The seasonal melting of ice entombed cryoconite holes on McMurdo Dry Valley glaciers provides oases for life in the harsh environmental conditions of the polar desert where surface air temperatures only occasionally exceed 0°C during the Austral summer. Here we follow temporal changes in cryoconite hole biogeochemistry on Canada Glacier from fully frozen conditions through the initial stages of spring thaw toward fully melted holes. The cryoconite holes had a mean isolation age from the glacial drainage system of 3.4 years, with an increasing mass of aqueous nutrients  with longer isolation age. During the initial melt there was a mean nine times enrichment in dissolved chloride relative to mean concentrations of the initial frozen holes indicative of an ionic pulse, with similar mean nine times enrichments in nitrite, ammonium, and dissolved organic matter. Nitrate was enriched twelve times and dissolved organic nitrogen six times, suggesting net nitrification, while lower enrichments for dissolved organic phosphorus and phosphate were consistent with net microbial phosphorus uptake. Rates of bacterial production were significantly elevated during the ionic pulse, likely due to the increased nutrient availability. There was no concomitant increase in photosynthesis rates, with a net depletion of dissolved inorganic carbon suggesting inorganic carbon limitation. Potential nitrogen fixation was detected in fully melted holes where it could be an important source of nitrogen to support microbial growth, but not during the ionic pulse where nitrogen availability was higher. This study demonstrates that ionic pulses significantly alter the timing and magnitude of microbial activity within entombed cryoconite holes, and adds credence to hypotheses that ionic enrichments during freeze-thaw can elevate rates of microbial growth and activity in other icy habitats, such as ice veins and subglacial regelation zones. The McMurdo Dry Valleys (MDV) are polar deserts comprising the largest ice-free expanse in Antarctica, and represent one of the coldest and driest ecosystems on Earth   have highly elevated ionic concentrations up to two orders of magnitude greater than in fully thawed holes, with large diurnal variations  in cryoconite holes through the start of the melt season on Canada Glacier in the MDV. This study will increase our understanding of cryoconite hole biogeochemistry, and help to define the role of cryoconite holes in the depauperate ecosystem of the MDV.T = 0, T + 3, T + 5, and T + 10 . Eight ice cores with no underlying cryoconite were also sampled on 25th November 2011. All cryoconite holes were completely frozen at T = 0, with increasing melt water at later time points. The same cryoconite holes were not sampled repeatedly; rather, eight different cryoconite holes were destructively sampled in the same local 10 m2 area of the glacier centered around the following coordinates: S 77°37.546′, E 162°57.620′. In all cases, samples were taken using a hand driven Kovacs ice corer. Prior to sampling, the width and length of the cryoconite holes  were measured using a ruler (±0.5 cm).The sample site was located on the surface of Canada Glacier, located within the 34 km long Taylor Valley of the MDV, Antarctica. Eight ice-lidded cryoconite holes were sampled at four different time points on 25th November, 28th November, 30th November, and 5th December 2011, hereafter termed After coring, ice cores and fully frozen cryoconite holes were transferred to large polyethylene bags for and transported to the field laboratory. In the laboratory, cores were melted in the laboratory and samples treated as below for water samples. In cryoconite holes where liquid water was present, the ice lid was removed and the water depth, sediment depth, and hole depth were measured with a ruler (±0.5 cm). Water was sampled using a 50 ml plastic syringe and transferred into 1L HDPE bottles for later ion and nutrient analysis and 65 ml glass BOD  bottles for dissolved inorganic carbon (DIC) and pH measurements later the same day. All HDPE bottles were rinsed three times with sample prior to filling. The BOD bottles were filled from the bottom using a length of Tygon® tubing attached to the syringe, and the bottle allowed to overflow 3× their volume prior to capping with no headspace. Cryoconite samples were collected with a stainless steel scoop into 50 ml Falcon tubes. Ice samples were collected in polyethylene bags, melted in the laboratory and the water treated as below for water samples.2 evolved using a portable PP systems EGM-4 infrared CO2 meter, with a coefficient of variation of 1%  and cations  were analyzed on a dual system Thermofisher Dionex 5000 capillary ion chromatograph with IonPac AS11-HC (0.4 × 250 mm) and CS16 (0.5 × 250 mm) columns at 30°C, using 32.2 mM KOH and 30 mM methanesulfonic acid (MSA) as eluents for anions and cations, respectively. Detection limits for Cl−, NO−3, and SO2−4 were 0.5, 0.6, and 0.2 μmoles L−1 respectively, with coefficients of variation of 0.8, 2.4, and 1.9% (n = 10). Detection limits for Na+, K+, Ca2+, Mg2+ were 2.7, 1.4, 1.0, and 0.1 μmoles L−1 respectively, with coefficients of variation of 1.8, 2.7, 1.9 and 1.9% (n = 10). Dissolved organic carbon (DOC) was analyzed on a Shimadzu TOC-V CSN analyzer. The detection limit was 10.1 μmoles L−1 with a coefficient of variation of 6.4% (n = 6). NO−2 and NH+4 were analyzed on a Bran + Luebbe Autoanalyzer 3, with detection limits of 0.09 and 0.4 μmoles L−1 respectively, and coefficients of variation of 1.5 and 9.2%, respectively. PO3−4, total dissolved phosphorus (TP(aq)) and total dissolved nitrogen (TN(aq)) were analyzed on a Lachat QuickChem 8500 Series 2 Flow Injector Analyzer using standard QuikChem® methods 31-115-01-1-I, 31-115-01-3-F and 31-107-04-3-A, respectively. Detection limits were 0.036, 1.4, and 1.0 μmoles L−1 for PO3−4, TP(aq) and TN(aq) respectively, with coefficients of variation of 0.5, 3.4, and 7.4% (n = 6). Dissolved organic nitrogen (DON) was defined as TN(aq) − [NH+4 + NO−2 + NO−3]. Dissolved organic phosphorus (DOP) was defined as TP(aq)-PO3−4.Dissolved anions  was analyzed using a Picarro L1120-i cavity ringdown spectrometer, with a precision of ±0.2‰. Inorganic carbon speciation in T + 10 cryoconite holes (the only time point where pH was reliably measured) was modeled using the computer program PHREEQ-C using its standard database.The δ−1 and 100 μg N g−1 respectively, with coefficients of variation for duplicate analyses of 9.5 and 5.3%. Exchangeable NH+4, NO−3, and NO−2 in wet cryoconite were extracted using a 2M KCl method  in dried cryoconite were analyzed using a three stage sequential digestion method using (1) 1 M MgCl2, (2) 0.1 M NaOH and (3) acid persulfate. Extracts were analyzed on a Shimadzu mini UV-vis spectrophotometer. The average coefficients of variation for duplicate cryoconite samples for the MgCl2, NaOH, and persulfate digestion steps were 10.1, 6.2, and 10.3%, respectively.Three cryoconite samples from each time point were analyzed for TOC, IC, and TN. All eight cryoconite samples from each time point were analyzed for solid phase nitrogen and phosphorus speciation. Dried cryoconite samples  were analyzed for total organic carbon (TOC) and total nitrogen (TN) on a Eurovector EA3000 Elemental Analyzer. Detection limits for TOC and TN were 100 μg C g−1 final concentration) and samples filtered onto black 0.22 μm polycarbonate filters (Millipore). Over 300 stained bacterial cells per slide were counted in duplicate using epifluorescence microscopy, and procedural blanks (sterilized deionized water) were subtracted from the sample counts. The same procedure was followed to enumerate photoautotrophic microbes using chlorophyll autofluorescence instead of staining the samples with DAPI   from biovolume.Three cryoconite samples from each time point were analyzed for total cell counts, autofluorescent counts, and biomass estimates. All eight samples at each time point were assayed for potential photosynthesis, bacterial production, and potential nitrogen fixation. Total and autofluorescent cell counts were enumerated by epifluorescent microscopy  was added. For each sample triplicate light, triplicate dark , and triplicate killed  vials were incubated for 1.5 h in an ice bath under a lamp. The average photosynthetically active radiation (PAR) at the surface of the ice bath measured using an Apogee PAR sensor and meter was 100 μmoles m−2 s−1 with a spatial variation of ±20 μmoles m−2 s−1. It should be noted that the actual PAR levels below ice lids in the cryoconite holes were not measured hence these rates of photosynthesis should be viewed as potential only. Vials were incubated for 1.5 h then removed from the ice bath and filtered through 0.45 μm cellulose nitrate filters. The pH of the filtrate was immediately raised to >pH 10 by the addition of pellets of NaOH prior to disposal. The filters were fumigated with 50% glutaraldehyde and 37% v/v HCl for >1 h, and the filters placed in scintillation vials. Vials were transported to the main laboratory (McMurdo), where 10 ml of Cytoscint ES scintillation cocktail was added and DPM values measured using a Beckman LS6000 scintillation counter, calibrated using external quench standards. Rates of photosynthesis (μg C g−1 h−1) were calculated following the procedures of Telling et al. , where 90 μl of 100% ice cold trichloroacetic acid was added to samples, and the tubes centrifuged at 16,000 g for 10 min. The pellet was washed sequentially with 5% trichloracetic acid and 80% ethanol and samples dried and weighed. Finally, 1 ml of Cytosint ES cocktail was added to samples, and DPM counted on a Beckman LS6000 scintillation counter calibrated using external quench standards. Bacterial production rates (μg C g−1 h−1) were calculated following the methods of Anesio et al.  between fully frozen cryoconite holes and surrounding ice cores as follows:Enrichment factors, where for example ion and nutrient concentrations in cryoconite holes are normalized to mean concentrations in the surrounding ice, can greatly facilitate the identification of biogeochemical sources and sinks , and mean Cice is the mean concentration in the ice cores.where T + 3, T + 5, T + 10) and fully frozen cryoconite holes (T = 0) to facilitate the identification of biogeochemical processes occurring during early melt freeze-thaw effects as follows (using the example for T + n):Similarly, we define enrichment factors between partially melted cryoconite holes .where 18O of the water as a more quantitative proxy than time for the percentage of ice melt within each individual hole, since the initial melt water should be enriched in the lighter 16O-H2O. 16O-H2O preferentially partitions into the water phase relative to the heavier 18O-H20  using Equation 3 from Fountain et al.  that the initial cryoconite water and additional annual ice melt have chloride concentrations equivalent to the mean chloride concentration of sediment free ice and, (c) that the ice lids contain no chloride  after normalizing ion and nutrient data to the surface area of the holes in order to negate the impact of concentration and dilution of nutrients by freeze-thaw.The isolation age of the cryoconite waters from the surrounding supraglacial water system was estimated using chloride mass balance, following the approach of Fountain et al. . Chloridn et al. , assuminT = 0 and T + 10 cryoconite holes with the measured nutrient demands of bacterial production and photosynthesis:Comparison of the rate of influx of nutrients from ice ablation into cryoconite holes with rates of microbial growth can give insight into potential microbial nutrient limitations  or TP(aq)] from ice ablation into the hole (μmoles m−2 h−1), based on the concentration of nutrients in the ice, Cice, and the ablation rate, Z , D is the estimated nutrient demand  from bacterial production or photosynthesis within the cryoconite hole (μmoles m−2 h−1), activity rate is the measured rate of bacterial production or photosynthesis in the cryconite (μmoles g−1 h−1), M is the dry mass of cryoconite within the hole , the Redfield coefficient, R, is 1 for C, 1/16 for N, and 1/106 for P, and A is the plan view area of the cryoconite hole (m2).where F is the nutrient flux [DOC, DIC, TN−1 (Table 6) to potential carbon fixed (μmoles C g−1) assuming a C:N molar ratio of 6.6:1 .Mean water depths in the holes increased with time to a maximum of 12.1 ± 4.9 cm at 2+ (20.7×), Mg2+ (8.2×), and SO2−4 (9.1×) were higher than those of Cl− , Na+ (2.0×), and K+ (2.6×). All dissolved nitrogen and phosphorus species  were depleted relative to Cl− with enrichment factors <2× (Table Ice core and aqueous geochemistry of cryoconite holes is summarized in Table  SO2−4 9.