Two torrefied microalgae (Chlamydomonas sp. JSC4 and Chlorella vulgaris ESP-31) were considered as a reducing agent. The kinetics dataset comprise of the activation energy (E), pre-exponential factor (A), and the reaction rate (k) while the thermodynamic dataset consists of the change in enthalpy (ΔH), change in Gibbs energy (ΔG), and change in entropy (ΔS). These kinetics and thermodynamics parameters are essential in understanding the reaction mechanisms of the reduction process of iron oxides enabling process optimization and improvement. Current literature lacks the kinetics and thermodynamics datasets for the reduction of iron oxides using the two torrefied microalgae as reductants. This work provides these datasets which are useful for the design of reactors in chemical looping combustion.The reduction of iron oxides transpires through the application of heat wherein a carbon source known as reductant is required. In order to design a chemical looping combustion using iron as an oxygen carrier and torrefied microalgae biomass as a reductant, the kinetics and thermodynamics dataset must be determined. Using the Arrhenius law of reaction, the kinetics dataset was obtained employing the three chemical reaction model such as the first order (C1), the reaction order 1.5 (C1.5), and the second-order (C2). The iron oxide reduction from hematite to metallic iron was sub-divided into three phases wherein phase 1 (Fe The figures and tables are described as follows.2 for the linear regression model fit for the three phases on the various blends of the hematite and the torrefied microalgae biomass. 22.12O3) to magnetite (Fe3O4), phase 2 from 595 C to 799°C for magnetite (Fe3O4) to wustite (FeO), and phase 3 from 800 C to 1200°C for wustite (FeO) to metallic iron (Fe)  is determined through the relation of the chemical reaction model shown in 2] and 1/T using the LINEST formula. The summary of the R2 model fit for the three reaction models on the three phases of reduction is shown in a), pre-exponential factor (A), and rate of reaction (k) are shown in The conversion degree α is determined through the TGA data provided in The model fit for the iron oxide reduction of the 1:1 blend of hematite and Chlamydomonas sp. is shown in From the linear equations shown in 2.3h is the Planks constant (=6.626 × 10−34 m2 kg s−1), KB is the Boltzmann constant (=1.381 × 10−23m2 kg s−2 K−1), and Tm is the maximum temperature with maximum thermogravimetric degradation. Using Eqs. The thermodynamics dataset was established using the following relation as described in Naqvi et al. .(6)ΔH=Ea


Seizure‐related 6 homolog (mouse)‐like 2 (SEZ6L2) was shown to be involved in transcription of a type 1 transmembrane protein for regulating cell fate. Until now, the expression and function of SEZ6L2 in various cancers, including colorectal cancer (CRC), were unclear. In the present study, we determined the expression of SEZ6L2 in a tissue microarray from patients with CRC and then, analysed the correlation between SEZ6L2 expression and the prognosis of the patients. Furthermore, the potential function of SEZ6L2 in CRC was determined using cell counting kit, colony formation assay and xenograft model in vitro and in vivo. Flow cytometry, Western blotting, immunohistochemical staining and a blocking experiment were employed to investigate the underlying mechanism of SEZ6L2 regulating CRC growth. Our results indicated that SEZ6L2 was significantly up‐regulated in tumour tissues of patients with CRC compared with adjacent normal tissues. Up‐regulation of SEZ6L2 was correlated with a poor prognosis in patients with CRC. In vitro experiments suggested that the knockdown of SEZ6L2 inhibits CRC cell growth and colony formation, but it has no significant impact on the invasion. The antitumour effects of shSEZ6L2 were also confirmed by a xenograft model. Investigations of the mechanisms indicated that the knockdown of SEZ6L2 impairs the growth of the CRC cells by inducing caspase‐dependent apoptosis, which was mediated by mitochondria‐related proteins. Furthermore, SEZ6L2 expression was inversely correlated with the expression of cytochrome C in malignant tissues in patients with CRC. Collectively, the present study indicates that SEZ6L2 is a potential prognosis biomarker and therapy target for CRC. It has been reported that mice deficient in SEZ6L2 suffer from motor ataxia, impaired cognition and abnormal neuronal innervation .In the present study, we aimed to investigate the expression and function of SEZ6L2 in CRC. A tissue microarray (TMA) containing 160 tissues from patients with CRC was performed for SEZ6L2 determination using immunohistochemical (IHC) staining, followed by an analysis of the correlation between SEZ6L2 expression and prognosis of the patients. Furthermore, the potential function of SEZ6L2 in CRC was determined by a cell counting kit, colon formation assay, and xenograft model in vitro and in vivo. Flow cytometry, Western blotting, IHC staining, and a blocking experiment were employed to investigate the underlying mechanism of SEZ6L2 regulating CRC growth. Collectively, these investigations would provide solid evidence for understanding the potential function and predicting role of SEZ6L2 in CRC.22.12 at 37°C for 2 weeks.The CRC cell lines and human intestinal epithelial cell (HIEC) were obtained from the American Type Culture Collection (ATCC). The cells were cultured in low‐glucose Dulbecco's modified Eagle's medium  with 10% foetal bovine serum , 1% antibiotics  and 5 µmol/L 2‐Mercaptoethanol (Sigma‐Aldrich Co.). The cells were infected with lentivirus‐shSEZ6L2 (GenePharma), and the stable infected cells were selected by puromycin . The z‐VAD‐FMK, dissolved in DMSO (Sigma‐Aldrich Co.) were added to treat the cells. All cells cultured in the atmosphere containing 5% CO2.2The cells were lysed using RIPA buffer  containing protease inhibitor cocktail (Merck Millipore). Protein concentrations were measured with a BCA assay kit (Thermo Fisher Scientific), and proteins were then separated by SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore). The membranes were then sequentially incubated with the appropriate primary antibodies  and horseradish peroxidase‐conjugated secondary antibody. The ECL kit (Merck Millipore) was used to detect the specific bands with iBright 2000 (Thermo Fisher). GAPDH, and COX IV was used as a loading control.2.33 cells per well in 96‐well plates. At 0, 24, 48 and 72 hours after cells plating, 10 μL Cell Counting Kit‐8  was added into each well, and the absorbance was measured at 450 nm at different time points with microplate reader (Thermo Fisher Scientific). For colony formation assay, the cells were plated into six‐well plates (1000 cells per well) and cultured for approximately 14 days. And then, after crystal violet staining, the colony formed in each well was counted and analysed.The cell viability assay and colony formation assay were performed as previous study indicated.2.44 cells were added to the upper chamber with serum‐free DMEM and incubated for 24 hours in the presence of 20% FBS in the lower chamber. The cells at the reverse side of a filter were fixed with 4% poly‐paraformaldehyde and stained with crystal violet. The cells were photographed with BX51 (Olympus), and the number of cells in each frame was counted and analysed.Cell invasion was determined using a 8 μm Transwell cell culture chamber (Merck Millipore). After starvation in serum‐free DMEM for 24 hours, 1 × 102.5The apoptosis of CRC cells was detected by Annexin V‐FITC/PI kit (Keygentec), following the instructions of manufacturers. Flow cytometry analysis was performed with BD LSRII Flow Cytometer (Becton Dickson), and data were analysed by BD FACSDiva software.2.62 × 0.52. All mice were kept in a specific pathogen free (SPF) room, with free access to food and water.For the xenograft model, stably infected HCT116 and HT29 cells were injected subcutaneously into the flank of 6‐8 weeks old female Balb/c nude mice (Hfkbio). The width and length of xenografts were measured every five days. The tumour volume was calculated as volume = length × width2.7DeadEndTM Fluorometric TUNEL System (Promega) was used to detect the apoptotic cells following the manufacturer's protocol. 4,6‐diamidino‐2‐phenylindole  was employed to detect the cell nuclei. The cell nuclei with dark green fluorescent staining were defined as apoptotic cells. The slides were photographed with BX51 (Olympus), and the number of cells in each frame was counted and analysed.2.8Tissue Micro Array (TMA) containing 160 CRC tissues was purchased from Outdo Bio. For IHC staining of the xenografts, tumour tissues were fixed, embedded and sectioned (4 μm thick). Immunohistochemistry (IHC) staining for TMA and xenograft tissues was performed in accordance with previous procedures.2.9The SEZ6L2 mRNA expression data in 470 CRC tissues and 41 normal colon tissues were downloaded from TCGA database, accompanied with the data of overall survival time of patients. The patients were also classified into two groups, high SEZ6L2 expression and low SEZ6L2 expression, based on the average expression of all patients. The Kaplan‐Meier method was employed to estimate the survival curves in two group patients in 12 years.2.10t test or one‐way analysis. The Kaplan‐Meier method was employed to estimate the survival curves, followed with the long‐rank test for comparing the difference. The correlation was analysed by Pearson's analysis. P value < .05 was considered to indicate statistical significance.Statistical analysis was performed using the SPSS Statistics software package . All of the data were performed as mean ± standard deviation. The differences were analysed by Student's 33.1To investigate the expression of SEZ6L2 in CRC tissues, two tissue microarrays, one containing 160 CRC tissues and the other containing 40 normal adjacent tissues, were performed for SEZ6L2 detection using immunohistochemical (IHC) staining. As shown in Figure 3.2Next, we aimed to determine the functional role of SEZ6L2 in CRC. The expression of SEZ6L2 in several CRC cell lines and human intestinal epithelial cells (HIEC) was detected by Western blotting. Our results indicated that SEZ6L2 was highly expressed in all detected CRC cells, including HCT116 and HT29 . Similar inhibition of tumour weights was also found in the shSEZ6L2‐1 group (53.9%) and the shSEZ6L2‐2 group (62.1%)  and 60.1% (shSEZ6L2‐2) . Collectively, SEZ6L2 is inversely correlated with cytochrome C expression in malignant tissues of patients with CRC.The expression of cytochrome C in tumour tissues of the HCT116 xenograft was determined by IHC staining. The results indicated that the knockdown of SEZ6L2 significantly promoted the expression of caspase 3 (Figure 4Colorectal cancer and many other cancers are often the result of molecular alterations that lead to the enhanced function of oncogenes or loss of function of tumour‐suppressor genes.Until now, the function of SEZ6L2 was poorly understood; however, studies have begun to reveal some of its basic functions. A previous study by Boonen et alApoptosis was induced through caspase‐dependent and caspase‐independent pathways in diverse cells.Collectively, our results demonstrated the prediction role of SEZ6L2 for the prognosis of patients with CRC and the antitumour effects of shSEZ6L2 in vitro and in vivo. The present study would provide a potential prognosis biomarker and therapy target for CRC. But further investigation is needed to demonstrated the direct binding targets of SEZ6L2, which may provide solid evidence for understanding the function of SEZ6L2 in cancers.All authors declare that there are no conflicts of interest.Ning An conducted all the experiments. Yaqin Zhao helped to draft the manuscript. Haitao Lan and Ming Zhang participated in the statistical analysis. Yuan Yin and Yi participated in the design of the study and finalized the manuscript. All authors read and approved the final manuscript.All experimental procedures were approved by the Institutional Animal Care and Use Committees of Sichuan University.Consent for publication is not applicable in this study, and no individual person's data were used.


