2 atmosphere at 37 °C.Characterization of Er2O3-NPsThe purchased powders of Er2O3-NPs were characterized using a charge coupled device diffractometer (XPERT-PRO, PANalytical, Netherlands) to determine its X-ray diffraction (XRD) pattern. Zeta potential and particles’ size distribution of Er2O3-NPs were also detected using Malvern Instrument Zeta sizer Nano Series (Malvern Instruments, Westborough, MA) equipped with a He–Ne laser (λ = 633 nm, max 5mW). Moreover, transmission electron microscopy (TEM) imaging was done to detect the shape and average particles’ size of Er2O3-NPs suspension.Sulforhodamine B (SRB) cytotoxicity assaySulforhodamine B (SRB) assay was conducted to assess the influence of Er2O3-NPs on the proliferation of cancerous Hep-G2 cells12. Aliquots of 100 µl of Hep-G2 cells suspension containing 5 × 103 cells were separately cultured in 96-well plates and incubated for 24 h in complete media. Hep-G2 Cells were then treated with five different concentrations of Er2O3-NPs (0.01, 0.1, 1, 10 and 100 µg/ml) incubated for 24 h or (0.1, 1, 10, 100 and 1000 µg/ml) incubated for 72 h. After 24 or 72 h of Er2O3-NPs exposure, cultured cells were fixed by replacing media with 10% trichloroacetic acid (TCA) and incubated for one hour at 4 °C. Cells were then washed five times with distilled water, SRB solution (0.4% w/v) was added and incubated cells in a dark place at room temperature for 10 min. All plates were washed three times with 1% acetic acid and allowed to air-dry overnight. Then, protein-bound SRB stain was dissolved by adding TRIS (10 mM) and the absorbance was measured at 540 nm using a BMG LABTECH-FLUO star Omega microplate reader (Ortenberg, Germany).Cells treatmentCancerous Hep-G2 cells were cultured at the appropriate conditions and dived into control and treated cells. The control cells were treated with an equal volume of the vehicle (DMSO; final concentration, ≤ 0.1%), while the treated cells were treated with the IC50 of Er2O3-NPs. All cells were left for 72 h after nanoparticles treatment and were harvested by brief trypsinization and centrifugation. Each treatment was conducted in triplicate. Cells were washed twice with ice-cold PBS and used for different molecular assays.Estimation of genomic DNA integrityThe impact of Er2O3-NPs exposure on the integrity of genomic DNA in cancerous Hep-G2 cells was estimated using alkaline Comet assay13,14. Treated and control cells were mixed with low melting agarose and spread on clean slides pre-coated with normal melting agarose. After drying, slides were incubated in cold lysis buffer for 24 h in dark and then electrophoresed in alkaline electrophoresis buffer. Electrophoresed DNA was neutralized in Tris buffer and fixed in cold absolute ethanol. For analysis slides were stained with ethidium bromide, examined using epi-fluorescent microscope at magnification 200× and fifty comet nuclei were analyzed per sample using Comet Score software.Estimation of intracellular ROS generationThe effect of Er2O3-NPs exposure on intracellular ROS production in cancer Hep-G2 cells was studied using 2,7-dichlorofluorescein diacetate dye15. Cultured cells were washed with phosphate buffered saline (PBS) and then 2,7-dichlorofluorescein diacetate dye was added. Mixed cells and dye were left for 30 min in dark and spread on clean slides. The resultant fluorescent dichlorofluorescein complex from interaction of intracellular ROS with dichlorofluorescein diacetate dye was examined under epi-fluorescent at 20× magnification.Measuring the expression levels of apoptotic and anti-apoptotic genesQuantitative real time Polymerase chain reaction (RT-PCR) was conducted to measure the mRNA expression levels of apoptotic (p53 and Bax) and anti-apoptotic (Bcl2) genes in control and treated Hep-G2 cells. Whole cellular RNA was extracted according to the instructions listed by the GeneJET RNA Purification Kit (Thermo scientific, USA) (Thermo scientific, USA) and using Nanodrop device purity and concentration of the extracted RNAs were determined. These RNAs were then reverse transcribed into complementary DNA (cDNA) using the instructions of the Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific, USA). For amplification, RT-PCR was performed using the previously designed primers shown in Table 116,17 by the 7500 Fast system (Applied Biosystem 7500, Clinilab, Egypt). A