The risk of hypoglycemia during and after exercise is a limitation to regular practice. In type 1 diabetes patients.To compare the influence of different intervals of insulin administration, to identify the proportion of oxidized energy substrates at different times of continuous aerobic exercise and evaluate the relationship between oxygen uptake and glycemic variation at the end of the exercise.Six patients with type 1 diabetes mellitus were evaluated: 6 pubertal, BMI Z-score 0.56±0.48; chronological age 13.7±2.6y; duration of disease 6.5±4.26y; mean HbA1c 9.13±1.7%. Patients took breakfast and insulin at the clinic . Continuous ergometric test at moderated intensity (60% VO2max) was performed for 30 min, one and two h after insulin (T1 and T2). Intensity was controlled through oxidation and heart rate measured at 10 and 30 min of exercise with K4b2® gas analyzer. Blood glucose levels were measured at the beginning, 10 and 30 min.Initial blood glucose was 299±48mg/dL, VO2max 40.7±9.63ml/Kg/min. No difference was found between intervals of insulin administration regarding the oxidation of substrates Figure . DiffereSubstrate oxidation wasn't influenced by insulin administration before exercise. A higher carbohydrate oxidation was observed at 10min of exercise and subsequent increase in fat oxidation at 30min. Fast insulin dosis and VO2max are a good predictor of glycemic variation at the end of continuous moderate aerobic exercise.


Sulfolobus solfataricus is a thermoacidophilic crenarcheote with a 3.0-Mb genome. Here, we report the genome sequence of S. solfataricus strain 98/2, along with several evolved derivatives generated through experimental microbial evolution for enhanced thermoacidophily. Sulfolobus solfataricus strain 98/2 is a thermoacidophilic chemoheterotrophic crenarcheote that grows optimally at 80°C and pH 3.0  is a deletion derivative of strain 98/2 called PBL2025  using Bowtie2 version 2.1.0 and SAMtools version 1.0 and corrected for the deleted region.High-molecular-weight genomic DNA was prepared from clonal cultures of the eviously , 4. The S. solfataricus 98/2 (SULA) genome is 2,727,337 bp in size. The genome sequence was annotated through NCBI’s online annotation pipeline and had a total of 2,924 genes, 178 pseudogenes, and 46 tRNA genes. The SULB and SULC strains had the same number of genes and tRNA genes but slightly different numbers of pseudogenes .The fully assembled S. solfataricus strain 98/2 (SULA) genome sequence is available at GenBank under the accession number CP011057. The evolved derivatives SULB and SULC are available under the accession numbers CP011055 and CP011056, respectively.The


Importantly, we identify HuR as a novel substrate of the ERK8 kinase pathway in response to H2O2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.Programmed cell death 4 (PDCD4) is a tumour suppressor implicated in cancer development and progression and was recently identified as a repressor of cap-independent translation of specific genes involved in the regulation of apoptosis. We show that the RNA-binding protein HuR binds to the PDCD4 3′UTR to protect it from miR-21-induced silencing. However, following H Notably, a hinge region between RRM2 and RRM3 contains a nucleocytoplasmic shuttling domain that shuttles HuR into the cytoplasm in response to cellular stressors such as UV, arsenite, and hydrogen peroxide (H2O2) UTP and a MAXIscript T7 kit (Ambion) as per the manufacturer's protocol. The RNA was run on a 5% acrylamide–8 M urea denaturing gel, excised and eluted in RNase free water overnight at 37°C. The RNA was then incubated with purified GST or GST-HuR in RNA binding buffer  for 30 minutes at room temperature then cross-linked at 250 mJ/μm2 in a Stratalinker. The complexes were treated with RNase T1 (1 U/μl), RNase A (10 μg/ml), and heparin (5 mg/ml) for 10 minutes. The samples were separated by SDS-PAGE gel, and exposed to X-ray film at −80°C overnight.The first 610 nt of the PDCD4 3′UTR containing the miR-21 binding site  was cloE. coli was transformed with the pGEX or pGEX-KG_HuR plasmid and grown overnight in 4 mL Luria-Bertani (LB) media containing ampicillin (100 mg/mL). The culture was added to 100 mL of LB media containing 100 mg/mL of ampicillin and grown to an OD of 0.7. Isopropylthio-β-galactoside (IPTG) was added to a final concentration of 1 mM and grown for 4 hours longer. The cultures were centrifuged at 5000 x g for 10 minutes at 4°C and supernatant discarded. The pellet was resuspended in 10 mL of ice-cold PBS and the samples were centrifuged again at 5000 x g for 10 minutes at 4°C. Samples were then lysed with 10 mL lysis buffer . Lysates were sonicated twice, 1% Triton X-100 was added, and samples were sonicated again. Samples were centrifuged at 13000 x g for 10 minutes at 4°C. Glutathione sepharose beads  were added to the supernatant and rotated at 4°C for 2 hours. Samples were washed 5 times with cold PBS and proteins were eluted using 20 mM L-glutathione, pH 8.0 in PBS rotating at 4°C for 1 hour.2O2 for 4 hours, washed twice with PBS and lysed in CHIP lysis buffer  for 30 minutes on ice. Lysates were spun at 13, 000 rpm for 15 minutes and supernatant was transferred to a new tube. Samples were incubated with 10 μg mouse anti-HuR or anti-mouse IgG for 2 hours at 4°C. Dynabeads Protein G (Novex by Life Technologies) were washed with CHIP buffer and added to samples (50 μL per sample) and rotated for 40 minutes at 4°C. Supernatant was removed and beads were washed 4 times with CHIP buffer. RNA was extracted with RNAzol as per manufacturer's protocol and qPCR was performed using the QuantiTect SYBR green PCR kit (Qiagen).HeLa cells were treated with 0.5 mM H2O2 or PBS as indicated and fixed with 3% formaldehyde in PBS for 10 minutes at room temperature. Cells were permeablized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature on a shaker, rinsed twice with PBS, and blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour. Primary antibody was added (mouse anti-HuR (1:500 dilution in 5% BSA in PBS) and incubated with cells overnight at 4°C, followed by 3 washes with PBS for 5 minutes each. Secondary antibody  was added for 1 hour followed by three 5 minute washes in PBS. Nuclei were stained with Hoechst 33342 (Pierce) for 5 minutes and washed with PBS twice. Coverslips were mounted on slides using Fluoromount (Sigma Aldrich). Confocal microscopy was performed using the 60X objective with immersion oil . Quantification of HuR nuclear and cytoplasmic distribution was done as described previously [Cells were grown on coverslips and treated with Heviously  with som32P-labelled, in vitro transcribed RNA probe in RNA binding buffer  for 30 minutes at room temperature with or without Cy5.5 3′-labelled miR21 RNA (Dharmacon). The complexes were separated on a 6% polyacrylamide gel and the gel was exposed to X-ray film at −80°C to detect autoradiography and subsequently scanned with the Li-Cor Odyssey infrared scanner  to detect the miR21 Cy5.5 signal.Recombinant GST or GST-HuR was incubated with 3VO4). Lysates were sonicated and centrifuged for 15 min at 13,000 x g at 4°C. Anti-Flag agarose beads (Sigma) were incubated with the lysate for 1 h at 4°C and washed 3 times in lysis buffer followed by a wash in kinase buffer . Kinase and substrate on beads were incubated in kinase buffer in the presence of  and 5 μCi of γ-32P-labelled ATP for 20 min at 30°C. Laemmli sample buffer was added, and samples were separated by SDS-PAGE, transferred to a PVDF membrane, and exposed to X-ray film. The membrane was subsequently analyzed by Western blotting.pCDNA3_Flag-HuR, pReciever_HA-ERK8, or pCDNA3_Flag empty vector were transfected into HeLa cells for 24 h and harvested in co-immunoprecipitation buffer  to determine p-value in repeated experiments. All results are shown as mean ± standard deviation. For RT-qPCR experiments, average RNA expression was calculated using data collected from three biological replicates and three technical replicates for each biological replicate. Unless otherwise noted, all results were obtained through a minimum of three independent experimental replications.


Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes.Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca Protein phosphatase 2A (PP2A) is a major Ser/Thr protein phosphatase that together with Protein phosphatase 1 accounts for more than 90% of all dephosphorylation events in any given tissue or eukaryotic cell. Reversible phosphorylation and dephosphorylation events are essential for the regulation of a multitude of cellular functions Each holoenzyme consists of a common core formed by the scaffolding (A) and the catalytic (C) subunit (two isoforms each) and associates with a variable regulatory B-subunit into a heterotrimeric complex The scaffolding subunit A (isoforms α and β) consists of 15 tandem huntingtin-elongation-A subunit TOR (HEAT) repeats which form an overall U-shaped conformation The structures of known members of the different regulatory B-subunits are highly diverse in terms of sequence and structure. Members of the B’’/PR72/PR70 subunits are encoded by three different genes  (The structure of the PR70 core fragment (residues 130–483) was solved by selenomethionine single anomalous dispersion (SAD) phasing and refined at 2 Å resolution yielding a final R= 16.2%) . The mod= 16.2%) . Calcine= 16.2%) . In addi= 16.2%) . A DALI = 16.2%) . Notably= 16.2%) , which e= 16.2%) , suggest= 16.2%) .2+ ion is seven. Just one of the interactions is contributed by a water molecule, and the coordination geometry is pentagonal bipyrimidal, which is the preferred coordination geometry for calcium The EF-hand is a helix-loop-helix motif , which often has the capacity to bind calcium. In a canonical EF-hand, the EF-loop is twelve residues in length and contains aspartic residues in positions 1, 3, 5, glycines in positions 4, 6, and a glutamate in position 12, which may in rare cases be substituted by an aspartate  [31], . SA2. CD unfolding curves were recorded with a JASCO J600A spectropolarimeter  at 230 nm. Data were analyzed according to a two state model to obtain apparent unfolding midpoints using the software GraFit5. It should be noted that thermal unfolding was irreversible under all conditions measured.CD thermal unfolding of different B-subunit constructs was followed over a temperature range from 15–70°C at a heating rate of 1°C/min in 10 mM Tris pH 7.5, 100 mM NaCl, 50 µM EGTA with increasing concentrations of CaCl2 was used. The heat generated after each ligand injection was obtained by integration of the calorimetric signal. Resulting binding isotherms were analyzed according to a 1∶1 binding model using the Origin software .ITC measurements were performed on an iTC200 instrument . Various B’’-constructs at a concentration of 20–40 µM in the calorimetric cell  were titrated with 200–300 µM of different Aα and Aβ constructs at 15°C. As buffer system, 20 mM Tris pH 7.5, 200 mM NaCl and 2 mM CaCl2, 0.5 mM TCEP, 1% glycerol at 15°C. Data were collected on the original gel filtration fractions from each SEC run, as well as on concentration series from fractions sampled from the later eluting half of each SEC elution peak (∼2–8-fold concentration). Fractions prior to the SEC void volume were used for buffer subtraction of the original unconcentrated gel filtration fractions, and concentrator eluates were used for buffer subtraction of each concentration series (2×−8×). Sequential exposures  were taken at 11.111 eV. Samples were radiation-sensitive, but for most samples, data from the first and second exposure measurements matched. For very radiation sensitive samples, the option “expose with mix” was applied. Here, 30 µl of sample were taken up in the Hamilton syringe and slowly pushed out during each exposure. No radiation-dependent aggregation was observed in sequential exposures. Data were analyzed using the ATSAS program suite SAXS data were collected at the SIBYLS 12.3.1 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory Figure S1Sequence alignment of B″ regulatory subunits. Secondary structural elements are indicated above the sequences. Conserved residues are highlighted and boxed in red. Residues that interact with the scaffolding subunit are highlighted with green circles. Residues that are involved in calcium binding are indicated by blue circles.(TIF)Click here for additional data file.Figure S2Structural overlays between the CBLD domains and individual EF-lobes of PR70 and the top scoring hits from DALI. All panels: PR70 is grey except for the calcium ions (yellow), and the DALI hits are blue except for calcium (orange). More information about the DALI hits can be found in A. KChIP1 on PR70 CBLD1. B. CBL2 on PR70 CBLD2. C. Calmodulin on EF-lobe 1. D. Calcium binding protein p22 on EF-lobe 2. E. CDC31p on EF-lobe 3. F. KChiP1 on EF-lobe 4.(TIF)Click here for additional data file.Figure S3Interaction interface between CBLD1 and CBLD2. The interface encompasses helix E4 from CBLD1, helices E5 and F6 from CBLD2 and the inter-domain linker. E4, E5 and F6 are colored as in (TIF)Click here for additional data file.


Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.Atrazine, a pre-emergent herbicide in the chloro- Measurements presented in Atrazine was observed to reduce the cell viability at the concentration of 1.0 to 10 µg/mL for 48 h in MCF-10A cells , possiblWe then performed apoptosis protein array analysis. The cells were treated with 0.1 µg/mL of atrazine for 6 h, and then, the relative levels of 35 apoptosis-related proteins were measured. Results showed that several apoptotic signaling pathway proteins were modulated following the 0.1 µg/mL of atrazine treatment for 6 h. Pro-apoptotic proteins, including phospho-Rad17 (Ser635) and TNFR1, increased, while Bad, p21 and p27 were inhibited , top. OnPixel density analysis , bottom To verify the involvement of enhanced p-Rad17 and TNFR, which are the most regulated proteins  in respoet al. [2+, reactive oxygen species (ROS), mitochondrial membrane potential and ATP generation play important roles in atrazine-induced apoptosis in carp ZC7901 cells. However, in our study, protein array analysis showed that some pro-apoptosis proteins in the mitochondrial pathway were actually inhibited by atrazine, including Bad and Bcl-2 . As expected, dose-response experiments showed that ATR, ATRIP and phospho-Chk1 were induced greatly by treatment of atrazine for 6 h . AtrazinATR responds to a wide variety of DNA damage architectures, including UV-induced base damage, replication stress and DNA DSBs . ATR phoThe DNA damage response ATR-CHK1 pathway is activated when the cells are under genotoxic stress . To furtImmunofluorescence imaging further strengthened our findings in Western blot analysis A,B. AfteAlthough p-RAD17, ATR, p-Chk1 and γH2AX were induced by atrazine in our study, activation of another tumor suppressor and important cell cycle checkpoint protein p53 was not detected in the apoptosis antibody array . This coin vivo. However, although the DSBs induced by atrazine are transient and can be efficiently repaired in MCF-10A cells, its potential genotoxicity should not be neglected due to the fact that DSB can be repaired and we did not assume that the repair was not genotoxic. In human cells, the major DSB repair pathway (NHEJ) is highly error prone. Furthermore, atrazine is not used as a pure active ingredient in application, but in the form of commercial formulations. These formulations contain substances or compounds that might interact with atrazine and elevate its adverse effects. For example, the commercial formula Gesaprim® and Gesaprim® adjuvant mixture and their metabolites have been shown to cause DNA damage with a comet and DNA diffusion assay [DNA DSBs are the most harmful initial event in cell carcinogenesis . Efficieon assay . Atrazinon assay  and enhaon assay . As γH2Aon assay , our dat® Aqueous One Solution Cell Proliferation Assay (MTS) kit was from Promega Company . The Human Apoptosis Array kit  was from R&D Systems . The Pierce BCA protein assay kit (Product No. 23227) and DAPI  were from Thermal Fisher Scientific .Atrazine (98% purity) from Superlco  was dissolved in 100% DMSO to make a stock solution of 10, 1.0, 0.1 and 0.01 mg/mL. The CellTiter 96Specific antibody against TNFR1 , phospho-Chk1  and ATRIP  were purchased from Cell Signaling Technology . Phospho-hRad17 (Ser635)  was from R&D System, Inc. ATR  was from Thermo Fisher Scientific, and phospho-H2AX  was from EMD Millipore . Secondary antibodies for immunofluorescence imaging of p-H2AX were from Life Technologies .2. The cells were maintained in Mammary Epithelium Basal Medium (MEGM)  added with 10 ng/mL hEGF, 5 µg/mL insulin, 0.5 µg/mL hydrocortisone gentamicin and amphotericin-B. Before use, the medium was completed with bovine pit extract (BPE)  at a final concentration of 0.4%. Cells were passaged twice a week. When treating the cells with atrazine, 1.0 μL of 10, 1.0, 0.1 and 0.01 mg/mL of atrazine stock solution was added into 1.0 mL of complete MEGM medium to prepare 10-, 1.0-, 0.1- and 0.01-µg/mL final treatment concentrations. An equal volume of DMSO was added as a vehicle control.MCF-10A cells from American Type Culture Collection  were grown as a monolayer in TC175 flasks at 37 °C in a humidified incubator supplied with 5% CO® Aqueous One Solution Cell Proliferation Assay (MTS) kit, according to the manufacturer’s instruction. Briefly, ninety-six well plates were seeded with 10,000 cells (per well) in 200 µL of completed MGEM medium. After cells were allowed to attach to the surface overnight at 37 °C, as described above, the medium was carefully removed. Medium with