Two typos were found in the Results section. The original, incorrect version has been replaced with the corrected version:Original, incorrect: “The majority of the patients were seen in the fourth decade of life (24%), which is followed by the fourth decade (20.7%) as shown in Table 1”New, correct: “The majority of the patients were seen in the fifth decade of life (24%), which is followed by the sixth decade (20.7%) as shown in Table 1”


Microbial communities, denitrification gene abundance and gas production in soils from tilled arable plots with contrasting fertilizer inputs  and regenerated woodland in the long-term Broadbalk field experiment were investigated. Soil was transferred to pots, kept bare or planted with wheat and after 6 weeks, transferred to sealed chambers with or without K15NO3 fertilizer for 4 days; N2O and N2 were measured daily. Concentrations of N2O were higher when fertilizer was added, lower in the presence of plants, whilst N2 increased over time and with plants. Prior soil treatment but not exposure to N-fertiliser or plants during the experiment influenced denitrification gene  relative abundance. Under our experimental conditions, denitrification generated mostly N2; N2O was around 2% of total gaseous N2 + N2O. Prior long-term soil management influenced the soil microbiome and abundance of denitrification genes. The production of N2O was driven by nitrate availability and N2 generation increased in the presence of plants.Factors influencing production of greenhouse gases nitrous oxide (N When soils are wet and O2 availability is limited but there is sufficient organic matter, a common condition in temperate agricultural soils, nitrate (NO3−) can act as an alternative electron acceptor and is reduced to N2O by many different groups of facultatively anaerobic bacteria3. This occurs with different types of fertilizer, whether organic, biological (e.g. legumes), urea or inorganic ammonia/nitrate compounds4. Some bacteria can further reduce N2O to N2 which is not environmentally harmful but nevertheless reduces nitrogen use efficiency, wasting N fertilizer inputs. In a survey of genome-sequenced prokaryotes, 7% contained denitrification genes exemplified by the nitrite reductase gene nirK, the alternate gene nirS or the gene encoding nitrous oxide reductase, nosZ5. However, not all prokaryotes with a nitrite reductase gene carry nosZ and in others the gene is not active. Consequently, for these organisms, the final denitrification step is the release of N2O. Also, there are also a substantial number of bacteria that contain nosZ without nirK or nirS and are assumed to sequester and reduce N2O, emitting N26. Many soil fungi can also denitrify: their contribution is minor when compared to that of bacteria in agricultural systems and arable soil but increases post-harvest and after incorporation of organic residues7. Fungi form only a small proportion of the soil microbiome under growing crops, and fungal denitrifiers contain nirK but not nosZ thus do not reduce any N2O they generate to N28. N2O generated by other processes including nitrification is at relatively low levels compared to the activity of denitrifying bacteria in anoxic, fertilized agricultural soils10.Sustainable agriculture for an increasing global population requires a balance between optimizing fertilizer inputs and minimizing adverse outcomes whilst increasing food production. Emissions of the greenhouse gas nitrous oxide (NnirK (encoding a Cu-containing enzyme) or nirS (encoding a cytochrome cd1) remains unclear11. The two forms can occur within different individual species of the same genus but (with rare exceptions) they are mutually exclusive12. Recent stable isotope probing experiments to identify denitrification genes in soil indicated that nirK was the most abundant and active, with greater sequence diversity than previously recognised13. The nosZ genes are also a diverse group and have been divided into two clades: nosZI in the Proteobacteria; nosZII in the Gemmatimodates, Bacteroidetes and Choroflexi14.The alternate nitrous oxide reductase genes have been known for many years but the significance of carrying either 2O fluxes from agricultural soils, some using intact or repacked soil cores under lab conditions, others made in the field, few have specifically investigated the role of crop plants or measured N2 emissions. Most are concerned with soil organic carbon (SOC), nitrogen and N-fertilizer forms and rates of application, soil moisture and temperature16. There is considerable information on the roles of these drivers but the effects of interaction between plant and soil factors including the microbiome on denitrification is less clear. A compilation of field data concluded that the presence of plants increased N2O emissions and also the ratio of N2:N2O17. Experiments in air-free systems with Argon or Helium replacing N2 have indicated that arable soils with young plants evolve more N2O and N2 than controls with no plants18. However, plants were also implicated in reduced soil moisture and less N fertilizer remaining in soil, leading to lower denitrification19. A review of 26 separate studies investigating cover crops in the field showed that 40% resulted in lower and 60% in higher emissions compared to control soils without cover crops20. Presence or absence of legumes, high soil N, rainfall and crop residues were all implicated in these differences. However, a more recent meta-analysis of 129 publications indicated that the presence of cover crops significantly reduced N2O emissions compared to bare soil21. In an experiment using soil cores with artificial root exudates, no N2O was detected in the control soil in contrast to that receiving exudates22 whereas a field experiment comparing bare soil  with non-legume cover crops found no significant difference in N2O emissions23. These contradictory results concerning N2O and N2 emissions from agricultural soil in the presence or absence of plants reinforce the need for a deeper understanding of the processes involved.Whilst there have been many surveys of N2O and N2 in soils taken from plots with contrasting long-term treatments which were anticipated to alter microbial communities with potential consequences for the relative abundance of denitrification genes. Furthermore, we aimed to examine the impact of growing plants and of applying KNO3 fertilizer on gas production. The soils were taken from the long-term Broadbalk Winter Wheat experiment at Rothamsted Research (UK), where different fertilizers and N-rates have been applied, resulting in soils with distinctly different edaphic properties. Using conditions designed to promote denitrification, soils from different long-term treatments, in pots with or without wheat plants, were treated with 15N-labelled fertilizer, control soils receiving no fertilizer and sealed in chambers with ambient air. The plants had been grown to the stem elongation stage where root exudation is at a maximum24 and the roots had colonised the pots so all soil was “rhizosphere”, for comparison with unplanted controls. The concentrations of N2 and N2O in these sealed chambers were measured daily over 4 days to provide a snapshot of denitrification activity in the rhizosphere and the control bulk soil; soil DNA and mRNA were extracted at this point to assess the abundance and activity of denitrification genes. From this we show the relative importance of antecedent soil treatments that influence edaphic factors including the soil microbiome, crop plants and N-fertilizer in promoting denitrification.In this paper, we report on a glasshouse experiment designed to measure production of N26. We chose plots that received farmyard manure (FYM) at 35 t ha−1, no N fertilizer (N0) or 288 kg N ha−1 annually (N6), together with the woodland soil (Wood). Table In the Broadbalk Experiment, running since 1843 at Rothamsted Research (UK), different fertilizers and N-rates have been applied consistently to winter wheat. In 1882, one section (previously referred to as “wilderness”) was taken out of cultivation and left to regenerate to woodlandBroadbalk is not fully replicated since it was set up before the advent of modern statistical design, hence each plot to be sampled was subdivided into three equally sized pseudoreplicates, each 9.3 m × 6 m in the arable plots and 9.3 m × 4 m in the FYM plot. The woodland area (80 m × 15 m) was also subdivided into equally sized pseudoreplicates. Ten soil cores (top 20 cm) were collected and pooled from each area in May 2014, generating three replicate soil samples for each of the four plots. Soil was sieved to 4 mm and stored at 4 °C for 14 days before transferring to pots; weighed subsamples were dried overnight at 80 °C to estimate the soil dry weight (dw).Triticum aestivum cv Cadenza) per pot, grown 6 weeks and given foliar feed if required (diagnosed by slight yellowing of leaves) and watered as normal with tap water. At 6 weeks post germination, wheat root exudation is at a maximum24; previous experiments showed wheat roots to fill the pots at this stage. The 24 unplanted pots were set up and kept in glasshouse alongside those with wheat in a randomised block design and were watered at the same time as the plants to maintain soil moisture. Soil bulk and particle density had been measured previously in the source soils27 and was used to calculate the pore space. This enabled subsequent manipulation of the water-filled pore space (wfps).A total of 48 pots were set up: three soil replicates from each of the 4 plots, with four treatments . Each Ø 10 cm pot contained 350 ml vol. soil; 24 pots were planted with 4 pre-germinated wheat seeds (2O), or a solution of K15NO3 (99% atom enrichment) in sd H2O as to give equivalent to 100 kg N ha−1 (i.e. 40 µg N g−1 dw soil) in each pot. Immediately after adding water or fertiliser, a 2 L polycarbonate bottle with the base removed was placed over each pot with a SubaSeal at the top, as shown in Supplementary Fig. When plants reached stem elongation but pre-flowering (c. 6 weeks post germination), the soils were adjusted to 95–100% wfps to create the anaerobic conditions conducive to denitrification, by adding sterile deionised water , with two ambient air samples taken at each sampling time. A single 22.5 ml air sample was taken at each timepoint for N2O analysis; for 15N isotope ratio sampling, a second 12 ml air sample was taken from each treatment fertilized with K15NO3 to measure 15N2-N and 15N atom% in N2O. The total (chamber plus pore space) volume and the soil dry weight in each pot was used to calculate N2O-N and 15N2-N g−1 dw soil for each replicate.The first samples (day 0) were taken immediately after sealing the chambers following the addition of K2O was measured using gas chromatography with an electron capture detector and an automated sample injection system; a TG2 trace gas analyser  interfaced to a Sercon 20–22 isotope ratio mass spectrometer (IRMS) was used to measure 15N enrichment of N2. Solutions of 6 and 30 atom% ammonium sulphate ((NH4)2SO4) were prepared and used to generate 6 and 30 atom% N2O28 and used as reference and quality control standards. Atmospheric air, with natural abundance 15N (0.3663 atom%) was used as the reference for N2. The N2 concentration in each sample was calculated from the ratios of the intensity of ion beams at mass to charge ratios 28, 29 and 30, using the equations of Stevens and Laughlin30.The N2 for subsequent DNA and RNA extraction using the RNA PowerSoil® isolation kit and RNA PowerSoil® DNA Elution Accessory Kit  following manufacturer’s instructions31. RNA samples were DNAse treated to remove DNA contamination using the DNase Max Kit , following the manufacturer’s protocol. Direct PCRs and gel electrophoresis were carried out on DNAse treated RNA to confirm all contaminating DNA had been removed. The quantity and quality of extracted DNA and DNAse-treated RNA were analysed by fluorometer Qubit® 2.0 dsDNA and RNA BR Assay Kits and Nanodrop microvolume spectrophotometer (Thermo Fisher Scientific).After 4 days, the chambers were removed. Soil samples were processed within 5 min to