× were hi SO2−4 9.× were hi18O-H2O during the first melt (T + 3) were lower relative to frozen cryoconite holes at T = 0 (Table − concentration in early melt waters (T + 3) was enriched 9.3× over fully frozen cryoconite holes (T = 0), alongside >9× increases in mean NO−3, NO−2, Mg2+, and DOC concentrations , while the mean DIC was depleted at T + 3 (0.5×) relative to T = 0. A plot of DIC against DOC indicates two distinct groups of samples: those with a relatively constant DOC level but varying DIC concentration  and those with a relatively constant DIC concentration and higher and varying DOC concentrations  between δ18O-H2O and log enrichment factors for Cl−, SO2−4, NO−3, NO−2, TN, Mg2+, Ca2+, DON, DOP, and DOC and a less strong (p < 0.05) correlation between δ18O-H20 and the log enrichment factor for K+  or between δ18O-H20 and log enrichment factor for DIC   concentrations varied from 0.05 to 12.9 μmoles L−1, with a mean of 2.6 ± 4.7 μmoles L−1.pH was only measured in cryoconite holes at (aq) in ice cores was 4.5 ± 1.3 (Table (aq) of cryoconite holes were elevated relative to the surrounding ice, bracketing the Redfield ratio at 5.9–9.6 (Table p < 0.01) between δ18O-H2O and DOC:TP(aq) but not between δ18O-H2O vs. DOC:TN or δ18O-H20 vs. TN:TP(aq)  , the remainder contained <0.1 mg N g−1 (Table The mean organic and inorganic carbon concentrations of cryoconite were 0.9 ± 0.4 and 0.5 ± 0.3 mg C gKCl varied with time. The lowest mean concentrations of NH+4KCl were at T = 0 (85 ng N g−1), with a maximum at T + 3 (962 ng N g−1) and reduced concentrations through T + 5 to a mean of 316 ng N g−1 at T + 10 , which fell to a mean of 3.1 ng N g−1 at T + 10 , with low concentrations of PMgCl2 (mean of 0.5 μg P g−1) and PNaOH (mean of 10.3 μg P g−1)  was 10.6 ± 6.8%  relative to other time points (0.9–6.5 μg C g−1 wet sediment)  and T + 10  (Table T + 3 and T + 5 (Table (aq) was ≤9μmoles L−1, and TNKCl was ≤330 ng N g−1  correlation between bacterial production and potential photosynthesis . There was a significant correlation of bacterial production with δ18O-H2O  but not between potential photosynthesis and δ18O-H2O   from chloride mass balance was 3.4 ± 3.6 (1σ) years , Na+ , K+ , Mg2+ , Ca2+  and SO2−4  with increasing isolation age , DON , DIN , DOP  and PO3−4  with increasing isolation age of the holes. In contrast, there were no significant (p < 0.05) relationships between isolation age and DON:DIN , DOP:PO3−4 , PMgCl2 , PNaOH , Presidual  or NKCl  (not shown), bacterial production  or photosynthesis   of the cryoconite holes , and below 0°C for all but a few days of the entire melt season  and phosphorus species (DOP and PO3−4) relative to Cl− are consistent with the microbiological uptake of nutrients  and nutrients  with increasing isolation age of the holes . It is possible that trends could be masked by the high natural variation in microbial activity rates in individual samples  were lower than the mean reported for cryoconite holes from a range of MDV glaciers   are similar to those reported previously from Canada Glacier using the 14C method of 17–58 ng C g−1 h−1   were close to the detection limit (Table NaOH (mean of 10.3 ± 3.4 μg P g−1). The majority of phosphorus in cryoconite was in the form of Presidual (mean of 1455 ± 241 μg P g−1), of which approximately 40% may be in the form of organic phosphorus  from ice ablation alone are unlikely to be able to sustain the measured rates of microbial activity, even with efficient recycling between the heterotrophic and autotrophic communities Table . Particuit Table  consisteit Table  there weT = 0 and T + 10; Table 2H4 m−2 d−1) were similar to those measured in open cryoconite holes on a Greenland glacier of <4.2–16.3 μmoles C2H4 m−2 d−1  of cryoconite holes coincident with more negative δ18O-H2O  Cl− has the second largest enrichment factor after Mg2+  , while microbial activity could influence the concentrations of C, N, and P species.Relative to frozen cryoconite holes at 2O Table . The meating T +  of cryocse Table . For exating T +  of cryoc+4 (9.7×), and NO−2 (9.7×) were similar to Cl− (9.3×)  , NH) Figure , suggestigure −3 2.2×  increased rates of bacterial production  and DOP (4.4×) during the ionic pulse were lower than for DOC and dissolved nitrogen species, resulting in higher TOC:TP(aq) and TN:TP(aq)ratios  elevated during the ionic pulse  rather than enrichment in mean DIC concentrations during the ionic pulse event (Table 2(aq) concentrations of this study (T + 10 only) ranged from 0.05 to 12.9μmoles L−1, with a mean of 2.6 μmoles L−1. Freshwater phototrophs typically have a higher affinity for CO2 than HCO−3 , with a median of 13 μmoles L−1  in cryoconite holes at T+10. There is also a weak but significant correlation (p < 0.05) between DIC and rates of photosynthesis , driving DIC (and Ca2+) concentrations down  may have promoted higher rates of photosynthesis in an early more nutrient enriched melt water, both consuming DIC and producing alkalinity to further drive carbonate equilibria toward carbonate precipitation  of basal ice forms thin films of water, perhaps tens of microns thick (Raiswell et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK , Akt and mTOR , but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the  In the present paper, we report for the first time that GDF15 increases Kv2.1 expression through TGFβRII to activate the Akt/mTOR pathway, which reveals an unanticipated role of the non-Smad-dependent pathway in the previously uncharacterized signalling mediated by GDF15. GDF15 , is a novel member of the TGFβ (transforming growth factor β) superfamily that was discovered in a screen for the conserved consensus sequences of TGFβs . GDF15 hin vitro and in vivo for embryonic and lesioned dopaminergic neurons in the midbrain and the substantia nigra and is dramatically up-regulated in cryolesioned cortical neurons  on ice for 30 min. After centrifugation (12000 g for 15 min at 4°C), the supernatant was mixed with 2× SDS loading buffer and boiled for 5 min. The proteins were separated by SDS/PAGE (10% gel) and were then transferred on to a PVDF membranes (Millipore). The membrane was blocked with 10% (w/v) non-fat dried skimmed milk powder and incubated at 4°C overnight with the following primary antibodies: mouse monoclonal antibody against Kv2.1 or Kv2.2 , rabbit polyclonal antibody against phosphorylated insulin receptor β subunit , rabbit monoclonal antibody against phosphorylated ERK1/2 or total ERK1/2 , rabbit monoclonal antibody against phosphorylated Akt , rabbit polyclonal antibody against phosphorylated mTOR , rabbit polyclonal antibody against TGFβR2 , mouse monoclonal antibody against phosphorylated tyrosine , or mouse monoclonal antibody against GAPDH . After extensive washing in TBST (TBS with 0.03% Tween 20), the membrane was incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG  for 2 h at room temperature. Chemiluminescent signals were generated using a SuperSignal West Pico trial kit (Pierce) and were detected by exposure to X-ray film or using the ChemiDoc XRS System (Bio-Rad Laboratories). The Quantity One software  was used for background subtraction and for the quantification of the immunoblot data.The cells were lysed in Hepes/NP40 (Nonidet P40) lysis buffer  hexanoate (Thermo Fisher Scientific) for 45 min on ice and were rinsed in ice-cold PBS containing 1 M glycine to quench the biotin reaction. The cultured neurons were lysed and then homogenized in modified radioimmune-precipitation assay buffer . The homogenates were centrifuged at 10000 g for 15 min at 4°C, and the supernatants were collected. The supernatants were incubated with 50 μl of NeutrAvidin–agarose (Thermo Fisher Scientific) overnight at 4°C and washed three times with radioimmune-precipitation assay buffer. The total and biotinylated surface proteins were detected using quantitative Western blots as described above.Surface biotinylation experiments were performed in cultured rat granule neurons . TTX, 4-AP, DRB , CHX (cycloheximide), U0126, rapamycin, LY364947, PP2 and poly-+ currents IK and IA. GDF15 treatment for 24 h failed to modulate the IA in CGNs. Thus we examined the effect of GDF15 on IK in the present study. IK was evoked by a 100 ms depolarization to +50 mV from a holding potential of −50 mV in the presence of 5 mM 4-AP, which suppresses the IA and permits better resolution of the IK. Incubation of the CGNs with GDF15 significantly enhanced the IK densities in a concentration-dependent manner. The data obtained from 94 neurons showed that incubation of the CGNs with 10, 100 and 300 ng/ml GDF15 for 24 h increased the current densities by −2.2% (n=27), 37.7%  and 45.6% (n=26) respectively , 14.3% (n=25) and 1.2% (n=28) respectively , where GK is the membrane K+ conductance, Vm is the membrane potential and Vrev is the reversal potential for K+. The G–V curve analysis indicated that GDF15 did not alter the half-maximal activation voltage of the IK (n=17) and GDF15-treated  neurons.We then examined whether the gating properties of the channels were altered, leading to the enhancement of the f the IK A betweenIK, currents were elicited using 10 s conditioning pre-pulses from −90 mV to +10 mV before a 400 ms test pulse of +10 mV. After normalizing each current amplitude to the maximal current amplitude obtained from the −90 mV pre-pulse as a function of the conditioning pre-pulse potential and after fitting the normalization with the function IK/IK-max=1/(1+exp[(Vm1/2−Vm)/k]), we obtained an inactivation curve for the IK and calculated the voltage at which the current amplitude was half-inactivated (Vh50). The half-maximal deactivation voltage of the IK in the control neurons  was similar to that in the GDF15-treated neurons   B. These IK amplitudes might be due to the up-regulation of channel expression levels. The primary α-subunits mediating the IK in CGNs are Kv2.1 and Kv2.2 [n=4). The levels of transferrin 2, a subtype of transferrin, in the membrane fractions were not affected. Additionally, the kinetics of Kv2.1 expression induction paralleled the increase in the IK amplitudes following GDF15 treatment  or 10 μM leupeptin (a lysosome inhibitor) for 30 min before treatment with GDF15. MG-132 alone did not mimic the GDF15-induced increase in the IK amplitude after incubation for 24 h, and it actually augmented the levels of total cellular Kv2.1 protein, similar to GDF15  is responsible for degrading most of the intracellular soluble proteins, and the lysosomes degrade most membrane and endocytosed proteins. Consequently, we explored whether these two pathways assisted GDF15 in regulating the to GDF15 B–5D. Com2448 (p-mTOR) began to increase at 30 min (146.4±5.8%) and then continued to increase to 228.7±3.6% by 24 h. For Akt phosphorylated at Ser473 (p-Akt), its phosphorylation level significantly changed by 26.9±1.6% after 15 min, and phosphorylation reached its peak after 3 h (n=4)  or from 36.4% to −17.6%  respectively. Similarly, LY294002 and rapamycin decreased the expression levels of the Kv2.1 α-subunit protein (n=4) and −47.3±2.2% (n=4) respectively. These data indicate that the mTOR pathway is also required for the GDF15-stimulated up-regulation of the IK density and increase in Kv2.1 α-subunit expression.Because our data