However, at low concentrations, H2O2 can regulate signaling pathways modulating cell growth, differentiation, and migration. A few mammalian aquaporins (AQPs) facilitate H2O2 diffusion across membranes and participate in tumorigenesis. AQP3 and AQP5 are strongly expressed in cancer tissues and AQP3-mediated H2O2 transport has been related to breast cancer cell migration, but studies with human AQP5 are lacking. Here, we report that, in addition to its established water permeation capacity, human AQP5 facilitates transmembrane H2O2 diffusion and modulates cell growth of AQP5-transformed yeast cells in response to oxidative stress. Mutagenesis studies revealed that residue His173 located in the selective filter is crucial for AQP5 permeability, and interactions with phosphorylated Ser183 may regulate permeation through pore blockage. Moreover, in human pancreatic cancer cells, the measured AQP5-mediated H2O2 influx rate indicates the presence of a highly efficient peroxiporin activity. Cell migration was similarly suppressed by AQP3 or AQP5 gene silencing and could be recovered by external oxidative stimuli. Altogether, these results unveiled a major role for AQP5 in dynamic fine-tuning of the intracellular H2O2 concentration, and consequently in activating signaling networks related to cell survival and cancer progression, highlighting AQP5 as a promising drug target for cancer therapies.Reactive oxygen species (ROS), including H AQPs are assembled in a homotetrameric structure in membranes, each monomer being a functional channel that facilitates a rapid bidirectional flux of water, and, in some cases, small uncharged solutes like glycerol, in response to osmotic or solute gradients . The thixiporins ,4,5,6,7.2O2 is a small molecule produced by aerobic metabolism or in response to extracellular insults, being the main ROS participating in oxidative sensing and signaling [2O2 can have a dual effect [2O2 across cell membranes may play a crucial role in the fine-tuning of cell oxidative status and its biological consequences.The importance of AQPs for water homeostasis and energy metabolism has been extensively reported ,9. More ignaling . Dependil effect . In thisSeveral studies have reported the overexpression of AQPs in human tumors of different origin, frequently correlated with tumor stage and aggressiveness , unveili2O2 membrane diffusion and is involved in oxidative cell response [2O2, and that the high level of expression and peroxiporin activity may play a key role in cellular adaption to oxidative stress, with impact on cancer cell migration. In the present study, we expressed human AQP5 in a yeast cell model, and first investigated its functional regulation by phosphorylation and pH, disclosing the important residues for water permeation as well as the ability of AQP5 to facilitate H2O2 diffusion across cell membranes. Secondly, we evaluated the contribution of AQP5 to cell survival under oxidative conditions and to cell adaptation to an oxidative environment. Finally, we measured the H2O2 influx rate mediated by AQP5 in human pancreatic cancer cells, and revealed its impact on cell migration under oxidative stress.In a previous study, we showed that rat AQP5 also mediates Hresponse . In addi2O2 and modulates cell resistance to oxidative stress. Moreover, in human pancreatic cancer cells, the measured AQP5-mediated H2O2 influx rate indicates a highly efficient peroxiporin activity. Cell migration was similarly suppressed by AQP3 or AQP5 gene silencing, and was promoted by AQP-facilitated H2O2 transport. Altogether, these results unveil a major role of AQP5 in cancer progression, highlighting its potential as a drug target for cancer therapies.We found that AQP5 expressed in yeast localizes to the plasma membrane and increases the membrane water permeability 10-fold compared to the control. Investigation of residues important for functional regulation revealed His173, localized in the selectivity filter, as crucial for channel permeability, independent of pH. Phosphorylation of Ser183 impaired AQP5 function, probably due to its proximity to His173, interacting with the negative charge of the phosphate group. Importantly, we showed for the first time that human AQP5 transports Hf) was 10-fold higher for AQP5-transformed yeast cells ((3.70 ± 0.31) × 10−3 cm s−1 and (0.37 ± 0.03) × 10−3 cm s−1 for AQP5 and control, respectively). Incubation with 0.5 mM HgCl2 markedly reduced water permeability of AQP5-transformed yeast cells (≈64%) without affecting control cells (a), which distinguishes passive water diffusion through lipid bilayer from AQP-mediated diffusion, was lower for AQP5 cells  compared to the control   were transformed with either the empty plasmid pUG35 (control cells) or the plasmid encoding hAQP5. Prior to functional analysis, expression and localization of AQP5 at the yeast plasma membrane were verified by fluorescence microscopy using GFP-tagging A. AQP5 fol cells C, indical mol−1) D. These Recent evidence supports the idea that human AQPs can be gated via different mechanisms, including pH and phosphorylation ,21. RegaWe generated point mutations in the AQP5 aromatic/arginine region and in intracellular loop D . MutatioPermeability experiments with AQP5 WT, mutants and control cells performed at two pHs (pH 5.1 and 7.4), showed that alteration of external pH did not affect water permeability, and that this is not dependent on protein phosphorylation. A recent study reported that phosphomimetic mutation of S156E increased membrane expression of AQP5 in HEK293 cells . In our To experimentally investigate the phosphorylation gating mechanism previously proposed in silico , we meas2O2 transport by a few human AQP isoforms, including AQP3 [2O2 transport [2O2 permeation through membranes.Several studies have reported Hing AQP3 , AQP8 [3ing AQP3 , and AQPing AQP3 ). Regardransport . Sequenc2 electrode). Briefly, hydrogen peroxide was added to cell suspensions, and H2O2 uptake was evaluated by monitoring its conversion into O2 with a Clark electrode after addition of catalase in excess to samples of the cell suspension. Since we found that human AQP5 is not pH-regulated, we performed the assays at a pH optimal for yeast cells (pH 5.1). As depicted in 2O2 consumption was three-fold higher for yeast cells expressing AQP5 compared to control cells. In addition, incubation with 0.5 mM HgCl2, shown above to inhibit AQP5 water permeability, reduced H2O2 consumption to the basal level corresponding to diffusion through the lipid bilayer, not affecting control cells. These data confirm that AQP5 can facilitate H2O2 membrane permeability. Since H2O2 uptake measured by the Clark electrode relays on its extracellular disappearance, and to assure that the high rate of H2O2 consumption observed for AQP5-yeast cells was due to cellular uptake, we loaded the cells with the ROS sensitive fluorescence probe H2-DFCDA and followed the intracellular increase in fluorescence after incubation with a range of H2O2 concentrations. As depicted in 2O2 concentration, largely facilitated by AQP5 expression.Thus, we measured the consumption of external hydrogen peroxide by yeast cells expressing human AQP5 using an electrochemical assay . This “overcoming” effect was not observed in control cells that were depleted of endogenous aquaporins, in which H2O2 slowly diffused through membrane lipids. As a control, catalase activity was also evaluated in non-treated cells and after incubation with 1 mM H2O2 for 60 min; absence of statistical differences indicate that this antioxidant scavenger is not responsible for the observed cellular resistance of AQP5-yeast cells.Subsequently, cells were exposed to short-term oxidative stress, i.e., in the presence of 1 mM Hd 60 min B. Result2O2 transport. Evaluation of relative gene expression by quantitative real-time RT-PCR demonstrated that both AQP3 and AQP5 were expressed in BxPC3, although with different levels of expression  still expressing AQP3, and cells silenced for AQP3 (siAQP3) still expressing AQP5, responded to H2O2 addition with a similar rate of fluorescence increase, approximately half the control , for AQP5 (siAQP5), and control cells transfected with a non-targeting construct (siCtrl).To investigate whether AQP5-mediated Hp < 0.01) and significant recovery after 24 h  (Studies on monolayers of BxPC3 control cells (siCtrl) showed a partial recovery of wound area after 12 h  A. Howeve2O2 stimulus on the viability of BxPC3 cells. As depicted, the addition of H2O2 up to 200 µM induced a maximal 30% loss of cell viability , but renders cells more resistant to short-term oxidative stress, evidencing the positive contribution of AQP5-mediated H2O2 are blocked by the permeability barrier imposed by biomembranes, and additional H2O2 is needed to force cell migration. It has been reported that during normal conditions (eustress) the gradient between extracellular and intracellular H2O2 is higher than 200-fold [2O2 permeability and prompting cellular processes like proliferation and migration [2O2 perfectly agrees with this notion.The recovery of the migration rate by external oxidative stimulus demonstrates that when AQP3 or AQP5 are downregulated, signaling events triggered by H200-fold ,32,33; tigration . The obs2O2 to activate signaling networks related to cell survival and proliferation, AQP5 can regulate cellular resistance to oxidative stress as well as facilitate cancer cell migration, and represents a promising target for the development of cancer therapies.Altogether, our findings demonstrate that AQP5 can play an important role in cancer cell survival. By allowing a dynamic fine-tuning of intracellular Hw/v glucose, 0.67% (DIFCO) yeast nitrogen base) supplemented with the adequate requirements for prototrophic growth [600 1.0).Transformed yeast strain was grown in YNB medium  using the Metafluor software .For subcellular localization of GFP-tagged AQP5 in 2. Cells were grown in RPMI1640 medium with 10% FBS and 1% penicillin/streptomycin. Medium was changed every 2–3 days and experiments were performed with 70% to 80% cell confluence.Biopsy xenograft of Pancreatic Carcinoma line-3 (BxPC3) was obtained from ATCC  and cultured at 37 °C in 5% CO® Negative Control siRNA #1  was employed as the negative control to ensure silencing specificity in all the experiments. Briefly, medium was removed and cells were supplied with Opti-MEM I reduced serum medium without antibiotics (Opti-MEM) . siRNA (30 pmol) were diluted in Opti-MEM and mixed with Lipofectamine™ RNAiMAX transfection reagent  pre-diluted in Opti-MEM according to the manufacturer’s instructions. After 5 min incubation at room temperature, the mix was added to the cells and incubated at 37 °C in 5% CO2. For double silencing, both AQP3-RNAiMAX and AQP5-RNAiMAX complexes were added to the cells. After 48 h of incubation, the knockdown efficiency was evaluated by quantitative real-time RT-PCR, as below.Short interfering RNA (siRNA) targeting human AQP3 (ID: s1521) and human AQP5 (ID: s1527) were purchased from Ambion. SilencerTotal RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen), according to the manufacturer’s protocol. The RNA was treated with RNase-free DNase I  to avoid contamination with genomic DNA. Extracted RNA was quantified with Nanodrop™ 2000c spectrophotometer. For template cDNA synthesis, 1 µg of total RNA was reverse transcribed in a 20 µL final volume using random hexamers primers  and 200 units of M-MLV reverse transcriptase (Invitrogen), as previously described .Real-time PCR reactions were carried out using a CFX96 Real-Time System C1000 , the TaqMan Universal PCR Master Mix , and the following specific TaqMan pre-designed gene expression primers: AQP1 (Hs01028916_m1), AQP3 (Hs01105469_g1), AQP5 (Hs00387048_m1), and ACTB (Hs99999903_m1) . The cDNA was amplified at the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.