preserve the integrity of the RNA and DNA. In pots with wheat, the roots had spread throughout the soil and soil adhered to the roots at sampling. This soil was shaken off and mixed, similarly for unplanted pots, soil was mixed, a subsample taken, sieved to 2 mm and frozen in liquid N32. The amplicon sequence data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB36852 (https://www.ebi.ac.uk/ena/browser/view/PRJEB36852).The bacterial and archaeal diversity was determined from the 12 samples taken at the time of field sampling by amplicon sequencing of 16S rRNA genes using the primers 515F/806R, sequenced on Illumina’s MiSeq platform and analysed to phylum (sub-phylum for Proteobacteria) level and to OTU (97% sequence identity) using the QIIME 1.8 pipeline. The method was described in detail previouslynirK and nirS) and the nitrous oxide reductase gene nosZ clades I and II are shown in supplementary Table −1 or 2–4 µl of RNA at 10 ng µl−1 and nuclease-free water  up to 10 µl, using a CFX384 Touch™ Real-Time PCR Detection System . The standards for each molecular target were obtained using a tenfold serial dilution of PCR products amplified from an environmental reference DNA and purified by gel extraction using the Wizard® SV Gel and PCR Clean Up System  following the manufacturer’s instruction then quantified by fluorometer Qubit® 2.0 dsDNA BR Assay Kit (Thermo Fisher Scientific). Standard curve template DNA and the negative/positive controls were amplified in triplicate. Amplification conditions for all qPCR assays consisted of an initial denaturation at 95 °C for 5 min followed by 40 (two step) cycles; 95 °C for 10 s and 60 °C for 30 s. The RT-qPCR program had an initial reverse-transcription step at 50 °C for 10 min. Each amplification was followed by melt curve analysis  to confirm the specificity of each assay. Efficiency of amplification for each primer set was > 82% with r2 ≥ 0.996 , nitrate (NO3−) and ammonia (NH4+) using a Skalar colorimetric continuous flow Analyzer. Results are given as μg g−1 dw soil.At the end of the pot trial, samples from each pot were oven-dried as described above, to measure moisture and estimate the final wfps. Extracts  were analysed for nitrite (NOP < 0.05), means were further tested using Tukey’s post-hoc method in the GenStat multiple comparison menu with 95% confidence. Where appropriate, standard errors of difference of means (s.e.d.) are indicated. When only two treatments were compared, Student’s t-test in the Excel data package was used. The statistics package PAST v. 3.1633 was used to analyse the relative abundance of 16S rRNA amplicons using: SIMPER to determine the percentage contribution of each phylum to each treatment; non-metric multidimensional scaling (NMDS) analysis at the OTUs level based on the Bray–Curtis similarity index; PERMANOVA to assess the significance of the NMDS plot and Spearman’s rank correlations of gene abundance, soil properties and gas emissions. Unless otherwise indicated, statistically significant differences are assumed to occur at P ≤ 0.05 and are referred to as “significant” throughout the text; results with no significant differences are referred to as NSD.GenStat 17th Edition  was used to perform ANOVA with soil origin, sampling date (i.e. time of incubation), presence or absence of plant and addition or not of fertilizer as factors, to compare values obtained from soil and gas analyses and from qPCR estimations of gene and transcript copy numbers. To check that each set of measured values met the assumptions of ANOVA and were normally distributed, residuals were plotted. If they did not show normal distribution, data was log-transformed and again checked for normal distribution of residuals. Where ANOVA results were significantly different (The soil properties shown in Table P ≤ 0.05) mean abundance in at least one soil, according to ANOVA , nosZI (5 × 107–2 × 108 copies) and nosZII (4 × 106–1 × 107 copies g−1 soil). The exception was nirS where N0, N6 and woodland soil had similar gene abundance (1 × 107 copies g−1 soil) and FYM significantly more with 4 × 107 copies g−1soil . Woodland also had a significantly higher ratio of nir:nosZ genes, 20:1 compared to 13:1 in the arable soils . This was influenced only by the origin of the soil, not the plant or fertilizer treatment  than the FYM soil with 1.8 × 108 copies g−1. The same pattern was seen with nirK transcripts: 2.4 × 105 copies in the woodland; 1.2 × 105 copies in the FYM soil . There was no significant difference between the two soils for nosZI transcripts which were much less abundant, mean 6 × 103 copies g−1 soil.The mean number of 16S rRNA transcripts overall in the woodland soil, 5.3 × 102−-N at the end of the experiment was below the limit of detection in most samples and is not included. The NH4+-N followed the same trend as the % N and bacterial abundance, significantly higher in the woodland soil  than 1.6 μg g−1 for bare soils. The NH4+-N is around tenfold less than the NO3−-N in unfertilized soils, indicating nitrifier activity in the aerobic soils prior to setting up the chambers whereby soil pore saturation to create anaerobic conditions is predicted to reduce nitrification rates.The concentration of soil NOoil Fig. a. Accord3–-N concentration to vary significantly between soils and to be influenced by both K15NO3 fertilizer additions and the presence of plants  than in the presence of plants (0.36 μg g−1 soil). Where K15NO3 was applied, differences due to soil of origin was not significant  than where plants were present (5.4 μg g−1).ANOVA showed the NOts Table . The NO315NO3 fertilizer addition had a significant influence  than 61.0% for all planted soils.The % water-filled pore space (wfps), set at an estimated 95% at the start of the experiment, had fallen to 60–80% in most soils by the end, and to 40% for the woodland soils with wheat Fig. . Water hce Table . The ove15NO3 fertilizer or water and sealing the chambers (day 0) were similar to ambient values and were not included in subsequent analyses . Subsequent samples were taken at 24 h intervals (day 1–4) until the experiment concluded, the chambers were dismantled, and the soil was sampled. ANOVA indicated that the presence/absence of plants and K15NO3 had a significant effect on N2O-N but not the sampling date either alone or in combination with the other factors; in contrast, CO2 levels were additionally influenced by sampling date  than with plants (1.1 ng g−1 soil). However, where K15NO3 was applied, the mean N2O-N was NSD in bare and planted soil. Over all treatments, the N2O-N measurements were highly variable with NSD between most means and overall differences due to the soil of origin were also NSD  less than the mean value where plants were present (16.8%). The data was used together with N2O-N measurements to show that the proportion of 15N in N2O-N was significantly less from the woodland soil when wheat was present  than without plants (239 ng g−1 N2-N). In the absence of plants there was NSD between the mean N2-N in the different soils but when plants were present, N6 produced significantly more N2 than the FYM and woodland soils  was dominated by N2-N with an overall mean of 390 ng g−1 soil compared to 4 ng g−1 for N2O-N. However, the relative abundance showed a significantly higher % N2O-N in bare N6 and woodland soils compared to those with wheat  and 15N atom% showed moderate negative correlation with NH4+-N  and, in the 15N-fertilized pots, 15N atom% (negative). The nir:nos ratio showed similar trends and additionally showed moderate negative correlation to N2O-N and, in the 15N-fertilized fertilized pots, to N2O-N as a % of (N2-N + N2O-N). Neither ratio showed significant correlation with N2-N. There were no statistically significant correlations between nosZI and nirK mRNA abundance and gaseous emissions.To investigate factors influencing gaseous emissions, Spearman’s rank correlation was derived for gas, soil and microbial parameters for all 48 pots where N2 emissions (prominent only in woodland soil without plants) correlated with NO3−-N; 16S rRNA, nirK and nosZI gene abundance; and with N2-N in the K15NO3 fertilized subset.The COnirK and nosZI, which did not appear to be increased by the regular application of N fertilizer. The denitrification genes are relatively common across different phyla, and it is likely that although the community composition varied, gene abundance was not limiting in these experiments. In contrast to the production of CO2, N2 and N2O did not show statistically significant correlation to 16S rRNA gene abundance, nor to any of the denitrification genes although there was a moderate negative correlation between nir:nos ratio and N2O-N, N2O%, and 15N atom%. This indicated that as the proportion of nitrous oxide reductase genes (nosZI + nosZII) increased compared to nitrite reductase genes (nirK + nirS), there was proportionally less N2O in the chambers as it was converted to N2.Microbial diversity was influenced by previous long-term treatment of the different plots, resulting in the distinctly different microbiome composition revealed by amplicon sequencing. These contrasting treatments: arable management with N-fertilizer (N6); no N (N0); organic FYM; and the woodland soil left untilled and unamended, provide very different scenarios and each appears to favour divergent combinations of phyla and subphyla as shown in Fig. 34, is one reason for the lack of consensus between different studies which attempt to define the most important genes. Edaphic factors such as pH are known to influence soil microorganisms: changing soil pH in different plots caused significant differences in denitrification gene abundance35. The relative abundance of nirK and nirS reported elsewhere appears to depend on soil properties and management: relatively more copies of nirS were reported in unfertilized bare fallow compared to nitrate or FYM-fertilized soil, where nirK was more abundant36. Our results, from different soils and treatments, are not directly comparable with these but are consistent with previous measurements in Broadbalk soil25. The relatively higher abundance of nosZI compared to nosZII is consistent with reports that nosZI occurs in Proteobacteria14 which together form the most abundant phyla in all Broadbalk soils. Recently, changes in the diversity and abundance of nirK and nosZ genes expressed in soil microcosms have been related to N2 and N2O emissions37. In our experiments, we could measure transcription of nirK and nosZI only in FYM and woodland soil and there were twice the number of copies of nosZI per nirK in FYM compared to woodland soil which could explain the lower N2O and N2O:N2 measured in the FYM compared to woodland soil in the absence of plants although correlations were not significant. When plants are present, differences in the uptake of the added 15N fertilizer that limit the amount left in soil as a denitrification substrate may change this relationship (see below). We were surprised that 16SrRNA gene abundance was not found to be significantly lower in bare soil compared to planted pots containing rhizosphere soil. Bacterial cell numbers are higher in the rhizosphere than bulk soil38 but the qPCR assays in our study were probably insufficiently sensitive to detect this, compared to the much larger differences due to the source of the soil.The biogeography of denitrifying bacteria and their genes, and differences in their relative abundances2O production. This agrees with the findings of a recent meta-analysis where fertilizer quantity and the presence of crops were the most important drivers influencing N2O emissions21. The relative abundance of the various genes involved in NO2− and N2O reduction did not appear to drive the gasses measured in our experiments, indicating that denitrification was not constrained by abundance of nirK, nirS, nosZI and nosZII in the arable soils although differences in relative abundances  might explain the significantly greater N2O production in bare, fertilized woodland compared to FYM soil. The relatively low N2O-N measured in woodland soils where wheat was grown could reflect the