indicated that GDF15 increased Kv2.1 protein synthesis and prevented its degradation, we first tried to determine whether the Akt/mTOR pathway, which has been reported to affect protein synthesis and CGN mutation ,23, was  h (n=4) A–6C. The reduced D from 37 protein E to 0.9±n=3) respectively at 60 min  pathway. After GDF15 treatment, the phosphorylation levels of ERK1 and ERK2 (p-ERK1 and p-ERK2) were significantly increased, reaching the maximal levels of 225.7% and 235.9%  receptor and p-CREB (cAMP-response-element-binding protein), two typical markers of CGN development, were also enhanced by GDF15 treatment (results not shown). Taken together, our observations suggest that the effect of GDF15 on Kv2.1 expression may be developmentally regulated and associated with neuronal maturation. Because multiple factors are associated with the developmental regulation of CGNs, further investigations are needed to confirm this speculation.The s system . Previoumulation ,15. Indee period . We obseto 7 DIC D and 3A,DIC CGNs D and 3A.IA densities, which is also associated with the development and maturation of CGNs [IK  activity [IK densities and Kv2.1 expression was similar to that caused by TGFβ1, the mechanism and biological significance seem quite different. We observed that the IK densities and Kv2.1 expression induced by TGFβ1 are sensitive to the narrow time-window of 7–8 DIC, in which the IK properties are close to those of mature CGNs [IK density and Kv2.1 expression only occur at 4–5 DIC (IK density, and this activity was associated with the cAMP/PKA pathway rather than with the Akt/mTOR pathway. These differences between TGFβ1 and GDF15 in the temporally co-ordinated manner and post-receptor signalling of Kv2.1 expression might be related to the cell surface expression profiles of both TGFβR1 and TGFβR2 or to distinct oligomeric interactions that occur during signalling. These observations are in accordance with the hypothesis postulated by Ehrlich et al. [Our previous study reported that TGFβ1 increases the activity . Howeverure CGNs , but the 4–5 DIC , when thh et al. ,52, who IK densities by increasing the expression of Kv2.1 in CGNs. The effect of GDF15 on Kv2.1 expression might be channelled through TGFβR2 to activate the Akt/mTOR pathway. Thus the GDF15–TGFβR2–Kv2.1–IK axis may play an important role in neuronal development and the maturation of CGNs. However, due to technical limitations, certain interesting questions, such as how phosphorylated TGFβR2 activates the Akt/mTOR pathway and why the time-window of GDF15 responsiveness is extremely narrow, cannot be completely explained in the present study. The present study could serve as a foundation to guide further in-depth studies of GDF15 function and mechanism.In conclusion, we showed that GDF15 potentiates the


SLCO1B1 polymorphisms are relevant in statin pharmacokinetics. Aim of this study was to investigate the genetic variability and haplotype profile of SLCO1B1 polymorphisms in Roma and Hungarian populations. Genotypes of 470 Roma and 442 Hungarian subjects for c.388A > G, c.521T > C and c.1498-1331T > C polymorphisms were determined by PCR-RFLP assay. Using these SNPs eight different haplotypes could be differentiated.p < 0.001). The most common SLCO1B1 haplotype was the ht8 (GTT) both in Roma (43.6 %) and in Hungarian (59.1 %) samples. The ht6 (GCT) was not present in Roma population samples Haplotype analyses showed striking differences between the Roma and Hungarian samples in ht4 , ht5  and ht8  haplotypes (p < 0.01), respectively. Linkage disequilibrium analysis showed that the studied variants are in different linkage disequilibrium patterns depending on the ethnic origin.Differences were found between Roma and Hungarians in SLCO1B1 388AA (24.5 vs. 45.5 %), GG (33.4 vs. 17.9 %) genotypes, AG + GG (75.5 vs. 54.5 %) carriers, in G allele frequency (0.545 vs. 0.362), respectively . The minor allele frequency of 521T > C and 1498-1331T > C SNPs are almost three times higher in Romas than in Indians . Observed allele frequency for 1498-1331T > C polymorphism reflects the measured average European rates in Hungarians. The results can be applied in population specific treatment algorithms when developing effective programs for statin therapy. SLCO1B1)  gene encodes for a membrane-bound sodium-independent organic anion transporter protein (OATP1B1) that is involved in active cellular influx of endogenous substrates (e.g. bile acids), xenobiotics and a wide panel of pharmaceutical compounds . OATP1B1 protein mediates active intracellular hepatic transport of anionic compounds, important in drug hepatic clearance and in general drug disposition [Solute carrier organic anion transporter family member 1B1  and encodes a 691 amino acid protein with 12 transmembrane helices [ helices , 4. OATPSLCO1B1 variants were recently associated with simvastatin-induced myopathies [SLCO1B1 gene spans fifteen exons and 190 common variants with minor allele frequency greater than 5 % (http://hapmap.ncbi.nlm.nih.gov/). Of these, two common non-synonymous SLCO1B1 variants have been well characterized: rs2306283  and rs4149056  [OATP1B1-dependent transport is an important step in mediating drug hepatic clearance. Statins (HMG-CoA reductase inhibitors) are widely prescribed for cardiovascular disease (CVD) risk reduction . OATP1B1opathies , implyin, V174A) . Recent , V174A) . The ass, V174A) . HoweverSLCO1B1 locus may have a sizable impact on pharmaceutical. Consequently, importance of SLCO1B1 genetic variants in clinical pharmacogenetics is underlined by the fact that there are some drugs (e.g. simvastatin) on the basis of the US Food and Drug Administration recommendations have already incorporated information regarding pharmacogenetic information about the SLCO1B1 in their drug label (www.fda.gov). Extensive literature and FDA warning labels indicate increased risk for myopathy in patients with specific genetic differences on the SLCO1B1 gene. Guidelines regarding the use of pharmacogenomic tests in dosing for simvastatin have been published in Clinical Pharmacology and Therapeutics by the Clinical Pharmacogenetics Implementation Consortium (CPIC) [Several studies indicate the wide substrate selectivity of OATP1B1 and demonstrate that sequence variation at the m (CPIC) .SLCO1B1 pharmacogene in drug dosing of statins, it is noteworthy to evaluate the SLCO1B1 genotype not only of single patients, but also the general frequency of the polymorphisms in the whole populations of different ethnic background. Beside the main population of Hungarians the Roma (Gypsy/Romani) minority forms the largest ethnic group in Hungary with specific genetic background [Regarding the high clinical importance of ckground , 16. Preckground , 18. Theckground .SLCO1B1 allelic variants in the constantly growing and world-wide spread populations of Roma people. The objective of this study was to determine the frequencies and haplotypes of SLCO1B1 variant alleles in Roma and Hungarian populations, to compare our data to other populations, mainly to Indians and Caucasians studied. Population based screening can be used for risk stratification in prevention programs at a population level.Accordingly, the genetic composition of Roma and Hungarian populations differ from the European Caucasians. Previous studies of several pharmacogenetically relevant genes already revealed differences in genetic structure of Roma and Hungarian populations –22. UntiSLCO1B1 A388G, T521C and T89595C polymorphisms in Roma and Hungarian populations are shown in Tables The allele and genotype frequencies of the SLCO1B1 388 AA (24.5 vs. 45.5 %), GG (33.4 vs. 17.9 %) genotypes, AG + GG (75.5 vs. 54.5 %) carriers and in G allele frequency (0.545 vs. 0.362) (p < 0.001), respectively. Furthermore, the frequency of SLCO1B1 521 TT (67.0 vs. 65.2 %) was significantly higher in Roma than in Hungarian population samples (p = 0.05). No significant difference was observed in SLCO1B1 T521C and the rs4363657 genotypes and minor allele frequencies between the Roma and Hungarian population samples.Significant differences were found comparing the Roma and Hungarian populations in the SLCO1B1 haplotype frequencies of Roma and Hungarians are summarized in Table SLCO1B1 haplotype was the ht8 (GTT) both in Roma (43.6 %) and in Hungarian (59.1 %) samples. The ht6 (GCT) was not present in Roma population samples. Haplotype analyses showed striking differences between the Roma and Hungarian samples in ht4 , ht5  and ht8  haplotypes (p < 0.01), respectively.The major haplotypes (ht) created by the examined SLCO1B1 variants are shown in Table Linkage disequilibrium analysis of SLCO1B1 rs2306283, rs4149056 and rs4363657 variant alleles showed that the studied alleles are in different linkage disequilibrium patterns according to the different ethnic origin of Roma and Hungarian populations  is the minor allele in Hungarians, however in Roma is the 388A was found to be the minor allele, like in Indians from Singapore, however, not in indigenous Indians from North of Indian subcontinent (Table Similarly to other Caucasian populations the SLCO1B1 rs4363657 noncoding polymorphism in Roma and Hungarian populations compared with data from the HapMap project are shown in Table http://hapmap.ncbi.nlm.nih.gov/). Interestingly, the 89595C allele frequency measured in Roma samples is almost three-fold higher than in Indians (Gujarati) and nearly similar to data was found in Africans. When comparing to other non-HapMap data, the C allele frequency of rs4363657 SNP in Hungarian (19.6 %) and Roma (18.5 %) populations found in our study was similar to that reported for Caucasian subjects, 15.4 % [The allele and genotype frequencies of , 15.4 %  and 13.0, 15.4 % , 27. TheSLCO1B1 rs2306283 SNP, Melo et al. found significant differences in allele frequencies when compared European Portugal population with the data of HapMap CEU population [SLCO1B1 and risk of myalgia/myopathy [Interestingly, regarding myopathy .SLCO1B1 T521C SNP (rs4149056) is common in non-African populations: Caucasian 8–20 %, Chinese 16 %, Japanese 10–16 % (Table SLCO1B1 polymorphisms found in the Hungarian population were similar to that observed in other Caucasian populations. The 521C variant representing SLCO1B1 haplotype (*15) is associated with rifampin-induced liver injury [SLCO1B1 markedly increases systemic exposure to simvastatin and the risk of muscle toxicity. The strength of the evidence is high for myopathy with simvastatin [The r injury . This noC SNP rs49056 is cSLCO1B1 haplotype was ht8 both in Roma and Hungarian populations. In both examined populations apart from ht8, the most frequent haplotype was ht4 (containing neither variant) followed by ht1 (including 521C and 89595C intronic variants). The ht6 was present in Hungarians with a low frequency (0.18), but was not detectable in Roma samples. Ht2, representing the T521C variant, is associated with decreased transporter activity that may potentially be associated with increased statin efficacy.The most common SLCO1B1 gene shows population specificity; therefore the examination of clinically relevant polymorphisms is important in different populations. The phenotype effect of the SLCO1B1 haplotypes on the transporter activity need to be investigated in future clinical studies of these populations, as well as the effect on statin treatment. Comparing the LD patterns of the two studied populations we can conclude, Roma samples showed higher degree of linkage disequilibrium of studied SNPs than Hungarians. As both of the studied populations present high degree of LD patterns, haplotypes analyses of SLCO1B1 gene exerts less advantage, especially because only the rs4149056 polymorphism have been described as clinically functional.If we consider, these results suggest that the pharmacogenetical pattern of http://bbmri-eric.eu/). The governance principles and maintenance management of the Biobank had been approved by the Hungarian National Research Ethics Committee . During the