comparative Ct (DDCt) method was conducted to measure the expression levels of amplified genes and GAPDH gene was used as a housekeeping gene. Results were expressed as mean ± S.D.Table 1 Sequences of the used primers in qRT-PCR.Full size tableAnalysis of cell cycle distributionDistribution of cell cycle was analyzed using flow cytometry. Control and treated cancer Hep-G2 cells with IC50 of Er2O3-NPs for 72 h were harvested, washed with PBS and re-suspended in 1 mL of PBS containing RNAase A (50 µg/mL) and propidium iodide (10 µg/mL) (PI). Cells were incubated for 20 min in dark at 37 C and analyzed for DNA contents using FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events are acquired and cell cycle distribution is calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).Estimation of apoptosis inductionApoptotic and necrotic cell populations were determined using Annexin V- Fluorescein isothiocyanate (FITC) apoptosis detection kit (Abcam Inc., Cambridge Science Park Cambridge, UK) coupled with two fluorescent channels flow cytometry. After treatment with Er2O3-NPs for 72 h and doxorubicin as a positive control, Hep-G2 cells were collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Harvested cells are incubated in dark with Annexin V-FITC/ propidium iodide (PI) solution for 30 min at room temperature, then injected via ACEA Novocyte flowcytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI). For each sample, 12,000 events were acquired and positive FITC and/or PI cells are quantified by quadrant analysis and calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).Statistical analysisResults of the current study are expressed as mean ± Standard Deviation (S.D) and were analyzed using the Statistical Package for the Social Sciences (SPSS) (version 20) at the significance level p  More

General description of the experimental areaThe experiment was performed for two years at the National Key Irrigation Experimental Station located on the Songnen Plain in Heping town, Qing’an County, Suihua, Heilongjiang, China, with a geographical location of 45° 63′ N and 125° 44′ E at an elevation of 450 m above sea level (Fig. 1). This region consists of plain topography and has a semiarid cold temperate continental monsoon climate, i.e., a typical cold region with a black soil distribution area. The average annual temperature is 2.5 °C, the average annual precipitation is 550 mm, the precipitation is concentrated from June to September of each year, and the average annual surface evaporation is 750 mm. The growth period of crops is 156–171 days, and there is a frost-free period of approximately 128 days year−122. The soil at the study site is albic paddy soil with a mean bulk density of 1.01 g/cm3 and a porosity of 61.8% prevails. The basic physicochemical properties of the soil were as follows: the mass ratio of organic matter was 41.8 g/kg, pH value was 6.45, total nitrogen mass ratio was 15.06 g/kg, total phosphorus mass ratio was 15.23 g/kg, total potassium mass ratio was 20.11 g/kg, mass ratio of alkaline hydrolysis nitrogen was 198.29 mg/kg, available phosphorus mass ratio was 36.22 mg/kg and available potassium mass ratio was 112.06 mg/kg.Figure 1Location of the study area. The map and inset map in this image were drawn by the authors using ArcGIS software. The software version used was ArcGIS software v.10.2, and its URL is http://www.esri.com/.Full size imageHumic acid fertilizerHumic acid fertilizer was produced by Yunnan Kunming Grey Environmental Protection Engineering Co., Ltd., China (Fig. 2). The organic matter was ≥ 61.4%, and the total nutrients (nitrogen, phosphorus and potassium) were ≥ 18.23%, of which N ≥ 3.63%, P2O5 ≥ 2.03%, and K2O ≥ 12.57%. The moisture content was ≤ 2.51%, the pH value was 5.7, the worm egg mortality rate was ≥ 95%, and the amount of faecal colibacillosis was ≤ 3%. The fertilizer contained numerous elements necessary for plants. The contents of harmful elements, including arsenic, mercury, lead, cadmium and chromium, were ≤ 2.8%, 0.01%, 7.6%, 0.1% and 4.7%, respectively; these were lower than the test standard.Figure 2Humic acid fertilizer in powder form.Full size imageExperimental design and observation methodsIrrigationIn this experiment, three irrigation practices, namely, control irrigation (C), wet irrigation (W) and flood irrigation (F), were designed (Table 1).Table 1 Different irrigation methods.Full size tableControl irrigation (C) of rice had no water layer in the rest of the growing stages, except for the shallow water layer at the regreen stage of rice, which was maintained at 0–30 mm, and the natural dryness in the yellow stage. The irrigation time and irrigation quota were determined by the root soil moisture content as the control index. The upper limit of irrigation was the saturated moisture content of the soil, the lower limit of soil moisture at each growth stage was the percentage of saturated moisture content, and the TPIME-PICO64/32 soil moisture analyser was used to determine the soil moisture content at 7:00 a.m. and 18:00 p.m., respectively. When the soil moisture content was close to or lower than the lower limit of irrigation, artificial irrigation occurred until the upper irrigation limit was reached. The soil moisture content was maintained between the upper irrigation limit and the lower irrigation limit of the corresponding fertility stage. Under the wet irrigation (W) and flood irrigation (F) conditions, it was necessary to read the depth of the water layer through bricks and a vertical ruler embedded in the field before and after 8:00 am every day to determine if irrigation was needed. If irrigation was needed, then the water metre was recorded before and after each irrigation. The difference between before and after was the amount of irrigation23.FertilizationIn our research, five fertilization methods were applied, as shown in Table 2. In this experiment, the rice cultivar “Suijing No. 18” was selected. Urea and humic acid fertilizer were applied according to the proportion of base fertilizer:tillering fertilizer:heading fertilizer (5:3:2). The amounts of phosphorus and potassium fertilizers were the same for all treatments, and P2O5 (45 kg ha−1) and K2O (80 kg ha−1) were used. Phosphorus was applied once as a basal application. Potassium fertilizer was applied twice: once as a basal fertilizer and at 8.5 leaf age (panicle primordium differentiation stage) at a 1:1 ratio22.Table 2 The fertilizer methods.Full size tableThis study was performed with a randomized complete block design with three replications. Three irrigation practices and five fertilizer methods were applied, for a total of 15 treatments as follows: CT1, CT2, CT3, CT4, CT5; WT1, WT2, WT3, WT4, WT5; FT1, FT2, FT3, FT4, and FT5 (C, W, and F represent control irrigation, wet irrigation, and flood irrigation; T represents fertilizer treatment).Measurements of the samplesA soil temperature sensor (HZTJ1-1) was buried in each experimental plot to monitor the temperature of each soil layer (5 cm, 10 cm, 15 cm, 20 cm and 25 cm depth). The transmission of photosynthetically active radiation was measured from 11:00 to 13:00 by using a SunScan Canopy Analysis System (Delta T Devices, Ltd., Cambridge, UK), and data during the crop-growing season were recorded every day24.Plant measurements were taken during the periods of tillering to ripening on days with no wind and good light. The fluorescence parameters were measured by a portable fluorescence measurement system (Li-6400XT, America). The detection light intensity was 1500 μmol m−2 s−1, and the saturated pulsed light intensity was 7200 μmolm−2 s−1. The functional leaves were dark adapted for 30 min, and then the maximum photosynthetic efficiency of PSII (Fv/Fm) was measured. Photochemical quenching (QP) and nonphotochemical quenching (NPQ) were measured with natural light. Simultaneously, the leaf chlorophyll relative content (SPAD) was monitored using SPAD 502 (Konica Minolta, Inc., Tokyo, Japan). For plant agronomic characteristics, the distance from the stem base to the stem tip was measured with a straight ruler to quantify plant height24.Statistical analysisExperimental data obtained for different parameters were analysed statistically using the analysis of variance technique as applicable to randomized complete block design. Duncan’s multiple range test was employed to assess differences between the treatment means at a 5% probability level. All statistical analyses were performed using SPSS 22.0 for Windows24.
Ethics approvalExperimental research and field studies on plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. We had appropriate permissions/licences to perform the experiment in the study area. More