drop to 40% wfps, conditions less conducive to denitrification. The proportion of 15N in N2O-N in bare woodland soil was lower than in the arable soils, and significantly lower where wheat was growing, indicating that some of the “extra” N2O in woodland soil arose from NO3−resulting from nitrification of NH4+ derived from the larger total N pool. The NH4+-N is likely to be a product of SOM mineralisation rather than a legacy of previous fertilization regimes; the results indicate that plants can stimulate the soil microbiome to increase this mineralisation. Nitrifying bacteria, archaea and fungi in soil can oxidize NH4 to NO2− and subsequently NO3−, especially in aerobic conditions.In water-saturated conditions conducive to denitrification, both nitrate fertilizer and plants had a major influence on N2O was produced when fertilizer was added to bare soil, and least in unfertilized soil with wheat, and the concentration measured in the chambers did not change significantly over the sampling period. In contrast, N2 increased over time, with more produced when plants were present. The 15N atom% in N2O was also higher in soil with plants. The increase in N2 indicates that N2O was being actively reduced during the experiment, that production and consumption of N2O was in equilibrium, and full denitrification from NO3– to N2 was more efficient in the presence of plants despite their competition for NO3–-N. Most microbes are more active in the presence of plants, stimulated by the energy-rich root exudates, and this may explain both increased 15N atom% in N2O and increased reduction of N2O to N2. In addition, this high degree of conversion of N2O to N2 may arise from the closed experimental system causing denitrifying microorganisms to remain in contact with N2O for longer than in open soil in the field, where N2O is likely to be rapidly lost to the atmosphere. In contrast to other experiments measuring the effect of small plants in enclosed chambers18, we used mature plants resulting in all soil being in contact with roots, effectively rhizosphere soil, for comparison to bulk soil in unplanted pots. This would have enhanced the plant effect, explaining the much higher conversion of N2O to N2 that we observed together with the reduction in soil NO3−-N which was rapidly assimilated by plants. The abundance of nosZ is reported to be influenced by resource availability, with relatively fewer copies detected in nutrient-poor environments and an increased proportion present in rhizosphere communities34. The relevance of other differences such as the presence of more copies of nirS in FYM soil, are unclear since they did not result in significant differences in gaseous emissions, although it should be acknowledged that the gene abundance was measured only at the end of the experiment and could have varied over the preceding 4 days.In our experiments, the most N2O and N2 are known to fluctuate and much greater replication is needed to demonstrate which factors apart from NO3− and wfps are driving denitrification. The closed design of our experiment with daily gas sampling did not allow measurement of gas fluxes but result indicate that the peak concentration of N2O was reached by 24 h and did not increase significantly after this time because it was being reduced to N2, which in contrast, increased each day in most treatments. Our observations that most N2O produced in soil is further reduced to N2 by active denitrifying bacteria is consistent with many reports. However, less N2 than N2O was measured in planted soil fertilized with KNO3 in a sealed system with He7 although the converse was observed in an earlier experiment with Ar18; fertilizer type and water saturation also influenced denitrification in these studies. Our system, in contrast, was designed to have an enhanced rhizosphere effect and growth of plants in a normal atmosphere. The maximum amount of K15NO3-N converted to N2 over the 4 days of our experiment was 4%, in the N6 soil, where 1.6 µg 15N2-N g−1 dw soil was detected after 4 days, derived from 40 µg K15NO3-N g−1 dw soil added when the chambers were sealed. With plants, ~ 5 µg NO3−-N g−1 soil remained after 4 days, suggesting that much more was taken up by the wheat than was available for denitrification. In other studies, N2O-N comprised 7% of (N2O-N + N2-N) emitted from cores of wet agricultural soil39 and 25% in saturated soils amended with artificial root exudates40. There are several reports that the ratio of N2O:N2 increases with the concentration of NO3− in soil41 but the concentration of K15NO3 in our experiments, 40 μg N g dw soil−1 equivalent to 100 kg N ha−1, was relatively modest (and decreased by 85% over 4 days in the presence of plants) compared with many other studies in vitro. For example, the in vitro study40 with model root exudate applied 100 μg N g dw soil−1 and application rates cited in field surveys were 160–300 kg N ha−1 year−129 and 200–500 kg N ha−1 year−141, respectively. In our pots, NO3− was more evenly distributed throughout the soil than in field applications, so avoiding localised high concentration activity hotspots. High concentrations of NO3− are reported to inhibit nitrous oxide reductase and hence the reduction of N2O to N243. In soil microcosms amended with with 50 μg NO3–-N g dw soil−1, N2O comprised > 1% (N2O + N2) after 48 h whereas adding 500 μg NO3–-N g dw soil−1 diminished N2 emissions by 38–90%42. Another study44 found fungal denitrification dominated initially, after high levels of straw and nitrate (200 mg NO3–-N g−1) were added to soil but when NO3–-N fell to 40 mg g−1 soil, N2 evolution dominated.Emissions of N2 production in the presence of 78% N2 in air, make it difficult to design experiments without adding isotopically labelled N fertilizer and plants reportedly do not flourish when N2 in air is replaced by Ar or He. The acetylene inhibition method previously used to prevent conversion of N2O to N2 has many disadvantages45. Our experiment was designed to determine the effect of well-developed wheat plants at peak root exudation on denitrification, and on the bacterial genes involved, following saturation of the soil, rather than following the development of the plant and denitrification rates over time. We were constrained by the need to sample gas from the chambers manually: future experiments using repeat robotic gas sampling would offer great advantages. Over the short 4 days enclosure, we observed that plants decreased soil NO3− and moisture levels and increased CO2, all of which may influence effect denitrification and could be investigated further.The constraints on measuring de novo N2O production in water-saturated soils and furthermore, it shows that the presence of plants promotes further reduction of N2O to N2, which is the major denitrification product. The relative abundance of the various genes implicated in denitrification did not play a major role in these experiments although long-term pre-treatment of soils had generated significant differences in the composition of their soil microbiomes.It would be interesting to attribute the denitrification genes that we detected to the organisms from which they originated. Further studies to sequence amplicons may reveal whether different groups are present and (with improved mRNA extraction) active in the different soils, coupled with estimates of gene abundance over time (within 24 h and over days), and improved monitoring of gaseous emissions may help to explain the high variability observed. Nevertheless, our work reported here confirms that N-substrate (nitrate) availability is the main driver for NSupplementary Information 1.


DMPK) gene causes myotonic dystrophy type 1 (DM1) and sequesters RNA processing proteins, such as the splicing factor muscleblind‐like 1 protein (MBNL1). Sequestration of splicing factors results in the mis‐splicing of some pre‐mRNAs. Small molecules that rescue the mis‐splicing in the DM1 cells have drawn attention as potential drugs to treat DM1. Herein we report a new molecule JM642 consisted of two 1,3‐diaminoisoquinoline chromophores having an auxiliary aromatic unit at the C5 position. JM642 alternates the splicing pattern of the pre‐mRNA of the Ldb3 gene in the DM1 cell model and Clcn1 and Atp2a1 genes in the DM1 mouse model. In vitro binding analysis by surface plasmon resonance (SPR) assay to the r(CUG) repeat and disruption of ribonuclear foci in the DM1 cell model suggested the binding of JM642 to the expanded r(CUG) repeat in vivo, eventually rescue the mis‐splicing.Expanded CUG repeat RNA in the dystrophia myotonia protein kinase  to include the exon 22. In vitro binding assay showed that JM642 bound to the CUG repeat RNA and disrupt the RNA foci in the cell. DMPK gene is the cause of the disease.[DMPK gene with the long CUG repeat sequesters the RNA‐binding proteins, such as the splicing factor MBNL1 in the nucleus.[Ldb3) gene, exon 11 is excluded from mRNA by about 80 % in the wild type, but exon 11‐included mRNA is produced about 50 % in DM1 cells.[Clcn1), the mRNA without exon 7a is dominant in wild type, whereas exon 7a‐included mRNA is abundant in DM1 cells.Atp2a1 gene coding sarcoplasmic reticulum calcium‐ATPase 1 (SERCA1) produces mRNA containing exon 22 in the wild type cells, whereas mRNA without exon 22 predominates in DM1 cells.Molecules modulating the splicing pattern of genes in DM1 cells have drawn attention as potential drugs treating this devastating neurological disorder.3, 4 DJM642 9‐immobilized sensor surface, and JM642 led to the disruption of ribonuclear foci in DM1 cell model expressing r(CUG)800 repeat, demonstrating that JM642 would be a useful molecular tool for the deeper understanding of the pathogenesis of DM1 and studies on the therapeutic potential of small molecules targeting DM1.These differences in the splicing patterns between the wild type and DM1 encouraged studies focused on the modulation of the splicing patterns. Besides prominent approaches using oligonucleotides,JM608 and its dimeric form JM642.  repeat and modulate the alternative splicing in DM1 cells.32, 33JM608 and JM642 on alternative splicing was investigated on pre‐mRNA of the Ldb3 gene in the C2C12 DM1 cell model conditionally expressing r(CUG)800 repeat RNA.800, the percentage of exon 11 exclusion in the Ldb3 gene was about 81±1.7 %, while the fraction in the DM1 cell model expressing r(CUG)800 was 53±1.9 %  at the concentrations higher than 30 μM. The effect of a monomer JM608 on the recovery in mis‐splicing was 58±1.9 % at 80 μM. For the reference, cytotoxicity of JM608 and JM642 to the C2C12 DM1 cell model was not apparent over the treatment range (data not shown).The effect of % Figure . After tJM642 on the mis‐splicing of pre‐mRNAs in the DM1 mouse model (HSALR), which expresses r(CUG)220 and exhibits the mis‐splicing of Clcn1 and Atp2a1 pre‐mRNAs.JM642 (10 mg kg−1 or 20 mg kg−1 per day) was administrated to the HSALR mice (n=3 in each group) by daily intraperitoneal (i.p.) injection for five days. The fraction of exon 7a exclusion for the Clcn1 gene was 85±0.53 % for the wild type mice and 44±2.4 % for the HSALR mice.  rescued the mis‐splicing in the Clcn1 gene, leading to an exclusion rate of 61±2.3 % (P=0.03) and 70±2.3 % (P=0.01), respectively, although an improvement of phenotypic myotonia was not apparent due to the partial rescue of splicing. The rescue effect of JM642 was also observed in Atp2a1 mis‐splicing. In the wild‐type mice, the inclusion rate for exon 22 is 100±0 %, whereas the inclusion fraction of exon 22 in the HSALR mice was 16±2.4 %  and 74±6.0 % (P<0.01), respectively. Toxicity was not observed within the mouse model over this treatment range. These results demonstrated the rescue effect of JM642 on mis‐splicing of Clcn1 and Atp2a1 pre‐mRNAs in DM1 in vivo.We then investigated the effect of model HSALR, which. Figure . Mis‐splmodel HSALR, which% Figure . After aJM642 and somewhat moderate effect of JM608 on the rescue in mis‐splicing of genes in DM1 cell and mouse models, we have investigated the origin of these biological effects. The current hypothesis on the expected therapeutic effects of small molecules in the treatment of DM1 stems from the competitive binding of small molecules with RNA‐binding proteins to the aberrantly expanded CUG repeat RNA in the nucleus.