collection and analysis of DNA samples and processing of the accompanying personal data the guidelines and regulations of the Helsinki Declaration in 1975 and the currently operative National regulations were followed. DNA of total of 470 healthy Roma samples  and 442 healthy Hungarian  were used in the study. Informed consent was obtained from all subjects.The DNA samples were from the central Biobank of the University of Pecs that is part of the National Biobank Network of Hungary, as well as the pan-European Biobanking and Biomolecular Resources Research Infrastructure (BBMRI)  gene including the rs2306283 , the rs4149056  and rs4363657  were analyzed. For primer design the sequences deposited into the GenBank (http://www.ncbi.nlm.nih.gov/genbank/) were used.Genomic DNA was isolated from peripheral leukocytes using routine salting out method, and was store with the patient’s informed consent. Three polymorphisms of the GC -3’ reverse. And the rs4363657 was detected with the help of the following primers: 5’-CAGTTTGCTAGTGTTTTGTTGAGG-3’ forward, 5’-ACCATCCAAGACGAACAAAGAG -3’ reverse. Underlining in the sequence indicates mismatch base introduced to generate artificial cleavage site. PCR amplification was carried out in a final volume of 50 μl on an MJ Research PTC 200 thermal cycler. PCR conditions were as follows: predenaturation for 2 min at 96 °C, followed by 30 cycles of denaturation for 30 s at 95 °C, annealing for 1 min by primer specific temperature, primer extension for 30 s at 72 °C, the final extension at 72 °C for 5 min. Annealing temperature for the A388G, T521C and T89595C primers were 57, 52 and 55 °C, respectively.Genotyping was carried out using polymerase chain reaction (PCR) followed by restriction endonuclease digestion (RFLP). For detection of the rs2306283 the following primers were used: 5’-CTGTGTTGTTAATGGGCGAA-3’ and 5’-GGGGAAGATAATGGTGCAAA-3’. The rs4149056 SNP was detected using the following set of primers: 5'- TTGTCAAAGTTTGCAAAGTG -3’ forward and 5’- GAAGCATATTACCCATGATen μl PCR products of the A388G, T521C and T89595C primers was digested by Taq1, Hin6I and KpnI restriction enzymes, respectively. Supplier of the restriction endonucleases is the Thermo Scientific, the incubation temperature was 65, 37 and 37 °C, respectively. The digested PCR products were separated by electrophoresis using a 3 % agarose gel stained with ethidium bromide and visualized by an UV transilluminator. In the amplicons of the A388G polymorphism was an obligatory cleavage site to enable us to monitor the efficacy of the digestion. In the samples with 388 AA genotype the Taq1 cleaves the 406-bp long PCR product in 159-bp and 247-bp fragments. If the 388 G allele was present in homozygouse form the 23-, 136-, 247-bp fragments could be detected. The 209-bp amplicon of the T521C SNP in the samples with CC genotype was digested into 21-bp and 188-bp long fragments. While in TT homozygotes the recognition site of the enzyme was missing, resulting in no digestion. However, all the homozygote variant samples from the PCR-RFLP assay were direct sequenced with the same primers utilizing an ABI 3500 genetic analyzer. Wild-type of 89595 TT yielded two fragments of 133 bp and 236 bp in length, and the variant 89595 CC homozygotes gave three fragments of 133 bp, 84 bp and 152 bp. The heterozygotes formed four fragments of 84 bp, 133 bp, 152 bp and 236 bp. The accuracy of the genotyping was evaluated by direct sequence analysis of randomly selected samples as described above.The method used for calculation of the Hardy-Weinberg equilibrium in different SNPs was described by G. H. Hardy and W. Weinberg in 1908 .p < 0.05) was assessed by χ2 test to compare the differences between studied groups. Statistical analyses were performed applying Excel for Windows and SPSS 11.5 package for Windows .Statistical significance  patterns. We required the minor allele frequency at each locus to be >0.05, with an Rsion 2.1 , 33. TheSLCO1B1 gene (Uniprot identifier: Q9Y6L6 is available at http://www.uniprot.org/uniprot/Q9Y6L6) for c.521T > C, c.388A > G and c.1498-1331T > C polymorphisms were rs4149056, rs2306283 and rs4363657, respectively based on the dbSNP database of NCBI .The identification numbers of the examined SNPs in


However, there are some reports implying that use of nano-ZrO2 is associated with cytotoxic effects like inhibiting the cell proliferation, DNA damage and apoptosis. In the present study, we examined whether nano-ZrO2 alters cell viability and glutathione peroxidase (GPx) activity in two neuronal cell lines.Today, special attention is paid to the use of zirconium dioxide nanoparticle -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and GPx activity was determined by quantifying the rate of oxidation of the reduced glutathione to the oxidized glutathione. The PC12 and N2a cells were cultured in the absence or presence of varying concentrations (31.25-2000 µg/mL) of nano-ZrONano-ZrO2 caused a significant reduction in cell viability and GPx activity after 12, 24 and 48 h, as compared with control group. These effects were concentration dependent and started from 250 µg/mL.2, at concentrations of > 250 µg/mL, has antiproliferative effects via reducing the cell defense mechanism against oxidative stress.The present study demonstrated that nano-ZrO Neurons2) and its product hydrogen peroxide (H2O2) can even produce more reactive species like hydroxyl radical, hypochlorous acid and singlet oxygen which can damage the components of extracellular matrix , Glutathione Peroxidase (GPx) and catalase (CAT) -23. The An imbalance between the productions of free radicals and the cell ability for detoxifying these radicals is involved in molecular mechanism of neurotoxicity -28. 2 has not been investigated before, this study was designed to elucidate the effect of these nanoparticles on viability and GPx enzyme activity of N2a and PC12 cells.Since, the cytotoxic effects of nano-ZrOCell lines and reagents2 (<100 nm in average size) were purchased from Sigma-Aldrich . PC12 and N2a cell lines, derived from rat pheochromocytoma and mouse neuroblastoma, respectively, were purchased from Pasteur Institute . High glucose (4.5 g/L) Dulbecco’s Modified Eagles Medium (DMEM) and fetal calf serum were purchased from Gibco . 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was obtained from Promega . Glutathione Peroxidase (GPx) Assay kit was purchased from Northwest Life Science Specialties, LLC .Commercial nano-ZrODispersion and characterization of the zirconia nanoparticles2 was suspended in media and dispersed by an ultrasonic bath for 60 min. Then the size of nanoparticles in suspension was characterized by Transmission Electron Microscopy , using one drop of sample on a carbocoated grid and high voltage (120K.V.) imagining TEM.Nano-ZrOCell culture and nanoparticle treatment2 cell culture flasks at 37 oC in a 5% CO2 humidified incubator. The test suspension of nano-ZrO2 was prepared using the culture media and dispersed for 20 min by using a sonicator  to prevent aggregation. The cells were treated with various concentrations of nanoparticles according to the time schedule. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, penicillin 100 IU/mL, and streptomycin 100 µg/mL. They were grown and maintained in 75 cmCell viability assay4 cells were seeded in each well of a 96-microwell plate and treated with various concentrations (0-2000 µg/mL) of nanoparticle suspension for 12, 24 and 48 h. At the end of the treatment, the MTS reagent, which is composed of the novel tetrazolium compound and an electron coupling reagent phenazine ethosulfate, was added to the cell media. After 3 h, cell viability was determined by measuring the optical absorbance at 490 nm using an ELISA microplate reader . The cytotoxicity of nano-ZrO2 was calculated using Graph Pad Software (Graph Pad prism 5 software) and presented as mean ± SEM of three independent experiments with three replicates for each concentration.The cell viability assay is based on the reduction of novel tetrazolium compound (MTS) by mitochondrial dehydrogenase in metabolically active cells to the water-soluble formazan . About 1Determination of glutathione peroxidase activityGlutathione peroxidase activity was determined using a sensitive kit produced based on the method of Paglia and Valentine . In this6 cells per mL media. After allowing the cells to be seeded for 24 h, nanoparticle suspension with different concentration  were added to each well for 12, 24 and 48 h. Then, Cells were washed with PBS, scraped and homogenized in 20 mM PBS containing 0.5 mM butylated hydroxyltoluene to prevent sample further oxidation. The homogenate was centrifuged at 3,000 g for 20 min at 4Cells were seeded in 6-well tissue culture plates with 10Statistical analysisThe results are presented as the mean ± SEM. The values were compared using the one-way analysis of variance (ANOVA) followed by Tukey’s post tests for multiple comparisons. The p<0.05 was considered to be statistically significant.Characterization of nano-ZrO22 was found to be less than 100 nm as shown in the images of Transmission Electron Microscopy (The size of the nano-ZrOcroscopy . Effect of nano-ZrO2 on cell viability2 on viability of N2a cells are shown in 2 at 2000 µg/mL decreased the cell viability to 32.76 ± 2.8 (P < 0.001), which was lowest viability amount among all the concentrations. Similarly, exposure of the N2a cells to the nanoparticles for 24 and 48 h showed a concentration-dependent decrease in cell viability. Again the cytotoxic effect was observed at concentrations more than 15.6 µg/mL (P < 0.05 and P<0.001) after 24 and 48 h exposure, respectively. No significant difference in cell viability was found between the three incubation times.The effect of nano-ZrO2 on viability of PC12 cells. After 12 h, the level of viability in the cells exposed to 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL of nano-ZrO2 was 80.90 ± 5.1, 69.66 ± 7.2, 66.23 ± 7.0, 69.29 ± 7.1, 51.75 ± 6.9, 48.45 ± 7.9, and 46.46 ± 3.5 %, respectively, which at concentrations more than 31.25 µg/mL was significantly (P < 0.05) lower than that of untreated cells (100 ± 5.2 %). Approximately, the same level of viability was observed when incubation time extended to 24 h. However, 48 h incubation with nano-ZrO2 leads to more reduction of cell surviving, so that, its cytotoxicity was started at concentrations of ≥15.6 µg/mL (P < 0.05).Effect of nano-ZrO2 on glutathione peroxidase activity2 on GPx enzyme is determined with decreasing its activity comparing to the untreated group which this reduction is started from 250 µg/mL (p < 0.001) after 12 h exposure of N2a cell line  after 12 h and 24 h exposure  and 5B a2 .Considering the increased usage of zirconia nanoparticles in many aspects of biological field, more attention should be considered to their safety or toxicity. The particles can enter the body via inhalation, skin penetration, ingestion or via metallic implant corrosion -33. It h2 particles have antiproliferative effect on N2a and PC12 cell cells. These neuronal cell lines have extensively used in researches to test possible neurotoxic or neuroprotective effects of compounds  and nitric oxide by Human THP-1 Macrophages (2 with cell membrane and/or accumulation of free radicals such as HO2•, O2•, HO• and carbon-centered radicals as a result of GPx activity decrease, can initiate oxidative damage and induce cell apoptosis (The correlation between oxidative stress and decrease of cell viability has been well documented -40. The rophages . Taken tpoptosis -46.2 at concentrations more than 250 µg/mL has neurotoxic effects through increasing the oxidative stress and this should be considered for therapeutic use. In conclusion, our study showed that nano-ZrO