different concentrations of atrazine or an equal volume of DMSO was added into the wells and incubated for 6, 12, 24 and 48 h. At the end of incubation, 20 μL of the cell proliferation reagent (MTS) were added to the medium and incubated for another 1.5 h at 37 °C. The absorbance of the treated samples against a blank control was measured using a microplate reader  with a test wavelength of 450 nm and a reference wavelength of 690 nm. Cell proliferation was performed in 4 wells, and each experiment was triplicated.Cell proliferation and cytotoxicity were measured using a CellTiter 966) were seeded in 60 mm × 15 mm sterile dishes in 6 mL of complete medium and incubated overnight at 37 °C, then treated with or without atrazine (0.1 µg/mL) for 6 h. The cells were harvested, and the cell pellets were lysed in 100 µL lysis buffer  and centrifuged at 16,000× g for 15 min at 4 °C. The supernatant were collected and kept at −80 °C until use or directly applied to the human apoptosis antibody array assay. The nitrocellulose membranes containing immobilized specific monoclonal antibodies against 35 apoptosis-related proteins were blocked with bovine serum albumin for 1 h on a rocking platform at room temperature. The membrane was then incubated with supernatant lysates of MCF-10A cells treated with or without atrazine (0.1 µg/mL) overnight at 4 °C on a rocking platform. After 3 washes (each wash for 5 min) of the membranes with 2 mL of wash buffer, the membranes were then incubated with the detection antibody cocktail for 1 h on a rocking platform, followed by 3 washes. Then, the membranes were incubated with streptavidin-horseradish peroxidase conjugate and by a chemiluminescent detection reagent. The membrane was then scanned using a G:Box Chemi , and pixel density was presented by quantifying the mean spot densities from three replicates.The expression profiles of 35 apoptosis-related proteins were detected and analyzed using a human apoptosis array kit following the manufacturer’s procedure. Briefly, MCF-10A cells (1 × 105) were seeded into a 6-well plate in 2 mL of complete culture medium and incubated overnight, then treated with atrazine. For time course experiments, the cells were treated with 0.1 μg/mL of atrazine for the indicated hours. For dose-response experiments, the cells were treated with indicated concentrations of atrazine for 6 h. Before harvest, the cells were washed with 5 mL of PBS twice, then lysed in 150 µL of lysis buffer (per well) containing 50 mM Tris (pH 7.4), 1% Triton X-100, 1% Nonidet P-40, 150 mM NaCl, 0.4 mM EDTA, 0.2 mM EGTA, 0.2 mM vanadate and 1% protease inhibitor cocktail . The lysates were centrifuged at 16,000× g for 15 min at 4 °C, and the supernatants were measured for protein concentration using a Pierce BCA protein assay kit. A total protein of 20 μg per sample was separated in 12% SDS-PAGE gel and transferred to PVDF membranes  by electrotransfer. The blots were subsequently blocked with 5% non-fat milk powder in TBS containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h and incubated at 4 °C overnight with primary antibodies of interest. After washing three times for 10 min with TBS-T, the membrane was incubated with horseradish-peroxidase-linked secondary antibodies of interest at room temperature for 1 h. The blots were visualized with the ECL Western blotting substrate  and imaged using a FluorChem M digital imager . The same membrane was re-probed with an anti-human actin antibody as an internal loading control. Band density on Western blot images was used as a measure of assayed protein level and analyzed by the AlphaView SA software.MCF-10A cells  were seeded into the petri dishes in 6 mL of complete MEGM medium. After 24 h of culturing to allow the cells to attach to the slips, cells were incubated with or without 0.1 µg/mL of atrazine for 0.5, 1 and 2 h, and then, cell layers in the slips were washed 3 times with 2 mL PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Afterward, the cell layers were washed twice with 2 mL of PBS. For immunofluorescence staining, the cells were permeabilized for 3 min in 0.25% Triton X-100 in PBS, washed twice by 2 mL of PBS and blocked for 1 h with 5% BSA in PBS. The cells were incubated at 4 °C overnight with primary antibodies against γH2AX (Ser139) at a 1:200 dilution, washed three times with PBS, followed by incubation with secondary antibodies of interest at a 1:400 dilution for 2 h at room temperature. The slides for γH2AX were mounted on cover slides with DAPI. Images were captured using a Carl Zeiss confocal microscopy.Cover slips coated with 40 µg/mL of poly-p < 0.05).All data are expressed as the mean ± SD (standard deviation). Differences among treatments were assessed by the one-way analysis of variance (ANOVA). If the variances among treatments were homogenous, data were subjected to the multiple comparison Dunnett’s test using a 5% significance level , but also induces the phosphorylation of H2AX and γH2AX foci. Atrazine at environmentally-detectable levels could trigger the DNA damage response ATR-CHK1 pathway. With highly sensitive assays, this study provided a direct molecular evidence of the most widely-used atrazine for its genotoxicity and possible carcinogenic properties in human mammary cells. Further investigations are needed to determine whether the atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur


We analyzed 340 type 2 diabetes patients whose serum GA and HbA1c levels had been repeatedly measured over 4 years. We assessed the pattern of changes with time in glycemic indices  and their relationship with beta-cell function. In all patients, glycemic indices decreased and maintained low levels around 15 and 27 months. However, from 39 months to 51 months, GA significantly increased but HbA1c tended to increase without statistical significance. We defined ΔGA/HbA1c as the difference between the nadir point (at 15 to 27 months) and the end point (at 39 to 51 months) and found that ΔGA/HbA1c was positively correlated with diabetes duration and negatively related to beta-cell function. In multivariable linear regression analyses, ΔGA/HbA1c was independently associated with diabetes duration. In conclusion, this study demonstrated that serum GA levels increase relative to HbA1c levels with time.We recently reported that glycated albumin (GA) is increased in subjects with longer duration of diabetes and with decreased insulin secretory function. Based on this, we investigated whether GA increases with time relative to glycated hemoglobin (HbA Measurement techniques included the hexokinase method for glucose and high-performance liquid chromatography using Variant II Turbo  for HbA1c. Serum GA was analyzed by an enzymatic method using an albumin-specific proteinase, ketoamine oxidase, an albumin assay reagent , and a Hitachi 7699 P module autoanalyzer . GA values were calculated from the ratio of GA to total serum albumin and expressed as a percentage. Serum C-peptide levels were measured in duplicate using an immunoradiometric assay method .The baseline glycemic indices . Differences were analyzed using Student's t-test for the continuous variables and the chi-square test for categorical variables.All continuous variables were presented as mean ± standard deviation (SD) or median (quartiles) or as mean ± standard error (SE) for variables on the graphs. Categorical variables were described as t-test were used to determine the significance of differences in glycemic indices according to duration of diabetes. We compared GA, HbA1c, and GA/HbA1c levels at baseline and 3, 15, 27, and 39 to 51 months after enrollment in all patients who had glycemic values available at that time point. Because all glycemic indices reached their lowest level between 15 and 27 months (arbitrarily defined nadir point), we defined ΔGA/HbA1c as the difference in GA/HbA1c between the end point (39 to 51 months) and nadir point (15 to 27 months). One-way ANOVA with Tukey correction was used to compare the differences of duration of diabetes and PCGR according to the tertiles of ΔGA/HbA1c ratio. Multivariable linear regression analysis was performed to determine the independent relationship of the studied variables including duration of diabetes associated with ΔGA/HbA1c increase. Statistical analyses were performed using PASW Statistics version 18.0 for Windows .Repeated measured analysis of variance (ANOVA) with Bonferroni correction and paired2 and the prevalence of hypertension was 57% (n = 195). Median duration of diabetes and levels of mean HbA1c were 1 (range: 0–5.0) year and 7.0% + 0.9%, respectively. We also measured baseline insulin secretory beta-cell function indices such as PCGR (3.24 ± 2.1), ΔC-peptide level (4.13 ± 2.6), and CGI (0.08 ± 0.4). All glycemic indices, GA (19.3 ± 6.6 versus 16.5 ± 4.9), HbA1c (7.7 ± 1.6 versus 7.0 ± 1.2), and GA/HbA1c (2.47 ± 0.5 versus 2.33 ± 0.4), were decreased at final follow-up compared to those at baseline. At the time of enrollment, the patients were being treated with metformin , sulfonylurea , DPP-IV inhibitors , or insulin .A total of 340 subjects  were enrolled in this study. The patient characteristics of the cohort are shown in 1c ratio (2.30 ± 0.4) improved and reached the nadir points on glucose control at 15 months (p = 0.028), but HbA1c tended to increase without statistical significance . The levels of GA at 27 and 39 months  as well as GA/HbA1c ratio at 15 and 27 months  were significantly lower than at 51 months  and the last time point (51 months) were not significant except for baseline  