[JM608 and JM642 could fit this hypothesis, we looked at the binding of these molecules to the CUG repeat RNA with the SPR assay. The biotin‐labeled r(CUG)9 repeat RNA and r(CCG)9 repeat RNA as control were immobilized through the tri‐ethylene glycol linker to the avidin‐coated sensor surface, and the analyte molecule was sequentially added with the increased concentration to the surface .Having observed the significant effect of  nucleus.2, 3 TJM608 and JM642 from the same sensor surface of r(CUG) repeat RNA were quite different in terms of the shape of the curve, which characterizes the association and dissociation kinetics as well as the affinity. The SPR profiles obtained for JM608 showed the rectangular shape indicating a rapid association and dissociation kinetics. ) of JM608 to the r(CUG)9 repeat RNA was determined 1.2 μm based on the assumed 1:1 binding isotherm.The SPR response curves obtained for . Figure  The loweJM642 were quite characteristic, showing a broad parabola shape without any plateau region. 9 observed in the SPR analysis may support the above speculation.  SPR responses on the r(CCG) repeat RNA surface were weak for both JM608 and JM642, even at 1.0 and 0.1 μm, respectively.In contrast, SPR response curves obtained for . Figure  The loweJM642 with RNA‐binding proteins on CUG repeat RNA, we have investigated the disruption of the ribonuclear foci in DM1 patient‐derived myoblast cells by JM642 treatment. Untreated DM1 myoblasts showed the formation of ribonuclear foci . Transcription across the expanded repeat was activated by Cre recombinase‐mediated excision of a transcription terminator cassette. C2C12 cells with recombination were selected using hygromycin B (300 μL mL−1). RNA was harvested after 2 days of incubation with JM608 and JM642. RNA extraction and analysis of the splicing pattern were carried out as described below. WST‐1 assay was performed according to the manufacturer's instructions .A conditional cell model for the analysis of MBNL1 splicing regulatory activity has been established, as reported previously.HSALR transgenic mice of line 20b (FVB inbred background) were described previously.JM642 at indicated dose and period by daily i.p. injection. After treatments, mice were sacrificed, and the rectus femoris (quadriceps) muscle was obtained for splicing analysis. RNA extraction and analysis of the splicing pattern were carried out as described below.Mouse handling and experimental procedures were performed following the Osaka University guidelines for the welfare of animals and were approved by the institutional review board. Homozygous Total RNA extraction from model cells, cDNA synthesis, and polymerase chain reaction (PCR) amplification were performed as described previously.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of UNC‐5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C‐mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions.Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR‐active nuclei. We report on the incorporation of  1C4 state and the B03 / 1S3 state.NMR spectroscopy was used to characterize the C‐mannose conformation in a thrombospondin repeat using a selective labeling approach. The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of the netrin receptor UNC‐5 allows interconversion between the canonical  Glycosylation plays an essential role in the structure and biological function of many proteins in living cells and viruses.1 carbon and the Cδ1 carbon atom of the amino acid side chain. C‐Man was first reported in human ribonuclease 2Among the many types of glycosylation, C‐mannosylation (C‐Man) of a tryptophan residue is a unique post‐translational modification which occurs in a variety of proteins. A single mannose is distinctively attached to the tryptophan indole sidechain via a carbon‐carbon linkage between the anomeric sugar C13C‐isotope labeled C‐Man glycoproteins; which allows specific investigation of the glycans present in proteins by solution NMR spectroscopy. This previously unknown labeling technique is based on the development of a Drosophila S2 cell line that cannot use glucose to generate mannose and are therefore dependent on the supplied mannose. The approach has been applied here to characterize the conformational dynamics of the C‐α‐mannose moieties of a thrombospondin repeat from the netrin receptor UNC‐5, which is involved in axon guidance by repulsion.[Considering the important role of glycans in protein function, the development of methods to produce and analyze glycosylated proteins is essential. Glycosylation is often incomplete and the proteins and glycans are frequently very dynamic and exhibit a high degree of conformational heterogeneity. Yet, solution NMR spectroscopy is particularly well suited for the analysis and structural studies of such inhomogeneous and complicated targets.epulsion.22, 23Drosophila S2 cell line, stably expressing a single thrombospondin type 1 repeat (TSR) of the netrin receptor UNC‐5, has been generated and described before.C. elegans allowed producing the C‐mannosylated form of the repeat. The TSR2 of UNC‐5 starts with the sequence LDGGWSSWSDWSAC, in which the first two tryptophans can be mannosylated by DPY‐1913C‐mannose cannot end up in other metabolites and the 13C‐mannose pool is not diluted.A m to 1 mm exogeneous mannose. The clone was routinely maintained in medium with 100 μm added mannose. To produce selective 13C‐labeled mannose in the UNC‐5 protein cells were cultured at a 300 mL scale and supplied with 500 μm13C‐isotope labeled mannose. Two mg of highly pure UNC‐5 TSR2 was produced that uniformly contained two C‐mannoses .The generated MPI knock‐out clone has a homozygous deletion of 55 bp in the gene sequence Figure . We esta13C‐labeled mannose in the UNC‐5 protein is shown in the 1H13C‐HSQC spectrum  were performed on this selective 13C‐isotope labeled probe to determine the exact sugar geometry and dynamics. Theoretical puckering studies exploring the kinetic energy landscape for several sugars shows that there are different favorable geometries and many possible interconversion pathways.[exo‐anomeric effects and the type of glycosidic linkage.[The successful incorporation of selective m Figure . Both mapathways.6, 7 T13C‐relaxation experiments  were performed on the selective labeled UNC‐5 protein sample as well as on free mannose, under the same conditions. Due to uniform isotope labelling of the sugar moiety, direct determination of 13C‐T2 is not possible. Therefore, the transverse 13C‐T2 relaxation rates were extracted from off‐resonance correction of the T1ρ values using T1. As expected, the rates and HetNOE values (supplementary Table 1) are clearly very different for the free and the covalently attached mannose, which correlates to the differences in their overall tumbling rate as well as their mobility. To evaluate the dynamical behavior, the relaxation data were analyzed using the model‐free method.[τc is very fast (about 125 ps), making separation of the overall motion and internal motion complicated and thus practically unfeasible. However, when the mannose moiety is covalently attached to the tryptophan, the dynamics are strongly reduced. The average order parameter for the C‐Man W5 is 0.91 and for the C‐Man W8 0.94, indicating that those moieties become rigidified when attached and reside within the environment of the UNC‐5 protein. Furthermore, the model‐free analysis points to the presence of little internal dynamics (τe in the order of about 60 ps) for the slightly more dynamic C‐Man W5.In order to probe the dynamical behavior of the mannose moieties, heteronuclear e method.29, 303J, the vicinal proton‐proton coupling constants. The dependence of the individual expected couplings on the corresponding endocyclic torsion angles of the pyranose conformationsThe conformation of the mannose six‐membered ring moiety was first probed by determination of 3J couplings indicates that those are mostly present in the 4C1 conformation (and possibly fluctuate to adjacent conformations such as E3 and 4H3). In contrast, the C‐mannose moieties of the UNC‐5 W5 and W8 mostly map towards the conformational space in the 1C4 hemisphere (including sugar pucker geometries such as B03 and 1S3). Remarkably, for the C‐Man W5 and W8, the observed intermediate 3J values  do not fully agree with either 1C4 or 4C1 conformation, as also observed by others.[1 carbon of the mannose to be in the equatorial position. The experimental coupling may possibly be an intermediate; probing an ensemble or exchange between two (or) more conformations that deviate around the H3‐C3‐C4‐H4 torsion angle (such as the B03 boat and 1S3 skew).For both free α‐mannose as well as β‐mannose, the analysis of the 3J couplings can be determined accurately, but for a covalently attached C‐mannose, which is in the context of the larger protein, the quantitative analysis becomes less accurate and less precise due to severe overlap, line broadening effects but also potential systematic errors.τc) and can be conveniently used for the study of larger biomolecules.[3J proton‐proton couplings. The selective 13C‐mannose labeled UNC‐5 protein sample allowed us to determine the CCRs for the C‐mannose moieties of the UNC‐5 W5 and W8 by means of quantitative 2D 1H13C‐Γ‐HCCH experiments . In addition to the 2D version, a 3D experiment was exploited, adding an additional carbon dimension, to resolve signal‐overlap (such as for the W8 C‐mannose H2 and H3 protons which appear at the same resonance). The obtained CCR data was analyzed in the same way as for the 3J coupling constants  coupling  likewise indicating possible mobility and/or existence of more conformational states that fast interconvert on the NMR timescale. The combined difference was also re‐examined without these values  and actually still shows analogous landscapes; clearly mapping the C‐mannose sugar geometry towards the conformational space in the 1C4 hemisphere.For free mannose, a small molecule, the vicinal r−6 and therefore substantially less sensitive to averaging compared to both, 3J couplings and CCR.Particularly important for biomolecular structure determination is the Nuclear Overhauser Effect (NOE), providing spatial distances between protons. For glycosylated proteins, the NOE buildup for the sugar is efficient because of the increased overall tumbling time due to the covalent attachment to the protein. The well‐established NOE can therefore be readily used to calculate and validate the geometry of the sugar. Moreover, fluctuations in the distance  would be reflected in an averaged NOE. However, NOE averaging is weighted by 1/13C‐selective labeled mannose facilitates the use of 13C‐resolved NOESY experiments, which is crucial to separate overlapping NOE cross peaks. NOEs were obtained from several 3D 1H1H13C‐NOESY‐HSQC spectra acquired with different mixing times (NOE build‐up) and from a 4D 1H13C1H13C‐NOESY‐HSQC spectrum  measured on the selective 13C‐labeled C‐Man UNC‐5 protein. From modelled structures of all 38 canonical states the expected proton‐proton distances were compared. Even though most combinations feature rather similar distances for all geometries close to the NOE distance error ranges (with only about 0.6 Ångstrom or less difference between the 4C1 and 1C4 conformation), taken together  couplings and CCR data) they can enclose the global shape of the sugar. The most indicative NOE to differentiate between the 4C1 and 1C4 conformation is the H3‐H5 distance . The NOE cross peak between these two protons is inconveniently very close to the diagonal (and for W8 strongly overlapping with H3‐H4), but this could be well resolved by using the 4D experiment. For W5, only a very weak NOE was observed between H3 and H5 and for W8 there was no cross peak detected, which is indicative for the 1C4 hemisphere and thus nicely in agreement with the previous findings. In addition, NOEs observed between the tryptophan and mannose (eg. Hβ#‐H1 and Hϵ1‐H2) resolve their relative orientation. The structure of the C‐Man tryptophan moieties was calculated using ARIA/CNS.