DNA topoisomerases play a key role in tumor proliferation. Chemotherapeutics targeting topoisomerases have been widely used in clinical oncology, but resistance and side effects, particularly cardiotoxicity, usually limit their application. Clinical data show that a decrease in topoisomerase (top) levels is the primary factor responsible for resistance, but in cells there is compensatory effect between the levels of top1 and top2α. Here, we validated cyclizing-berberine A35, which is a dual top inhibitor and preferentially targets top2α. The impact on the top2α catalytic cycle indicated that A35 could intercalate into DNA but did not interfere with DNA-top binding and top2α ATPase activity. A35 could facilitate DNA-top2α cleavage complex formation by enhancing pre-strand and post-strand cleavage and inhibiting religation, suggesting this compound can be a topoisomerase poison and had a district mechanism from other topoisomerase inhibitors. TARDIS and comet assays showed that A35 could induce cell DNA breakage and DNA-top complexes but had no effect on the cardiac toxicity inducer top2β. Silencing top1 augmented DNA break and silencing top2α decreased DNA break. Further validation in H9c2 cardiac cells showed A35 did not disturb cell proliferation and mitochondrial membrane potency. Additionally, an assay with nude mice further demonstrated A35 did not damage the heart. Our work identifies A35 as a novel skeleton compound dually inhibits topoisomerases, and predominantly and specially targets top2α by interfering with all cleavage steps and its no cardiac toxicity was verified by cardiac cells and mice heart. A35 could be a novel and effective targeting topoisomerase agent. DNA topoisomerases are essential enzymes for cells to modulate DNA topology by regulating the over- or under-winding of DNA strands during cellular processes such as DNA transcription, replication, or recombination . Top1 isGiven that they are highly expressed in aggressive cancer cells and are essential to cancer cell survival, top1 and top2α are potential drug targets for treating human malignancies. Compared with top1, top2α is more essential for cell viability because only top2α can drive the separation of two DNA duplexes after replication and its deletion is lethal for cell survival –8. ChemoBerberine  and repetitive cytotoxicity assays, cyclizing-berberine A35 (replacement of 9-methoxyl with an ester moiety) Figure , right eIn the present study, we evaluated the novel skeleton compound A35 in a cell-free assay, a cell assay and in further animal experiments and demonstrated that A35 could target top1 and particularly top2α by increasing pre-strand and post-strand cleavage and inhibiting the religation. Additionally, we also illustrated that A35 could specifically target top2α and not top2β; meanwhile, we confirmed that A35 did not induce cardiac cell injury in rat cardiomyocyte cells and nude mouse hearts.Given that A35 possesses a greater number of aromatic rings and its structure is similar to known topoisomerase2 inhibitors , 23, we Given that top2α is indispensable for cell division and was strongly inhibited by A35, in the following experiment we focused on investigating the effects of A35 on top2α. There are several steps in the top2α catalytic cycle. As a consequence, multiple independent approaches are required to determine the mechanisms underlying the drug-mediated inhibition of top2α. For A35, its intercalation into DNA is attributed to its polar structure, and this intercalation might lead to the topoisomerase DNA binding site being occupied or distortion of the DNA backbone; this can then interfere with top2-DNA binding. The ability of A35 to intercalate DNA was assessed using a top1 unwinding assay as described , pBR322DGiven that A35 could intercalate into DNA, we utilized an EMSA assay , 32 to mThe effect of A35 on top2α ATP enzyme activity was examined by thin layer chromatography with [γ-32P]-ATP and results showed that novobiocin (a known top2α ATP enzyme inhibitor)  significThen we evaluated the effect of A35 on the DNA cleavage. A cleavage assay in the presence of 8 U top2α, 0.2 μg pBR322 and increasing concentrations of A35 was performed, and the results showed that cleaved linear DNA was formed and the cleaved band presented in a dose-dependent manner Figure . TogetheUsually pre-strand cleavage and post-strand cleavage participated in the top2α-mediated DNA cleavage process . Interfe2+, incubated with increasing concentrations of A35 led to cleaved DNA increased  in the reaction buffer . The addd Figure , and ind2+. After using EDTA to chelate the excess Ca2+ ions, Mg2+ reintroduction triggered the religation of the linear band -dGTP in reaction 2 buffer  with 0.5 units of DNA polymerase I . Unincorporated 32P-dGTP was removed using mini Quick Spin DNA columns (Roche), and the eluate containing the 3′-end-labeled DNA substrate was collected. Approximately 3 nM of radiolabeled DNA substrate was incubated with recombinant top1 (60U) in 20 μl of reaction buffer  at 25°C for 30 min in the presence of various drug concentrations. The reactions were terminated by adding SDS  followed by the addition of two volumes of loading dye . Aliquots of each reaction mixture were subjected to 16% denaturing PAGE. Gels were dried and visualized by using a phosphoimager.DNA cleavage assays were performed as described previously –59 A 117To examine the effects of A35 on DNA intercalation, a top1-based assay was carried out according to the literature . In brieThe effect of A35 on the binding of top2α to DNA was evaluated using an electrophoretic mobility shift (EMSA) kit (Invitrogen) as previously described  accordin32P]-ATP by thin layer chromatography as previously described [32P]-ATP  and continued to incubate at 37°C. Aliquots (2 μl) were withdrawn at various time points  and loaded onto pre-washed polyethyleneimine-impregnated cellulose plates (Sigma-Aldrich) and air-dried. Reaction mixtures were resolved by developing plates with freshly prepared NH4HCO3 (0.4 M). Plates were air-dried and exposed to autoradiographic film. Spots corresponding to free phosphate were excised from the thin layer chromatography plates and quantified using a scintillation counter.ATPase activity of top2α was examined by measuring the liberated phosphate of [γ-escribed , 61. BriDNA cleavage assay was performed by using a Top2α Drug Screening Kit (Topogen), 0.2 μg pBR322 DNA plasmid was incubated with top2α or top2β which was obtained and purified as described previously  in 20 μl2 and 0.03 μg/μl bovine serum albumin. Reactions were incubated for 20 min at 37°C and terminated by addition of 2 μl of 10% SDS, followed by addition of 1.5 μl of 0.25M EDTA and continued to culture for 5 min at 37°C. Then, reactions were digested with 8 μg of proteinase K and incubated for 60 min at 56°C. DNA products were separated on a 1.4% gel containing 0.7 μg/μl ethidium bromide.Top2α-mediated plasmid DNA cleavage in the absence of nucleotide triphosphate was performed as described with slight modifications , 63. TopA top2α-mediated post-strand passage cleavage assay was performed as described above for pre-strand passage cleavage with the exception that 1 mM AMPPNP (Sigma) was added in the reactions .2 and 0.03 μg/μl of bovine serum albumin for 10 min at 37°C. Immediately after, various concentrations A35 or control were added, followed by the addition of 2 μl of 0.1 M EDTA to the reactions. Then, the reactions were re-initiated by the addition of 0.1 M MgCl2 (2 μl) and transferred immediately onto ice. At the indicated time points , reactions were terminated by the addition of 1% w/v SDS and incubation continued at 37°C for 5 min. The reactions were then incubated with proteinase K (8 μg) for 30 min at 56°C. DNA products were separated on an agarose gel (1.4% w/v) containing ethidium bromide (0.5 μg/μl).Top2α-mediated religation of DNA in the absence of ATP was performed as described previously . Top2α  for DSB detection or an alkaline comet assay for single-strand break detection as described in the manufacturer's procedures and the literature . The treThe TARDIS assay is used to determine cleavable complex formation and has been described in detail . BrieflyTopoisomerase knockdown experiments were carried out in the same manner as described previously . Small iWhole-cell lysates were used for immunoblotting as described previously .5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocar bocyanine iodide (JC-1) dye (sigma) exhibits potential-dependent accumulation in the mitochondria, and JC-1 selectively enters the mitochondria and spontaneously forms complexes known as J-aggregates. If the membrane potential is disturbed, the dye remains in the monomeric form, emitting only green fluorescence. Thus, this dye was employed to detect changes in the mitochondrial membrane potential (ΔΨm). JC-1 dye was added to the culture medium at 10 μg/ml and incubated for 15 min at 37°C. After mounting on the slides, the cells were immediately examined under a fluorescence microscope (Olympus).in vivo efficacy of A35 was evaluated with HepG2 xenografts in nude mouse . First, 1 × 107 HepG2 cells suspended in 200 μl of PBS were inoculated s.c. in the right armpits of nude mice. After 3 weeks, the tumors were removed from the nude mice and dissected aseptically in sterile saline. Pieces of tumor tissue (2 mm3 in size) were then transplanted into the right armpits of nude mice with a trocar. Tumor-bearing mice were randomly divided into 3 groups (n = 5) when the tumor size was about 100 mm3. A35 (10 or 20 mg/kg) was administered by intraperitoneal injection once a day until the mice were sacrificed. Tumor size was measured every 3 days, and tumor volume was determined as length × width2/2. Mice were killed when the tumor volumes of the control group reached 1000 mm3; the tumor loads and hearts were isolated and used in further assays.The Hearts from A35-administered mice were excised and preserved in liquid nitrogen until frozen heart sections were produced. In the cardiotoxicity positive control group, DOX was administered by intraperitoneal injection at 0.5 mg/kg once every two days. H&E staining was performed as described in . Slides


Myocardial perfusion cardiovascular magnetic resonance (CMR) with vasodilator stress has high diagnostic accuracy for detecting coronary artery disease (CAD). Current CMR perfusion pulse sequences use largely fixed acquisition parameters designed to acquire at least three slices every heart beat, optimized for the heart rates that typically occur during pharmacological stress. In patients with lower heart rates there can be a significant amount of unused potential imaging time Figure . In thosA new perfusion method which automatically adapts the acquisition duration to maximise spatial resolution whist maintaining 3 slice imaging at every heart beat was developed Figure . Ten heaAdaptive perfusion CMR was feasible in all subjects. Mean stress heart rate (HR) was 89 ± 11 in the fixed resolution group and 90 ± 18 in the adaptive resolution group. There was no statistical difference in the haemodynamic data between the two groups. The standard perfusion sequence acquired in-plane resolution was 2.42 × 2.42 mm and the mean HR adaptive sequence resolution was 1.91 × 1.91 mm ± 0.41 (range 1.53-2.89)(p = 0.001). In two cases the stress HR was too high for alternate R-R interval imaging with the fixed resolution sequence resulting in alternate heart beat imaging. This did not occur with the adaptive sequence which adjusted the resolution was adapted (to 2.84 × 2.84 and 2.89 × 2.89 mm respectively). The mean DRA width was 3.0 ± 0.6 mm (95% CI: 2.57-3.51) with the standard perfusion sequence and 2.1 ± 0.6 mm (95% CI: 1.65-2.57) with the adaptive sequence (p < 0.001)[Figure Optimising the use of available imaging time during CMR myocardial perfusion imaging with heart rate adaptive shot acquisition duration is feasible and improves the acquired resolution and reduces dark rim artifact whilst maintaining image quality. The effect on diagnostic performance of perfusion CMR should be investigated.SP is funded by a British Heart Foundation fellowship (Fs/10/62/28409) SP and JPG received an educational research grant from Philips Healthcare.