–ΔCt method (adapted from Reference [The relative quantification of gene expression was determined using the 2eference ). Using 6 cells/well and were allowed to adhere for 24 h prior to AQP silencing. After 48 h incubation with silencing reagent, a wound was made with an even trace in the middle of the monolayer using a sterile 10 µL pipette tip. After washing three times with phosphate buffered saline (PBS) to remove cell debris, cells were incubated with vehicle or with 100 µM H2O2 prepared in low serum (2% FBS) RPMI medium . The cells were then incubated at 37 °C in a 5% CO2 incubator, and images of the wound were captured at intervals of 2 h. The distance of the wound was measured under a light microscope and analyzed using the software ImageJ (https://imagej.net). Wound closure was normalized to original wound area at time 0. All samples were tested in triplicate, and the data are expressed as the mean ± SEM.BxPC3 cells were seeded in six well microplates at a density of 0.15 × 10600 1.0 were harvested by centrifugation , washed three times and resuspended in ice cold sorbitol (1.4 M) K+-citrate (50 mM pH 5.1 or 7.4) buffer up to a concentration of 0.3 g mL−1 wet weight, and cells were incubated on ice for at least 90 min. Prior to permeability assays, cells were preloaded with the non-fluorescent precursor 5-(and-6)-carboxyfluorescein diacetate , which is intracellularly hydrolyzed, yielding the impermeable fluorescent form (CF). Cells were then diluted (1:10) in 1.4 M sorbitol buffer and immediately used for experiments.For water permeability assays, yeast transformants grown up to ODPermeability assays were performed by stopped-flow fluorescence spectroscopy, as previously described , using a2O2 consumption was measured in intact yeast cells. Cells were harvested by centrifugation , resuspended in fresh growth media and incubated at 30 °C with orbital shaking. Hydrogen peroxide (50 µM) was added to intact yeast cells, and the consumption of H2O2 was measured in samples of the cell suspension by following O2 release with an oxygen electrode  after the addition of catalase [2O2 consumption is reported as a first order rate constant (s−1) obtained from the slope of a semi-logarithmic plot of H2O2 concentration versus time.The Hcatalase . H2O2 co2O2 influx, oxidation kinetics of non-fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate  to fluorescent 2′,7′-dichlorofluorescein was measured until 60 min from H2O2 administration, according to previously described methods [To evaluate H methods .600 1.0 were harvested by centrifugation  , washed three times with phosphate buffer 0.1 M pH 5 and resuspended in the same buffer to OD600 1.4. Cells were then incubated with 5 µM H2-DCFDA for 45 min at 30 °C and washed one time with phosphate buffer 0.1 M. Cells were transferred to black multi-well plates and incubated with several concentrations of H2O2 (0.5–50 mM). Fluorescence intensity was measured after addition of H2O2 over time until 60 min in a microplate reader at an excitation/emission of 485/520 nm . As control, H2O2 non-treated cells were incubated with 5 µM H2-DCFDA and fluorescence intensity was followed. The intracellular ROS accumulation was calculated from the slope of a plot of fluorescence intensity versus time and normalized to non-treated cells.Yeast transformants grown up to OD6 cells/well, and were allowed to adhere for 24 h prior to AQP silencing. After 48 h incubation with silencing reagent, H2O2 transport was measured in individual adherent cells on a coverslip. Briefly, cells were loaded with 10 µM H2-DCFDA for 30 min at 37 °C in 5% CO2. Next, cells were washed twice with Ringer Buffer (RB) pH 7.4  and the coverslips with the cells were mounted in a closed perfusion chamber  on the stage of a Zeiss Axiovert 200 inverted microscope, using a 40× epifluorescence oil immersion objective. Fluorescence was excited at wavelength 495/10 nm; emission fluorescence was collected with a 515/10 nm band pass filter. Data were recorded and analyzed using the Metafluor Software  connected to a CCD camera . Cells were equilibrated in RB pH 7.4 for 2 min, and then 100 µM H2O2, freshly prepared in RB, was added directly to the cells. H2-DCFDA fluorescence was scanned every 10 s. For inhibition studies, cells were incubated with 10 µM Auphen for 15 min or 0.1 mM HgCl2 for 5 min at 37 °C in 5% CO2. H2O2 consumption is reported as a first order rate constant obtained from the slope of a semi-logarithmic plot of H2O2 concentration versus time.BxPC3 cells were seeded in six well microplates at a density of 0.15 × 102O2 was freshly prepared at the time of inoculation for oxidative stress experiments. Yeast strains were grown in liquid YNB medium, with orbital shaking, at 30 °C up to OD600 ≈ 1.0 corresponding to 1 × 107 cells/mL. Cells were harvested by centrifugation   and resuspended to OD600 1.0 in fresh growth media and incubated with 50 µM curcumin or 50 µM naringenin at 30 °C with orbital shaking for 60 min. Cells were then harvested by centrifugation   and resuspended to OD600 ≈ 10, and multi-well plates were prepared with serial 10-fold dilutions of the original concentrated culture up to 10–8; 3 µL suspensions were spotted with replica platter for 96 well plates device on plates containing YNB solid medium with and without H2O2 and incubated at 28 °C. Differences in growth phenotypes of yeast strains were recorded after 2 days of incubation.Qualitative growth assay was performed on solid YNB medium, supplemented with 2% (w/v) glucose, containing hydrogen peroxide. Solid YNB medium with 1 mM H600 1.0). Cells were harvested by centrifugation   and resuspended to OD600 1.0 in fresh growth media and incubated with 1 mM H2O2 at 30 °C with orbital shaking for 15 and 60 min. Multi-well plates were prepared with serial 10-fold dilutions of each strain up to 10−6 and 3 µl suspensions were spotted on solid YPD plates. As a control for maximum viability, cells without treatment were also diluted and plated as described above. YPD plates were then incubated for 2 days at 28 °C until visible growth was observed and colonies were counted. Results are expressed as percentage of the time 0 (non-treated cells) colony number.Quantitative growth assay was performed on solid YPD medium. Yeast cells were grown overnight to mid exponential phase  , washed once with K+-citrate 50 mM pH 5.1 buffer, and resuspended to OD600 1.0 in fresh growth media and incubated with 1mM H2O2 at 30 °C with orbital shaking for 60 min. Cells were then harvested by centrifugation   and dry pellets were stored at −80 °C until analysis. Dry pellet was dissolved in phosphate buffered saline (PBS) and disrupted mechanically by vigorous agitation with acid washed glass beads for seven 1 minute intervals with cooling intervals between each agitation cycle. After disruption, cell lysates were cleared by centrifugation  (VWR™ Micro 1207 Centrifuge) and the supernatants were used for assays. Prior to performing the assays, protein concentration of cell lysates was determined according to Bradford, using bovine serum albumin as a standard [For antioxidant measurements, yeast transformants grown up to ODstandard .2O2 degradation in cell lysate, which occurs mostly by catalase activity, as it has one of the highest turnover numbers among all the enzymes. For catalase activity assay, 40 μL of supernatant was mixed with H2O2  for the start of the reaction. Different dilutions of hydrogen peroxide (0–75 mM) were used for standards. The reaction was stopped after 5 min by addition of ammonium molybdate , and color development was measured spectrophotometrically in a plate reader at 405 nm . One unit of catalase activity is defined as the amount of enzyme needed for degradation of 1 µmol of H2O2/min at 25 °C. Catalase activity was expressed as units of catalase per milligram of proteins in cell lysate (U mg−1).The catalase activity was measured by the modified method of Goth . This mep-values < 0.05 were considered statistically significant.All the experiments were performed in biological and technical triplicates. Results were expressed as mean ± SEM of at least three independent experiments. Statistical analysis between groups was performed by two-way ANOVA and non-parametric Mann–Whitney test using the Graph Prism software . 2O2 through cell membranes and contributing to cell proliferation and migration. Selective targeting of AQP5 may open new perspectives for anti-cancer drug development.This work unequivocally demonstrates that human AQP5 is involved in redox biology, facilitating the passive diffusion of H