[1C4 chair and a state adjoining the B03 boat and 1S3 skew geometry . This NOE based finding is well in agreement with the conformational landscapes indicated by the 3J couplings and CCR data  and is therefore potentially also applicable to other kind of glycosylation modifications containing mannose (O‐ and N‐glycosylation) or fucose, which is derived from mannose. Notably, the Drosophila S2 cell line used in this work appeared to be especially suitable for the expression as yeast and mammalian cells show mannose toxicity after deletion of the MPI enzyme.[m of mannose.A new strategy is presented to prepare selective I enzyme.40, 414C1 and 1C4 conformations. Therefore, we evaluated the conformation of C‐mannose by using the selective 13C‐ mannose labeled UNC‐5 TSR2. Thermal denaturation studies on the protein showed that C‐Man plays an important role in the protein stability (possibly by assisting the formation of the Trp‐Arg ladder) and simulations performed with C‐mannoses in either 1C4 and 4C1 chair arrangements indicated similar effects.Preceding NMR42, 4313C‐isotope labeled mannose which allowed us to nicely resolve the signals of the mannose moieties  and perform NMR experiments that require the presence of the 13C label. In particular, the selective 13C‐labeling enables the investigation of the C‐mannose dynamics by heteronuclear 13C‐relaxation experiments and more detailed structural conformational studies by using cross‐correlated relaxation and 13C‐resolved NOESY experiments. Investigation of the sugar dynamics indicated that the UNC‐5 C‐mannoses are mostly rigid and therefore likely integrated in the stable structure of the folded protein. This is well in agreement with the observed increased thermal stability of the C‐mannosylated protein in comparison with the unmodified protein. The analysis of our 3J, CCR and NOE data shows that the C‐mannose puckers span a range of conformational space in the 1C4 hemisphere allowing interconversion between the canonical 1C4 and the B03 / 1S3 state. This conformation differs from the free mannose, which is most stable in the 4C1 hemisphere, but is well in agreement with previous NMR studies and simulations and retains the tryptophan indole moiety in the preferential equatorial position.[03 boat is a stable conformation4C1 conformation1C4 chair conformation and the flexibility of the ring permits a rotation of 180° around the C1‐Cδ1 axisUsing our novel labeling strategy, the UNC‐5 thrombospondin repeat was expressed at high yields with incorporation of uniform The exact role and mechanism of the sugar structure will only be fully understood when incorporating these observations in the larger context of the biological functions of the protein and the relevant dynamical aspects of its structure and molecular interplay, which are supposed to be affected or regulated by glycosylation. Our study using the UNC‐5 thrombospondin repeat shows a striking difference of the pyranose ring conformation between the free form and the C‐linked mannose. Other targets and glycosylation modifications may share this feature but could also show a substantially different behavior.26, 27The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Using stable isotope analysis of incremental dentin segments, we reconstruct breastfeeding, weaning, and childhood dietary patterns of Eastern Zhou period (771–221 BC) individuals from the Central Plains of China. Previous isotopic research on the Eastern Zhou demonstrated dietary difference between male and female diets in adulthood via bone collagen analysis. To understand the development of gendered dietary patterns we must examine the early life period. We aim to identify the timing of the weaning process, whether childhood diets were the same as adulthood diets, and if there were differences between the diets of boys and girls during childhood.13C and δ15N isotope data from 23 individuals from two Eastern Zhou archaeological sites (Xiyasi 西亚斯and Changxinyuan 畅馨苑).We present incremental dentin and bone collagen δ3 foods, such as wheat and soybean, for almost all children, while later adulthood diets indicate greater incorporation of C4 foods (millets), particularly for males.Weaning was completed between ages 2.5 and 4 years. Females were weaned slightly earlier than males. Early childhood diets show significant incorporation of C3 foods than expected, suggesting that grains such as wheat may have been adopted in these communities as foods for children. Nevertheless, dietary differentiation between females and males began in childhood, with boys eating more millets (C4 foods) than girls. The findings suggest that feeding children was a significant aspect of socialization and cultural gendering of individuals in ancient China.Childhood diets included greater amounts of C Resource availability during infancy and childhood shapes the energy allocation toward each of these functions and determines the organism's survival into adulthood, adult health status, as well as the reproductive success of the individual  city of Zhenghan (郑韩故城), located in the Central Plains region of China  and nitrogen (δ15N) are now commonly used in archaeological studies because these elements provide important insights into human history and evolution, especially related to dietary patterns and paleoenvironmental reconstructions   and δ13C (decline by around 1‰) in tooth and bone samples to demarcate the completion of the weaning process and the shift to a diet without significant amounts of breast milk  and where various protein sources may be introduced to children's diets . Additionally, if children are weaned onto diets that are unique to the childhood period , then isotopic profiles will not match the isotopic values of that same individual in later adult bone collagen samples, or the specified adult group dietary mean values , potentially confounding interpretation of an exact age where breast milk was no longer consumed. Finally, identifying smaller contributions by particular food groups will be isotopically masked by the more dominant food(s), such that if breast milk is retained in the diet but consumed in very small amounts (or consumed irregularly) it likely will not register in dentin chemistry. Therefore, we can at best make an estimate of a general time period where breast milk was significantly reduced and therefore other foods became more primary in the child's diet , and where diets were usually isotopically constrained with very minimal mixing across CDue to differential renewal/turnover rates of bodily tissues, we can chemically analyze various tissues to document dietary inputs from particular periods of time during the life‐course. For example, teeth develop during very specific windows of childhood and record the chemical signature of the diet consumed during that time of growth  , suggesting that children ate more plant‐based diets.Bioarchaeologists have generally used two strategies to study childhood dietary practices: analysis of bones from individuals who died during youth ; while others analyze teeth from humans of any age to capture discrete periods of early life and compare that to a bone sample to look at diachronic intra‐individual dietary patterns , while their childhood diets ranged from δ13C –19.6 to −15.0‰, containing an unexpected signal indicating some consumption of C4 foods (millets)  along with a bone sample from the same person. By studying individuals who died in adulthood, variables such as sex could be inferred  and used as a factor to study how sex may relate to weaning patterns, childhood diet, and adulthood diet. We employ the incremental method of sampling human dentition, which divides a tooth into small segments from crown to root with associated age estimations for each section  and Eastern Zhou (771–221 BC) periods. The Eastern Zhou is subsequently divided into two phases: the Spring and Autumn phase (771–476 BC) and the Warring States phase (475–221 BC). Eastern Zhou society was socially stratified with centralized power  but major states were increasingly factionalized and battled over time, culminating in the defeat of the Zhou dynasty and the unification of China by Qin Shihuang in 221 BC Feng, . During Panicum miliaceum; foxtail millet: Setaria italica) were grown on the Yellow River floodplain, starting from about 10,000 years ago, with agricultural intensification of their cultivation in subsequent millennia  was domesticated to the south of this area, in the Yangtze River valley region  and barley (Hordeum vulgare) were introduced into the Central Plains of China around the late Neolithic and were increasingly incorporated into human diets over time and Changxinyuan , while archaeological millet samples had δ13C values ranging from −11.8 to −9.6‰ (again with significant overlap of the two species). The difference in carbon isotope values is primarily derived from temporal variation in atmospheric 13C, the Suess effect, with modern atmospheric δ13C about 1.5‰ more negative than preindustrialization due to the combustion of fossil fuels  and encompassing the common millet range (−10.3 to −9.2‰). They also noted that the nitrogen isotope values overlapped, with common millet spanning δ15N 3.3 to 6.6‰ and foxtail millet ranging from δ15N 3.9 to 6.9‰  of faunal bones have previously been analyzed for stable carbon and nitrogen isotope data  and average δ15N of 6.2‰ (SD = 1.0‰), suggesting most pigs consumed a mixed diet of C3 and C4 plants, and that they may have been foddered on significant amounts of millet plants.Zooarchaeological studies of Central Plains archaeological sites have indicated the importance of various domesticated and wild animals, including terrestrial and aquatic species. The faunal assemblages of Eastern Zhou period sites are dominated by pig bones, with some contributions from dog, cattle, horse, sheep, and smaller amounts of fish, deer, turtle, bird, and other animals , with males consuming more C4 foods and females consuming more C3 foods.Expanding on these findings of dietary differences between females and males, we set to test whether male–female dietary inequality can be traced to the earliest phases of childhood. Male–female dietary differences may manifest as different timing of weaning as well as differences in weaning and childhood diets. We sampled 23 individuals from the Eastern Zhou period urban mortuary populations of Xiyasi (西亚斯) and Changxinyuan (畅馨苑), for a single, early developing, permanent tooth. We hypothesized that: (a) children were weaned between 2 and 4 years of age, showing a similar pattern to other archaeological populations that have been studied in China  and eight adult individuals from Changxinyuan had a tooth for analysis . Additionally, there were four Changxinyuan individuals without existing bone collagen data so we included a bone sample from each for isotopic analysis (Table Tooth samples were selected based on multiple criteria to try to sample across the mortuary populations: (a) preference for individuals who had been included in the previous bone collagen isotopic study by Dong et al., is Table . Age andis Table .n = 14) and canines (n = 9), which were divided into 1 mm increments after demineralization for isotopic analysis  did not yield sufficient samples for analysis, meaning there are occasional time gaps in the longitudinal tooth data  were prepared for collagen following the same method . All archaeological collagen samples  and δ15N ranging from 6.6 to 10.1‰ (average δ15N = 8.1‰), and Changxinyuan samples' δ13C ranging from −12.3 to −9.6‰ (average δ13C = −10.7‰) and δ15N ranging from 6.1 to 7.9‰ (average δ15N = 7.1‰).The corresponding bone collagen dietary data .15N values for the first dentin increment and a subsequent decline in δ15N in the earliest forming increments over the first few years of life indicating the ubiquity of breastfeeding in early life for Eastern Zhou children . Most individuals also show a significant change in δ13C over their first few years of life, with changes between 