and GA/HbA5 months . From 15 Figures . Howeverbaseline . In sum,me point .1c ratio significantly increased from the nadir point to the final follow-up point, which was designated as ΔGA/HbA1c (1c (β coefficient (STD β) = 0.187, p = 0.001), mean GA , and mean HbA1c  were positively associated with ΔGA/HbA1c. On the other hand, beta-cell function indices were negatively related to ΔGA/HbA1c. In particular, PCGR  was more strongly associated with ΔGA/HbA1c than ΔC-peptide .Since the GA/HbAGA/HbA1c , we triebA1c  and lower levels of PCGR   to investigate the impact of diabetes duration on ΔGA/HbA1c ratio and PCGR. The ΔGA/HbA1c ratios (expressed as percentages) were significantly elevated in patients with diabetes of duration >5 years compared to other groups . After additional inclusion of PCGR in model 2, PCGR showed significant correlation with ΔGA/HbA1c , but history of DPP-IV inhibitor use lost its significance. In model 3, duration of diabetes was further adjusted and the significant correlation of PCGR with ΔGA/HbA1c disappeared . However, duration of diabetes was still independently associated with ΔGA/HbA1c . Moreover, this association remained significant even after adjustment for glycemic status of subjects .Multivariable linear regression models were applied to determine the clinical and laboratory variables associated with ΔGA/HbA1c . We focuhttp://dx.doi.org/10.1155/2015/576306). PCGR showed the strongest relationship with mean GA . It also had significant correlation with duration of diabetes  and insulin use  (model 1). To evaluate the association between PCGR and ΔGA/HbA1c, model 2 was developed, which showed a significant negative relationship .Additionally, we conducted multiple linear regression analyses to determine variables associated with PCGR at baseline  at enrollment. Lastly, we conducted mixed meal tests to obtain basal and stimulated C-peptide levels, which were then used to calculate PCGR as a measure of beta-cell function. That allowed for standardization of the stimulation calories and glucose content. Because it can be easily calculated and is a reliable indicator of beta-cell function, the PCGR is being used more frequently to help determine the optimal antidiabetic drug treatment [r = 0.808, p < 0.001) which strongly predicted beta-cell function . In multivariable linear regression analyses, PCGR was also associated with ΔGA/HbA1c. However, because the duration of diabetes strongly affects ΔGA/HbA1c, after adjusting for duration of diabetes, the association between ΔGA/HbA1c and PCGR disappeared , sex , body mass index (kg/m2), waist circumference (cm), hypertension , and estimated glomerular filtration rate (ml/min/1.73m2). **Model 2 was additionally adjusted for ΔGA/HbA1c . Supplementary Figure 1 Correlation between ΔC-peptide and PCGRSupplementary Table 1 Analyses to determine the variables associated with PCGR


But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca2+] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This “perimeter release model” provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca •2+ channels form clusters with highly variable numbers of channelsCa•EGTA sensitivity suggests that synaptic vesicles are tightly coupled to clusters•2+ channel number per cluster alters synaptic efficacy, but not precisionCa•A perimeter model accounts for synaptic efficacy and precision during development 2+ channels and synaptic vesicles critically influences synaptic transmission. Nakamura et al. find that action potentials evoke release of vesicles coupled to the perimeter of Ca2+ channel clusters, explaining synaptic efficacy and precision during development.Nanoscale topography of presynaptic Ca However “loose”  or “tigh “loose” . Detaile 5–10 nm . However2+ channel antibodies and freeze-fracture replica labeling electron microscopy (EM) have established that VGCCs form clusters at the AZ of central mammalian synapses . In another set of labeling samples using a different batch of antibodies (La2), a higher particle density was observed (8.6/μm2 at P7 and 21.7/μm2 at P14).The calyx of Held synapse undergoes both morphological and functional changes during the second postnatal week, when rodents start to hear sounds . To exampe VGCCs . Large cred face A. Specif3, Because the gold particles appeared to form clusters, we tested this possibility by comparing the gold particle distribution with a randomly distributed particle model C–S1F. Tov2.1 labeling followed by RIM staining revealed that the majority of Cav2.1 clusters were closely associated with RIM particles  between clusters was similar, but not identical, across ages (La1): 899 ± 60 nm for P7, 779 ± 32 nm for P14, and 880 ± 38 nm for P21  to P14  and then remained constant for P21 . These data indicate that the density of Cav2.1 remains similar from P7 to P21, but the number of Cav2.1 per cluster and the cluster area increase with development from P7 to P14.The number of particles per cluster in La1 samples varied over a wide range, from 2 to 27 D with an2+] changes was altered during the hearing acquisition period, we recorded local Ca2+ transients in response to single APs using a high-speed confocal spot detection method and low-affinity Ca2+ indicator Oregon green BAPTA-5N (XY axis) and 650 nm (Z axis)  and slow (>10 ms) component, consistent with localized detection of a [Ca2+] domain  . In P7 c] domain . In conttic face B and S4,tic face .2+ transients were also observed for confocal spot locations along the synaptic face, albeit less frequently than in P7 calyces , and exhibited a similar spatial dependence  to the Ca2+ entry site , thus mimicking the AP measured at P7 (2+ transient amplitude (7) or P14 (AP14) calyces. The shorter duration AP14 waveform reduced the time to peak of the Ca2+ current (ICa) by 51% ± 1%  as compared to using AP7 waveforms. Ca2+ transients recorded from the same spot location were 56% smaller when elicited using AP14 than when elicited by AP7  with either 0.1 mM or 2 mM EGTA-containing pipette solutions  matched that of the experimental data  and a Cav2.3-specific blocker SNX-482 , suggesting the VGCC-sensor distance is similar among VGCC subtypes. Consistent with these results, we observed no significant difference in the spatial distribution of Ca2+ entry estimated with confocal line-scans with and without these subtype-specific VGCC blockers (v2.2 and Cav2.3 have a similar distribution to Cav2.1 before hearing onset.The sensitivity of excitatory postsynaptic currents (EPSCs) to presynaptic EGTA has been used to assay the VGCC-sensor distance at the calyx of Held . We reviblockers , suggest2+ buffers remained intact. Bath application of EGTA-AM (10 μM) for 15 min reduced the EPSC amplitude by 35% ± 11%  following termination of Ca2+ entry  across the entire synaptic face  at different locations with respect to the VGCC cluster, we found Pv = 1 within those voxels containing a VGCC, 0.8–1 for voxels within the cluster center, and 0.01 for voxels 55 and 92 nm from the edge of a cluster with 4 and 12 open VGCC, respectively. Increasing the intraterminal [EGTA] from 0.1 to 10 mM inhibited Pv by 20% at the cluster center. Only when Ca2+ sensors were positioned outside the cluster .To simulate vesicular release, we drove a five-state Case model  using thproteins . Computi2+ sensor for vesicular release is located within a short distance from the edge of VGCC clusters. We call this topography the perimeter release model and refer to the distance between the closest open VGCC and the Ca2+ sensor as the perimeter coupling distance (PCD). Simulations using an AP7 waveform and a Ca2+ sensor with higher Ca2+ affinity for P8–P10 rats  is based on single-channel conductance (3.3 pS in 2 mM [Ca2+]) measured for Cav2.1 at the calyx of Held (v2.2 (2.7 pS) at an autonomic preganglionic synapse  and P14 were performed using APeach age . We usedeach age D. Surprieach age E. In coneach age  predicteeach age C. The sheach age B can be each age F and 8A.each age G, similaeach age B and 8C.2+ sensor . This finding could account for spontaneous EPSCs (Whether vesicular release requires opening of one  or multius EPSCs  but not us EPSCs . Our res2+ current measurements (see v2.1 channels per cluster was 18 (=11.1/0.62) on average and varied between 3 and 73 across clusters. At P7 we estimate that there are 10 Cav2.1 channels per cluster, but because Cav2.1 channels comprise 53% of the VGCCs at P7  are located 15–30 nm from the outer perimeter of VGCC clusters . SDS-FRLthan AZs  and 2. Tthan AZs H did notthan AZs , unless v2.1 channels also form clusters within AZs at hippocampal  amplifier (Molecular Devices). Presynaptic internal solution exchange was performed using pipette perfusion, as described previously (2+] changes with Oregon green BAPTA-5N added to presynaptic pipette solution. All experiments were performed at room temperature (22°C–24°C). Data analysis was performed with IgorPro 6.3 (WaveMetrics) using NeuroMatic. Deconvolution of EPSCs was performed as describe previously (Whole-cell patch-clamp recordings were made from calyces of Held and MNTB neurons of acute brainstem slices prepared from P7–P22 Wistar rats. Presynaptic AP, Ieviously . Confocaeviously . All val2+ diffusion and binding with Oregon green BAPTA-5N and buffers in the vicinity of VGCC clusters was simulated using a finite-difference method (2+ transients. Nanoscale simulations were performed without the Ca2+ indicator and then used to drive a five-state Ca2+-dependent release model (Cae method  in orderse model . All sim