Plasmodium falciparum k13 gene are associated with artemisinin (ART) resistance. However, it is unclear whether the F446I mutation, the most prevalent allele at the China–Myanmar border and north of Myanmar, is associated with ART resistance. Therefore, the aim of this study was to investigate the role of this mutation in ART resistance by generating transgenic parasites expressing the F446I mutant allele.Mutations in the 0–3 h) was used to evaluate ART susceptibility of the transgenic parasites in vitro.The transgenic parasites carrying the F446I or C580Y mutation in both 3D7 and FCC1/HN isolates were generated by single crossing-over recombination and verified using PCR and gene sequencing. The ring-stage survival assay of 0–3 h (RSAF446I mut, 3D7C580Y mut, FCC1/HNF446I mut and FCC1/HNC580Y mut were successfully generated. These parasite lines showed no changes in the expression level of k13 when compared with their parent parasite isolates. However, introduction of the F446I mutation in k13 of the 3D7 and FCC1/HN isolates led to elevated ring survival rates detected using RSA0–3 h when subjected to both 700 and 20 nM concentrations of dihydroartemisinin. The survival rates were similar to those detected in the parasite lines with the C580Y mutation.Four transgenic parasite lines named 3D7k13 led to increased ring survival, suggesting that this mutation may be associated with ART resistance and could be used as a molecular marker for monitoring ART-resistant parasites. The results also highlights the importance of surveillance of F446I mutants for containing the resistant parasite.Insertion of the F446I mutation in  Plasmodium falciparum parasites in western Cambodia; a delay in clearance time was observed in infected patients who had received the standard 3-day treatment [Artemisinin (ART) is a quick, effective anti-malarial drug that was discovered in China , 2. Sincreatment , 5. The reatment , 7. The 0–3 h) that is well correlated with in vivo parasite clearance rates [P. falciparum  has been identified as a molecular marker for monitoring ART-resistant parasites [k13 is associated with slow parasite clearance rates in vivo and high parasite survival rates in vitro [k13 in the Thai–Cambodian/Thai–Myanmar border areas are C580Y, Y493H, R539T, and I543T, while that in the northern Myanmar and China–Myanmar border areas is F446I [0–3 h survival rates [k13 [k13 at the China–Myanmar border and in northern Myanmar. Previous study reported that of 392 isolates from the China–Myanmar border, 128 had the F446I mutation with a prevalence of 32.7% [k13 gene of 2 distinct laboratory strains, 3D7 and FCC1/HN, of P. falciparum and investigate the impact of the mutation on ART susceptibility.Currently, tools available for the detection and surveillance of ART-resistant malaria parasites include measurement of in vivo parasite clearance rates and ring-stage survival assay of 0–3 h , and 5 mg/ml AlbuMAXII . Plasmodium falciparum lines were propagated in human O+ erythrocytes  and maintained at 37 °C in 5% CO2 and 90% N2. The parasites were synchronized using 5% d-sorbitol, as described previously [The parasite culture was maintained in vitro in RPMI 1640  with 10 mM HEPES , 20 μg/ml hypoxanthine , 50 μg/ml gentamicin , 0.17% NaHCOPfK13F446I and pARL-PfK13C580Y recombinant plasmid. The full-length k13 gene with F446I mutation and 1563 bp of the C-terminal of k13 gene was amplified using overlapping PCR. The primers for amplifying fragments with F446I were as follows: 1350 bp 5′-terminal fragment was amplified by primer pair of PARL-F (GCagatctATGGAAGGAGAAAAAGTAAAAACAAAAG) and K13-446-R (TCCACCTATACAAATTACTAATGGG); while 831 bp 3′-terminal fragment was amplified by primer pair of K13-446-F (CCCATTAGTAATTTGTATAGGTGGA) and PARL-R (GCggtaccTTATATATTTGCTATTAAAACGGAGTG). The two fragments were combined by PCR through overlapping sequences to generate the full length k13 gene with F446I mutation by primer pair of PARL-F and PARL-R. Similar strategy was used to generate the1563 fragment of k13 gene with C580Y mutation: the 1128 bp upstream fragment was amplified by primer pair of PARL-A-F (GCagatctAACGGAATTAAGTGATGCTAGTG) and K13-580-R (AGCAACATACATAGCTGATGA) while the 435 bp downstream fragment by K13-580-F (GCTATGTATGTTGCTTTTGAT) and PARL-R. The resulting fragments were cloned into the pARL-GFP plasmid by using the restriction sites BglII and KpnI  to generate recombinant plasmids of pARL-PfK13F446I and pARL-PfK13C580Y. The recombinant plasmids were purified using the Endo Free Plasmid Maxi Kit .The pARL-GFP plasmid containing the drug screening gene hDHFR was used to construct the pARL-P. falciparum 3D7 and FCC1/HN (isolated from Hainan Province and established for in vitro continuous cultivation in 1978) parasites were transfected with the recombinant plasmids as described previously [P. falciparum parasite culture with a high synchronous ring stage was prepared at 5% parasitaemia. 270 μl Cytomix buffer  was mixed with 100 μg of the plasmid, and then with 50% haematocrit of the infected erythrocytes. The mixture was transferred to a 0.2-cm cuvette (Bio-Rad) and electroporated using the Bio-Rad MicroPulser with voltage 310 V and 950 μF. The transfected culture was selected using 2.5 nM WR99210  after 48 h until the WR99210-resistant parasites were obtained. DNA was extracted using QIAmp Mini kits  and detected by PCR using primers hDHFR, P3/P4, P4/P5, and P3/P6. The primers for identification of transfected parasites were as follows: hDHFR-F: TTAATGTATTATTCCAATGTGCATGAT, hDHFR-R: GTACTTAATGCCTTTCTCCTCCTG; P3: ACCATGATTACGCCAAGC; P4: AAAACGAACATTAAGCTGCC; P5: AACAAGGCGTAAATAT TCGTGT; P6: ATAAGAACATTTAAAATTTCTTCATT. The sequences of the modified k13 gene were confirmed by DNA sequencing. RNA extracted from the F446I mutant parasite was digested by DNaseI  for 15 min in room temperature before transcribed to cDNA. The full-length of k13 gene was amplified from the cDNA of F446I mutant parasite and subcloned to pEASY-T1 cloning Vector . The primers for amplification were as follows: K13-F: ATGGAAGGAGAAAAAGTAAAAACAAAAG; K13-R: TTATATATTTGCTATTAAAACGGAGTG. The RNA of F446I mutant parasite after digestion was used as template to amplified k13 gene as a no-RT control. The k13 gene in the pEASY-T1-K13 recombinant plasmid was sequenced using the primers of M13 as follows: M13F-GTAAAACGACGGCCAGT; M13R-CAGGAAACAGCTATGAC.Synchronous eviously . Brieflyk13 protein was produced by GenScript Corporation (USA). Briefly, a synthetic peptide (MEGEKVKTKANSISC) was conjugated with keyhole limpet haemocyanin and formulated with adjuvant. Rabbits were immunized for 4 times at 2-week intervals. The polyclonal antibodies were affinity-purified with peptides.The antibody against 2, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 0.65% NP-40, 1 mM DTT, and 1× protease inhibitor cocktail). The mixture was incubated for 1 h at 4 °C and centrifuged at 14,000 rpm to obtain the supernatant. The supernatant was separated by 10% SDS-PAGE gel and detected using PfK13 polyclonal antibody (1:200). The Actin (Abcam-ab40854) was used as the loading control (1:1000). The secondary antibody was IRDye800CW goat anti-rabbit (LI-COR) diluted at 1:5000.Western blot was performed as described previously . Briefly0–3 h was performed as described previously [P. falciparum parasites were synchronized 2 times with 5% sorbitol to obtain tightly synchronous early-ring stage. The initial culture was prepared at 1% parasitaemia and 2% haematocrit, and then was exposed to 700 or 20 nM DHA  for 6 h. Then, the culture mixture was washed 3 times with non-serum 1640 culture medium, and cultured again for 66 h. The viable parasites were counted using a microscope in Giemsa-stained thin smears. The survival rate was calculated using Ps/Pc × 100%, where Ps is the parasitaemia in the DHA treatment group, while Pc is the parasitaemia in the control group. The counting was performed by two different experienced laboratories. Each assay was repeated for 3 times independently, with each group setting three technical repetitions. Each technical repetition was counted for 40,000 erythrocytes.RSAeviously . Brieflyk13 gene were produced with either F446I or C580Y mutations by using PCR. For construction of the replacement F446I, 2 DNA fragments covering the entire k13 gene (2181 bp) were amplified from the genomic DNA of P. falciparum. The two fragments were combined using PCR with an overlapping sequence of 15 bp in which a point mutation of T to A was introduced, resulting in changes to the corresponding amino acid residue from phenylalanine (F) to isoleucine (I) (designated as PfK13F446I mut gene). The full-length PfK13F446I mut gene contains the 5′ cleavage site of KpnI and 3′ cleavage site of BglII, which allowed cloning of the entire gene into the vector pARL-GFP through corresponding sites in the vector and generation of the recombinant plasmid designated as pARL-K13F446I. The substitution of T to A in the PfK13F446I mut gene was verified by DNA sequencing. The overall strategy for the preparation of PfK13F446I mut is outlined in Fig. To generate the recombinant plasmids for the transfection, the fragments of PfK13C580Y mut gene. However, in this case, only a truncated k13 gene (1563 bp) was amplified instead of the full gene. This is because of the position of the C580Y mutation near the C-termini and that only a partial sequence of the gene is needed for single crossing-over recombination. Similarly, two fragments covering the truncated k13 gene were amplified and combined using PCR. A point mutation of G to A at nucleotide position 1739 was introduced through the overlapped neighbouring sequences of the 2 fragments, resulting in a change from cysteine (C) to tyrosine (Y) (designated as PfK13C580Y gene).A similar strategy was used for the generation of the P. falciparum, 3D7 (designated as 3D7wt) and FCC1/HN (FCC1/HNwt), were used for the transfection. Parasites in the high synchronous ring stage were transfected with individual recombinant plasmids by electroporation, as described above , indicating that only the mutant allele is expressed.As shown in Fig. k13, the k13 expression of each transgenic parasite clone were determined with western blot. As shown in Fig. k13 in both 3D7C580Y mut and 3D7F446I mut clones were similar to those in the 3D7wt line. Similar expression levels of k13 were detected in FCC1/HNwt and the transgenic parasite lines FCC1/HNC580Y mut and FCC1/HNF446I mut.To investigate whether the integration of the mutant alleles affected the expression of 0–3 h was performed to evaluate the transgenic parasite lines expressing F446I and C580Y mutants of k13 for their susceptibility to DHA. First, ring survival rates of both transgenic parasites and their parent lines were measured when parasites were exposed to elevated concentrations of DHA. As shown in Fig. wt and 3D7F446I mut parasite lines were observed (p > 0.05), while at 11, 22, and 44 nM DHA, the ring survival rates of 3D7F446I mut were significantly higher than that of 3D7wt . At the concentrations beyond 44 nM, i.e., from 88 to 700 nM, no parasite was observed in the 3D7wt parasite line, and different ring survival rates were detected in the 3D7F446I mut line. A similar result was obtained for the FCC1/HNF446I mut line when compared with its parent parasite strain  when 700 nM DHA was used, while the ring survival rates for the transgenic parasite lines of both 3D7F446I mut and 3D7C580Ymut were similar at 0.73 ± 0.10% and 0.91 ± 0.11%, respectively . Similar results were obtained after measuring the ring survival