2 and 6‰ noted across the individuals studied. This variability in δ13C during early life indicates that carbon sources in these very young childrens' diets were shifting, either because their mother's diet was changing , or through the introduction of complementary weaning foods from different C3/C4 sources, or a combination of both.All the isotopic profiles show elevated δn = 7 Xiyasi and n = 2 Changxinyuan) have dentin isotopic profiles that follow the “typical” weaning curves that other studies have identified, with a 2–4‰ drop in δ15N which then generally flattens out, and a concurrent ~1‰ drop in δ13C  do not have isotopic profiles that neatly fit this “typical” weaning model, instead many show a protracted decline in δ15N over many years and significant fluctuations in δ13C, clouding the ability to make a very clear identification of the cessation of breast milk in the diet. For these individuals we made an estimation of the age of weaning based on patterns of δ15N declining in the early‐forming increments and concurrent changes in the δ13C in the isotopic profiles for these 14 individuals. We found that those estimated weaning ages ranged from about 2 to 5 years old, with an average estimated age of 3.6 years , but that there is a pattern of difference between age of weaning for Xiyasi females (average age 3.3 years) and Xiyasi males (average age 4 years). On Figure Interestingly, two historical sources provide further evidence to support the general weaning patterns observed in these Chinese archaeological isotopic studies to date. According to Analects of Confucius, which were initially recorded during the Warring States period (475–221 BC), but assumed their final form during the mid‐Han Dynasty (206 BC–AD 220), Confucius taught that deceased parents must be mourned for a 3‐year period, as this is the same length of time that parents care intensely for their children after birth: 宰我出，子曰:“予之不仁也!子生三年，然后免于父母之怀，夫三年之丧，天下之通丧也。予也有三年之爱于其父母乎?”, an influential physician of Sui and Tang Dynasties, and which is the earliest known document that discusses breastfeeding, weaning, and childhood feeding. They note that the Qianjinfang indicates that infants should be exclusively breastfed for 6 months, followed by the introduction of complementary foods . In most individuals' isotopic profiles . This suggests relative stability in dietary protein sources after age ~5 years old through adulthood until death, though there may have been fluctuations during years that our samples do not account for. A few of the individuals from the Changxinyuan cemetery , show more protracted declines in δ15N across their dentin profile, continuing past the estimated age of cessation of breastfeeding. This suggests their dietary protein intake was almost continuously changing across their childhood. However, even these individuals have their final dentin δ15N values approaching their bone collagen δ15N values, potentially suggesting relative stability in dietary protein from that point in later childhood onward in their lifetime.For most of the individuals studied, after weaning concludes the δs Figure , we see 15N post‐age 5 years was 7.5‰, and the Xiyasi males average δ15N was slightly higher at 7.8‰ . However, we caution that the Changxinyuan group is a small sample and therefore these findings may not be representative of the greater population dietary patterns.We calculated the average of the dentin isotope values for each individual after age 5 Tables  and S2 a4.2.213C dentin values, indicating urban Eastern Zhou children were fed across both C3 and C4 plant groups  which became registered in their bone collagen before death.During childhood, we see significant variability in δ13C values of boys are slightly higher than the values of girls, indicating that boys are being fed more C4 foods than their sisters, cousins, and female peers. The average dentin δ13C value of Xiyasi boys (after age 5) was −13.6‰ while the average Xiyasi girl dentin δ13C after age 5 was −14.8‰, indicating greater consumption of C4 foods by Xiyasi boys than girls. We can see this pattern in the loess curve plotted in Figure 4 foods for boys. Within the Changxinyuan group, two of the males  show a different pattern, with dentin δ13C values that are mostly less negative during childhood than their adulthood bone collagen values, indicating greater C4 (millet) consumption during childhood followed by a slightly more mixed diet with more C3 inputs during adulthood. There is a very notable difference in the δ13C values between boys and girls for Changxinyuan: the average δ13C for girl's dentin (after age 5) is −14.2‰, while the average δ13C for boy's dentin (after age 5) is −9.8‰. The carbon loess curves . The first tooth section analyzed was formed during early infancy and therefore the high nitrogen value is a product of something that occurred in the first months after birth. There are two main theories that are typically proposed to explain high nitrogen values in infants: (a) the mother had a protein‐rich diet, potentially consuming marine food sources , or (b) the infant experienced an intense period of physiological stress  we think this is unlikely given that the boy's second dentin sample drops significantly and then continues to slowly decline and stabilize, suggesting that breast milk was still a portion of his diet and therefore his mother's nitrogen values were similar to the rest of the adult female population (around 7–8‰). We believe the high nitrogen isotope value reflects a brief but significant period of stress after birth where the infant was being breast‐fed but also was catabolizing his own bodily tissues to provide him sufficient energy requirements for survival  has a δ5Feeding children goes beyond biological necessity and into the realms of sociopolitical action: what one feeds a developing body needs to be both nutritionally adequate and culturally acceptable  are separated to show the trends across each group.Click here for additional data file.


They exhibit interconnected open mesopores allowing fast ion transport and robust cycle stability with nearly 100% coulombic efficiency, along with rich redox‐reactive sites allowing high capacity even at a high rate of ≈90 C. Moreover, assembling the NMTiO2 anode with the nitrogen‐doped graphene (NG) cathode in an asymmetric full cell shows a high energy density exceeding its counterpart symmetric cell by more than threefold as well as robust cycle stability over 10 000 cycles. Additionally, it gives a high‐power density close to 26 000 W kg−1 outperforming that of a conventional sodium‐ion battery by several hundred fold, so that full cells can be charged within a few tens of seconds by the flexible photovoltaic charging and universal serial bus charging modules.Sodium‐ion energy storage is of the most attractive candidate for commercialization adoption due to the safety and cost demands of large‐scale energy storage systems, but its low energy density, slow charging capability, and poor cycle stability are yet to be overcome. Here, a strategy is reported to realize high‐performance sodium‐ion energy storage using battery‐type anode and capacitor‐type cathode materials. First, nitrogen‐doped mesoporous titanium dioxide (NMTiO −1 outperforming that of a sodium‐ion battery so that full cells can be charged within a few tens of seconds.Hybrid sodium‐ion energy storage is reported with high energy density exceeding that of a symmetric supercapacitor by more than threefold, robust cycle stability over 10 000 cycles, and high power density close to 26 000 W kg The problem is that it gives poor electronic conductivity and ionic diffusivity, thus resulting in fast capacity fading and short cycle life.Beyond the increasing demand for electric vehicles (EVs) and portable devices,2 (NMTiO2) structure synthesized through the controlled pyrolysis of a metal–organic framework (MOF) and also a nitrogen‐doped graphene (NG) were utilized as anode and cathode materials, respectively. The nitrogen‐doped mesoporous NMTiO2 structures are shown to give excellent energy density, high power density allowing ultrafast charging, and robust cycle stability over 10 000 cycles. An MOF has the secondary building units (SBUs) that could play potentially as active sites for electrochemical redox reactions. Also, its tunable porosity and functionality could offer fast ion transport pathways by enabling the facile accessibility of ion carriers to active sites. Moreover, the unique structures and properties of the NMTiO2 can be summarized as follows: 1) it contains open mesopores (≈4 nm) enabling an easy penetration of electrochemical Na‐ion carriers between the electrolyte and active sites; 2) ultrafine nanocrystals with nitrogen‐doped TiO2 units can be encapsulated inside its interior parts, which establish excellent stability at the fast charging rate such as ≈90 C (30 000 mA g−1) with negligible capacity reduction over a long cycle life; and 3) the heterogeneous N atoms in its matrix lead to enhanced electrochemical ion sorption/desorption during repeated charge–discharge cycles. Also, a NG, having many heterogeneous N atoms in the carbon matrix and facilitating the accessibility of anions, was employed as the cathode material to achieve high capacity. Furthermore, the NMTiO2//NG full cell is configured into a hybrid asymmetric configuration to demonstrate high energy density and robust cycle stability. Additionally, the flexible photovoltaic charging and USB charging modules are realized to demonstrate the ultrafast charging capability of NMTiO2//NG full‐cell devices.Herein, we realize hybrid sodium‐based electrochemical energy storages on a new paradigm strategy, where both a nitrogen‐doped mesoporous TiO2‐MIL‐125 (Ti) represented by the chemical formula of Ti8O8(OH)4(C6H3C2O4NH2)6 with a short reaction duration. During the synthesis, the functional groups of the NH2− ions and Ti4+ ions in ligands (C6H3C2O4NH2)6 and SBUs (Ti8O8(OH)4) serve as the heterogeneous dopants and metal resources, respectively. In addition, the nanocages in MOFs play an important role in introducing mesopores.Figure2 units was successfully synthesized through the simple pyrolysis of MOFs in air condition, and Figure 2 NPs. It is found that the diffusion of sodium ions in a salt‐containing electrolyte is facilitated by the introduced mesopores in the NMTiO2. Furthermore, the nitrogen‐doped graphene was synthesized as a cathode material by plasma treatment with nitrogen gas  and methanol (MeOH) , and then put into a 35 mL glass tube, which is capped by a rubber septum and placed in a microwave synthesizer. Next, the mixture was heated to 150 °C, held for 1 h, and then cooled to room temperature. The yellow product was washed with DMF and methanol several times. Also, the powder was dried and evacuated in vacuum oven at 60 °C. After then, the powder of the NH2‐MIL‐125 (Ti) was placed in an alumina boat under the steady air‐flow condition. Moreover, the sample was annealed to 350 °C, kept for 2 h, and then cooled to room temperature. The product is a light‐yellow‐colored powder, which is ascribed to nitrogen dopants playing to result in the decreased bandgap from 3.24 to 2.78 eV.2 were investigated by UV–vis absorption spectra and real photo images, and Figure S1 (Supporting Information) shows the shift of observation spectra owing to the reduced bandgap by ≈0.54 eV and the light yellow color on nitrogen doping. It is notable that the nitrogen‐doped TiO2 NPs are formed, and the mesopores between particles are introduced during the thermal oxidation process. Mesopores in the negative electrode . The rectangular parallelepiped shapes with round edges are clearly observed. The mesoporous NMTiO2, which was synthesized at a low temperature through the pyrolysis of the NH2‐MIL‐125 (Ti), has a uniform shape and size, and its morphology is quite similar to that for the NH2‐MIL‐125 (Ti) . The transmission electron microscope (TEM) and scanning TEM (STEM) images  and X‐ray photoelectron spectroscopy (XPS) analyses . The STEM–EDS mapping images further show that each constituent is uniformly distributed throughout the sample. The