Previous studies suggested that bisphosphonate zoledronic acid exerts an anti-tumor effect by interacting with the microenvironment. In this study, we aimed to elucidate the mechanism behind the anti-breast cancer effect of zoledronic acid.in vitro human breast cancer cell survival, but did affect human stromal cell survival. Breast cancer cell death in co-culture with stromal cells was analyzed in vitro by fluorescent microscopy and flowcytometry analysis. In co-culture, the addition of stromal cells to breast cancer cells induced tumor cell death by zoledronic acid, which was abolished by transforming growth factor (TGF)-β. In the in vivo chicken chorioallantoic membrane model, zoledronic acid reduced the breast cancer cells fraction per tumor only in the presence of human stromal cells. Zoledronic acid decreased TGF-β excretion by stromal cells and co-cultures. Moreover, supernatant of zoledronic acid treated stromal cells reduced phospho-Smad2 protein levels in breast cancer cells. Thus, zoledronic acid exerts an anti-breast cancer effect via stromal cells, accompanied by decreased stromal TGF-β excretion and reduced TGF-β signaling in cancer cells.Here we showed that zoledronic acid did not influence  Breast cancer accounts for the highest cancer incidence and cancer mortality among women worldwide . DespiteBisphosphonates, currently used as supportive treatment in breast cancer patients with bone metastases, have a potential anti-cancer effect via microenvironmenal cells. A recent meta-analysis  concludeIn vitro culture models are mostly too simplified and traditional mouse models fall short in this setting, since mouse stromal infiltration into human cell line xenografts as well as into patient derived xenografts occur to a high extent [It would be of great benefit for further optimization when this indirect anti-tumor effect would be further clarified. For this, optimal preclinical models containing both human cancer cells and human stromal components are essential , 8. At ph extent , 10.in vivo in an immune deprived setting. By using in vitro and in vivo models consisting of human stromal cells as well as human breast cancer cells, we studied the role of stromal cells in breast cancer bisphosphonate sensitivity. Our research provides functional evidence of the role of stromal cells in zoledronic acid (ZOL) mediated breast cancer cell death.We have optimized the chorioallantoic membrane (CAM) model, which makes it possible to study the direct interactions between human tumor cells and human stromal cells 50 of 486, 194, 155 and 86 μM respectively. However, ZOL affected stromal cell survival at concentrations far below the concentrations directly affecting breast cancer cell lines: IC50 for human stromal cell line Hs27a was reached at a concentration of only 8 μM. In addition, the macrophage cell line RAW 264.7 was also very sensitive to ZOL treatment, with an IC50 of 19 μM . This effect was ZOL dose-dependent in the co-culture group, increasing breast cancer cell death to 21.6 ± 0.6 % for 100 μM (P < 0.01) and 27.6 ± 7.8 % (P < 0.001) for 500 μM. In mono-culture, increasing the dose of ZOL did not increase breast cancer cell death (9.6 ± 1.6 % for 100 μM and 10.3 ± 1.7 % for 500 μM of ZOL). At 48 hours, the stromal-dependent breast cancer cell death induced by ZOL was even more pronounced than at 24 hours . And the effect became more pronounced as the dose of ZOL increased, with breast cancer cell death of 6.5 ± 2 % for 50 μM, 11.8 ± 2.3 % for 100 μM and 18.4 ± 3.3 % for 500 μM in the mono-culture group versus 37.0 ± 0.4 % for 50 μM, 38.0 ± 3.4 % for 100 μM and 44.0 ± 4.6 % for 500 μM in the co-culture group . In mono-cultures of SCP2, ZOL increased breast cancer cell death after 48 hours compared to control from 4.3 ± 1.4 % to 11.8 ± 2.3 % (P < 0.05) for 100 μM and 18.4 ± 3.3 % (P < 0.001) for 500 μM ZOL . This effect was ZOL dose-dependent in the co-culture group, increasing breast cancer cell death to 11.4 ± 1.4 % for 50 μM (P < 0.001), 11.6 ± 2.9 % for 100 μM (P < 0.001). For 500 μM no difference was seen in SCP2 cell death with and without Hs27a cells (39.1 ± 10.5 % vs 26.1 ± 5.1).Breast cancer cells death after ZOL treatment was also determined by flowcytometry analysis. SCP2 cells were labeled with DiI and cell death was determined by LIVE/DEAD stain uptake. In the presence of stromal cells, SCP2 cell death was induced after treatment with ZOL. At 24 hours Figure , 10 μM oin vivo CAM assay, we investigated the effect of ZOL in two different breast cancer suptypes; ER positive (MCF-7) and triple negative (SCP2) breast cancer. Tumors grown on the CAM assay consisted of tumor cells only, tumor cells mixed with Hs27a stromal cells or Hs27a cells only.In the in vivo CAM assay, vehicle-treated tumors containing SCP2 plus Hs27a cells were heavier  and larger  compared to tumors containing only SCP2 cells . The tumor size was not affected by ZOL compared to vehicle treatment for tumors consisting of only SCP2 cells. However, tumors containing SCP2 plus Hs27a cells were sensitive to ZOL. On day 14, these tumors weighed less and were smaller when treated with ZOL compared to vehicle-treated SCP2 plus Hs27a tumors   Figure  and 3B.vs 21.4 ± 6.2 mg P < 0.01) and larger (54.4 ± 15.5 mm3vs 18.6 ± 5.6 mm3P < 0.001)   Figure  and 3D c) Figure  and 3D. ) Figure .P < 0.01 SCP2, P < 0.05 MCF-7). Moreover, the size of ZOL-treated SCP2 and MCF-7 co-culture tumors was reduced compared to the size of their respective mono-culture tumors -β1 excretion levels, both active and latent, in supernatant of stromal cells and co-cultures were reduced by 48 hours treatment with ZOL (Suppl. Table 1). Total TGF-β1 levels in supernatant of stromal cells decreased after treatment with 50, 100 and 500 μM ZOL Figure . In the in vitro  for 10 μM ZOL, 35.6 ± 2.9 % to 13.3 ± 2.5 % (P < 0.001) for 50 μM ZOL, 37.7 ± 2.9 % to 15.3 ± 3.8 % (P < 0.001) for 100 μM ZOL and 47.8 ± 8.6 % to 32.9 ± 1.9% (P < 0.001) for 500 μM ZOL. The addition of pure active TGF-β1 to monocultures of SCP2 cells treated with ZOL did not have this effect.ZOL treatment resulted in dose-dependent increased death of SCP2 cells in co-culture with Hs27a cells o Figure . The addvs 4.06 ± 0.8% P < 0.01)  Figure . In SCP2Active TGF-β signaling activity in SCP2 breast cancer cells, measured by pSmad2 levels, was increased after incubation with Hs27a supernatant Figure . As a coOur study provides functional proof that the bisphosphonate ZOL can exert an anti-breast cancer effect only in the presence of stromal cells. We investigated this in various breast cancer subtypes and two microenvironmental cell lines. These results complement research by Junankar et al. showing that the bisphosphonates risedronate and pamidronate target macrophages in the breast cancer microenvironment of mouse 4T1 xenografts . In theiThese results suggest that bisphosphonate treatment not only modulates the bone environment, but also affects non-bone disease. This is in line with clinical data. A recent meta-analysis showed that breast cancer recurrence decreased in early breast cancer patients treated with a bisphosphonate . The cle50 levels observed in the MTT assay.Bisphosphonates inhibit osteoclast activity by inhibiting protein prenylation . As inhiin vitro – without the presence of stromal cells – affected neither TGF-β signaling nor tumor cell death. Our study explains this apparent paradox by demonstrating the mediating role of the stromal cells. TGF-β transcriptional activity can also be inhibited by ER activation [In our co-culture model, treatment with zoledronic acid led to reduction of total TGF-β levels and active signaling activity. Moreover, treatment of breast cancer cells with an anti-TGF-β antibody instead of zoledronic acid induced breast cancer cell death only in the co-culture condition. These results indicate that reduced TGF-β signaling in breast cancer cells, as observed in co-culture after zoledronic acid treatment, can cause increased cell death of breast cancer cells. However, our results do not rule out that, apart from TGF-β, other factors could also be involved in this tumor-stroma interaction after zoledronic acid treatment. Moreover, the reason to why breast cancer cells, in co-culture compared to mono-culture after zoledronic acid treatment, are more dependent on TGF-β signaling remains to be elucidated. Previous research suggested a strong role for the microenvironment with respect to bisphosphonate sensitivity and TGF-β signaling . In micetivation , therebytivation , 15. If,To our knowledge, ours is the first study to evaluate the effect of ZOL on the breast cancer-stroma interaction by assessing the separate components. This analysis was made possible by using the CAM model. Due to its low immunogenicity, xenografts can be grown on the CAM model up to day 18 of embryonic development . MoreoveIn conclusion, the anti-breast cancer effect of ZOL is dependent on the presence of stromal cells. This is accompanied by decreased stromal cell TGF-β excretion and reduced TGF-β signaling in cancer cells.2 and were routinely tested for Mycoplasma. Short tandem repeat profiling (BaseClear) was used to authenticate the cell lines.The human cell lines used in this study were representing different breast cancer subtypes and two microenvironmental cell lines. ER positive breast cancer cell line MCF-7 and immortalized stromal cell line Hs27a (American Type Culture Collection (ATCC)) were cultured in Roswell Park Memorial Institute (RPMI) medium (Invitrogen), supplemented with 10% fetal calf serum (FCS). Hs27a cells were cultured