rates of FCC1/HN F446Imut and FCC1/HN C580Y mut lines when compared with the FCC1/HNwt strain  from the above-mentioned experiment were selected to further measure the ring survival rates of the F446I and C580Y mutant parasite lines. No parasite of the wild type strain of 3D7wt  at 20 nM DHA. The sequencing of the PfK13 gene of the 3D7F446I mut-F1 clone showed an additional mutation at nucleotide position 1156 from G to T, resulting in a change in amino acid residue from valine to leucine at position 386 (data not show). It is rare for multisite mutation to occur in k13 in field isolates. Further investigation is required to understand whether more than 1 mutation in k13 affects the ART susceptibility of the parasite.For generating transgenic 3D7 parasites with the F446I mutation, 7 clones were obtained by sub-cloning the WR99210-resistant parasite by limiting dilution. Then these clones were sequenced to confirm the presence of F446I. All the clones showed increased levels of ring survival from 17.6 to 29.5% at 20 nM DHA when compared with 3.15% survival of 3D70–3 h [k13 [k13 mutations, such as C580Y, R539T, Y493H, and I543T, have been reported to be associated with the resistance of parasites to ART [k13 is F446I. In previous study, the result showed that the prevalence of this mutation is 32.7% in the China–Myanmar border area and this mutation was not detected in Thai/Cambodia and Thai/Myanmar border regions [ART resistance is evaluated in vivo by delayed parasite clearance and in vitro by high ring survival rates with RSA0–3 h . The res3 h [k13 , 10. Soms to ART , 19, 20. regions . From 20 regions  conducte regions ; however regions . A recen regions .0–3 h. Of them, only 6 had a high ring survival rate  that was defined as the association with delayed parasite clearance in vivo, and the ring survival rates of the other isolates were below the P95 value. The statistical analysis indicated that the F446I mutant was not associated with high ring-stage survival. Thus, there is much debate on whether the F446I mutation is associated with ART resistance. In this study, F446I mutation in k13 was introduced to the ART-sensitive 3D7 and FCC1/HN strains, and the resulting transgenic parasite lines FCC1/HN F446I mut and 3D7 F446Imut showed increased ring survival rates at both 20 and 700 nM DHA concentrations. Moreover, the survival rates of the transgenic parasites with the F446I mutation were similar to those of the parasites with the C580Y mutation demonstrated to be correlated with ART resistance. Thus, the result suggested the F446I mutation may be associated with ART resistance.In previous study, the ring survival rate of 27 isolates with F446I mutation were measured by using RSAk13 is a primary marker for ART resistance and the most prevalent mutation in western Cambodia where the resistant parasite was first reported. Several transgenic parasite clones have been generated in which the C580Y mutation was introduced into the k13 gene of various parasite isolates by using the CRISPR–Cas9 technology or zinc-finger nucleases [k13 C580Y showed survival rates of 11–15%, while another transgenic FCB parasite clone expressing the C580Y allele showed a survival rate of 1.9% when compared with the survival rate of 0.3% in the parent FCB isolate. We generated both FCC1/HNC580Y mut and 3D7C580Y mut transgenic lines that showed 0.63 ± 0.07% and 0.91 ± 0.11% RSA0–3 h survival at 700 nM DHA concentration, whereas the survival rates of their parent parasites were 0%. Interestingly, when the DHA concentration reduced to 20 nM, both transgenic lines showed 17.5 ± 0.80% and 33.08 ± 1.20% survival rates, whereas their parent parasites showed only 2.86 ± 0.44% and 3.15 ± 0.13% survival rates, respectively. It could be concluded that the introduction of both C580Y and F446I mutations in k13 of 2 distinct laboratory parasite strains resulted in increased ring survival indicating that, similar to the C580Y substitution, F446I may play role in parasite response to ART.The C580Y mutation in ucleases , 13. Thek13 is the most prevalent allele at the China–Myanmar border and north of Myanmar. In this study, this mutation is associated with the increased ring survival rates by genetically introducing the mutation into the k13 gene of distinct ART-sensitive laboratory parasite strains, suggesting that the F446I mutation be used as a molecular marker for monitoring ART-resistant parasites and highlighting the importance of surveillance of F446I mutants for resistant parasites.In summary, the F446I mutation in


N2O emissions from soils irrigated at different salinity levels varied in a similar pattern which was triggered by soil moisture dynamics. Yet, the magnitudes of pulse N2O fluxes were significantly varied, with the peak flux at 5 g/L irrigation salinity level being much higher than at 2 and 8 g/L. Compared to fresh water irrigated soils, cumulative N2O fluxes were reduced by 22.7% and 39.6% (N0), 29.1% and 39.2% (N120) for soils irrigated with 2 and 8 g/L saline water, while they were increased by 87.7% (N0) and 58.3% (N120) for soils irrigated with 5 g/L saline water. These results suggested that the effect degree of salinity on consumption and production of N2O might vary among irrigation salinity ranges. As such, desalinating brackish water to a low salinity level (such as 2 g/L) before it is used for irrigation might be helpful for solving water resources crises and mitigating soil N2O emissions.To reveal the effect of irrigation salinity on soil nitrous oxide (N The rise of atmospheric N2O concentrations is primarily due to unreasonable agricultural management activities [2O emissions [2O emissions from soil [Due to a shortage of irrigation water, brackish water , or marginal saline water, has been adopted as an alternative of fresh water for agricultural irrigation in some areas of the world ,2,3,4,5.tivities ,12. Soilmissions ,14,15. Trom soil .2O emissions [2O fluxes from a drip-irrigated cotton field without nitrogen (N) fertilizer application increased with irrigation salinity from 0.4 to 8.0 ds/m. Yet, other studies [2O emission significantly, either from soils or industrial waste-water sludge. Furthermore, some researchers [2O emissions varied among soil conditions. For instance, at under 65% water hold capacity (WHC), soil N2O fluxes at 11.7 g/L salinity level were greater than at 35.1 g/L, while it showed the opposite behavior at under 40% WHC [2O flux at 8.0 ds/m irrigation salinity level was greater than at 0.4 ds/m, but it showed opposite results under 360 kg N/ha [2O emissions, few researchers have focused on N2O emissions from soils irrigated with brackish or saline water. The connection between N2O emissions and irrigation salinity is still unclear.Increasing salt-affected agricultural land has attracted interest to understand the influence of salinity on soil Nmissions ,20,21,22 studies ,20 showeearchers ,22 found 40% WHC . Moreove2O fluxes from soils irrigated with water at different salinity levels, and discuss its responses to variations of soil moisture and salinity.Thus, pot experiments were conducted on a silty clay soil treated with or without N application, under three irrigation salinity levels. The primary objective of this study was to quantify N3 for 0–5 cm layer and 1.33 g/cm3 for 5–50 cm layer, according to the local soil’s native bulk density. Columns were left undisturbed for over 60 days to avoid the influences of soil disturbance on N2O emission. Detailed soil properties are listed in Experiments were conducted from May to August 2016 on a silty clay soil collected from a plastic greenhouse located at Nanjing Vegetables Scientific Institute in 2012 . A soil sample was air-dried, ground, sieved, homogenized, and finally packed into cylindrical soil columns  to a bulk density of 1.25 g/cm2 at a mass ratio of 1:1 to fresh water. Urea (N = 46.2%) was used as the N source. Fresh water (Ec = 0.3 ds/m), either with N0 and N120 application, was set as a control (namely treatments CK and CK + N). All treatments  were replicated six times (three for gas sampling and another three for soil sampling). The soil was watered twice, at 22:00 on 19 July and 22:00 on 3 August 2016. For each treatment, 3530 and 3210 mL of fresh or saline water was supplied to replenish surface (0–20 cm) soil moisture to field capacity for the first and second irrigation. Air temperatures after the first irrigation ranged from 33.1 to 38.4 °C, which was on average 8.3% higher than that after the second irrigation (29.9–38.4 °C).The soil sample was irrigated at three salinity levels: 2, 5 and 8 g/L , either for zero (N0) or field N application levels . Saline water was produced by adding NaCl and CaClGas samples from each soil column were collected daily during the observation period using the static chamber , which was made of 8 mm polyvinyl chloride (PVC) and equipped with a thermometer and an electric fan at the top for measuring air temperature and mixing air inside . A rubbe2O concentration was analyzed within 48 h after sampling using a gas chromatograph system  with an electron capture detector (ECD). N2O fluxes were calculated according to the linear increment in N2O concentration within the chamber [2O fluxes were calculated by integrating daily values along the sampling period. N chamber . CumulatSoil samples (approximately 3–5 g each) from 20 cm depth in 5 cm increment were collected at 10:00 h every 2 days using stainless steel samplers (O.D. = 10 cm). Soil moisture content was determined gravimetrically by drying at 105 °C for 24 h, and water-filled pore space (WFPS) was calculated as the percentage of soil volumetric moisture content relative to the soil porosity. Soil electrical conductivity (Ec) was measured (soil:water = 1:5) using an electrical conductivity meter . 