XPS spectra of the NMTiO2 sample show the distinct peaks corresponding to C 1s, O 1s, Ti 2p, and N 1s at ≈283, 529, 463, and 398 eV, respectively. Furthermore, the STEM–EDS analysis of the NG  presents the uniformly distributed C, O, and N species as the constituent elements of the NG, while the XPS analysis  of the NG for C 1s (≈284 eV), O 1s (≈531 eV), and N 1s (≈399 eV) demonstrates the sharp peak of N 1s indicating the existence of rich nitrogen atoms in the graphene sheets, so that they provide rich active sites for the electrochemical reactions with the ions in the electrolyte. In addition, the high‐resolution STEM image  structures was collected via the X‐ray diffraction (XRD). The high‐crystalline NH2‐MIL‐125 (Ti) was also confirmed from the sharp diffraction peaks attributed to the micropores in Figure S8a (Supporting Information).2 (JCPDS card No. 21–1272). However, for the lower‐temperature‐annealed sample, the diffraction peaks of the rutile TiO2 (JCPDS card No. 21–1276) were not observed and the peaks are on more broad shapes and lower intensities, implying that the NMTiO2 could be synthesized to have a smaller particle size and a single anatase phase in the lower temperature. Also, a nitrogen sorption analysis at 77 K  shows that NH2‐MIL‐125 (Ti) has a high surface area (1146.3 m2 g−1). The nitrogen dopants in the NMTiO2 were further investigated by XPS analyses and the Fourier transform infrared spectroscopy (FT‐IR). The N 1s peak at 399.2 eV  under the air flow conditions. In the first range below ≈335 °C that is the crystallization temperature of titanium ions, we find that small amounts of surface‐absorbed water molecules and impurities were removed. After then, the crystallization of titanium ions and the combustion of carbon species were observed to be occurring up to ≈550 °C. These results imply that the thermal oxidation of titanium ions could be proceeded above 335 °C. The electrochemical performances for NMTiO2 and NG were also evaluated in coin‐type half‐cells with the sodium foils as both counter and reference electrodes. The STEM images of NMTIO2 samples  and a typical hysteresis isotherm feature (type IV).2 frameworks annealed at low temperatures include more active sites for the electrochemical reactions. We find that the pores with an average size of ≈4 nm were significantly increased under the pyrolysis condition at the lowest temperature (350 °C). In addition, the electrochemical reactions of Na ions with the negative materials were characterized through the cyclic voltammetry (CV) measurements . The CV curves and rate performances support that the samples annealed at 350 °C for 30 min show the highest reversible capacity, thus, better than those at a higher temperature for a longer time. This indicates that the Na‐ion interaction into NMTIO2 nanocrystals should be mediated by open mesoporous channels introduced within ultrafine nanocrystals. In this reason, the sample annealed at 350 °C during the 30 min was selected as an anode material for a hybrid full‐cell device. In the charging and discharging processes, the discharging capacity is observed to be slightly higher than the charging capacity because the sodium insertion occurs during the discharging process increasing the voltage. The electrochemical impedance spectroscopy (EIS) analysis . The galvanostatic intermittent titration technique (GITT) measurements were also analyzed to determine the diffusion kinetics of Na+. Figure S12 (Supporting Information) shows the time–voltage profile of NMTiO2 during charging/discharging and the voltage plateaus for phase transition by sodium‐ion insertion were clearly observed during charging and discharging reactions. The difference of discharging behaviors was also shown in Figure S12b,c (Supporting Information). The discharging time of the 350 °C sample exhibits to be much longer than that of the 500 °C sample. Moreover, the 350 °C sample was found to accommodate the higher concentration of Na+ ions up to 1 mole fraction. From the time–voltage profiles of titrations in the voltage plateau region during discharging , we also observed that the 350 °C sample shows the much smaller ΔτE of the transient voltage change during a titration current flux after the relaxation period. Consequently, these support that the nitrogen‐doped mesoporous TiO2 synthesized at the low annealing temperature can give the facilitated Na‐ion diffusion between the surface and the electrolyte.To investigate the morphological influence on the cell performance, we synthesized the products at different processing temperatures and reaction times such as from 350 to 500 °C and from 30 min to 2 h, respectively. Figure S10 (Supporting Information) shows the thermal oxidation behavior of NHes Figure3a show s Figure  clarifies Figure  at a scae Figure ,e. The ms Figure  was also−1.Figure S13 (Supporting Information) also clarifies the electrochemical performance of the NG cathode in a half‐cell configuration investigated with the Na foil as a counter electrode in the working potential window of 3–4.5 V. The CV curve shows a typical capacitive behavior and the gravimetric measurements demonstrate the maximum capacity of ≈78 mAh g2//NG sodium‐ion full‐cell device  outperforming that of a conventional sodium‐ion battery by more than 400‐folds. The results clarify that the NMTiO2//NG full cells outperform the other full‐cell devices of same materials such as TiO2//NaLi0.2Ni0.25Mn0.75O,2Ti6O13//Na3V2(PO4)2F3,2(PO4)3//Na3V2(PO4)F3,4Ti5O12//Na3V2(PO3)3,2Ti3O7//Na2/3(Ni1/3Mn2/3)O2,2Ti3O7//GF (graphene oxide (GO) film)2//NG full cell exceeds that of 25.6 Wh kg−1 for the symmetric NG//NG full cell by more than threefold  with the high capacity retention and nearly 100% coulombic efficiency . Additionally, we demonstrated the photovoltaic charging module and USB chargeable light emitting display (LED) device  were dissolved in a mixed solvent of N,N‐dimethylformarmide and methanol , and then put into a 35 mL glass tube, which was capped by a rubber septum and placed in a microwave oven . The mixture was heated to 150 °C, held for 1 h, and then cooled to room temperature. The yellow powder product was separated by centrifugation. After washing with DMF several times, the product was stored in methanol. The product was dried and evacuated in vacuum oven at 60 °C for 1 day.Titanium isopropoxide (0.6 mmol) and 2‐aminoterephthalic acid  were placed in an alumina boat, placed within box furnace with a fixed air flow. Under the steady air flow, the samples could be decomposed by thermal oxidation reaction. The sample was annealed to 350 °C, kept for 2 h, and then cooled to room temperature. The product appeared as a light‐yellow powder. In order to obtain a highly porous structure, this annealing procedure was conducted at a relatively low temperature and long reaction time considering the crystallization temperature.The powder products of the NH3) were dissolved in concentrated sulfuric acid , and the mixture was stirred during 20 min. Then, potassium permanganate  was slowly added into the mixture in the ice bath blow 20 °C, where it was maintained for 15 min, and then it was stirred for 4 h under 40 °C. After then, deionized water (100 mL) was slowly added into the mixture, where it was maintained for 1 h. The mixture was cooled to room temperature and hydrogen peroxide  was added into the mixture. The mixture was washed via vacuum filtration with hydrochloric acid (HCl), acetone, and DI water, respectively. The product was dried via freeze drying for further use. Finally, the GO solution (1 mg mL−1) was chemically reduced with hydrazine (1 µg mL−1) as a reduction agent in oil bath at 80 °C. Then, the product was filtered and dried following the same method as for a GO product. The RGO solution was drop‐casted onto a glass, and put into plasma‐enhanced chemical vapor deposition (PECVD). Then, hydrogen and nitrogen gas plasmas were sequentially applied for 3 and 10 min, respectively.The NG was prepared via the nitrogen plasma treatment of a reduced graphene oxide (RGO), where GO nanosheets were synthesized by Hummers' method. The graphite flake (5 g) and sodium nitrate . The chemical bonding information for the functional groups present in samples was also elucidated by using an FTIR spectroscopy  analysis. The NMTiO2 and reference samples were ground with KBr using a mortar and a pestle in the ratio of 1:100 in weight, and then the mixture was pressurized through the hand‐operated pressurization to the thin pellet having the width of 6 mm. The spectra were obtained at 2 cm−1 with 50 scans per spectrum in the range of 500–4000 cm−1. The thermal behavior of NH2‐MIL‐125 (Ti) during the thermal oxidation procedure was investigated by thermogravimetric analysis (NETZCH TG 209 F1 Libra) in a range of 20–700 °C under 5 °C min−1 heating rate and air flow conditions. To investigate the changes on nitrogen doping effects, the diffused absorption spectra were obtained by a VARIAN Cary‐300 UV–vis spectrophotometer using powder samples as prepared.The morphology and structures of the samples were analyzed by the field emission SEM  and the high‐resolution STEM–EDS . The TEM specimens were prepared by dropping the dispersed samples in volatile solvents on the TEM grid (Ted Pella Inc.) The X‐ray diffraction data were collected using a SmartLab θ–2θ diffractometer in reflectance Bragg–Brentano geometry. The diffractometer used a Johansson‐type Ge(111) monochromator filtering Cu Kα1 radiation at 1200 W  to minimize line broadening and was equipped with a high‐speed 1D detector (D/teX Ultra). The 2θ range was 3°–90°. The surface chemical states were investigated by the XPS (Thermo VG Scientific Sigma Probe) analysis. Moreover, the porosity was analyzed through N2 and NG were characterized by using the 2032‐type coin cells in which Celgard 2400 and Na foil were used as separators and counter/reference electrodes, respectively. On the sample preparation, the active material, super P, and polyvinylidene fluoride (PVDF) (80:10:10 in weight) were dispersed in N‐methyl‐2‐pyroolidinone (NMP) to form a slurry. Then, the slurry was cast onto the Cu or Al foil using the doctor blade technique. The cast electrodes were dried in a vacuum oven at 80 °C overnight. The standard organic electrolyte was used in which the 1 m NaClO4 was dissolved in ethylene carbonate (EC) and diethyl carbonate (DEC) (EC:DEC = 1:1 in volume). The entire cell preparation steps were conducted in an argon‐filled glove box with the moisture content and oxygen levels less than 1 ppm. All of the electrochemical performance measurements were done at room temperature using a potentiostat/galvanostat/EIS system . Moreover, electrochemical analyses of the half‐cells were measured in an operating potential range of 1–3 V (anode) and 3–4.5 V (cathode) versus Na/Na+, respectively. The impedance analysis was conducted in a frequency range from 0.01 Hz to 1000 kHz with the amplitude of 5 mV. The GITT measurements were carried out at ≈0.1 C in a period of 1 min. Before the measurements, the pre‐sodiation procedure was conducted by attaching the NMTiO2 electrode with the Na foil during 1 h. The mass loading was measured by the ultra‐microbalance . The electrochemical performances of full‐cell devices were determined at an average mass loading amount of 3–4.5 mg cm−2 for the total mass of anode and cathode materials.The electrochemical properties of the NMTiO2 composite as the anode and the NG as the cathode, respectively. The NG cathode was prepared in the same manner as the anode. The electrochemical analysis was carried out using the same method as in the half‐cell. The electrochemical measurements of the NMTiO2//NG full cells were carried out in an operating potential range of 1–3.8 V. The electrochemical performances of full‐cell devices were determined on the mass loading of 3–12 mg cm−2 for the total mass of anode and cathode electrodes. The overall cell energy/power densities were calculated based on the total mass of cathode and anode materials .Sodium‐ion energy storage full cells were assembled using the NMTiOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.