up to a maximum of 30 passages during which these cells remained phenotypically stable and viable. Macrophage cell line RAW 264.7 was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% FCS and 1% glutamine. The inflammatory triple negative breast cancer cell line SUM-149 (Asterand) was cultured in HAM's Nutrient Mixture-F12 (HAM) supplemented with 5% FCS, 5 μg/mL insulin and 1 μg/mL hydrocortisone. Triple negative breast cancer cell line MDA-MB-231 (ATCC), the daughter cell line MDA-MB-231-H2N (H2N) (transfected to stably overexpress HER2) , luciferHs27a, RAW 264.7, MCF-7, MDA-MB-231, H2N, SCP2 and SUM-149 cells were plated in 96-well plates with a density of 2,000 – 12,500 cells/well. ZOL (SelleckChem) was added in different concentrations  and cells were incubated for 96 hours. Thereafter, 3--2,5- diphenyltetrazolium bromide (MTT) was added and formazan production as read out of cell number and metabolic activity was measured as described previously .in vitro co-culture model was described previously [This fluorescence-based eviously . To distversus fragmented nuclei). For each condition, 6 - 12 fields of view were counted and the average percentage of dead cells was calculated.After incubation, the plates were centrifuged at 300 g for 7 minutes. Glass slides were collected, fixed with methanol:acetone (1:1) and stained with 1:1000 4′,6-diamidino-2-phenylindole (DAPI). The results were analyzed by fluorescent microscopy. Tumor cell viability was assessed with nuclear DAPI staining based on observation of the nuclear structure  or a pan-TGF-Δ antibody (10 ng/mL) (R&D systems) for 24 hours. Cell death was determined by flowcytometry analysis after incubation with fluorescent LIVE/DEAD stain (Life Technologies) by manufacturer's protocol as described before . Cells pin vivo, the CAM model was used as described previously [6 cells in 50 μL culture media and Matrigel (BD Biosciences) (1:1). On day 10 of embryonic development, a 4 mm latex ring  was placed on the CAM and 15 μL of ZOL (200 μM) or vehicle was pipetted in the ring. The dose ZOL used in the CAM assay was based on our in vitro experiments and concurs with a dosage of 16 μg/kg for an egg weighing on average 0.05 kg. This is a low dose compared to the clinic, where a patient receives a dose of 4 mg concurring with a dosage of 53 μg/kg for a patient weighing 75 kg.To study the effect of ZOL eviously . Two bren = 15, MCF-7 n = 6) or vehicle , group 2) Hs27a cells treated with ZOL (n = 8) or vehicle (n = 7) and group 3) a mixture of breast cancer cells, SCP2 or MCF-7, with Hs27a cells (1:1) treated with ZOL  or vehicle . On day 14 of embryonic development, tumors were harvested, weighed and measured with a caliper.Each experiment comprised 3 subgroups; group 1) breast cancer cells only, SCP2 or MCF-7 cells treated with ZOL  were stained with an antibody against cytokeratin (CK) 8, 18 and 19 (Abcam 1:100). Substitution of the 1Total TGF-β1 levels were determined in supernatant of Hs27a cells in mono-culture or in co-culture with MCF-7, SCP2 or H2N cells after 24 and 48 hours incubation with ZOL. Cell culture supernatant was removed, centrifuged for 15 minutes at 240 g to remove any residual cells or cell remnants, and subsequently stored frozen in aliquots. Total TGF-β1 levels were measured with enzyme-linked immunosorbent assays (ELISA) according to manufacturer's instructions . The absorbance of each well was measured by a microplate reader (Bio-Rad).As read out for TGF-β signaling activity, protein expression of phosphorylated Smad2 was measured by Western blot. Hs27a cells were grown as monolayer and treated with ZOL (10 - 500 μM) for 48 hours. Subsequently, 500,000 SCP2 cells were plated in 6 wells plates and incubated for 24 hours. Pure active TGF-β1 (5 ng/mL) or harvested supernatant of Hs27a cells was added to the confluent SCP2 cells. After 1 hour incubation, the supernatant was removed. Total cell lysates were size fractionated and transferred to a membrane as described previously [plus (Roche Diagnostics). Band density was evaluated by ImageJ software.Membranes were exposed to primary antibodies . Binding of antibodies was determined using horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO) and visualized with Lumi-lightP < 0.05.Data are presented as mean ± SD. Statistical analysis was performed using the one-way or two-way ANOVA test with Tukey's or Bonferroni's post hoc test . Differences were considered significant when


Small GTPases are monomeric guanine nucleotide-binding proteins. In plants, ROPs regulate plant cell polarity, plant cell differentiation and development as well as biotic and abiotic stress signaling pathways.Arabidopsis AtRop1 in potato led to H2O2 accumulation associated with the reduced development of Phytophthora infestans Montagne de Bary and smaller lesions on infected potato leaves. The expression of the Strboh-D gene, a NADPH oxidase homologue in potato, was analyzed by RT-PCR. Expression of this gene was maintained in DN-AtRop1 transgenic plants after infection with P. infestans. In transgenic potato lines, the transcript levels of salicylic acid (SA) and jasmonic acid (JA) marker genes  were analyzed. The Lox gene was induced dramatically whereas expression of Npr1, a gene up-regulated by SA, decreased slightly in DN-AtRop1 transgenic plants after infection with P. infestans.We report the subcellular localization of the AtRop1 protein at the plasma membrane in tobacco epidermal cells using GFP fusions. Additionally, transient and stable expression of a dominant negative form (DN) of the P. infestans. This is associated with increased NADPH oxidase-mediated H2O2 production and JA signaling.In conclusion, our results indicate that DN-AtROP1 affects potato resistance to  Solanum tuberosum) is the fourth largest crop in the world. Due to the high altitude, cold temperature and limited virus vectoring, Inner Mongolia has become the largest potato producing province in China. However, due to the absence of resistance genes to P. infestans in most cultivated potato varieties, potato late blight causes dramatic yield losses in Inner Mongolia [P. infestans and generate resistant cultivars through the combination of traditional and molecular breeding approaches. Numerous studies have investigated the molecular basis of quantitative resistance to pathogens [PR, StPK1, and StLRPK1 are involved in resistance to potato late blight [P. infestans in potato is lacking.Potato  production, cell cycle and differentiation [Small GTPases are monomeric guanine nucleotide binding proteins . Rho GTPntiation -18. Planntiation -21. Planntiation ,22,23. Lntiation -21. ROPsntiation . To datentiation ,25,26, 7ntiation . The prontiation ,22,23,28Magneporthe grisea via H2O2 accumulation, achieved through the regulation of NADPH oxidase activity [2O2 production [Pseudomonas syringae pv. Tomato (Pst) DC3000 ( P. syringae). However, AtROP10 has the opposite effect on resistance to bacteria [MsRac1 gene results in cell death, thus leading to the development of brown necrotic lesions [Medicago truncatula plants indicates that MtROP9 plays a key role in ROS-mediated early infection signaling [Rho-related GTPases are clearly involved in the establishment of plant defense. In rice, OsRac1 positively regulates the defense response to activity -32. OsRaactivity -36, but activity . Overexpoduction ,39. In Abacteria . In barlbacteria . Howeverbacteria ,42,43. I lesions . In addiignaling . All of 2−), hydrogen peroxide (H2O2), hydroxyl radical (HO·) and singlet oxygen (1O2), which generated by plasma membrane NADPH oxidases play pivotal roles in the defense response, and are thought to act as secondary messengers for the induction of resistance responses, such as the increased expression of defense genes and the induction of hypersensitive cell death known as the hypersensitive response (HR) [Nicotiana benthamiana inhibiting ROS accumulation led to reduced resistance to P. infestans [Hyaloperonospora arabidopsidis [Reactive oxygen species(ROS) including superoxide (Onse (HR) ,47. Rapinse (HR) ,48. In Nnfestans . In Arabdopsidis . Increasdopsidis .PR-1, PAD4, EDS1, and PDF1.2 [NPR1) is up-regulated by SA accumulation during pathogen infection and is routinely used as a marker gene to track SA-mediated signaling pathways [P. infestans; SA reduction renders plants more susceptible to P. infestans possibly due to lower PR gene expression [P. infestans [Lox gene is highly correlated with JA accumulation in response to various stresses [Arabidopsis thaliana mutants impaired in the JA signaling pathway are always compromised in their resistance to necrotrophic pathogens [AOC) or OPDA reductase 3 (OPR3) genes, both coding for enzymes involved in JA biosynthesis, also perturbs the potato response to Pep-13, a pathogen-associated molecular pattern (PAMP) from P. infestans, by reducing the accumulation of ROS and hypersensitive cell death. SA accumulation has been observed in JA deficient plants [Many studies have shown that SA  and JA (Jasmonic acid) are key signaling molecules which regulate plant resistance by inducing the expression of a series of defense genes,such as d PDF1.2 . SA playd PDF1.2 . The nonpathways . A recenpression . Increasnfestans . JA is anfestans ,58. In pnfestans . Lipoxygstresses . Arabidoathogens . JA acumathogens . Silencit plants , indicatArabidopsis and its role in the establishment of resistance to potato late blight. Our results demonstrate that transient or stable overexpression of an inactive form of AtRop1 (DN-AtRop1) enhances potato resistance to