Previous reports have shown that denosumab treatment of osteoporotic patients decreases bone resorption and fracture risk, but there have been no clinical studies on changes in bone turnover markers, 1,25(OH)Here, we report such findings in 22 patients  with osteoporosis following 4 months of treatment. Bone metabolism had been inhibited by prior BP administration in the BP pre-treated group.2D3 and PTH were significantly increased at 1 week and decreased gradually thereafter, but these did not change notably in the BP pre-treated group.The bone resorption markers serum tartrate-resistant acid phosphatase type 5b and urinary type I collagen cross-linked N-telopeptide were significantly decreased from baseline values for the entire study period in both groups. The bone formation marker bone alkaline phosphatase was significantly decreased at 4 months in the denosumab alone group only, and N-terminal propeptide of type 1 procollagen was significantly decreased at 2 and 4 months in the denosumab alone group versus no remarkable change in the BP pre-treated group. In the denosumab alone group, 1,25(OH)2D3 and PTH were significantly increased by denosumab but these did not change in the BP pre-treated group.Our results suggest that treatment with denosumab causes a strong inhibitory effect on bone resorption markers and mild inhibitory effect on bone formation markers. 1,25(OH)NCT02156960. Registered 31 May 2014.Current Controlled Trials  Osteoporosis is a major health concern, especially in elderly women, that carries an increased incidence of bone fracture and ensuing morbidity. Thus, the prevention of fractures is the primary therapeutic goal for this condition .Recent treatments for osteoporosis have been based on our current understanding of bone biology. Receptor activator of nuclear factor-kB ligand (RANKL) is a cytokine that is essential for osteoclast differentiation, activation, and survival . DenosumIn one report, denosumab therapy resulted in a significant, early, and sustained increase in bone mineral density (BMD) and enhanced bone strength in the improvement of both cortical and trabecular bones . Another2D3. The status of vitamin D in the body is typically evaluated using the stored form of serum 25(OH)D 2D3 is tightly regulated by parathyroid hormone (PTH), fibroblast growth factor (FGF)-23, and other hormones, as well as by cytokines 2D3 and PTH during osteoporosis treatment with denosumab.The major form of vitamin D in the serum is 25(OH)D, which is its principal storage conformation whose concentration is approximately 1000 times higher than that of 1,25(OH)ytokines . As it iWhile both are anti-resorptive drugs, BP and denosumab have different action mechanisms in osteoporosis. BP functions after bone deposition and has a long half-life and duration of anti-absorptive effects. Therefore, bone metabolism may be affected by BP pre-treatment, although there have been no reports on whether prior treatment with BP affects serum PTH or vitamin D levels during treatment with denosumab.2D3, and PTH in Japanese osteoporotic patients.In this study, we examined the clinical results of 4 months of denosumab treatment with or without BP pre-treatment on bone turnover markers, serum Ca, 1,25(OH)Twenty-two patients with osteoporosis (16 women and 6 men) were recruited for this study at our institutions between July 2013 and April 2014. The cohort’s average age was 74.6 years. The patients were divided into 2 groups of 11 patients each for the denosumab alone  age: 76.3 ± 7.0 years in women and 69.8 ± 9.2 years in men) and BP pre-treated  groups. All patients were diagnosed as having primary osteoporosis. Patients in the denosumab alone group had no history of medication that may have affected bone or calcium (Ca) metabolism, while those in the BP pre-treated group had been taking oral BP for at least 6 months prior to this study. The diagnosis of primary osteoporosis was made in accordance with the revised criteria established by the Japanese Society of Bone and Mineral Research . We gave2D3 (20.0–60.0 pg/mL) were measured by immunoradiometric assays. Each marker was measured just prior to denosumab administration and at 1 week, 1, 2, and 4 months of denosumab treatment. After overnight fasting, serum and first void urine samples were collected between 8:30 a.m. and 10:00 a.m. Immunoassays were performed by SRL, Inc. .Serum Ca was corrected with serum albumin (the reference range 8.5–10.2 mg/dL). Serum bone alkaline phosphatase (BAP)  and N-terminal propeptide of type 1 procollagen (P1NP)  were measured as bone formation markers using a chemiluminescent enzyme immunoassay and an antibody radioimmunoassay, respectively. Serum tartrate-resistant acid phosphatase (TRACP)-5b (the reference range in women 120–420 mU/dL) and urine N-terminal telopeptide of type I collagen (NTX)   were measured as markers of bone resorption using the enzyme-linked immunosorbent assay (ELISA). Serum whole PTH (9–39 pg/mL) and 1,25(OH)Bone mineral density (BMD) was measured using a Dual-energy X-ray Absorption (DXA) fan-beam bone densitometer  at the L1-4 levels of the posteroanterior spine and bilateral hips.t-test. P-values of < 0.05 were considered to be statistically significant. Statistical analyses were performed using the statistical package R, version 3.0.1 .In both groups, we compared the changes in each marker at each time point  using linear mixed models and Holm’s correction method for multiple comparisons. Each marker value was individually adopted as a response variable: the timing of the measurement was used as a fixed effect, while the individuality of the measurement was adopted as a random effect. Comparisons between the markers of both groups at each measuring point were performed using Welch’s The authors received no hospitality, honoraria, or travel expenses via the companies that market denosumab. This study was approved by the institutional ethical review board at Shinshu University School of Medicine and Show Inan General Hospital prior to its start and written informed consent was obtained from all subjects.Serum Ca level did not change significantly in either group. The changes in adjusted Ca were within the reference range. However, whereas Ca was decreased at 1 week and 1 month before returning to baseline levels at 2 months in the denosumab alone group, it was increased in the BP pre-treatment group at 1 week and 1 month Fig. .Fig. 1LoSerum TRACP-5b and urinary NTX were significantly lower in the BP pre-treated group before denosumab administration. However, the resorption markers were nonetheless significantly inhibited by denosumab at each time point in both groups Fig.  and b. ISimilarly to bone resorption markers, in the BP pre-treated group, BAP and P1NP were at notably lower values compared with the denosumab alone group at treatment onset. In the denosumab alone group, BAP rose slightly at 1 month but then gradually decreased until 4 months after initiation of treatment to a significant degree. P1NP was markedly decreased at 1 week and became significantly lower at 2 and 4 months Fig. . In the 2D3 was significantly increased from as early as 1 week to 2 months, peaked at 1 week after denosumab administration, and then gradually decreased. Similarly, whole PTH was significantly increased at 1 week and slowly decreased thereafter ter Fig.  and c.2D3 level was increased slightly up to 4 months. Whole PTH level did not increase, and in fact decreased slightly, but not significantly, during the observation period iod Fig.  and c.2D3 or whole PTH between groups. However, values of 1,25(OH)2D3 at 1 week to 4 months and whole PTH at 1 week and 1 month became significantly higher in the denosumab alone group than in BP pre-treated group oup Fig.  and c.2D3 and PTH at 1 week, followed by a gradual decrease, and 3) mild inhibitory effects on bone formation markers during the observation period. On the other hand, in the BP pre-treated group, denosumab administration resulted in: 1) further significant inhibition of urinary NTX and serum TRACP-5b, 2) a slight decrease in BAP and P1NP, 3) a slight increase in Ca at 1 week and 1 month, and 4) no marked changes in 1,25(OH)2D3 or PTH.In the present study, denosumab administration in the denosumab alone group caused: 1) strong inhibitory effects on bone resorption from as early as 1 week, 2) a slight decrease in Ca at 1 week and 1 month and a significant increase in 1,25(OH)We noted that the bone resorption markers serum TRACP-5b and urinary NTX were significantly lower in the BP pre-treatment group at study onset, which indicated that prior therapy with BP had effectively modulated bone resorption. However, these markers decreased significantly from as early as 1 week and leveled off at 1 week for urinary NTX and 1 month for TRACP-5b. Hence, denosumab has strong inhibitory effects on bone resorption at an early stage after therapy commencement, regardless of BP pre-treatment.With respect to bone formation markers, both BAP and P1NP also showed lower, albeit insignificant, values in the BP pre-treated group. In the denosumab alone group, BAP decreased steadily and P1NP decreased slowly after an initial marked drop. At 4 months, both values had decreased to comparable levels in both groups. However, P1NP at 4 months remained significantly higher in the denosmub alone group. In the BP pre-treated group, the values of serum BP and P1NP decreased slightly.Generally, bone resorption and bone formation change in parallel due to the phenomenon of coupling . However2D3 increases intestinal Ca absorption and resorption of Ca from bone, and therefore plays a prominent role in Ca regulation along with PTH [2D3 [2D3 that must be synthesized de novo in response to PTH [2D3 is comparatively short, the regulation of Ca, PTH, and 1,25(OH)2D3 levels is usually strictly regulated in the body.Ca status is strictly regulated by intestinal Ca absorption, bone resorption, and renal re-absorption. 1,25(OH)with PTH , 14. PTH2D3 and PTH levels transiently increased after alendonate administration by a yet unknown mechanism [2D3 and PTH caused by BP therapy were decreased Ca. Furthermore, increased 1-25(OH)2D3 caused: 1) PTH receptor increase [2D3 levels [2D3 and PTH also significantly increased after denosumab treatment. It is conceivable that a similar mechanism is involved by which denosumab strongly inhibits bone resorption, resulting in immediate and significant 1,25(OH)2D3 and PTH increases.Shiraki et al. have reported that serum 1,25(OH)echanism . We specincrease , 2) acce3 levels . In our 2D3 did not increase and PTH tended to decrease. However, the mechanisms for such phenomena remain unknown.The most important finding in this study was that in the BP pre-treated group, regardless of further inhibiton of bone resorptive markers by denosumab therapy, 1,25(OH)The limitations of this study are 1) a small sample size, 2) short follow-up period, and 3) only a tendency of serum Ca changes may have been demonstrated due to the small cohort.2D3 and PTH were temporarily increased by denosumab treatment in the denosumab alone group. On the other hand, the values of these parameters did not change further as bone absorptive markers became significantly inhibited in the BP pre-treated group.In conclusion, denosumab has a strong inhibitory effect on bone resorption markers, although its inhibitory effects on bone formation markers are weak. Levels of 1,25(OH)