2O fluxes (n = 3) among treatments in non-pulse or pulse period. A paired t test was conducted to evaluate the difference among soil depths or treatments soil-related factors  with time series .Statistical analysis was performed using SPSS 19.0 . A Fisher’s least significant difference (LSD) test was performed to determine the difference in soil average or cumulative Np > 0.1).No significant differences were found in soil temperature among treatments with fresh or saline water irrigation . For allSoil moisture at 0–15 cm layers varied in the similar pattern and fell within same ranges in both saline water and fresh water irrigated soils, either under N0 or N120 level . Soil mop < 0.05) than that of fresh water irrigated soils (0.06–0.2 ds/m), either under N0 or N120 level. For S2 + N, S5 + N and S8 + N treatments, soil Ec mainly varied in the ranges of 0.2–2.2 ds/m, 0.3–3.8 ds/m, 0.5–5.4 ds/m, respectively. It increased remarkably with irrigation salinity from 2 to 8 g/L, with averages from S5 + N and S8 + N treatments 59.1% and 139.8%, i.e., significantly higher than from S2 + N treatment (p < 0.05). For soils with N0 application , soil Ec was slightly lower than that of soils with N120 application, but varied in a similar pattern.In contrast to soil moisture, soil Ec was low immediately after irrigation and increased gradually along with the decrease of soil moisture . General2O fluxes from saline water irrigated soils were quite different among salinity levels, even though their temporal courses triggered by the soil wetting-drying process were much similar. Under the N120 level, N2O fluxes from soil irrigated at 5 g/L salinity level were much higher than at 2 and 8 g/L levels after both irrigations. Under the N0 level, a similar phenomenon was found for soil N2O fluxes after first irrigation, yet it remained almost the same among salinity levels after the second irrigation , and then declined to 66.5, 41.9, and 71.7 μg N2O/(m2h). Irrigation significantly stimulated N2O emission from soils, with peak N2O fluxes after second irrigation 32.3–140.9 times greater than observed on 2 August prior to the second irrigation. Compared to CK + N treatment, the peak N2O flux was enhanced by 8.2% for S5 + N treatment, while it was reduced by 10.2% and 28.8% for S2 + N and S8 + N treatments. As a result, the average N2O flux from S5 + N treatment increased by 1406.1 μg N2O/(m2 h), and from S2 + N and S8 + N treatments decreased by 702.1 and 944.7 μg N2O/(m2h), compared to CK + N  and 2078.2–4424.2 μg N2O/(m2h). The peak N2O fluxes from S2 and S5 soils were 5.7% and 66.5% higher than CK, yet the flux from S8 soil was 40.5% lower. Consequently, the average N2O fluxes from S2 and S8 treatments were reduced by 22.6% and 39.6%, while from S5 treatment was increased by 87.7% (p < 0.01), compared to CK. Across salinity levels, N2O fluxes were significantly lower in unfertilized soils, decreasing by 39.1% on average (p < 0.05)  .2O fluxes from saline water irrigated soils under N0 level ranged from 586.3 to 1822.4 mg/m2, with fluxes at 5 g/L salinity level as the highest . Yet for 5 g/L saline water irrigated soil, it increased by 73.0% on average. Over the whole experimental period (30 days), cumulative N highest . The app2O emissions [2O emissions during drying processes were confirmed from saline water irrigated soils in the current experiment  were much higher from soils irrigated at 5 g/L salinity level than at 2 and 8 g/L, while conditions of soil WFPS and temperature kept almost the same ranges .Generally, pulse Nropriate . In curre ranges . It impl2O emissions, whereas their conclusions were contradictory to each other [2O fluxes from 2 and 8 g/L (Ec of 3.6 and 12.7 ds/m) saline water irrigated soils were significantly lower than from fresh water irrigated soils (Ec of 0.3 ds/m), while from 5 g/L (Ec of 8.1 ds/m) saline water irrigated soil, it enhanced remarkably. This phenomenon likely suggested that the effect degree of salinity on consumption and production of N2O might vary among irrigation salinity ranges. Some previous results once indicated that the activity of N2O reductase, which reduces N2O to N2 in nitrogen transformation, was inhibited to a lesser degree under low rather than medium salinity [2O emission at moderate rather than low salinity levels. Furthermore, the activities of ammonia-oxidizing bacteria and ammonia-oxidizing archaea were significantly reduced under high salinity levels, which may result in the inhibition of the first step of the nitrification (ammonia oxidation) process, and thus, possible reduced N2O emission levels [2O emissions under high salinity level. These results also indicated that reducing salinity of brackish water from 5 to 2 g/L could be helpful for N2O mitigation.Previous researches had investigated the impact of salinity on Nch other ,21,22,31ch other , or showch other ,21,31. Isalinity ,33,34. Tn levels . This mi2O emissions from soils irrigated with water at different salinity levels. Results indicated that the response of N2O emissions to salinity might vary among irrigation salinity ranges, and irrigation with desalinated brackish water could be a potential measure for N2O mitigation. But unfortunately, the N2O emissions were collected from a soil with low initial salinity (Ec = 0.4 ds/m), and the irrigation salinity level did not set in a finer classification . Thereafter, more investigation should be done to reveal the effect of salts on N2O emissions under different initial soil salinity conditions and higher irrigation salinity levels, and to figure out a suitable irrigation salinity level, which is helpful for mitigating N2O emissions while meeting the requirement of crop growth.With increasing shortage of agricultural water resources, brackish water  is widely applied for irrigation ,36. The 2O emissions from soils irrigated with saline water were found to be triggered by soil moisture dynamics, and the magnitude of pulse N2O fluxes for 5 g/L treatment were significantly higher than those for 2 and 8 g/L treatments. These results implied that soil wetting-drying processes dominated the temporal process of N2O emissions from saline water irrigated soils, while irrigation salinity and N level affected its magnitudes remarkably. Compared to fresh water irrigation, 2 and 8 g/L saline water resulted in lower N2O emissions from soils either under N0 or N120 level, while 5 g/L saline water irrigation enhanced it significantly. It can be inferred that the sensitivity of N2O production and consumption to irrigation salinity might be different among salinity ranges, and reducing the salinity of brackish water might be a potential measure for solving agricultural water crises and reducing soil N2O emissions.Pulse N


Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by transposon tagging mutagenesis with the Ac element. We describe the generation of the mutagenized population and the screening to isolate new lpa1 mutants. In particular, we developed a fast, cheap and non-disrupting screening method based on the different density of lpa1 seed compared to the wild type. This assay allowed the isolation of the lpa1-5525 mutant characterized by a new mutation in the lpa1 locus associated with a lower amount of phytic phosphorus in the seeds in comparison with the wild type.Phytic acid, or myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases to make available P together with all the other cations needed for seed germination. When seeds are used as food or feed, phytic acid and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Therefore, reducing the amount of phytic acid is one strategy in breeding programs aimed to improve the nutritional properties of major crops. In this work, we present data on the isolation of a new maize ( After incubation of 1 h at room temperature the blue-colored phosphomolybdate complex was observed: the intensity of the blue color is directly proportional to the free phosphate content. The free phosphate content was quantified by using a spectrophotometer (λ 650 nm) and adopting a series of calibration standards obtained from a stock solution of KH2PO4.The Chen assay  was perf−1 frequency. For each sample, in a beaker we added 20 mL of hydrochloric acid (0.66 M) to 1 g of flour that was vigorously stirred overnight at room temperature. 1 mL of extract was transferred into a 1.5 mL microfuge tube and centrifuged at 13,000 rpm for 10 min; then 0.5 mL of supernatant was transferred in a fresh microfuge tube and neutralized by the addition of 0.5 mL of sodium hydroxide solution (0.75 M). The neutralized extracts were submitted to enzymatic dephosphorylation reaction using the solutions supplied by the kit and trichloroacetic acid (50% w/v). The reactions were done in duplicate, to determine free phosphorus and total phosphorus. For colorimetric determination of phosphorus, 1 mL of sample extract was transferred into a 1.5 mL microfuge tube with 0.50 mL of color reagent . The samples were mixed by vortex and incubated in a water bath at 40 °C for 1 h.In order to measure the content of phytic acid we used Megazyme’s kit K-PHYT 11/15 (Astori-Tecnica). Flour samples were obtained using a ball mill , grinding the seeds for 1 minute at 21 oscillations sThe standard phosphorus solutions were prepared as described in manufacturers’ instructions, with the only modification that after preparation the standards were not treated as samples, i.e., incubated at 40 °C, but were left at room temperature for 1 hour. The phytic acid content was quantified by using a spectrophotometer (λ 655 nm). The data obtained with the Megazyme software were expressed as g phytic acid/100 g of flour that we converted into mg phytic P/g of flour.ZmMRP4 sequence-specific amplification polymorphism (S-SAP) markers that allowed the identification of the lpa1-1 versus Lpa1 allele. The allele-specific forward primers were designed on the single nucleotide substitution polymorphism in the ZmMRP4 10th exon [Lpa1 wild type–specific forward primer was ZmMRP430L (5’-GTACTCGATGAGGCGACAGC-3’), whereas lpa1-1 mutation-specific forward primer was ZmMRP432L (5’-GTACTCGATGAGGCGACAGTG-3’). The reverse primer ZmMRP410R (5’-CCTCTCTATATACAGCTCGAC-3’) was used to amplify both wild type and lpa1-1 alleles.A molecular analysis was performed using 0th exon ,36. The Lpa1/lpa1-1 allele-specific amplifications contained an aliquot of genomic DNA, 1 × Green Go Taq buffer , 2.5 µM MgCl2, 0.2 µM each of dATP, dCTP, dGTP, and dTTP, 0.3 µM of forward ZmMRP430L/ZmMRP432L-specific primer, 0.3 µM of reverse ZmMRP410R primer and 1.25 unit of Go Taq Flexy DNA polymerase (Promega), in a final volume of 25 µL. The reaction mixture of the Lpa1 and lpa1-1 amplicons were 468 bp long. The amplicons were loaded on 1% (w/v) agarose gels and visualized by ethidium bromide staining under ultraviolet light.The reaction mix underwent an initial denaturation step at 94 °C for 2 min, 37 cycles of denaturation at 94 °C for 45 s, annealing at 62 °C for 1 min, extension at 72 °C for 1.5 min. Extension at 72 °C for 5 min was performed to complete the reaction. The ZmMRP4 locus was amplified in wild type and in putative new lpa1 mutant using an high fidelity long range DNA polymerase  with the primers 5L (5’-TGGTGAGGGGATCAGAGACG-3’)  and ZM 2R (5’-GAACTTCCAAAGGCAAGGGACA-3’) , ZM2F (5’-GGAAAAGTGAGCTCCAAAGTTTA-3’)  and 51R (5’-AAGCATCAGCTTCGGGTAATGT-3’) . The primers positions are referred to the ATG on the genomic sequence.The amplicons obtained were run on 1% agarose gel and visualized by ethidium bromide staining under UV light.