P. infestans infection, a resistance associated with the accumulation of H2O2 mediated by a NADPH oxidase homologue gene. This suggests that H2O2 plays a crucial role in AtRop1-mediated potato resistance to P. infestans infection. Additionally, preliminary results indicate the involvement of JA in AtRop1-mediated potato resistance to late blight.We analysed AtRop1, a small G protein in To determine the subcellular localization of AtRop1, a mGFP:AtRop1 construct was transiently expressed in tobacco epidermal cells and the GFP signal detected under fluorescence microscopy. As shown in Figure P. infestans, Agrobacterium tumefaciens (LB4404) strains carrying either CA-AtRop1 (constitutively active) or DN-AtRop1 (dominant negative) were infiltrated into potato leaves. Twenty-four hours later, P. infestans (5 × 105 zoospore/mL) was inoculated at the infiltration sites and the lesion sizes were measured at different time points after inoculation. As shown in Figure . These results suggest that AtRop1 may be involved in potato resistance to P. infestans.To determine the importance of AtRop1 in the potato response to P. infestans mycelium around the inoculation sites was firstly detected using trypan blue staining. As shown in Figure P. infestans. This showed that P. infestans mycelium development at the CA-AtRop1 and DN-AtRop1 transient expression sites was significantly lower than control sites, with DN-AtRop1 expression sites showing the lowest biomass levels  was detected by real-time PCR. Quantitative analysis indicated reduced development of P. infestans in the three DN-AtRop1 transgenic lines compared to the control plants  showed that the accumulation of H2O2 followed the same temporal pattern in the DN-AtRop1 and control lines, but levels were significantly higher in DN-AtRop1 transgenic lines  is the main regulator of H2O2 production in these transgenic lines. RT-PCR was performed using primers derived from Strboh-D, as shown in Figure Strboh-D gene remained constant at 24 hpi, decreasing slightly by 72 hpi, to almost half the control level by 96hpi   promotes H2O2 production [DN-OsRac1) leads to the suppression of H2O2 [Rac1 (DN-hRac1) in soybean or OsRac1 in rice cell suspensions also leads to the suppression of H2O2 production in response to an elicitor treatment. This suggests the existence of a positive regulation mechanism by small G proteins on plant resistance mediated by H2O2 accumulation [DN-OsRac1) in tobacco. This led to induced H2O2 accumulation after infection with Tobacco mosaic virus (TMV) [NtRac5 (Rac5 cloned from Nicotiana tabacum) in tobacco led to lower levels of H2O2 [AtRop1 (DN-AtRop1) gene in potato led to a decrease in the development of P. infestans at the infection sites. This suggests a negative regulation mechanism of AtRop1 on potato resistance to late blight. H2O2 was found to be a key player in the signaling pathway associated with AtRop1-mediated potato resistance. The overexpression of a negative form of AtRop1 resulted in the accumulation of H2O2, and enhanced potato resistance to P. infestans infection. H2O2 is known to be a key player during the establishment of plant resistance [2O2, which triggers the HR in the infected areas, was observed in plants infected by non-host or avirulent strains of pathogens. In our study, overexpression of DN-AtRop1 promoted the accumulation of H2O2 after P. infestans infection, which possibly triggered an HR in the infected area, thus limiting the development of P. infestans, and resulting in smaller lesions   and over of H2O2 .We showesistance ,63. The nfection .2O2 can be achieved either by mitochondrial electron transport during aerobic respiration or by oxidoreductases and metal-catalyzed oxidation. The activation of a membrane-associated NADPH oxidase constitutes the main source of H2O2 accumulation [P. infestans [StRboh in potato transgenic lines was observed after inoculation with P. infestans. However, its expression was suppressed dramatically in control plants. This indicates that the accumulation of H2O2 in the transgenic lines was likely to be caused by the sustained expression of the membrane-localisted StRboh. Meanwhile, the plasma membrane localization of both AtRop1 and StRboh suggests that the PM may be the platform connecting AtRop1 and StRboh, thus facilitating the accumulation of H2O2. Catalase is a major H2O2 scavenging enzyme inside plant cells, and hydrolyses H2O2 into H2O and O2 to protect plant cells from deleterious effects of H2O2 [P. infestans(data not shown), suggesting that H2O2 accumulation is mainly caused by potato homologues of PM-localized NADPH oxidases.Generally, the production of Hmulation ,69. Stronfestans . In our  of H2O2 . HoweverP. infestans, the transcript level of marker genes in these two pathways was estimated by RT-PCR. Our data showed that Lox gene expression was induced in DN-AtRop1 transgenic lines during P. infestans infection, indicating that the JA pathway was activated by DN-AtRop1 overexpression, whereas the SA signaling pathway was partially suppressed in this study.SA and JA are important signaling molecules in plant responses to pathogens. To check whether they were involved in the increased resistance of DN-AtRop1 transgenic lines to P. infestans invasion of the resistant potato cultivar Zihuabai [P. infestans, but was also involved in the establishment of systemic acquired resistance of potato induced by BABA [P. infestans. A recent study established that JA and SA signaling pathways act antagonistically in potato [Npr1 transcript levels and the concomitant increase in JA after inoculation with P. infestans suggest a conservation of the antagonistic effects of SA and JA. In addition to SA and JA, there are other hormones involved in plant resistance, such as ethylene (ET), brassinosteroids, Cytokinins (CKs), and auxin. Among them, the brassinosteroids and CKs were shown to enhance the plant resistance to pathogens through NPR1 [Npr1 transcript levels after infection with P. infestans, indicating that AtRop1 may positively regulated the NPR1 expression in potato.Our data differ from previous reports showing that the SA signaling pathway was activated, and the JA signaling pathway was inhibited to a certain extent during Zihuabai . In addi by BABA ,73. Howe by BABA . So, AtRn potato . The decugh NPR1 -76, a maugh NPR1 , while augh NPR1 . Our dat2O2 accumulation. We propose that H2O2 accumulation in turn limits P. infestans development, leads to smaller lesion size and enhances potato resistance to late blight. Our results also indicate that JA may be involved in AtRop1-mediated potato resistance to P. infestans.We verified that AtRop1 could regulate potato resistance to late blight. The DN-AtRop1 appears to positively impact potato resistance to late blight, through activation of membrane-associated NADPH oxidase, thus facilitating HEcoRI and SacI, then blunt-end and ligated with the empty vector pBI121 to construct three expression vectors: pBIG-CARop1, pBIG-DNRop1 and pBIGRop1. The PCR identification of A. tumefaciens containing pBIG-CARop1, pBIG-DNRop1 and pBIG-Rop1 were performed by using the GFP primers 5′-GAC GTA AAC GGC CAC AAG TT-3′ and 5′-GAA CTC CAG CAG GAC CAT GT-3′.The constructs pLG-CARop1, DNRop1 and Rop1, and the pBI121empty vector were kindly provided by Dr. Zhao Yan . The constructs were digested with Agrobacterium tumefaciens (strain LB4404) containing the expression vector (pBIG-CARop1/pBIG-DNRop1/ pBIG-Rop1) and the empty vector (GFP only) were grown for approximately 48 h in 5 ml LB medium contained 10 mM MES (pH 5.7), 20 μM acetosyringone and 100 mg/L rifampicin. Agrobacterium were collected and resuspended in 5% sucrose at a concentration of OD600 = 2.0, followed by infiltration into tobacco leaves using a syringe without the needle. After transient expression, the tobacco leaves were placed in the dark for 72 h. The GFP signal was detected in tobacco epidermal cells via confocal microscope (Carl Zeiss LSM-510).Phytophthora infestans mating type A1 was kindly provided by Prof. Zhang Ruofang (Potato Engineering Center of Inner Mongolia University). After culturing on sterilized oat grains at 18°C for 2–3 weeks, the sporangia of P. infestans were washed with sterile water and kept at 4°C for 4–6 hours to facilitate zoospore release. Zoospores were filtered through 1–2 layers of sterilized gauze, and the concentration determined using a haemocytometer under the microscope. The solution of zoospores was diluted to 5 × 105 zoospore/mL for all inoculations [5/mL) and incubated at 20°C with 85% humidity for resistance evaluation and determination of H2O2 accumulation [2O2 production and assays of mycelium biomass.The potato leaves were inoculated with 20 μL zoospore suspension (5 × 10mulation . ZoosporAgrobacterium tumefaciens (strain LB4404) carrying the AtRop1 mutants (CA-AtRop1 and DN-AtRop1) were grown for approximately 48 h in LB medium contained 10 mM MES (pH 5.7) and 20 μM acetosyringone. Following centrifugation, bacteria were resuspended in a infiltration medium that contained 10 mM MgCl2, 10 mM MES (pH 5.7) and 150 μM acetosyringone at a concentration of OD600 = 0.5 . The bacteria were infiltrated into detached potato leaves using a syringe without needle. Twenty-four hours later, 20 Μl of the zoospore suspension(5 × 105/mL)was inoculated at the infiltration sites. The resistance of potato to P. infestans was evaluated using lesion area of the inoculated leaves at different time points. Lesion area was estimated by counting the length × width of the lesion. Every experiment was repeated three