At least four sdw1/denso alleles have been reported and HvGA20ox2 is considered as the candidate gene. Up to date, results of studies have not univocally proven the genetic relationship between sdw1/denso and HvGA20ox2.HvGA20ox2 and Mloc_56463 genes was identified at the sdw1 locus from a semi-dwarf mutant Riso no. 9265. Expression of the genes encoding gibberellin biosynthesis (HvGA20ox1 and HvGA3ox2) were increased in the mutant compared to the wild type Bomi, while the expression of GA catabolic gene HvGA2ox3 was decreased. Over-expression of HvGA20ox2 could rescue the semi-dwarf phenotype and increase GAs concentration.In the present study, a complete deletion of Morex_contig_40861 including both 2, acted as GA 20-oxidase, is the functional gene for the sdw1/denso semi-dwarfism. Lose of HvGA20ox2 is partially compensated by HvGA20ox1 and further feedback is regulated by gibberellin. We also deduced that the sdw1/denso allele itself affects later heading owing to its reduced endogenous GAs concentration.We confirmed that a GA biosynthetic enzyme HvGA20oxThe online version of this article (doi:10.1186/s12864-015-2116-x) contains supplementary material, which is available to authorized users. Plant height is one of the most important agronomic traits in cereal crops that not only determines plant architecture, but also is closely associated with grain yield. A reduction in plant height usually leads to strong straw and high resistance to lodging, as happened in semi-dwarf rice and wheat cultivars. However, reduced plant height means lowering of canopy, which favors the epidemic spread of fungal diseases, resulting in an undesired increase of fungicide use, and a reduction of yield potential mainly due to smaller grain weight . For optsd1 in rice is one of the most famous genes and has been widely used to produce semi-dwarf phenotype in both japonica and indica rice. It was reported that sd1 encoded one of the GA 20-oxidases and was involved in the last steps of gibberellin biosynthesis [sdw1/denso, which was postulated as homologous to ga5 in Arabidopsis and sd1 in rice [sdw1/denso gene is a 10-20 cm reduction of plant height, depending on environmental conditions [sdw1/denso gene, such as late heading and maturity, decreased thousand-grain weight and high screening [sdw1/denso, both positive and negative were reported depending on different genetic backgrounds [sdw1/denso has been introduced to numerous cultivars. For example, newly released more than 150 European cultivars carried the denso allele [The Green Revolution genes have been identified and isolated in the semi-dwarf varieties by reversed genetics. Among them, ynthesis –6. Semi-ynthesis . One of  in rice , 9. The creening . As for kgrounds , 15. Howo allele .denso in 1946, as named accordingly [denso and sdw1, respectively. Interestingly, the denso mutant has been used to develop malting barley, while the sdw1 has been limited to feed barley [sdw1 allele was found in a M2 generation from the variety Bomi, treated by neutrons in the Stockholm reactor and named as Riso no. 9265. Although these mutants are characterized by semi-dwarf phenotype, all of them are sensitive to GA3 [There are at least four independent alleles based on the allelism test done up to date , 17. Oned barley , 12. Thee to GA3 .sdw1/denso was first mapped on the 3HL as a phenotypic trait [GA20ox2 homolog was identified using PCR method with the primers designed from the conserved domain of rice sd1 and considered as a candidate gene of sdw1/denso [HvGA20ox2 in denso mutant was reduced by 4-fold, but almost 60-fold in the sdw1 mutant, compared to the control [HvGA20ox2 as the functional gene. In addition, the function of HvGA20ox2 was predicted based on its functional domain, being highly similar with those of sd1 (OsGA20ox2) and ga5 (AtGA20ox1). No evidence showed its function in vitro or in vivo. In the present work we investigated the sdw1 allele of Riso no. 9265 and identified a complete deletion contig, which includes two genes Mloc_56462 (HvGA20ox2) and Mloc_56463 (a putative methyltransferase PMT26). The function of the HvGA20ox2 gene was further analyzed using genetically-transformed Arabidopsis. Furthermore, real-time PCR assays revealed that transcript level of GA synthesis-related genes were significantly different between Bomi and Riso no. 9265. The current results showed that the semi-dwarf phenotype of Riso no. 9265 is attributed to the deletion of HvGA20ox2.In 1993, w1/denso . Meanwhi control . HoweverAt maturity, plant height and length of spike, culm and internodes of Bomi and its semi-dwarf mutant Riso no. 9265 were measured Table . Plant hHvGA20ox2 (Mloc_56462) was identified after blast against Gramene (http://www.gramene.org/) using the sequences of HvGA20ox2 from our previous study [HvGA20ox2 were designed to amplify its genomic sequence. In Bomi, both primer pairs produced single band with different sizes. One is 2974 bp and the other is 3165 bp. However, no PCR product was obtained in Riso no. 9265 . One Morex_contig_40861, covering 21596 bp was identified, and it contains HvGA20ox2 and Mloc_56463 genes. Several primers covering Morex_contig_40861 were designed  and a semi-dwarf mutant ga5-3. T1 transgenic plants were selected by BASTA analysis and PCR method. We obtained eleven and six transgenic lines in Col-gl1 and ga5-3 background, respectively. All of them displayed a higher growth rate. Homozygous T3 plants of transgenic Col-gl1 lines (OE-1 and OE-2) and transgenic ga5-3 lines (OE-3 and OE-4) were taken randomly for further analysis. The four transgenic plants differed greatly in the expression levels of HvGA20ox2. However, transcripts of barley GA oxidase-encoding gene were not found in both wild type and semi-dwarf mutant ga5-3  showed GA-overdose phenotype although they differed in the expression level of er Table . In addiga5-3, we characterized the GA-affecting traits in ga5-3 transgenic lines (OE-3 and OE-4), including hypocotyl length, internodes length and number, flowering time, and final plant height. The results showed that ga5-3 transgenic lines (OE-3 and OE-4) also had the GA-overdose phenotype, with longer hypocotyls and internodes, more internode number, flowering earlier in comparison with those of wild type or ga5-3 mutant  was a candidate gene of sdw1/denso and different expression level of HvGA20ox2 was observed between denso and sdw1 (Jotun) alleles [HvGA20ox2 and Mloc_56463 genes was completely deleted in the mutant Riso no. 9265. Because of the incomplete barley reference genomes and repetitive nature of the barley genome [HvGA20ox2 has gibberellin 20-oxidase activity and may be involved in GA biosynthetic pathway. Moreover, the sdw1/denso mutant is GA sensitive and exogenous application of 10 ppm GA3 restored its plant height [sdw1/denso mutants are GA deficiency. On the contrary, Mloc_56463 is a putative methyltransferase and has not been reported or predicted as a GA-related gene. Thus, HvGA20ox2 is considered as the only candidate responsible for the semi-dwarfism in Riso no. 9265.Previously, we reported that barley  alleles , 15. Hert height , which iArabidopsis, GA20-oxidase genes are differentially expressed and involved in different developmental processes controlled by GA, but the plants that constitutively express each of three GA20oxs  behave as the control treated with GA [HvGA20ox2 transgenic Arabidopsis  were up-regulated and GA catabolic gene (HvGA2ox3) was down-regulated in Riso no. 9265 compared with Bomi, suggesting a type of feedback regulation from the low bioactive GAs because of the null mutation of HvGA20ox2. Feedback regulation of GA20ox and GA3ox and feed-forward regulation of GA2ox genes expression have been also shown in GA-deficient Arabidopsis mutants [sd1, the expression of OsGA20ox1 was increased in stem to compensate the sd1 effect [HvGA20ox1 could compensate the effect of HvGA20ox2 in the stem at least partially, and the feedback or feed-forward regulation of the GA dioxygenase helps maintaining a relative stable endogenous GA level. Consequently, Riso no. 9265 exhibited the observed plant height.It was reported that GA promoted stem elongation and was found in the young tissues , 23. HvGe length . Once jo mutants –29. In r1 effect . As a resdw1/denso, and conclude that it encodes a GA biosynthetic enzyme, GA20ox, on the basis of the following results. Firstly, sdw1/denso mutant responds to exogenous GA3 [sdw1 allele is a complete lose of HvGA20ox2 activity due to the deletion of HvGA20ox2 gene in Riso no. 9265  are up-regulated and the expression of GA catabolic gene HvGA2ox3 is down-regulated in Riso no. 9265 in comparison with those in wild type Bomi, respectively. We conclude that sdw1/denso encodes one of the GA biosynthetic enzymes, GA 20-oxidase and the deletion of HvGA20ox2 as well as the compensatory of HvGA20ox1 and feedback regulation of gibberellin results in semi-dwarf phenotype of Riso no. 9265. We also deduced that sdw1/denso allele itself evoked later heading due to its reduced endogenous GAs concentration.The current study showed a complete deletion of over 21 kb DNA fragment including both Semi-dwarf mutant Riso no. 9265, kindly provided by Dr. Chengdao Li of Murdoch University, Australia and its wild parent Bomi, kindly provided by Dr. Jing Zhang of Chinese Academy of Agricultural Sciences, China were used in this study. Both genotypes were planted in 2 × 6 m plots. The heading date was recorded as the number of days after sowed when 50 % of the spike emerged from the sheath. At maturity, 20 plants of each genotype were harvested at random for measurement of plant height, spike and internode length. Meanwhile, main stems of each genotype were taken at the initial jointing stage for measuring the expression levels of gibberellin biosynthetic and catabolic genes.HvGA20ox2 isolated from barley was conducted using two-pair primers: (1) forward primer (sdwF10) 5’ CTAGCTCACACACCTCTCATCTCAT 3’, and reverse primer (sdwR10) 5’ GTTCCCGACAAAAATTCCGTGT 3’; (2) forward primer (sdwF9) 5’ CTCTCCCGCACACTCACTCGCAAC 3’, and reverse primer (sdwR13) 5’ GCGGTGAGGGGGCATGCATAT 3’. PCR reaction was comprised of 50 ng of template DNA, 0.3 μM of each primer, 1 × PCR buffer, 0.4 mm dNTP and 0.2 U PCR enzyme  in a final volume of 10 μl. PCR cycling conditions consisted of an initial denaturation step of 94 °C for 3 min, followed by 30-35 cycles of 98 °C for 10 s, 60 °C for 30s, and 68 °C for 3 min.Seedlings at tillering stage were sampled and DNA was extracted according to Murray and Thompson  with smaHvGA20ox2 were listed in Additional file 1: Table S1. PCR reaction was comprised of 1 × Taq Mix (Bio life), 0.3 μM of each primer and 50 ng of template DNA. The following PCR amplification profile was used : denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 0.5-2 min depending on the size of amplilcons, and a final extension at 72 °C for 5 min. The amplification products were run in 1 % agrose gels and sequenced by Biosune Biotechnology Co. Ltd.Primers of Morex_contig_40861 and neighbor genes of HvGA20ox2 was amplified from Bomi by PCR. The primers were designed as 5’ AGTACTCGAGCTCACACACCTCTCATCTCAT 3’ and 5’CTATGGATCCGAATCAGCCCGTGGAT 3’ with XhoI and BamH I sites, respectively. The amplified PCR product was confirmed by sequencing and cloned into T4 vector for further manipulation. Insert was confirmed by sequencing. The CDS paragraph of HvGA20ox2 digested with XhoI/ BamH I was cloned into the XhoI/ BamH Isite located between the 35S CaMV promoter and ocs terminator of the pFGC5941 binary vector, named as pFGC5941- HvGA20ox2. The binary vector was transformed into Agrobacterium tumefaciens by electroporation, and the floral dip method was used to transform Col-gl1  and a semi-dwarf mutant ga5-3  [HvGA20ox2). The constructs were also verified by sequencing. For analysis of HvGA20ox2 gene expression level in transgenic plants, 7-day-old Col-gl1, ga5-3 and T3 transgenic seedlings were harvested for RNA extraction.For plasmid construction, full-length CDS of k016701) . T1 tranArabidopsis were stratified at 4 °C for 1-2 d and sown on soil at 24 °C under LDs (16 h of light). Plants for hypocotyl and root length measurements were grown vertically on one half times Murashige and Skoog media  containing 1 % MES, 3 % sucrose and 0.46 % Gelrite with pH 5.8 under LDs, being measured after 7 d. Flowering time was scored as the days when buds could be detected with naked eyes. Other measurements were performed on the plants that had stopped flowering.The seeds of ga5-3 using Spin Column Plant total RNA Purification Kit(Sanggon Biotech (Shanghai) Co., Ltd). cDNA was prepared from 1 μg RNA using AMV First Strand cDNA Synthesis Kit(Sanggon Biotech (Shanghai) Co., Ltd). qPCR reactions were performed using SYBR Green (SG Fast qPCR Master Mix(High Rox), BBI ) and the Applied Biosystems Stepone plus Real-time PCR System. The Real-time PCR assays were performed in triplicate for each cDNA sample. For determining transcription levels of barley GA20ox2 and genes encoding final biosynthesis of GA, HvACTIN and HvGAPDH for barley, and At1g13320 and At4g26410 for Arabidopsis were employed as reference genes [RNA was extracted from the stems of Bomi and Riso no. 9265, as well as 7-day-old seedlings of T3 transgenic lines, Col-gl1 and ce genes , 36. AddArabidopsis tissue (7-day-old seedling) was homogenized in liquid nitrogen and extracted at 4 °C in cold 80 % (v/v) methanol containing 1 mM butylated hydroxytoluene for 4 h. The extracts were collected after centrifugation at 8000 g at 4 °C for 20 min. The residues were suspended in the same extraction solution and stored at 4 °C for 1 h, and then centrifuged again at 8000 g at 4 °C for 20 min. The two resulting supernatants were combined and passed through a C18 Sep-Pak cartridge . The efflux was collected and dried in N2. The residues was then dissolved in 0.01 M phosphate buffer solution (pH 7.5) and concentrations of GA1+3 and GA4+7 were determined in an indrect enzyme-linked immuno sorbent assay (ELISA) using GA3 and GA4 antibody, respectively. The ELISA KIT was obtained from Professor Baomin Wang  and the methods were described in previous publications [Approximately 0.5 g fresh ications , 38.t test. In order to avoid heterogeneity of variance, a natural log transformation was applied to hypocotyl length,and a square transformation was applied to flowering time (days), plant height, root and internode length. Least significant difference (LSD) at 5 % probability was used to assess the difference between genotypes. For statistical analysis of qPCR data, cycle threshold (CT) values were used to determine Δ CT values (Δ CT = CTtarget –CTreference), and expression levels of target genes relative to reference gene were determined as 2Δ CT-. For comparison of GA concentration and the expression levels of GA-regulated transcripts between transgenic and wild type plants, ANOVA was used. Analysis of qRT-PCR efficiency showed that all amplicons of the genes used in this study were within the optimal range of 98.7-102.4 %.Phenotypic differences of Bomi and Riso no. 9265 were tested by Student’s In this study we only used plant material including barley and Arabidopsis. Based on the rule of BMC Genomics, no ethics statement was required for the collection of genetic material.