The XRD, FESEM and XPS analyses were indicated that the vanadium is successfully doped in the crystal lattice of LiNi0.84Co0.10Mn0.06O2 with high crystallinity. 0.05 mol% vanadium doped LiNi0.84Co0.10Mn0.06O2 exhibits superior initial discharge capacity of 204.4 mAh g−1, cycling retention of 88.1% after 80 cycles and rate capability of 86.2% at 2 C compared to those of pristine sample. It can be inferred that the vanadium doping can stabilize the crystal structure and improve the lithium-ion kinetics of the layered cathode materials.Ni-rich layered LiNi However, the long-term cycling stability is proportionally deteriorated with increasing Ni contents. It can be caused by surface side reactions, phase transitions during charge-discharge, the reduction of Ni4+ and oxygen release, resulting in deterioration the crystal structure. This phenomenon suffers a decrease in electrochemical performance, and hinder commercialization3.Lithium-ion batteries (LIBs) are being widely used for portable device, uninterruptible power supplies (UPS), hybrid electric vehicles (HEVs), electric vehicles (EVs) and so on. The recent demand for LIBs has been considerably increasing and the research for LIBs focuses on high capacity, long cycle life and high-rate capability. Recently, Ni-rich layered oxide has received a lot of attention due to their higher specific capacity, lower cost and lower toxic than other cathode materials4, dopants5, core-shell6, concentration-gradient structures7 and single crystalline materials9. Among them, doping is an effective strategy to improve stability of layered structure because the dopant ions help suppression of the phase transition and the voltage fading. So far, lots of single dopants have been explored such as Al10, Ti11, Zr12, Mg13, Mo14, Na15, Sn16, B17, Fe18 and Cr19 to improve the electrochemical performance of layered cathode materials. Vanadium was also studied as one of the dopant by many researchers because vanadium can take various valence state, leading to the stability of the crystal and increase of Li-ion diffusion coefficient23. These can significantly improve the capacity, coulombic efficiency, cycleability and rate capability. To the best of our knowledge, the effect of the vanadium doped Ni-rich NCM have still not been studied.Many efforts have been made to solve these problems and modify cathodes using coatings0.84Co0.10Mn0.06O2 (hereafter V-doped NCM) by solid state method to stabilize the crystal structure and deliver superior electrochemical performances.Therefore, in this paper, we synthesized well-crystallized V-doped Ni-rich LiNi0.84Co0.10Mn0.06O2, the Ni0.84Co0.10Mn0.06(OH)2 precursor was prepared by a co-precipitation method. An aqueous solution of NiSO4·6H2O, CoSO4·7H2O and MnSO4·H2O was used as starting materials. Simultaneously, a NaOH solution and NH4OH solution as a chelating agent were also used. The prepared spherical Ni0.84Co0.10Mn0.06(OH)2 precursor was mixed with LiOH·H2O at a molar ratio of 1: 1.05 and V2O5 powder as a vanadium source with the molar ratios of 0, 0.005, 0.01 and 0.02 mol%. After that, the powders were calcined at 500 °C for 5 h and 760 °C for 15 h in air. V-doped NCM was prepared as illustrated in Fig. For the synthesis of LiNiThe crystal structure of the materials was characterized by X-ray diffraction , in the 2θ range of 10°–80°. The morphology of the samples was observed by field emission scanning electron microscopy  and elemental mappings of the samples were measured by an energy dispersive X-ray detector . The chemical state of transition was conducted by X-ray photoelectron spectroscopy .6 in a mixed solution containing ethylene carbonate (EC)/dimethyl carbonate (DMC)/ethylmethyl carbonate (EMC)  was employed as electrolyte. The assembly of all coin cells was carried out in an argon-filled glove box.For the electrochemical performance, the cathode electrode was prepared by mixing 96 wt% active material, 2 wt% super P and 2 wt% polyvinylidene fluoride (PVDF) binder. The mixed slurry was coated on Al foil (16 μm in thickness) and dried at 100 °C for 10 h in a vacuum oven. After that, the electrode was punched into disks and then dried at 120 °C for 10 h. A 2032 coin cells were fabricated with pristine and V-doped NCM as a cathode and lithium foil (500 μm in thickness) as an anode. A polyethylene  was used as a separator and 1 M LiPF−1. The electrochemical impedance spectroscopy (EIS) measurement was conducted with a VSP300 impedance analyzer using the frequency range of 1 MHz to 10 mHz with 5 mV amplitude.Charge-discharge test was carried out galvanostatically under the voltage range of 3.0–4.3 V and various current density using electrochemical equipment  at 25 °C. Cyclic voltammetry (CV) of the samples was carried out by multi potentiostat  between 3.0 and 4.3 V at a scan rate of 0.1 mV s2 structure with the space group R-3m24. It can be inferred that V-doped NCM maintains the crystal structure of pristine NCM since XRD patterns of V-doped NCM are similar to that of pristine NCM due to low doping concentration26. The V-doped NCM has a clear peak splitting of the (006)/(102) and (108)/(110), indicating well-ordered layered structure27. The (003) peak shifts to lower angle with increasing V content by Bragg’s law (nλ = 2dsinθ), indicating vanadium was successfully incorporated into the NCM cathode. It can be inferred that the V5+ ions, having larger ionic radius (0.59 Å) than those of Ni2+ (0.56 Å), Co3+ (0.54 Å) and Mn4+ (0.53 Å), were substituted into the lattice of the NCM28. It can also be explained by charge compensation that dopant can change the valence states of transitional metal with different ionic radii via vanadium doping29. Therefore, the lattice parameters increase linearly with an increase in doping concentration, enabling smooth and fast kinetics of Li ions30. The I(003)/(104) ratio indirectly means the degree of cation mixing in the layered structure, resulting from similar ionic radius of Li+ (0.76 Å) and Ni2+ (0.69 Å)31. There is a little difference in the I(003)/(104) ratios and the ratios of pristine, 0.005 mol% (V-0.005 NCM), 0.01 mol% (V-0.01 NCM) and 0.02 mol% (V-0.02 NCM) doped NCM were 1.31, 1.35, 1.29 and 1.25, respectively. The V-0.005 NCM shows the highest I(003)/(104) ratio of 1.35, indicating the low cation mixing with lithium ions at the 3a site, transition-metal ions at the 3b site32. We can assume that the cation mixing is decreased by replacing Ni in NCM with V, beneficial to Li ion diffusion. However, excessive V doping above 0.01 mol% shows a lower I(003)/(104) ratio, indicating high Li+/Ni2+ cation mixing, since it can lead to deformation of the NCM original structure30. Therefore, we can infer that suitable vanadium substitution can reduce the Li/Ni cation mixing with well-ordered layered structure.The XRD patterns of pristine and V-doped NCM are shown in Fig. 33.Figure Figure 3/2 and Ni2p1/2. The Ni2p3/2 peak appears at 855.8 eV, which means a high oxidation state35. The binding energies of Co2p3/2, Co2p1/2 appear at 781.8 eV, 795.4 eV, indicating the dominant Co3+ cation. The Mn spectrum has the binding energy of 644.3 eV for the Mn2p2/3, indicating Mn4+ cation36. It agrees with the previous report for the NCM cathode materials37. Although no peaks corresponding to dopant vanadium are observed at the XRD , respectively in a potential range of 3.0–4.3 V at 25 °C. All the samples displayed typical charge-discharge profiles of Ni-rich NCM materials40. The pristine delivered the highest discharge capacity (204.6 mAh g−1) and coulombic efficiency (89.6%). The initial discharge capacity and columbic efficiency slightly decreased with increasing the V content. It can be ascribed to the partially substituted electrochemically inactive V ions, occupying the electrochemically active transition-metal site (3b site), as reported earlier by Zhu et al.22. There was only a very slight difference between pristine and V-0.005 NCM. It was reported that the most of vanadium can enter the transition-metal site in the crystal lattice28. The small amount of vanadium dopant remaining in the NCM generates Li3VO4 impurity at the surface, resulting in low electrochemical activity due to reduced active materials, as shown in Fig. 2(b)22. However, V-doped NCM samples showed the superior cycle stability than that of pristine, as shown in Fig. 22. In addition, less Ni2+ ions occupy the Li+ sites for the NCM, leading to the NCM structure more stable. Thus, substituted vanadium ions into transition metal sites contribute to the structural stability during long-term cycling. The results are summarized in Table For electrochemical tests, the loading level of the electrode was adjusted about 14.5 mg/cmves Fig.  and cyclves Fig.  of prist41.Figure s) in the high frequency is not considered because the same electrolyte was used in this paper42. The semicircle at high frequency is related to resistance of solid electrolyte interface (RSEI) and the semicircle in the middle frequency represents the charge transfer resistance (Rct) at the interface between the electrode and electrolyte. The straight line with the slope of 45° at low frequencies, corresponding to the Warburg impedance, is related to Li+ diffusion in the bulk electrode43. We can observe that V-doped NCM exhibited lower RSEI and Rct than those of pristine NCM, as shown in Table SEI value of V-doped NCM is closely related to the stable surface chemistry in NCM since the total bonding energy of metal-oxygen on the surface alleviates the surface degradation, as mentioned in Fig. ct is the key factor for the cathode impedance of cell, determining the electrochemical activity44. As shown EIS spectra, the V-0.005 NCM represented the lowest Rct. Therefore, EIS result indicates that vanadium substitution can effectively suppress the increase in RSEI and Rct which could facilitate efficient lithium ion intercalation, resulting in superior rate capability and cyclability.To investigate the effect of the vanadium doping on the electrolyte and NCM interfacial resistance, electrochemical impedance spectroscopy (EIS) were measured after 80 cycles, as shown in Fig. −1. The oxidation/reduction peaks of pristine and V-0.005 NCM were observed around 3.82 V/3.67 V and 3.77 V/3.69 V, respectively, corresponding to Ni2+/Ni4+ 21. Also, the redox peaks were observed around 4.25 V/4.14 V and 4.23 V/4.15 V, respectively, which are corresponding to Co3+/Co4+ 45. As can be seen in Fig. 48. These low Rct and polarization values ensure slow capacity fading during long term cycling.To demonstrate the reason for the improved performances of V-0.005 NCM, CV measurements were conducted. Figure 0.84Co0.10Mn0.06O2 cathode material was synthesized by solid-state reaction. V-doped NCM samples exhibited better cyclic stability and rate capability (at rates as high as 2 C) compared to pristine NCM. Among them, the 0.005 M vanadium doped NCM showed the excellent structural stability and best electrochemical performances. It can be inferred that the amount of vanadium that can be substituted into the transition metal sites is limited and residual vanadium produces deleterious Li3VO4 impurity. The introduction of an appropriate amount of vanadium dopant not only provides smooth and rapid lithium ion insertion-extraction by large lattice parameters but also increase the electronic conductivity of NCM. More importantly, it has a positive effect on strong bonding between vanadium and oxygen, enabling remarkable structural stability. As a result, we can conclude that V-0.005 NCM can be regarded as a promising cathode for next-generation LIBs.In this paper, V-doped LiNi


Microbial communities became dominated by Firmicutes bacteria within 16 h, while few changes were observed in the fungal communities. Changes in N-biogeochemistry were rapid and denitrification enzyme activity (DEA) increased up to 25-fold with the highest rates occurring in microcosms at pH 8.3 that had been incubated for 24-hour prior to measuring DEA. Nitrous oxide reductase was inactive in the pH 4.7 controls but at pH 8.3 the reduction rates exceeded 3,000 ng N2–N g−1 h−1 in the presence of native DOM. Evidence for dissimilatory nitrate reduction to ammonium and/or organic matter mineralisation was observed with ammonium increasing to concentrations up to 10 times the original native soil concentrations while significant concentrations of nitrate were utilised. Pure isolates from the microcosms were dominated by Bacillus spp. and exhibited varying nitrate reductive potential.Rapid and transient changes in pH frequently occur in soil, impacting dissolved organic matter (DOM) and other chemical attributes such as redox and oxygen conditions. Although we have detailed knowledge on microbial adaptation to long-term pH changes, little is known about the response of soil microbial communities to rapid pH change, nor how excess DOM might affect key aspects of microbial N processing. We used potassium hydroxide (KOH) to induce a range of soil pH changes likely to be observed after livestock urine or urea fertilizer application to soil. We also focus on nitrate reductive processes by incubating microcosms under anaerobic conditions for up to 48 h. Soil pH was elevated from 4.7 to 6.7, 8.3 or 8.8, and up to 240-fold higher DOM was mobilized by KOH compared to the controls. This increased microbial metabolism but there was no correlation between DOM concentrations and CO Denitrification efficiency is primarily affected by soil pH because pH influences carbon supply and associated metabolisms but also impacts the activity of denitrification enzymes adapted to specific pH conditions and the function of N2O reductase (N2O-R)  . Dissimilability . Again, eduction . AlthougThe long-term effects of pH on microbial community structure and abundance have been studied at local to global scales, but only broad conclusions can be drawn . For exa2)2) is added to the soil (as fertiliser or urine), it quickly undergoes hydrolysis in the presence of urease enzymes: − ions produced during this process cause substantial pH increases, to values >7.5 over the course of a few days, coupled with the pH mediated release of dissolved organic matter (DOM)  . During tabolism —suitablent study , using KFirmicutes increased by 38% after urine addition while Proteobacteria decreased (18%), but fine resolution of taxonomic groups contributing to denitrification or any other N-cycling process was not achieved.In addition to allowing documentation of soil physicochemical response, urine patches represent a natural laboratory setting for investigating microbial community structural and functional response to rapid pH change. A few studies have reported changes in soil microbial population structures in response to urine (or synthetic urine), but have not investigated the taxonomic profiles of these populations in detail . In the 4OH proxy  and all chemicals used were ACS reagent grade, unless otherwise stated.−1; total N, 2.4 g kg−1; NO3-N, 20 mg kg−1 and NH4-N, 4 mg kg−1. Further details about the soil and sampling site can be found in c kg−1 soil). These treatments were selected based on results from previous experiments  at Lincoln, Canterbury, New Zealand. The basic chemical characteristics of the soil were: pH 5.6; total C, 27 g kgeriments  with the minimal .A total of 240 soil microcosms were prepared, covering 4 KOH treatments and 5 incubation times. This provided twelve analytical replicate microcosms for each KOH rate ×  incubation time combination; where, four microcosms were designated for soil chemical analysis prior to DEA assays, two were designated for nucleic acid extraction and microbial culturing work (prior to DEA), and the remaining six microcosms were used for DEA assessments  .2  over a 30 min period until O2 was <0.03%. The microcosms were then incubated at 20 °C on an orbital shaking platform (150 rpm) for 16, 24, 32, 40 or 48 h. After each incubation the headspace was sampled for N2O and CO2.Microcosms were prepared as described by 2 (acidified with 2 mL of 2M HCl to dissolve any carbonates). The remaining slurry from these four microcosms was centrifuged , and the supernatants filtered (<0.45 µm) then frozen (−20 °C), pending pH, EC, DOC, DON and NOThe following sampling protocol was followed: for chemical analyses, 5 mL of slurry was collected from four microcosms to determine dissolved CO3-N kg−1  and respiration rates were calculated from the linear relationship between evolved N2O or CO2 and time. The extracts were analyzed for pH (ThermoScientific Orion™ AquaPro™ pH combination electrode) and electrical conductivity (Eutech Instruments PC510 conductivity meter). Dissolved organic C was determined using a Total Organic Carbon Analyzer . Total N was determined by persulfate oxidation, as described by Analytical methods are described in A 1 mL aliquot of soil slurry was centrifuged at 14,000 rpm for 5 min. DNA from the resulting pellet was extracted with the MoBio Powersoil DNA kit . The V3–V4 variable regions of the bacterial 16S rRNA was amplified with the 341f and 785r primer pair . The funPCR amplifications contained ∼10 ng DNA template, 10 nmol each primer, 1×  mastermix, and 0.5 U KAPA3G polymerase , in a final volume of 20 µl. Reactions were performed in duplicate. Cycling parameters were 94 °C for 2 min; 30 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s. Duplicate reactions were combined and purified with AMPure XP beads . Purified amplicons were quantified by gel electrophoresis and UV absorbance (NanoDrop ND-1000). Amplicons were 2 ×300 bp paired end sequenced on an Illumina MiSeq platform .http://www2.dpes.gu.se/project/unite/UNITE) (http://biom-format.org/) (USEARCH v8.0.1517  was usede/UNITE) . Biom (Oat.org/)  in USEARX + 1) transformations were applied.The phyloseq  and ggplTo understand gradients and group structures across treatments, matrix plots of standardised data were prepared using a reduced sample set of 20 OTUs, with the OTUs retained having the greatest contribution to total counts for the individual samples compared at each pH value, or in the case of fungi, across all pH values. Using reduced sample sets allows simplification of the matrix plots by removing those organisms accounting for a negligible proportion of the total number of OTUs. Samples in the matrix plots were clustered using Bray Curtis similarity  while the 20 OTUs presented were clustered according to similarity based on an index of association across samples tested.Previous research by 3 (30g L−1 + 0.5 g L−1 KNO3). Plates were incubated under anaerobic conditions (Whitney jars with gas packs) at 24 °C for 2 to 6 days.Ten µL sample of slurry from each pH adjusted microcosm was serially diluted (10 µL into 1 mL followed by three dilutions of 10 µL into 100 µL) and plated onto TSB or 1/10 diluted TSB containing KNORepresentative colonies of different morphology were selected and re-streaked onto the same media and grown under anaerobic conditions at 24 °C. Isolates were stored in 20% glycerol at −80 °C, with a selection of isolates identified by amplification and Sanger sequencing of the 16S ribosomal RNA gene using the 27F and 1492R primers .3. 20 mL cultures were then initiated with 1/100 dilutions of the overnight cultures, and grown anaerobically (O2 replaced with N2) at 24 °C. After 48 h, gas samples were extracted with a gas-tight Hamilton syringe and analysed for the presence of N2O and CO2 by gas chromatography (as outlined above) and 10 mL samples of the bacterial cultures were removed for NOCells were grown aerobically overnight in TSB with KNOc kg−1 KOH had average pH values of 4.7, 6.7, 8.3 and 8.8 respectively. During the 48 h incubation EC reached ∼6.4 mS cm−1 in all microcosms except the pH 6.7 microcosms which reached ∼5.7 mS cm−1  occurring during the first 16 h of incubation. During the 48 h incubation DON in the control microcosms increased from 2.4 to 7.4 mg kg−1, while in the pH 6.7, 8.3 and 8.8 microcosms DON increased ∼9, 140 and 223-fold in response to KOH addition, respectively (R2 = 0.98)  .2 production) was associated with DOM increases but the amount of DOC (or DON) solubilised via pH change was not a good predictor of respiration. Respiration in the control and pH 6.7 microcosms generally followed an increasing trend for the first 32 h before decreasing (2-C g−1 h−1). Respiration in the pH 8.8 microcosms followed an opposing trend, declining slightly during the first 32 h, followed by recovery.Higher respiration . In the pH 8.8 microcosms, NH−1 during the first 16 h of incubation and remaining at that concentration until after 32 h, when a further increase from 30 to 50 mg kg−1 occurred by 48 h .The control microcosms had the highest concentrations of Ncubation . The pH 2O g−1 h −1 recorded for the control, pH 6.7, 8.3 and 8.8 treatments respectively .Microcosms incubated for 16 h had the lowest DEA rates with 80, 540, 34 and 0.2 ng Nectively . DEA remfor 48 h . In contproduced . DEA rat2O-R activity during the DEA assays based on the N2O/(N2O + N2) ratio. These showed that N2O reduction was absent in the control microcosms while in the pH 6.7, 8.3 and 8.8 treatments between 56 and 100% of the N2O produced was reduced to N2 depending on incubation time  profiles during the DEA assays were mirrored by decreases in NO−1 occurred in the pH 8.3 microcosms after 32 h incubation and in the 8.8 microcosms after all incubation times . It is acknowledged that lower soil respiration in the presence of acetylene will reflect both the absence of any CO2 derived from metabolic N2O reduction but possibly also general impediment of other anaerobic metabolisms. It is unlikely that acetylene would have served as dominant carbon source during the 4 h DEA incubation given the excess DOC available and time required to adapt to using acetylene   project has been deposited at DDBJ/EMBL/GenBank under the accession d fungi) . Approxid fungi) .The phylum level bacterial profile from the control (pH 4.7) microcosms after 16 h incubation was made up of Acidobacteria (∼7%), Actinobacteria (∼12%), Bacteroidetes (∼7%), Proteobacteria (∼20%), Firmicutes (∼5%), Planctomycetes (∼5%) and phyla that had abundances of >5% including Verrucomicrobia, Chloroflexi, Gemmatimonadetes and Armatimonadetes. Up to 35% of OTUs could not be classified . After 2Tepidibacter sp.). These two OTUs then decreased later in the incubation, partially displaced by other Clostridium spp. (OTUs 16 and 26)   . The inc and 26) . Across  and 41) .Bacillus, Paenibacillus, Clostridium and Alkaliphilus spp.  and a low representation of Actinobacteria and Bacteroidetes compared with the pH 4.7 control microcosms. Across the incubation period, the dominance of Firmicutes increased further, initially driven by the same Bacilliales and Clostridiales OTUs (4 and 1), but at a much higher relative ‘abundance’ than observed in the pH 6.7 treatment. Their expansion coincided with the highest DEA rates, with the average ‘abundance’ of OTU_4 being ∼20% of total at this time and OTU_1 being ∼23% , Fig. S3lus spp. . CommuniBacillus, Paenibacillus, Clostridium and Alkaliphilus spp.   .An identical analysis of rarefied bacterial and fungal OTUs datasets is presented in 2O, and five showed moderate utilisation of NO2O. One isolate showed production of NH2O and two isolates exhibited respiration but did not appear to utilise N. Of the 33 isolates, 22 were selected for identification by ribosomal 16S gene DNA sequencing. All were from the Firmicutes phylum, of which three were Paenibacillus spp. and one was a Brevibacillus sp.  compared with isolates that exhibited high NO4 and N2O production. Relevant 16S sequences were submitted to NCBI with accession numbers assigned between MH211426 and MH211463, submission number SUB3915485 (Bacillus spp. and three Acidovorax spp. (All isolates except one of the B3915485 . Six orgrax spp. .+ (KOH) and NH4OH—product of urea hydrolysis) are much more effective in solubilising organic matter than divalent cations such as Ca2+ (Ca(OH)2) (KOH additions and resulting pH elevation caused the concentration of DOM in soil microcosms to greatly increase (150-fold) compared to the controls. Added hydroxyl ions displaced negatively-charged organic molecules into solution. Previous work has shown that monovalent cations like KCa(OH)2) . The amoCa(OH)2) , as werer) over oligotrophs (K) , pH is known to be a dominant environmental variable that shapes soil microbial communities . Changesophs (K) , but at ophs (K) .Previous analysis of the soil used in this study suggests that a mix of carbon sources are released as pH increases, of which 45% are bioavailable. These range from labile hexose and pentose sugars to more recalcitrant polyphenolic molecules . The lacFlavobacteriales), specifically in the control and pH 6.7 microcosms (∼5.5-fold-increase in DOM with ∼2-fold increase in respired CO2), suggesting that the microbial community does respond to higher concentrations of bioavailable C at pH values <7.Proteobacteria are abundant in high C soils  with β aRhizobiales), Actinobacteria and Acidobacteria OTUs at all pH and DOM values, while a few β- and γ-Proteobacteria OTUs increased. Our results suggest that the chemical changes induced by KOH addition to soil are comparable to soils where pH and DOM are elevated due to higher urea inputs. Niche differentiation occurs in soil where higher bovine density (and presumably urea inputs) induces increases in pH and total organic carbon, with Actinobacteria, α-Proteobacteria and Verrucomicrobia decreasing and Bacteriodetes increasing  and then followed by Clostridiales (up to 53%),  have been observed in alkaline soil crusts (pH 8.5) that were rehydrated and incubated under dark anoxic conditions  that are highly responsive to more recalcitrant C-sources, are either alkaliphilic or alkalotolerant, and show fermentative type metabolisms  identified in this study suggest a wider range of pH tolerance  indicates no fungal biomass change  of low NO2O-R and nitrate reductase  ratio in the control microcosms equalled 1, indicating that this treatment did not have active N2O-R. 2O-R depends on the post-transcriptional pH being greater than 6.1, which is consistent with our results. In an agricultural environment this raises interesting ecological questions, because urea hydrolysis happens to elevate pH for several days which would immediately alleviate any post-transcriptional interference of nos Z expression and allow rapid production of functional N2O-R.In contrast to the KOH amended microcosms, the N2 within 16 h and maximum rates after 24 h which indicates that suitable redox conditions for denitrification were established quickly in our microcosms and a corresponding rapid genetic and enzymatic response followed. The predominant electron acceptors in a weakly reducing environment are O2, NO2) (2O flux and relative soil gas diffusivity (Dp/DO)  occurring within <10 h (We observe full denitrification of NOe (MnO2)  with the (Dp/DO) . Biologiand 42 h . Recent in <10 h .Bacillus spp. and they have been shown to be numerically important culturable members of denitrifying communities in agricultural soils (Paenibacillus (pH 8.3 microcosms) are also capable of heterotrophic nitrification, dissimilatory NOPaenibacillus, some Bacteriodetes (as observed in the pH 6.7 microcosms) have N2O-R and have been observed to fully denitrify NO2  . Using tfication .Bacillus species are well known as nitrate reducers and N2O emitters, but many strains do not produce N2O after NOBacillus species with varying concentrations of N2O produced  is derived from labile (nucleophilic) organic N such as amines  influence the competition between denitrifiers and DNRA bacteria through competition for fermentative C-substrates (electron donors). Higher ratios between substrates and nitrate leads to a combination of fermentation and DNRA (both fermentative and respiratory) with no denitrification. When the ratio between substrates and nitrate lowers, denitrification takes a larger role until it eventually out-competes both fermentation and DNRA . Comparial nosZ’ .−1, but were not out of the ordinary when compared to other studies investigating urine additions to soil, nor is the associated release of excess SOM  with some research indicating that excess N-fertilisation (and possibly intensification) leads to soil C declines .At soil pH values representing expected deviations induced by either urine or urea prills, we observed large increases in DOM, respiration and DEA potential within <24 h of pH change. DEA potential was such that in order to balance the concentrations of NH10.7717/peerj.6090/supp-1Supplemental Information 1This is a supplemental file containing pertinent figures and tables organized in ascending numerical order as cited in the text of the main publication. Each figure or table in this file contains explanatory information describing what each represents.Click here for additional data file.10.7717/peerj.6090/supp-2Supplemental Information 2This file contains raw data underpinning Fig. S2Click here for additional data file.10.7717/peerj.6090/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.6090/supp-4Supplemental Information 42O and N2 production).This file contains data used to determine optimal incubation time periods to investigate DEA that allowed for expression of denitrification gene activity (biogeochemical detection of activity via NClick here for additional data file.10.7717/peerj.6090/supp-5Supplemental Information 52 measured and that liberated from carbonates via acidification.This file contains raw data of both COClick here for additional data file.10.7717/peerj.6090/supp-6Supplemental Information 6This file contains data from experiments 3 and 4 summarized into one file with averages and standard deviations calculated and exploratory graphs.Click here for additional data file.10.7717/peerj.6090/supp-7Supplemental Information 7Data represents raw sequence counts which were then standardised by total in PRIMER 7 prior to analysis.Click here for additional data file.10.7717/peerj.6090/supp-8Supplemental Information 8This data represents the OTU information with Megablast ID.Click here for additional data file.