times.2O2. The DAB solution was freshly prepared to avoid auto-oxidation. Leaf segments were floated in 0.1% (w/v) DAB solution (pH 3.8) and kept at 25°C in dark. After 8 h, the leaves were transferred into 95% ethanol and bleached by boiling for 30 min. Leaves were then stored in glycerol-ethanol (1/4 v/v) solution until photographed. The brown color due to DAB polymerization represented the accumulation level of H2O2 [The staining method 3, 3 ′-diaminobenzidine (DAB) was used to detect the accumulation of H of H2O2 . Color q2O2. The H2O2 and titanium sulfate generated yellow peroxidate-Ti compound precipitate. This problem was solved by treating with sulphuric acid; the color represents the concentration of H2O2,. Values were collected at OD415 nm and were linearly related to H2O2 concentration [2O2, 30% standard H2O2was reacted with 5% titanium sulfate (w/v) in reaction buffer that contained ammonia and acetone, then solved with 2 M sulphuric acid. The regression equation was obtained through the formula: Y = 0.8144X + 0.025, R2 = 0.9953 by using values collected at OD415 nm through spectrophotometry(TU-1901)and used as standard curve. Then, 0.1 g of ground potato leaves were collected at 0, 24, 36, 48, 72 and 96 hpi, in liquid nitrogen and the powder transferred into 1.5 mL centrifuge tubes containing 1 mL prechilled acetone(4°C)to extract H2O2 . After centrifugation for 10 min at 6000 rpm, the 100 μL supernatant was used to react with 5% titanium sulfate (w/v). After the mixture was solved in 2 M sulphuric acid, the H2O2 concentration over time  was detected using a UV Spectrophotometer at OD415 nm.The titanium sulfate standard curve method was used to quantify the concentration of Hntration . To geneP. infestans mycelium. Infected potato leaf segments were transferred into 95% ethanol and boiled for 30 min to bleach the leaves. Leaves were then infiltrated with 0.5% (w/v) trypan blue solution for 4 hours at 25°C. After washing the samples with water for three times, the mycelium was visualized under the microscope [Trypan blue staining was used to detect the development of croscope .P. infestans on potato plants using a lightcycler 480II in combination with the qPCR kit SYBR Select Master Mix . DNA was extracted by using the hexa-decyl tri-methyl ammonium bromide (CTAB) method [P.infestans-specific repetitive DNA sequence [EF-1α gene as an endogenous control.Quantitative real time PCR was applied to determine the growth of ) method . The prisequence  and primSolanum tuberosum cv “Shepody” were grown on Murashige and Skoog (MS) medium in vitro at a photo flux density of 3000–4000 Lx, 60-85% relative humidity, and a 16 h/12 h day and night cycle at 25°C. A single colony of Agrobacteria LB4404 was cultured in a shaker culture of 10 mL of LB medium containing kanamycin (100 mg/L) and rifampin (100 mg/L) and kept at 28°C, 220 rpm for 48 h. The leaf veins of 3 ~ 4 weeks old potato seedlings leaves were cut, then floated in MS liquid medium that contained bacterial suspension  and kept in the dark for 2–3 days. The leaves were transferred onto MS solid medium after drying on sterile filter paper. After 2 days, the leaves were transformed onto MS medium contained 0.5 mg/L indolylacetic acid (IAA), 2.0 mg/L 6-benzyl aminopurine (6-BA), 2.5 mg/L gibberellic acid (GA3), 25 mg/L hygromycin and 500 mg/L cefotaxime to induce adventitious buds. Four weeks later, regenerated potato shoots were grown on regeneration medium containing 25 mg/L hygromycin and 500 mg/L cefotaxime for selection. The transgenic plants were identified by Southern blot and Western blot.Potato plants Potato genomic DNA was extracted using the CTAB method . For DNAThe GFP probe for Southern blotting was generated according to the instruction of DIG DNA Labeling and Detection Kit II  based on its sequence. After genomic DNA was digested with EcoR I and Sac I, the products were separated on 1.0% agarose gels, then transferred onto a nylon membrane. The membrane was washed with 2× SSC , then twice with 0.1% SDS (5 min), followed by washing twice with 0.5 × SSC once and twice with 0.1% SDS (15 min) at 65°C. The membrane was baked at 80°C for 2 h to cross-link the DNA with the nylon membrane. The hybridization of the membrane with the labeled GFP probe was performed overnight in hybridization buffer (provided by DIG DNA Labeling and Detection Kit II) at 42°C. After hybridization, the membrane was washed with washing buffer  for 5 min; followed by incubation with blocking solution (provided by DIG DNA Labeling and Detection Kit II) for 30 min. The membrane was then incubated with Anti-Digoxigenin-AP (1:5000 dilutions) for 30 min. The membrane was washed with washing buffer twice, and equilibrated in detection buffer  for 5 min. The membrane was stained with 1 ml CSPD, and incubated at 25°C for 5 min, then analysed ueing a Chemi XT4 .3PO4, 2.5% sucrose, 0.1% β-mercaptoethanol, and 0.5 mM phenylmethyl sulfonylfluoride. The crude extract was centrifuged at 4°C, 12,000 rpm for 20 min. The supernatant was collected and mixed with loading buffer. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% non-fat dry milk in PBST buffer and incubated with an anti-GFP monoclonal antibody  and HRP-conjugated anti-rat secondary antibody  to detect the signal by Chemi XT4 .Transgenic potato leaves were ground in liquid nitrogen and the powder transferred into 1 mL protein isolation buffer (pH 6.0), which contained 5 mM KStRboh-D-F(5-AGC TGC AGA ATA CGC AGC GTT GA-3)StRboh-D-R(5-GGC ATT GAA ACC GGT GAG CTT GT-3)Npr1-F(5-TGC TGC CAT GCG TAA CGA ACC A-3)Npr1-R(5-TGG ACC AAA ACT TGG CCC CAC A-3)Lox-F(5-AGA ACT TTG CTC TTC TTG CAA G-3)Lox-R(5-GGT AAT ATT CAT TGT GTC CCG-3)GAPDH-F (5-TGG ACA ATG GAA GCA CCA TGA GC-3)GAPDH-R (5-TGC TTG ACC TGC TGT CAC CAA GA-3).Total RNA was extracted from potato transgenic leaves using the RNA iso Reagent . The quality of RNA was examined on a 1.2% agarose gel. The cDNA for RT-PCR was generated using AMV transcriptase  according to the manufacturer’s instructions. The specific primers used for RT-PCR analysis were as follows:


Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110  were grown in aseptic jars. After 30 days, shoot decapitation , soil addition , or both (DS) were applied. N2O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N2O from the soybean rhizosphere. The N2O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N2O emission. A 15N tracer experiment indicated that N2O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N2O emission was compared between a nirK mutant  and ΔnosZ. The N2O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N2O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N2O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N2O from nodule N via their denitrification processes (N2O source), whereas nosZ-competent B. japonicum exclusively takes up N2O (N2O sink). Net N2O flux from soybean rhizosphere is likely determined by the balance of N2O source and sink.A model system developed to produce N The aimBradyrhizobium japonicum cells were grown at 30°C in HM salt medium  mL−1, 100 μg spectinomycin (Sp) mL−1, 100 μg streptomycin (Sm) mL−1, 100 μg kanamycin (Km) mL−1, and 100 μg polymyxin B mL−1; for E. coli, 50 μg Tc mL−1, 50 μg Sp mL−1, 50 μg Sm mL−1, 50 μg Km mL−1, and 50 μg ampicillin mL−1.The bacterial strains and plasmids are listed in t medium  supplemei medium . AntibioE. coli were performed as described previously  was extracted twice with 30 mL distilled water to remove nitrate and nitrite. The suspension was shaken for 10 min in centrifuge tubes and then centrifuged at 5,555×g for 15 min . The pellet was resuspended in 30 mL distilled water.Soil was collected from an experimental field at Tohoku University . This gray lowland soil had pH).Because the 2O to N2 , 43, and nodules  and 5A, nirK, a small quantity of N2O was released . These results strongly suggest that the nosZ gene of B. japonicum is involved in the uptake of N2O that is released from degraded nodules. In nosZ gene to N2O flux is shown as “CZ1”. In the absence of nosZ, there was a significant difference in N2O flux between ΔnosZ and ΔnirKΔnosZ  and ΔnirKΔnosZ . These The term “rhizosphere” was first coined in 1904 by Lorenz Hiltner in Germany, who had a special interest in complicated N transformations around leguminous nodules with higher N contents in fields . In a se2O emission from degraded nodules in the soybean rhizosphere is due to B. japonicum and other soil microbes. When plants were inoculated with B. japonicum nosZ− strains and treated with shoot decapitation and soil addition (DS), N2O was markedly produced  and other soil microbes , and nosZ-competent B. japonicum (nosZ+ strains) takes up N2O (N2O sink)  .2O from degraded nodules? Prokaryotic denitrification, fungal denitrification, ammonium oxidation, and nitrate ammonification have been nominated as soil microbial sources of N2O , suggesting that it is unlikely to be due to nitrification. KNO3 addition also enhanced the contribution of B. japonicum to N2O emission in relation to the other soil microbes  and N2O uptake by B. japonicum (nosZ+)  .nosZ+ strains of B. japonicum as inoculants has been expected to reduce N2O emissions from soybean fields (nosZ+ strains produced no N2O and were able to take up N2O from degraded nodules (2O reduction by nosZ-carrying inoculants was shown in a soil-filled pot planted with soybeans (B. japonicum mutants with increased N2OR activity (2O flux from soybean rhizosphere.The use of n fields , 43. Ind nodules . Recentlsoybeans . Thus, Bactivity  might be