We report the properties of a series of electrodeposited Ge-Sb-Te alloys with various compositions. It is shown that the Sb/Ge ratio can be varied in a controlled way by changing the electrodeposition potential. This method opens up the prospect of depositing Ge-Sb-Te super-lattice structures by electrodeposition. Material and electrical characteristics of various compositions have been investigated in detail, showing up to three orders of magnitude resistance ratio between the amorphous and crystalline states and endurance up to 1000 cycles. The recent development of GeTe/Sb2Te3 super-lattice structure-based interfacial phase-change memory data storage devices has shown even better performance with reduced switching energies, improved write-erase cycle lifetimes and faster switching speeds [GeCl5], [NnBu4][SbCl4] and [NnBu4]2[TeCl6], were prepared and purified for the ternary electrodeposition. All electrochemical experiments and the electrolyte preparation were performed inside a glove box in order to exclude moisture and oxygen (<5 ppm). Electrolytes were prepared in anhydrous CH2Cl2 using 0.1 mol dm−3 tetrabutylammonium chloride ([NnBu4]Cl) as the supporting electrolyte. A Pt gauze was used as the counter electrode, and for the reference electrode, a home-made Ag/AgCl reference electrode, using 0.1 mol dm−3 [NnBu4]Cl in CH2Cl2, was used. All experiments were performed in a standard one-compartment electrochemical cell within a wire mesh Faraday cage.Three precursors, [N2Sb2Te5 target was used for calibration. High-resolution SEM measurements were carried out with a field emission SEM (Jeol JSM 7500F). Films were imaged in high-vacuum mode. Annealing was performed using a rapid thermal annealer (Jipelec JetFirst) in a N2 atmosphere.The deposited GST films were investigated using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). A Zeiss EVO LS 25 microscope equipped with an Oxford INCA x-act X-ray detector was used for the SEM and EDX analyses. For EDX quantification measurements, a sputtered thin film from a Ge2/Si substrate by reactive sputtering at room temperature. After the electrodeposition of GeSbTe films, another TiN film was sputtered on top of the GeSbTe films and subsequently patterned by photolithography and plasma etching to form the top electrode of the memory cell.Electrodeposition was performed on planar TiN substrates in order to provide a technologically relevant substrate. A 200-nm TiN film was deposited onto a SiOAll electrical measurements were performed with a Keithley 4200 semiconductor characterisation system. During the measurements, the programming voltage bias was applied to the top electrode, while keeping the bottom electrode grounded. The probe/point contacts to the top and bottom electrodes of the devices were realised through a pair of Wentworth probe needles, using a Wentworth Laboratories AVT 702 semi-automatic prober. For electrical pulsing, two ns-pulsing measuring units (PMUs) were integrated within the same characterisation setup. The resistance of the devices after applying set or reset voltage pulses was measured at 0.1 V (DC).2Sb2Te5. The two peaks at approximately −0.45 and −1.0 V and the shoulder at −1.50 V correspond with the reduction peaks for the individual Sb, Te and Ge reagents, respectively. The different threshold voltage for deposition of the different materials opens up the opportunity to vary the composition of the compound by varying the deposition potential. Seven electrolytes with the same Ge and Sb concentration (10:10 mM) and varying Te concentration (3–10 mM) were deposited at two different voltages (−1.5 and −1.75 V). The compositions of the material vary from Ge2.3Sb5.0Te2.7 (−1.5 V) to Ge4.0Sb4.5Te1.5 (−1.75 V) for 3 mM Te and from Ge0.9Sb2.6Te6.5 (−1.5 V) to Ge0.9Sb1.6Te7.5 (−1.75 V) for 10 mM Te. The key effect of the deposition potential can be monitored by the Sb/Ge compositional ratio in the deposited film when the precursor ratio is constant, as shown in Fig. Figure −2, which corresponds to a film thickness of ca. 200 nm. Energy-dispersive X-ray (EDX) spectra for all films are displayed in Fig. 2 layers, with no noticeable impurities except for some carbon content. Quantification of EDX results indicates that the film compositions are approximately (a) Ge0.5Sb1.0Te8.5, (b) Ge2.4Sb2.0Te5.6, (c) Ge3.5Sb1.0Te5.5 and (d) Ge5.0Sb3.5Te1.5, as tabulated in Table 2Sb2Te5 film of the same thickness.In concentration-dependent electrodeposition, all depositions were performed potentiostatically at −1.75 V, more cathodic than the germanium deposition potential, to ensure significant deposition of all three elements. The film thickness was controlled by the total electrodeposition charge density of 0.4 C cm0.5Sb1.0Te8.5 film is characterised by small granules with noticeable irregular nucleation on the surface , where the current suddenly increases to the compliance value (1 mA). The back sweep then shows linear I-V behaviour, which is consistent with the material having switched from the amorphous state to crystalline state. Similar switching features can be observed for the other three compositions. It is observed that a higher Ge content in the GeSbTe material leads to a higher threshold voltage in the memory cell, as shown in Fig. Et) depends on the intrinsic material properties, including the optical band-gap, where the higher the optical band-gap corresponds to the higher threshold switching field [Vth is not possible as the re-amorphized areas are different for each composition.The electrical memory switching properties of the electrodeposited GST thin films were investigated by fabricating vertical memory devices with TiN contacts on top of the GeSbTe films Fig. . The samice Fig. , this hing field , 25. Theng field  and hencng field ; the mel0.5Sb1.0Te8.5 thin-film device displays stable endurances over 100 cycles. A dramatic decrease of reset resistance was observed after ca. 160 cycles which indicated the failure of this device. This is a typical “stuck-set” failure in phase-change memory, which is normally caused by either the heater seasoning or stoichiometric shifts of the phase-change material [2.4Sb2.0Te5.6 memory cell shows a similar performance. Substantial improvements of endurance performance can be observed for the Ge3.5Sb1.0Te5.5- and Ge5.0Sb3.5Te1.5-based memory cells, where the number of cycles increases to over 400 and 1000, respectively. However, it should be noted that this improvement of endurance comes with a decrease in the resistance ratio as a trade-off.The representative endurance performance of the four memory cells based on electrodeposited GeSbTe films were characterised by electrical pulsing as shown in Fig. material . The Ge25.0Sb3.5Te1.5-based memory cell. This leads to the differences in the resistance ratio as shown in Fig. ca. 103) is observed for both Ge0.5Sb1.0Te8.5- and Ge2.4Sb2.0Te5.6-based memory cells. For Ge3.5Sb1.0Te5.5- and Ge5.0Sb3.5Te1.5-based memory cells, the resistance ratios drop to ca. 102 due to the lower reset resistances.The distribution of the resistance for the four memory cells at both set and reset states is illustrated in the box plot in Fig. 0.5Sb1.0Te8.5, Ge2.4Sb2.0Te5.6, Ge3.5Sb1.0Te5.5 and Ge5.0Sb3.5Te1.5). Phase-change memory cells based on the four films have shown promising switching properties with high-resistance ratio (three orders of magnitude) and good durability.The phase-change memory properties of electrodeposited GeSbTe thin films have been measured for various compositions. The composition of the resultant films can be tuned by varying the deposition potential in a single electrolyte, making their future application in depositing super-lattice structures possible. Film composition modulation was also achieved by varying the electrolyte concentrations with deposition of four device-quality GeSbTe thin films (Ge


II atom in the title compound, bonded to four N atoms of the azamacrocylic ligand and two N atoms of the thiocyanate ions, shows a slightly distorted octahedral coordination geometry. In the crystal, molecules are connected by hydrogen bonds, forming chains along the b-axis direction.The Ni 2(C16H38N6)], has been determined from synchrotron data. The asymmetric unit consists of two halves of the complex molecules which have their NiII atoms located on inversion centres. The NiII ions show a tetragonally distorted octahedral coordination geometry, with four secondary amine N atoms of the azamacrocyclic ligand in the equatorial plane and two N atoms of the thiocyanate anions in the axial positions. The average equatorial Ni—N bond length [2.070 (5) Å] is shorter than the average axial Ni—N bond length [2.107 (18) Å]. Only half of the macrocyclic ligand N—H groups are involved in hydrogen bonding. The complex molecules are connected via intermolecular N—H⋯S hydrogen bonds into two symmetry-independent one-dimensional polymeric structures extending along the b-axis direction. One of the n-butyl substituents of the macrocycle exhibits conformational disorder with a refined occupancy ratio of 0.630:0.370.The crystal structure of the title compound, [Ni(NCS) The axial bond lengths, Ni1A—Nax and Ni1B—Nax are 2.119 (1) and 2.093 (1) Å, respectively. The axial bonds are longer than the equatorial bonds, which can be attributed either to a large Jahn–Teller distortion effect of the NiII ion and/or to a ring contraction of the azamacrocyclic ligand 16H38N6)](ClO4)2, was prepared by a slightly modified method reported by Jung et al. (198916H38N6)](ClO4)2  was slowly added a MeCN solution (5 mL) containing sodium thiocyanate  at room temperature. A pale-pink precipitate was formed, which was filtered off, washed with MeCN, and diethyl ether, and dried in air. Single crystals of the title compound were obtained by layering of the MeCN solution of sodium thiocyanate on the MeCN solution of [Ni(C16H38N6)](ClO4)2 for several days. Yield: 0.062 g (49%). FT–IR : 3304, 3243, 2929, 2867, 2069, 1468, 1386, 1273, 1204, 1070, 925.The title compound  values of 1.2 or 1.5Ueq of the parent atoms. The C7A and C8A atoms of the macrocyclic ligand were refined as disordered over two sets of sites  with refined occupancies of 0.630 and 0.370, respectively. The bond lengths and angles of the disordered part were restrained to ensure proper geometry using DFIX and DANG instructions of SHELXL2014  I. DOI: 10.1107/S205698901501110X/gk2635Isup2.hklStructure factors: contains datablock(s) I. DOI: 1405450CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: