In anterior cruciate ligament reconstruction, quadrupled semitendinosus (Quad ST) grafts have potential advantages over doubled semitendinosus–gracilis (ST/G) including larger diameter and gracilis preservation, however the ideal tibial fixation method of the resultant shorter Quad ST graft remains elusive if a fixed-loop suspensory fixation device is used on the femur. We investigated whether the tibial fixation biomechanical properties of a Quad ST fixed indirectly with polyethylene terephthalate tape tied over a screw in a full outside-in created tunnel was superior to a ST/G graft fixed with an interference screw.In a controlled laboratory study, six cadaveric matched pairs of each construct were subjected to cyclic loading to mimic physiologic loading during rehabilitation. This included preconditioning cycling, cyclic loading to 220 N for 500 cycles, then cyclic loading to 500 N for 500 cycles.High standard deviations across the measured parameters occurred with no significant difference between measured parameters of elongation for the different constructs. Elongation of the Quad-ST construct was greater at 10 and 100 cycles, but not statistically different. Four of the six Quad-ST constructs failed below 100 cycles, compared with two failures below 100 cycles in the ST/G construct. There was a strong correlation between cycles to failure and bone mineral density for the Quad ST-tape constructs.Tibial fixation of Quad ST with a tied tape–screw construct in a full-length tunnel was not biomechanically superior to ST/G graft fixed with an interference screw, exhibited greater nonsignificant construct elongation with earlier failure, and was more reliant on bone mineral density.In vitro laboratory study. Currently, the ideal anterior cruciate ligament reconstruction (ACLR) graft and fixation choice remains elusive, with all common graft options having some disadvantages. Patella tendon autografts have become less popular in recent years , possiblGracilis harvest in addition to semitendinosus has been linked to knee flexor weakness by some authors , 8 and lMagnussen et al.  recentlyThe aim of this study is to investigate an alternative method of tape–screw construct fixation using full-length outside-in created tibial tunnels with tape tied over the screw for additional fixation. This alternative method would offer a graft tibial fixation option to surgeons who wished to preoperatively utilize a quadrupled ST graft to avoid harvesting the gracilis, or intraoperatively if a harvested gracilis tendon was inadequate in diameter or length to be used, while utilizing a fixed suspensory fixation device on the femur.Two alternative ACLR tibial fixation methods were evaluated by matched-pair cadaveric laboratory biomechanical analysis: a ST/G graft with interference screw (ST/G-screw) and a Quad ST with 5-mm braided PET tape and screw (Quad ST-tape). Ethics approval was obtained from the institution’s Human Ethics Committee.Six matched pairs of seronegative cadaveric tibiae were thawed to room temperature and dissected (average age = 53 ± 5 years). All soft tissue was then removed. Then specimens were stored at room temperature prior to preparation, covered with gauze moistened with 0.9 % saline solution so as not to affect their mechanical properties . The bonThe tibiae were evenly assigned to each matched cadaveric hamstring ACL fixation graft construct. Each of the 12 graft preparations was performed by a 15-year postfellowship trained orthopedic surgeon (C.J.V.). Remnant muscle fibers were scraped from the tendons with scissors held at 90° to the tendon long axis. Six ST/G grafts were prepared using a Graftmaster III Graft Preparation System Board . The ST/G tendons were proximally doubled over a 20-mm Endobutton CL, and the distal tendon ends were sutured with no. 5 Ethibond  in a locking pattern. A proximal #1 Ethibond  tripled circumferential suture was applied 15 mm distal to the proximal loop  that could load the graft construct to 500 N. The Quad ST-tape construct preparation and suturing technique were the same on the tibial side as previously described for other Quad ST-tape techniques . The ST The grafts were marked 50 mm from the proximal end, which corresponds to the 35-mm segment length of the articular ACL, plus 15 mm of femoral tunnel graft. All graft construct diameters and lengths were measured using an ACL Graft Sizer .The tibia ACL footprint was identified, and the planned intraarticular tunnel exit site was marked with a surgical marker, centered at the footprint of the native ACL. The distal tibial tunnel position was also marked, standardized at 6 cm distal to the tibial articular surface on the anteromedial tibial cortex. All tunnels were prepared using the Acufex ACL Aimer . The tibial tunnel was drilled obliquely “outside-in” with a tibial tunnel guide set at 45° from the medial metaphysis into the anatomic footprint of the ACL. The planned length of the tunnels was 45 mm, and the diameter was a function of the measured graft diameter.The grafts were drawn through the tibial tunnel, distally to proximally using traction on the Endobutton sutures, until 50 mm of the graft was above the proximal tibial tunnel aperture.To fix the ST/G grafts to the tibiae, a Biosure interference screw in polyetheretherketone (PEEK)  was inserted into the tibia tunnel parallel to the hamstring tendons over a guide wire with manual traction to the distal no. 5 Ethibond sutures. The screw size was based on the surgeon’s usual surgical technique strategy, matching the screw diameter to the graft and tunnel diameter, or downsizing the screw if the graft was between sizes, to maximum diameter of 8 mm. The 30-mm-long screws were inserted until there heads were flush with the tibial cortex.The Biosure screw has a flat head, is slightly conical, and incorporates a consistent wall thickness throughout the length of the screw with a tapered body for easier insertion .In Quad ST-tape constructs, a 10- or 11-mm-diameter × 25-mm-length PEEK Biosure interference screw, oversized to tunnel size, was inserted into the tibial tunnel between the two tape distal ends. Manual traction was applied to the distal tape ends before and during screw insertion Fig. . The scrEach tibia was mounted on the base of the testing machine  so that the tibial tunnel was coaxial with the line of pull of the linear actuator Fig. . The Endt-test, where possible. When nonpaired comparison was undertaken, a standard t-test was utilized.Two high-definition cameras were set up orthogonal to the line of pull, and images captured at the following points: at 50 N prior to cyclic loading, at the peak of each loading cycle for the first 500 cycles at 220 N, at the 50 N load between the two cyclic loading regimes, at the peak of each loading cycle for the 500 cycles at 500 N, and at 50 N on completion of cyclic loading. The following results were captured: number of cycles to failure, elongation under initial load, elongation at 10 and 100 cycles, and slip of the unloaded graft after 500 cycles  and used to calculate the displacements. The program allows the user to select common landmarks on bodies of interest and calculates the number of pixels between them. In this case, we selected a point in the graft construct and a point on the tibia. These raw data were scaled by finding the number of pixels over a known distance in the same images. In this case, the pin used to hold the femoral end of the construct was 8 mm, so it was used to calibrate the data. The displacement values described above were calculated by measuring the difference between the relevant readings.n cycles was defined as the difference between the displacement at the maximum load of the nth cycle and the displacement at the maximum load of the first cycle (peak to peak) . Two-tailed paired Student p = 0.0041).Table All 12 constructs failed during cyclic testing, hence no load-to-failure tests were conducted. Eleven of the constructs failed during the first of the 500-N cycles, hence comparison of displacement results was only conducted for the first phase of cyclic loading, where the peak load was 220 N. The number of cycles to failure for each specimen is shown in Fig. In the Quad ST-tape group, four out of six constructs failed below 100 cycles, and three of those failed in the first cycle. In contrast, the ST/G construct had two failures below 100 cycles, with the remainder of failures occurring after 385 cycles. The distribution of these failure data is presented in Fig. The displacement results for the ST/G and Quad ST-tape constructs, respectively, were as follows: elongation under initial load of 2.26 mm SD 3.58 mm) and 4.32 mm (SD 3.73 mm)   (Table The average BMD for the ST/G and Quad-ST tape group was 0.92 and 0.91 g/cme common . The num.9) Fig. . The oppThe mode of failure for all specimens was displacement greater than 5 mm, with the modes of catastrophic failure for the two constructs being consistent Table  but dissThere were no statistically significant differences between the parameters measured for the two tibial fixation methods, however there were high standard deviations, a common observation in ACL fixation literature , 23, 29.It is important to note that the image-processing method used cannot isolate the cause of the early displacement failures, hence it was not possible to ascertain whether this failure was due to initial laxity in the tape, a poor interface between the interference screw, tape, and bone, or the elasticity of the graft–tape interface.The tunnel friction forces are difficult to define, as they depend on several factors including the normal force, the friction coefficient between the materials, and their contact area. Matching the tibial tunnel diameter to the ST/G diameter resulted in friction along the entire length of the graft–tunnel interface, playing a role in resisting the tensile loading for this graft compared with the shorter Quad ST-tape, which had much less graft tunnel contact. Friction along the graft–tunnel interface may have led to longer survival of two low BMD ST/G specimens compared with the matched pair with Quad-ST tape, suggesting that interference fixation would be preferable in patients with lower BMD.n = 2). In contrast, the same parameters showed weak correlation with BMD for the ST/G construct.The stability of the ST/G reconstruction also depends on the size and density of the tendons, with poor-quality tendons being more likely to deform plastically or slip past the screw . It is pThe difference in mode of catastrophic failure is also noteworthy. The ST/G reconstructions characteristically failed when the graft slipped around the interference screw; i.e., both the static friction along the graft–tunnel interface and the anchoring with the interference screw failed to counter the tension load. The majority of Quad ST-tape constructs failed when the knotted tape slipped around the screw into the cancellous bone. The interference screws used had a different design from other reports for a partial inside-out technique tape fixation technique, which may have resulted in the heterogeneous fixation results in our study. Full-length tunnels were used to mimic an ACLR technique using fixed suspensory loop femoral fixation with tibial tape–screw fixation. If our tunnels had been drilled partial length inside-out, our results may have been different; however, given that the majority of Quad ST-tape failures were due to graft slippage around the screw, despite the tape being tied over the screw head, it remains uncertain if the graft slippage still would not have occurred. If the tunnels had been drilled partial length inside-out so that an isthmus of bone remained at the screw tip, this may have provided additional friction to the tape. However, as noted above, we attempted to add to the tape–screw fixation by tying a knot over the screw in compensation for the full-length tunnel.One other issue of concern is the tendency of the Quad ST-tape construct to fail by elongation earlier than the ST-G construct. While the sample size was not sufficient to clearly determine which failure results were outliers, elongation as a material property of the braided 5-mm PET tape we utilized has been reported by other authors in situations other than ACL fixation , but notOne of the strengths of this study is the use of matched human tissue for both the bony and soft components. Magen et al.  suggesteThe main weakness of the study is the limited number of matched constructs, which when combined with the high variability of measured parameters resulted in insufficient samples to accurately determine the significance of the results when failure due to 5 mm of relative displacement was considered as the critical parameter.Tibial fixation of ACL quadrupled ST grafts tied over a tape–screw construct with a full outside-in created tunnel was not superior to ST/G grafts fixed with an interference screw.


Investigation of organic compounds in ice cores can potentially unlock a wealth of new information in these climate archives. We present results from the first ever ice core drilled on sub‐Antarctic island Bouvet, representing a climatologically important but understudied region. We analyze a suite of novel and more familiar organic compounds in the ice core, alongside commonly measured ions. Methanesulfonic acid shows a significant, positive correlation to winter sea ice concentration, as does a fatty acid compound, oleic acid. Both may be sourced from spring phytoplankton blooms, which are larger following greater sea ice extent in the preceding winter. Oxalate, formate, and acetate are positively correlated to sea ice concentration in summer, but sources of these require further investigation. This study demonstrates the potential application of organic compounds from the marine biosphere in generating multiproxy sea ice records, which is critical in improving our understanding of past sea ice changes. We analyzed a suite of organic compounds,alongside commonly measured ions, in the first ever shallow ice core from Bouvet IslandMultiple marine‐sourced organic compounds are detected throughout the coreOrganic compounds, alongside MSA, show significant positive correlations with sea ice Bouvet 22.1Bouvet is a volcanic SAI at 3°E, 54°S. The Bouvet ice core was collected as part of the Sub‐Antarctic Ice Core Expedition project on board the Antarctic Circumnavigation Expedition cruise in 2017 , formate (formic acid), and acetate (acetic acid), were cut and measured as discreet samples of 5‐cm resolution using a Dionex Integrion reagent‐free ion chromatograph in a class‐100 cleanroom  with varimax rotation was carried out using annual average data for all compounds Tables , and S2 Input data were log transformed to normalize data distribution. Reliability of the initial PCA output was checked using a resampling method where repeat PCAs were run for the data set removing each compound from the matrix one at a time. The variance explained by each output component  was compared for each repeat run, determining whether one input compound is having a disproportionate impact on the output components. For the full data matrix PCA, principal components and variance explained were as follows: PC1 61%, PC2 18%, PC3 8%, PC4 6%, and all other components ≤2%; for all resampling PCAs, PC1 65–59%, PC2 20–15%, PC3 8–6%, PC4 6–4%, and all other components ≤2%. The low variation in PC% demonstrates no extremes in component values are caused by the removal of any one compound. Therefore, all are retained in the input data matrix.Significance of components was decided based on Kaiser's Rule Kaiser, . The PCA2.3.22 data cell. Spatial correlation plots are generated using NSIDC SIC in the Royal Netherlands Meteorological Institute (KNMI) Climate Explorer (https://climexp.knmi.nl/start.cgi). The concept here is to correlate a compound record over each grid cell in the region with the SIC of that corresponding grid cell, thus telling us where the ice core record obtained correlates with SIC. The correlation uses annual resolution compound time series and SIC for the 2001–2016 time period, calculating with detrended SIC data in 3‐month bins, one starting at each of the 12 months of the year. Temporal autocorrelation  is accounted for by adjusting the number of degrees of freedom to account for lag‐1 autocorrelation in the data where degrees of freedom are larger than 1. Spatial autocorrelation  effects are discussed in section We use two measures of sea ice conditions in this study. The first is Bootstrap (version 3.1) sea ice concentrations (SIC) from Nimbus‐7 scanning multichannel microwave radiometer and Defense Meteorological Satellite Program Special Sensor Microwave Imager‐Special Sensor Microwave Imager/Sounder. Data are available from the National Snow and Ice Data Centre (NSIDC) from 1979 onward Comiso,  and calcSecondly, sea ice extent (SIE) is generated from the daily SIC data, calculating the area of all data points where sea ice was above a concentration threshold of 15% within the region. The median average SIE displayed in Figure A geometric mean regression technique was used to calculate correlation coefficients between winter SIE (defined as the September average) and organic compound concentrations following the method of Abram et al.  from Smi2.3.3The Numerical Atmospheric‐dispersion Modelling Environment back trajectory model was run for 1‐month periods for each of February and September in representative years 2008, 2009, and 2010. These months are those in which a compound had significant correlations to SIC , indicative of terrestrial sources , herein winter‐to‐spring. This region lies along the sea ice margin during maximum winter SIE and extends to the west along the sea ice margin. The correlation between oleic acid and SIC shows a very similar region of significance during the same period.p < 0.05) by chance and to do so in coherent regions owing to spatial autocorrelation in the sea ice variability. We find, however, that much greater than 5% of grid points are significantly correlated to our records. Further, the coherent regions of significant correlation are immediately adjacent to our site and almost solely up‐wind , reinforces a link between the two compounds. Correlation coefficients between compounds and September SIE averaged over the 45–70°S, 50°W to 10°E area are r = 0.45 (p < 0.05) for oleic acid/SIE and r = 0.40 for MSA/SIE but p > 0.05, showing positive correlations with lower confidence in the case of MSA/SIE.At Bouvet Island, a location on the edge of median maximum SIE, SIE variations may be particularly important in controlling phytoplankton blooms in the area. In years with lower ice extent, for example, the island may not actually be within the sea ice zone. To investigate this further, Figure While the long‐term trend for SIE over the record 2001–2016 does not significantly increase or decrease, concentrations of both oleic acid and MSA show a slight increase. This may appear dominated by a sharp increase in concentration of both compounds in sample year 2016, but trend lines excluding this data point maintain a slightly reduced but still positive trend for 2001–2015. It has been shown previously that high diffusivity of MSA in ice samples may lead to a smearing of original concentrations of the compound deposited on the ice. However, this will not affect the record of compound variability along an ice core record if storage of samples has been consistent . ACE and Elizabeth Thomas received funding from École Polytechnique Fédérale de Lausanne, the Swiss Polar Institute, and Ferring Pharmaceuticals Inc. Joel Pedro acknowledges support from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007e2013)/ERC Grant Agreement 610055 as part of the ice2ice project.Supporting Information S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


The solubility limit of Mg2+ in BMT was determined by XRD to be x = 0.05, and evidence was found for occupation of the A site by Mg2+. Electron paramagnetic resonance (EPR) was employed as a key technique to investigate the effect of Ts on oxygen vacancies in BM1T. The structure of BM1T changed from pseudocubic at Ts = 1200 °C to tetragonal at 1300 °C to mixed phases of hexagonal and tetragonal at 1400 °C. When Ts ≥ 1300 °C, a g = 1.956 EPR signal was observed at T = −188 °C and assigned as ionized oxygen vacancies. Mg2+ exhibited amphoteric behavior of substituting for the double cation sites. When Ts = 1400 °C, B-site Mg2+ and oxygen vacancies mainly existed in the hexagonal phase and A-site Mg2+ was dominant in the tetragonal phase. The higher tan δ was attributed to the higher concentrations of oxygen vacancies and Ti3+ in the hexagonal phase.Nominal (Ba Singly doped magnesium (Mg2+) and Mg and rare earth co-doped BaTiO3 (ABO3) compounds that satisfy X7R or X8R specification have found applications in multilayer ceramic capacitors (MLCCs) [BaTiOnesium Mg+ and Mg  (MLCCs) ,5,6.3, for example, Mn2+ [2+ was also considered to be substituted for the B site as an acceptor because 6-CN Mg2+ is closer to Ti4+ in ionic size, and the defect notation was written as 3 [Many transition metal ions with lower valence states occupy the B site when doped in BaTiOle, Mn2+ ,8. Similand Vink . Considen BaTiO3 ,11,12,133 is still lacking; and (2) the possibility of occupying the A site for Mg2+ has not been determined. Our previous study confirmed the amphoteric behavior of Dy3+ in BaTiO3, which can occupy both A and B sites [3+ at the A site is 1.19 Å [2+ (1.23 Å) with the same coordinate number (CN). Ionic radii with different CN are given in At present, two scientific problems remain unsettled and need further investigation: (1) direct evidence for observing  B sites ,15,16. Ts 1.19 Å , which i3+ and the similar ionic radii between Dy3+ and Mg2+ indicate that although the structures and properties of B-site Mg-doped BaTiO3 have been studied, the possibility of Mg2+ occupying the A site should not be excluded. Although MgTiO3 has a distorted rhombohedral structure [3, the difference in crystalline structure between MgTiO3 and BaTiO3 may not be a key factor for Mg2+ to enter the A site. Thus, the amphoteric nature of Mg2+ in BaTiO3 is still a scientific problem to be clarified.The amphoteric behavior of Dytructure , which i3−MgTiO3 (BMT) solid solutions were prepared at different sintering temperatures. At a lower sintering temperature (Ts) such as 1150 °C, Mg2+ was considered to segregate to the surfaces of the grains and play an important role in the formation of the core–shell structure [2+ into the BaTiO3 lattice. The site occupation and amphoteric behavior of Mg2+ and the dependence of Ts were investigated. The electron paramagnetic resonance (EPR) technique was employed to detect the existence of In this work, BaTiOtructure . Therefox1−Mgx)TiO3 (x = 0.015) (BM1T) were prepared by the mixed-oxide method, described elsewhere [3, MgO, and TiO2 powders. The molded pellets were sintered at 1200, 1300, and 1400 °C, respectively, for 12 h in air to form ceramics. In addition, (Bax1−Mgx)TiO3  (BMT) were prepared at 1200 °C for 12 h to investigate the occupation of Mg2+ at the A site.Nominal  data were collected using a DX-2700 X-ray diffractometer . The lattice parameters were calculated by MS Modeling  using Rietveld refinement in the Reflex Package and Cu Kα1 radiation (λ = 1.540562 Å). Scanning electron microscope (SEM) investigations were performed using an EVOMA 10 SEM  operated at 15 kV. The sample surfaces were first polished and then thermally etched at the same sintering temperatures for a few minutes before SEM measurement. The dielectric properties were investigated at 1 kHz, from −75 to 200 °C, at a heating rate of 2 °C/min using a Concept 41 dielectric/impedance spectrometer (Novocontrol) with an applied voltage of 1 V. Temperature-dependent electron paramagnetic resonance (EPR) measurements were performed using an A300-10/12 X-band spectrometer  operating at 9.43 GHz. The EPR cavity of the spectrometer was replaced with an ER 4102ST cavity.x1−Mgx)TiO3 (x = 0.015) (BM1T) ceramics prepared at Ts = 1200–1400 °C are shown in Ts = 1200 °C exhibited a pseudocubic perovskite structure (space group: Pm3m) marked by a symmetric and broad characteristic (200) peak at ~45° /(200) splitting (P4mm), similar to the tetragonal BaTiO3 (JCPDS Cards No. 5–626) /(200) peaks and the (204) peak (3 (space group: P63/mmc) (JCPDS Cards No. 34–129)  b. When T04) peak a, inset  34–129) b, i.e., Ts.SEM images of BM1T are shown in x1−Mgx)TiO3 (x = 0.015‒0.20) (BMT) ceramics sintered at Ts = 1200 °C are shown in x = 0.05. The secondary phases of the hexagonal BaMg6Ti6O19 [3 appeared in BMT when x ≥ 0.07. Thus, the solubility limit of Mg2+ in BMT sintered at Ts = 1200 °C was determined by XRD to be x = 0.05. The variation in unit cell volume (V0) as a function of x for BMT is shown in the inset in x ≤ 0.05, V0 decreased linearly with increasing x. In the multiphasic region of x > 0.05, V0 increased.XRD patterns of nominal (Baε’) and dielectric loss (tan δ) for BM1T are shown in ε’–T curve of BM1T sintered at Ts = 1200 °C was smooth and even, showing a rounded hill at around Tm = 110 °C. The Curie peak of BaTiO3 was dramatically suppressed due to Mg doping, and this ceramic satisfied the X8S specification (|(ε’−ε’RT)/ε’RT| ≤ 22% in a temperature range from −55 to 125 °C) with ε’RT = 1200. BM1T exhibited a very low tan δ (0.0176) at room temperature and lower tan δ (<0.05) in a T range of −55 to 110 °C. Subsequently, tan δ increased with increasing T.Temperature dependencies of the dielectric permittivity  transition at dielectric peak temperature Tm = 96 °C and an orthorhombic–tetragonal (o–t) transition at T2 = 12 °C.When Ts was increased to 1400 °C, the bimodal feature in the ε’–T curve became more distinct and t–c and o–t transitions occurred at Tm = 106 and 14 °C, respectively. The ε’RT decreased and tan δ increased rapidly above T = 50 °C.As Ts = 1200 °C, only the g = 2.004 signal existed over the measuring temperature (T) range of −188 to 150 °C TiO3 [2+ on the A site.Temperature-dependent EPR spectra for BM1T are shown in o 150 °C a. This sacancies . The pai.15)TiO3 , which mTs = 1300 °C, except for the g = 2.004 signal, two additional signals at g = 1.974 and 1.956 observed at T = −188 °C (When  −188 °C b were as•+e→VO•)  vacancieTs = 1400 °C existed in mixed forms of the hexagonal and tetragonal phases. Five EPR signals appeared below T = −100 °C and their intensity increased with decreasing T  [Ts = 1400 °C, the electrons in BM1T can be trapped by Ti4+ ions to cause a reduction from Ti4+ to Ti3+. It has been reported that the (Bax1−Cax)TiO3 (x = 0.03) ceramic sintered at Ts = 1500 °C showed a more ordered tetragonal structure, and only a Ti3+-related signal at g = 1.932 was observed at T = −188 °C [Ts = 1300 °C  ,22. This −188 °C ,22. Howe 1300 °C b. In the2+ (1.61 Å) and Mg2+ (1.23 Å) and 6-CN ionic size between Ti4+ (0.605 Å) and Mg2+ (0.72 Å), a continuous decrease in V0 with x (≤0.05) for BM1T sintered at Ts = 1200 °C  is equal to the tetragonal BaTiO3 . This implies that Mg2+ tends to remain close to the surfaces of the grains and plays an important role in the temperature-stable X8S behavior in BM1T, as suggested by Chang et al. [2+ exists only at the A site as On the basis of a simple comparison of 12-CN ionic size between Ba 1200 °C , inset ph phases . The app•• pairs ,11,12,13observed a. On theTi4+ 0.60 Å and Mg2+ in (Bax1−Mgx)TiO3 was 0.15. They neglected a small amount of the secondary phases of BaMg6Ti6O19 and MgTiO3, which also appeared in their samples with x ≥ 0.05 [2+ may exist at the A site.El Ghadraoui et al. indicated that the solubility limit of Mgx ≥ 0.05 . Their rTs was increased to 1300 °C, 2+ ions transferred from the A site to the B site during the cooling process of ceramic sintering, accompanied by the creation of When detected b, revealTs = 1400 °C, more Mg2+ ions enter the B site. The concentration of 1−xMnx)O3 with x < 0.03 [3 play the same role in the formation of the hexagonal phase. Thus, When tragonal c. The hex < 0.03 ,27. The exagonal . This im2+ ions in BM1T sintered at Ts ≥ 1300 °C exhibited amphoteric behavior, i.e., Mg2+ existed at the A site as As a whole, Mg3. It is not easy to create Ts is lower than 1200 °C and tan δ at Ts = 1300 °C is astonishingly low over the T range of −55 to 150 °C (tan δ ≤ 0.03).The Ts can create Ts = 1400 °C. The high value of tan δ is attributed to the high concentrations of The increase in  in BM1T .2+ in (Bax1−Mgx)TiO3 ceramics sintered at 1200 °C was 0.05, and rhombohedral MgTiO3 and hexagonal BaMg6Ti6O19 phases were observed with higher doping content. The evolution of unit cell volume provided sufficient evidence for the A-site occupation of Mg2+. The x = 0.015 ceramic had a pseudocubic crystal structure when the sintered temperature was 1200 °C and exhibited a temperature-stable X8S dielectric specification with ε′RT = 1200. The structure transformed into a tetragonal phase when sintered at 1300 °C, and tetragonal and hexagonal phases coexisted when sintered at 1400 °C.The solubility of Mgx = 0.015 sintered above 1300 °C, the g = 1.956 signal observed at T = −188 °C was assigned as ionized oxygen vacancies (2+ acted as an amphoteric doping ion with g = 1.934 and 1.942 originated from the hexagonal phase in x = 0.015 and were related to Ti3+ which, along with For


Early in the melt season (June–July), vertical thinning gradually ceases as the glacier increasingly thickens. During late summer to midwinter (August–February), vertical thickening occurs linearly throughout the entire ice column, with strain rates averaging 0.016 a−1. We show that these complex variations are unrelated to topographic setting and localized basal slip and hypothesize that this seasonality is driven by far‐field perturbations in the glacier's force balance, in this case generated by variations in basal hydrology near the glacier's terminus and propagated tens of kilometers upstream through transient basal lubrication longitudinal coupling.Temporal variations in ice sheet flow directly impact the internal structure within ice sheets through englacial deformation. Large‐scale changes in the vertical stratigraphy within ice sheets have been previously conducted on centennial to millennial timescales; however, intra‐annual changes in the morphology of internal layers have yet to be explored. Over a period of 2 years, we use autonomous phase‐sensitive radio‐echo sounding to track the daily displacement of internal layers on Store Glacier, West Greenland, to millimeter accuracy. At a site located ∼30 km from the calving terminus, where the ice is ∼600 m thick and flows at ∼700 m/a, we measure distinct seasonal variations in vertical velocities and vertical strain rates over a 2‐year period. Prior to the melt season (March–June), we observe increasingly nonlinear englacial deformation with negative vertical strain rates  in the upper half of the ice column of approximately −0.03 a Autonomous phase‐sensitive radar can be used to track the fine‐scale motion of glacier internal layersVertical velocity profiles of internal layers evolve seasonally in a region of fast flowChanges in the local strain field are driven by downstream far‐field longitudinal stress coupling Spatially, the flow of ice masses is modulated by variations in basal traction, which arise from variations in bed topography  provide the first noninvasive, direct, and continuous measurements of vertical velocity and englacial strain  is the third fastest outlet glacier in West Greenland , we deployed three ApRES arrays  at site S30 on Store Glacier Figure  maintainThe three array sites were separated from each other by up to 450 m and made measurements at a sampling interval ranging from 1 to 8 hr Figure , and theFrom these records, we measured and modeled the vertical displacement and velocity of these reflectors relative to the starting location of the ApRES antennas and calculated vertical strain‐rate profiles from time series of the vertical velocity of internal reflectors. Through this process, we standardized the time step of each time series by downscaling the temporal resolution to 24 hr, resulting in continuous daily measurements of ice column vertical deformation.λc = 0.43 m in cold ice), forming a “chirp,” with an ensemble of chirps known as a “burst”  technique, where a carrier wave is continuously transmitted over a spectrum of frequencies, sampling over periods much greater than the target two‐way travel time  of individual internal reflectors relative to the radar antennas by comparing their internal phase between successive bursts Figure . Then, t3.2.1To calculate the vertical displacement of internal reflectors and their associated phase noise, we follow the (i) phase processing procedures described by Brennan et al. a and the6 s−1 , whereas the phase provides an indication of the “fine range” (Rf) of the reflector within the specified coarse range bin n through the following relations . In equation B is the system bandwidth (200–400 MHz = 200 MHz), c is the speed of light, p is the pad factor, and εr is the dielectric constant of ice, which we assume to be 3.1 . All data processed here used a pad factor of 10; variation of the pad factor did not significantly alter results. In equation c is the phase of the deramped signal at the coarse range bin center. The sum of the coarse range and fine range will, when the above conditions are satisfied, give an estimate of the total range from the radar to the specified reflector, which is precise to ∼3‐mm root mean square at a distance of 1.8 km  weighting the deramped waveform in the time domain using a Blackman window; (ii) increasing the series to a multiple of its original length by appending trailing zeros (“zero padding”); (iii) time shifting the signal so that the phase center aligns with the start of the sample; and (iv) Fast Fourier‐Transforming (FFT) the modified time series to transform the waveform into the frequency domain. Due to the proportionality of frequency and time‐of‐flight in deramped frequency‐modulated continuous‐wave observations  rather than at the antenna (ϕd). This phase shift is essential if offsets are calculated using phase differencing procedures to determine changes in range to millimeter precision. Mathematically, the amount of phase shift is represented by weighting the FFT‐processed deramped waveform by a reference array equal to the phase conjugate of the expected phase at the center of the range bin. The reference array is described by equation 17 of Brennan et al. ϕrefωc is the chirp center frequency in terms of angular velocity (rad/s), K = 2πB/T is the chirp gradient (rad/s2) that represents the frequency‐modulated sweep rate, where, within the equation, B is the system bandwidth (200–400 MHz), and T is the total pulse duration.Here For the phase differencing procedure, burst pairs from each range‐processed spectrum (profile) were then processed iteratively with a daily (24‐hr) time step through complex cross correlation of the burst spectra pair  to correctly identify the amount of displacement between reflector pairs. The magnitude of f⋆g is the coherence, which indicates the similarity of the segments to each other, and is scaled from 0 for incoherent segments to 1 for segment pairs with a constant phase difference. The phase of f⋆g is the amplitude‐weighted (vector‐averaged) phase difference between the segments.Here the complex correlation γ^fgn,Λ^=argmaxΛγfgn,Λ. Here the maximization of the coherence and its corresponding lag vector is represented by the hat operator ^. Therefore, the coarse‐range displacement of the segment n is the amount of lag Λ^ that corresponds to the maximum coherence γ^fg, scaled by the coarse‐range resolution δcn=c for Λ^ in the equation calculating the referenced and centered phase within the coarse range bin δfnFinally, the total displacement of the segments is the sum of the coarse‐ and fine‐range displacements: 3.2.2Depth‐dependent profiles of vertical strain were calculated by fitting a model to each internal layer's vertical velocity profile. As these profiles vary seasonally and on timescales as short as a single day, we used quadratic best‐fit models Figures a and 3b δmR is the modeled displacement, and the model parameters a, b, and c were solved using a robust nonlinear least‐squares search. The use of a quadratic model, though simple, easily identifies the degree of nonlinearity within vertical velocity profiles through the first model parameter a, which represents the curvature of the model with depth R. When the value of a approaches zero, the model depicts an increasingly linear strain regime through depth  when equation Lastly, the depth‐dependent derivative of equation Using equation σn is the standard deviation of each measured segment n. This ensures that prominent internal layers influence the fitted curve more than weak internal layers.The ApRES range error from phase noise increases with increasing depth, due to progressive attenuation in the received signal power with increasing depth. Therefore, to address the heteroskedastic behavior in range, observations were weighted  to the basal reflector  were measured during 2014 and 2015 using dual‐frequency GPS receivers (Trimble 5700 and R7) installed on 4.9‐m‐long poles drilled 3.9 m into the ice surface. The GPS receivers were powered by a 50–100 Ah battery, solar panels, and a wind generator and sampled at a 10‐s interval. Data from the receivers were processed kinematically , where ux is taken along the direction of flow. We interpolated through these sparse measurements using a temperature‐dependent flow model  cumulatively over the entire ice column to calculate the deformational velocity time series ud for the period 04 August to 25 September 2014 when the tilt sensor string was operational. Subtracting ud from the time‐varying surface velocity us , and μ the sediment internal friction angle. c0 can be assumed to be negligible for deforming till due to the low clay content  and μ (30°) were set to those of Trapridge till , summer and autumn (deployment 14b), and a whole year (deployment 15). The vertical velocities varied substantially with depth and over time at and between each site Figure . During While the upper 400 m of the measured ice column during the Spring of 2014 at deployment 14a revealed negative vertical velocities, where internal reflectors move increasingly closer to the antennas located at the ice surface  correlation values. Although the redeployment of the ApRES in July 2015 (deployment 15) did not show the same trend in near‐surface thinning observed the previous year in the deployment 14a time series, it confirmed that strain thickening again dominated the ice column from July to December for the second year in a row , suggesting almost‐linear strain thickening along the ice column  begins to descend in depth through the rest of the winter and into spring  through successive months, reaching close to 0 during May and June. Only from mid‐June does the vertical velocity profile revert back to a purely thickening ice column through the course of the summer season, as evidenced by the increase in TSR‾ values , the curvature of the strain profile n Figure . Indeed,n Figure b as welln Figure c. After g Figure c. The des Figure .R2 > 0.90) outside the melt season, they were unable to capture fully the nonlinearity in strain behavior during late spring and early summer, as evidenced by the markedly lower R2 values through this period local variations in topographic setting;(b)local variations in basal traction; and(c)far‐field variations in glacier dynamics.Variations in the vertical strain rate within an ice column are often attributed to spatial and temporal variations in processes occurring at the base of the glacier. These can consist of changes in (i) basal topography, (ii) basal mass balance, influenced by the sediment and water content of basal ice, coupled with the presence and pressure of meltwater at the ice/sediment interface, and (iii) basal traction  is located over a local 3‐km‐wide depression within a regional bedrock high Figure c. This rR2 = 0.045; Figure Examination of the surrounding basal topography show that ice is flowing over a local depression Figure c. The ea5.2pi). Together with GPS measurements at the ice surface, the ice velocity exhibits moderate seasonal changes at S30, where small variations in basal water pressure were concomitant with large fluctuations in surface ice velocity and uplift. These observations suggest an inefficient basal hydrological system with water flowing both at the ice‐sediment interface and within the basal sediment layer itself, resulting in a situation where basal motion at S30 is sensitive to the influx of surface meltwater to the subglacial environment.Basal conditions at S30 have been examined through seismic analysis by Hofstede et al. , who obsp = 0.360; Figure p = 0.430; Figure Discrete ice flow acceleration events occurred during the melt season, when the influx of surface meltwater to the subglacial environment generated fluctuations in surface ice velocities and surface uplift  within the quadratic model was allowed to fluctuate (equation R2 > 0.75), we acknowledge that, during (i)  from July to late January and early February the following year. In late February or early March, we find vertical velocity to gradually induce vertical thinning in the upper half of the ice column, while the lower half continues to thicken. At this point the curvature of the best‐fit model for the observed strain begins to increase. Eventually, in the months preceding the onset of summer melt (April–June), the two different strain regimes become so different that model fails to capture the nonlinearity of the thickening deeper section (R2 ≤ 0.85). While we cannot fully explain this depth transition in strain regime, we infer that it is associated with a relatively thick layer of Wisconsin‐age ice present in the lowest ∼100 m of the ice column at S30 (Doyle et al., The distinct response of local strain at site S30 to temporal variability of ice flow taking place tens of kilometers away indicates that the transport of meltwater along subglacial drainage paths causes flow variations from the terminus all the way to 30‐km inland, thereby connecting site S30 with the fastest flowing sections downglacier Figure . From miSupporting Information S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.


In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα AMP-activated protein kinase (AMPK) is activated in response to intracellular energy depletion, e.g., during insulin resistance when cellular glucose uptake is limited—especially in contracting skeletal muscle  or in cu2+ levels and inducing depolymerization of the actin cytoskeleton methylene]-4,5-dihydro-4-oxo-2-thiazolyl]amino]benzoic acid , amurensin G , 5-((6-chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo [d]imidazol-2-yl)oxy)-2-methylbenzoic acid , or SNP was assessed in segments pre-contracted with phenylephrine to 80% of their maximal contraction due to KCl in the presence and absence of L-NAME. Relaxation was expressed as the percentage of phenylephrine precontraction. Removal of the endothelium was performed by intraluminal application of CHAPS  into the aortae. Aortae and carotid arteries were prepared free of adhering tissue and cut into 2.0-mm segments. Aortic rings were mounted in standard 10-mL organ bath chambers, stretched to 1 Human endothelial cells: Human umbilical vein endothelial cells were isolated and cultured as previously described [escribed  and usedMurine pulmonary endothelial cells: Mouse lungs were freshly processed as previously described [escribed .Adenoviral particles expressing the C-terminal Flag-tagged human full-length eNOS were used to transduce cultured umbilical vein endothelial cells as described previously .32PγATP was used to radioactively label the protein. Proteins were separated by SDS-PAGE, and the gel was exposed to X-ray film after drying. The eNOS wild-type or mutant proteins with C-terminal myc or Flag tags were overexpressed by transfection in HEK cells or adenoviral transduction in human umbilical vein endothelial cells, and after two days, cells were lysed and eNOS was immunoprecipitated by c-myc or Flag immunoprecipitation (IP). The immunoprecipitated proteins were used as a substrate for kinase assays with purified AMPKα1/β1/γ1 subunits  . The lysHuman umbilical vein endothelial cells  were transduced with lentiviral particles mediating the expression of Cas9 (Lenti-Cas9-2A-Blast was provided by Jason Moffat (Addgene plasmid # 73310)) and selected by blasticidin (10 µg/mL). Thereafter, a second lentiviral transduction with guide RNAs directed against AMPKα1  was performed, and puromycin (1 µg/mL) was used to select for double-transduced cells. The efficiency of the knockdown was analyzed by Western blotting. v/v); NaF, 25 mmol/L; Na4P2O7, 10 mmol/L; 2 μg/mL each of leupeptin, pepstatin A, antipain, aprotinin, chymostatin, and trypsin inhibitor, and phenylmethylsulfonyl fluoride (PMSF), 40 μg/mL). Detergent-soluble proteins were heated with SDS-PAGE sample buffer and separated by SDS-PAGE, and specific proteins were detected by immunoblotting. To assess the phosphorylation of proteins, either equal amounts of protein from each sample were loaded twice and one membrane incubated with the phospho-specific antibody and the other with an antibody recognizing total protein, or blots were reprobed with the appropriate antibody. Cells were lysed in Triton X-100 lysis buffer , 2 mmol/L; ethylenediaminetetraacetic acid (EDTA) 2 mmol/L; Triton X-100, 1% . Statistical evaluation was done using Student’s


Large, plume-like internal ice-layer-structures have been observed in radar images from both Antarctica and Greenland, rising from the ice-sheet base to up to half of the ice thickness. Their origins are not yet understood. Here, we simulate their genesis by basal freeze-on using numerical ice-flow modelling and analyse the transient evolution of the emerging ice-plume and the surrounding ice-layer structure as a function of both freeze-on rate and ice flux. We find good agreement between radar observations, modelled ice-plume geometry and internal layer structure, and further show that plume height relates primarily to ice-flux and only secondarily to freeze-on. An in-depth analysis, performed for Northern Greenland of observed spatial plume distribution related to ice flow, basal topography and water availability supports our findings regarding ice flux and suggests freeze-on is controlled by ascending subglacial water flow. Our results imply that widespread basal freeze-on strongly affects ice stratigraphy and consequently ice-core interpretations. Subsurface ice-sheet radar images reveal large plume-shaped bodies rising from the base, with their origin not yet understood. Here, the authors show that freeze-on of water at the ice-sheet base combined with ice-flux explains the vertical extent, shape and structure of the observed plumes. Over the past decade, the relevance of RES data has been widely recognised and spatial coverage over Antarctica and Greenland has been substantially increased by NASA’s Operation IceBridge4. Recent RES data provide internal ice layers at an improved vertical resolution over the whole ice-sheet thickness, permitting observations of the deepest layers6. What had been identified as mountains in previous RES profiles7 has now been recognised to be large-scale plume-like internal ice-layer structures, observed for example in large numbers in the Gamburtsev mountains, East Antarctica, as well as in Northern Greenland9  enables the detection of the bed and internal ice layers of an ice sheet, with the internal reflections, assumed to be isochrones, that correspond to former ice-sheet surfacesnd9 Fig. a. Such ind9 Fig. ; the for16. However, none of these model results reproduce the observed RES layer architecture over the whole depth of the ice sheet, concentrating on the dominant near-basal structures. Bons et al.14 have obtained some fold structure using a two-dimensional (2-D) structural model at crystal scale, and Wolovick et al.15 and Wolovick and Creyts16 produced overturned folds that resemble in parts the observed near-basal fold structures by applying transient changes in slipperiness. However, as previously shown by Leysinger Vieli et al.17, the layer structure over the whole ice column is of importance, since a local change in boundary condition or flow mode deflects the isochrones over the column, and leads to patterns that can look distinctively different for transects along or oblique to ice flow. This suggests that in order to evaluate a process by means of layer stratigraphy, both modelled and observed ice stratigraphy need to be compared over the whole ice column along transects of similar angle to ice flow.Only two of the suggested processes have been tested by means of numerical modelling11, and favours basal freeze-on of ascending water at the base of the ice as an explanation. Under the assumption of uniform, constant and low effective pressure Peff , the mechanism behind glaciohydraulic freeze-on is the pressure-induced depression of the melting point (termed here PiDMP), stemming from the Clausius–Clapeyron relation, which describes the relationship between pressure and melting point. As water flows uphill along the hydraulic gradient (this is possible for bed inclines no steeper than 11 times the surface slope18), reduction in ice overburden leads to an increase in its melting point temperature20. In consequence, water will freeze onto the base with the released latent heat keeping the temperature of the uphill flowing water close to its melting temperature.In Antarctica the occurrence of plume-like structures has been found to be strongly related to basal topography22. Neither do we know the basal topography well enough to evaluate if there are warm-based deeper parts surrounded by frozen areas24. In addition, does the community’s limited understanding of basal hydrology prevent us from predicting its thermodynamics consequences, in particular answering whether it is possible for meltwater channels to advance through freeze-on areas so as to connect isolated melt patches.The basal temperature of the Greenland ice sheet is only poorly constrained, as we do not know the geothermal heat flux or how it varies on a kilometre to 100 km scale. Analysis combining independent data are needed to obtain reconstructions for the basal temperatureHere, we examine the hypothesis of basal freeze-on and present the consequent isochrone layer architecture obtained using mathematical models of various complexities and spatial dimensions; the results closely resemble the layer architecture observed in RES profiles. Observed plume-like features map well with locations that are favourable in topography for the PiDMP process and match areas where water flow is likely, additionally corroborating the hypothesis. We further establish that low ice flux and contrasts in ice rheology are conducive to larger plumes. Our findings highlight important implications of the basal freeze-on process for ice dynamics and ice-core drilling.23, which affects both Φ and the computed water path). According to theory, PiDMP depends dominantly on basal water flux and topography, whereas the thermal state of the ice is of secondary importance. Our spatial analysis thus supports the hypothesis of basal freeze-on due to ascending water for Greenland.Although in Greenland the relief of the subglacial topography is less pronounced than in Antarctica, we find that plume-like structures  are predominantly located on the flanks of the main subglacial basin in regions of moderate ice flow and surface slope Fig. . To veri25 . We simulate local freeze-on by adding mass at the ice-sheet base. The geometrical setting for a typical plume  while assuming no basal sliding.Here we test by numerical modelling the hypothesis of basal freeze-on causing plume formation and investigate how such accreted ice at the ice-sheet base influences the internal layer architecture. We use a forward, 3-D, time-dependent, numerical ice-flow model conceived as a stream-tube model  using finite differences. It uses simplified mechanics based on the shallow-ice approximation (SIA), which ignores all horizontal deviatoric stress gradients and is therefore based on the assumption of small horizontal deviatoric stress gradients. The model is capable of continuously tracking tracers and isochrone layers within the iceume Fig.  is takenFirst, we ran our model to reach the steady-state solution for the undisturbed case of uniform surface accumulation without basal accretion, which results in surface and bed parallel internal layers and a non-linear age-depth relationship Fig. . Followiume Fig. . Subsequmentclass2pt{minimume Fig. .The internal meteoric ice-layer architecture follows the plume shape. Typically, near-basal internal layers are bent sharply upwards at the upper end of the plume, and folded at the downstream end of the plume, resembling the observed RES structure  model solving the equations describing the mechanics of ice flow (full-Stokes equations) and a freely evolving surface (see Methods section ‘2-D FS ice-flow model’). By solving all stress gradients, the FS model avoids the difficulties associated with the simplified mechanics of the SIA model. As the FS model comes at a high computational cost, we use it here in plane flow only, for validation purposes. These additional experiments confirm the plume dynamics and isochrones patterning obtained by the SIA-based model Fig. .Fig. 4FuA further complication is the jump in age that would occur in nature between the fresh accreted ice and the oldest meteoric ice. For both SIA and FS models this jump leads to numerical diffusion of age. In order to follow the age discontinuity and to test independently the isochrone structure obtained by solving the age advection equation , we traQ) can be expressed by the balance flux (B), which is the sum of the accumulation rate .Fig. 5PQ consists of ice flux arising from meteoric accumulation (M) and from freeze-on (F). In a vertical ice column in plane flow, the total ice flux Q is the sum of the meteoric ice flux M, which comes from the surface, and the freeze-on (or accreted) ice flux F, which originates at the bed  We define the fraction of the freeze-on flux component as the ice-flux ratioh; see Methods section ‘Ice-flux relationship with basal freeze-on’, Eq. , and isn’, Eqs.  and 12)Q consiston’, Eq. ).F increases only over the accretion area for both the transient and the steady-state case, while the meteoric ice flux M is steadily increasing  is proportional to R .Of secondary importance for the plume height is the distribution of the horizontal speed with depth 14). For o R Fig. . For thepth Fig. . This plB  comes from the meteoric ice flux M inducing in general a decrease in the ice-flux ratio R along flow . This effect is amplified in regions with small surface-accumulation areas, which further reduce the meteoric ice flux M; such regions exist for instance near ice divides or where flow is divergent, typically upstream of rising beds29. Downstream, plume heights generally decrease owing to the increasing meteoric ice flux M, which is a consequence of both contributing accumulation area and flow convergence. In addition, basal sliding becomes more important downstream, affecting the distribution of horizontal velocity with depth, thus further decreasing the plume height , since where meteoric ice flux ght Fig. .31. Predicted freeze-on rates are in general very low  have been previously suggested as a result of PiDMP, controlled by the bed slope (αb), surface slope (αs), and the water flux (Qw) when αb has opposite sign to αs20 . The PiDMP process leads to an increase in freeze-on rate Qw and bed slope αb, and to a decrease in αs20.In general, subglacial hydrology at the base of the ice sheet is poorly constrained as a consequence of the logistic difficulties of achieving sufficient observations. However, recent modelling studies suggest the existence of major meltwater pathways, subglacial lakes and, related to the mechanism described here as PiDMP, widely occurring basal freeze-onQw is unmeasured, we use the relationship between the bed and surface slope to quantify the effect of PiDMP on the freeze-on rate S = αb/αs, and find that for slope ratios ranging between −2 ≥ S ≥ − 11, the freeze-on rate Qw but also for increasing αs  to tens of centimetres (for large Qw) and even metres per year (for both large αs and Qw). As the calculated bed and surface slope depend heavily on the grid size used, it is readily conceivable that locally conditions arise in Φ and Qw, which lead to freeze-on rates comparable to the ones used in our experiments . The efnts Fig. .Fig. 7CaS has the consequence that water can flow up steeper adverse bed slopes αb > 0 for a steeper inclined surface slope αs < 0 (or vice versa), leading to a greater PiDMP effect owing to larger spatial gradients in overburden and thus melting point. This dependence on slope is of importance in the interior of Greenland, where the ice-sheet surface in general exhibits larger slopes (αs ≤ − 0.4° or αs ≤ −0.007) than in Antarctica (αs ≤ −0.2° or αs ≤ −0.0035)32, allowing for larger freeze-on rates Qw  and moderate ice velocities . Since the surface slope is nearly flat, Φ is also low (<10−3), commonly leading to bed slopes that are too steep for water to flow uphill, and restricting plume formation. Away from the divide, the first large plumes (>H/3) start to form in regions where the basal meltwater is likely convergent, leading to an increasing basal water flux Qw, with the total ice flux Q and freeze-on index Φ still being generally small. Further along, towards the margin both Φ and Qw tend to increase (while Q is still moderate) and allow for larger plume heights. In regions of fast flow (large Q), the available basal water flux and Φ are in general too low to produce freeze-on rates R required for large plumes. These observations fit well with the modelling results by Dow et al.31 producing larger freeze-on rates along important water flow paths and towards the margin.A detailed observational analysis for Greenland locates large plumes along possible water paths that cross areas of high freeze-on indices . Towards the onset of fast flow, the existing plume-like structures concentrate due to convergent flow ; Figs. 33.The enhanced availability of basal meltwater generated from basal sliding and water flow, combined with adverse bed slopes, is key to the formation of large plumes in regions of moderate ice flow. Meanwhile, the along-flow reduction of water flux M and, as a consequence, the total ice flux Q (hence increasing R) and high availability of surface meltwater reaching the bed34, larger plumes are again possible.In fast-flow areas, the large ice flux together with increased basal sliding  prevent in general high-rising plumes Fig. . An exce9, reflecting different ice temperature and fabric, and consequently affecting the relationship between ice-flux ratio R and relative height h  for casA) for ice flow36  by factors of 0.1, 0.5, 2 and 10 in four separate experiments we increase or reduce the stiffness of the accreted ice respectively, in order to compare it with the base  case, which uses an uniform viscosity throughout the ice sheet. For the same amount of accreted ice with increased stiffness (factors 0.1 and 0.5) we find that plumes as expected rise higher than in the base case , plume structures can be formed by the accretion process.To investigate the effect of a rheology contrast between the meteoric and accreted ice on plume growth, we perform a simple experiment, using the FS model. We use the same experimental set-up as described earlier, with the exception of a modified accreted ice viscosity. By changing the rate factor  and softer ice (downstream of accretion area), freeze-on rates 4 from 2010 to 2014 over northern Greenland  and a cross-track resolution ranging between 420 and 740 m25. As it uses the SIA, which ignores all horizontal deviatoric stress gradients, we use an FS model, which solves all stress gradients (full-Stokes equations) and therefore gets around the difficulties of the SIA model. Owing to its high computational cost, we use the FS model in this paper only in plane flow and the SIA-based model mainly in two dimensions.In this paper, the model used in general to investigate the ice-sheet isochrone architecture is a forward, 3-D, time-dependent, finite-difference stream-tube model (surface and bed are fixed in time), using simplified flow physics, capable of calculating tracers and isochrone layers within the ice25.As an input, the model requires surface and basal topography, accumulation and melt (freeze-on) rate to calculate the velocities using balance fluxes, prescribing the velocity distribution in the vertical by a shape function. Here we use a surface that is fixed in time, and a shape function for velocity and ice flux that arise from the SIA and prescribe balance fluxes at the inlet marginal boundaries (Dirichlet boundary condition)38 solving the Stokes equations describing ice-flow mechanics . We use this computationally much more expensive flow model in two dimensions only to compare the internal layers qualitatively with the ones obtained by the simplified model. To avoid the comparison of code dependencies, we ran the finite-element Elmer/Ice code both by solving for the SIA and the FS using a fixed surface—as for the case of the simplified model, applying Dirichlet boundary conditions as before. Additionally, we ran the FS with a freely adjustable surface. The differences in the modelled 2-D isochrone structures are small for both FS experiments, with largest differences at the ice divide and, for the free-surface case, towards the surface  for the 2-D vertical plane-flow model or r =  in the 3-D space, with z pointing upwards, and the time coordinate t. The horizontal coordinates are denoted by μ = (x) and μ = , respectively. The surface is given by z = s, the bed by z = b, and the total ice thickness by H = s − b.The ice flow is calculated using a Cartesian coordinate system u is the ice-flow velocity vector, X the age, Q =  the total horizontal ice flux, which is the same as the balance flux B in steady-state, M =  the ice flux arising from meteoric accumulation, F =  the ice flux arising from freeze-on, The relevant equations for the steady-state stream-tube model are:By adding Eqs.  and 5)  5) and cX are obtained by solving the time-dependent age equation:Transient internal layers of age −1 39) as background melt rate, except along freeze-on patches.To avoid arithmetical issues that arise in steady state from predicted infinite ages at the ice-sheet base, we prescribe a small basal melt rate  = B(x) in steady state.At any position us can be split into the contribution coming from basal sliding ub and ice deformation ud. For the SIA , the surface velocity isτb = ρgHαs is the basal shear stress, A is the rate factor and n the flow-law exponent (used here as n = 3)36. It follows that the partial flux z isThe surface velocity Q between the bed and the surface z = H is thereforeThe total flux ud = 0), and internal deformation, ID , the depth-averaged velocity For the specific cases of uniform plug flow, PF /H and the normalised partial flux ω = q(ζ)/Q40, obtained by dividing Eq. . For un(see Eq. ) originah, given the flux ratio, R, from the balance flux components originating from freeze-on F and M ,,16), resQ = 6000 m3 a−1 m−1 using Eq. (R = 0.0035 (solving ω(h) in Eq. . DividiGw is the heat generated by subglacial water flow, Gs is the heat generated by sliding, Gg is the geothermal flux, Gp is the heat required to maintain the flowing water at pressure melting point, and Gf is the heat released by freezing or used by melting; the heat Gc conducted away in the ice is ignored in this paper. The signs of all the G components are consistent, that is, a positive sign is heat being added to the system, a negative sign is removal of heat. By ignoring Gc we underestimate the freeze-on rate.It is beyond the scope of the paper to calculate the ice-sheet heat balance at the base including the effects of surface accumulation, but the calculation or reliable estimation of G components (labelled with corresponding subscript), we obtain a general expression for the freeze-on rate L is the volumetric latent heat of freezing, Qw is the water flux, ∇ψ is the hydrostatic-potential gradient, T the basal tangential traction, u is the ice velocity, kr and ki is the conductivity of the rock and ice material respectively, ∂zθ is the vertical temperature gradient, and b−,b+ refer to elevations an infinitesimally small distance below and above the bed respectively. Further, Qw = Qw ⋅ ∇ψ/|∇ψ| is the water flux taken along the steepest gradient of the hydrostatic potential, J is the volumetric heat capacity of water, C is the Clapeyron constant for water, ρi is the ice density, g is the gravitational acceleration and ∂xH = (αs − αb), with αs and αb as the surface and bed gradient, respectively, is defined along the steepest surface gradient. Note the relationship of the water flux Qw = −κ∇ψ with the hydraulic conductivity κ along the hydrostatic potential, ensuring that Gw > 0. However, in this paper we do not need to know κ, since we assume a basal water pressure Pw that is near or at the ice overburden pressure Pi = ρigH leading to a spatial uniformity of the effective pressure (defined as Peff = Pi − Pw).Rearranging Eq.  using thGc is generally negative; terms p and c can, if sufficiently large, permit Gc we underestimate the freeze-on rate.The terms in Eq.  in the f20, we express the freeze-on rate Qw, bed and surface slope αs and αb, respectively, along the steepest gradient. Further, we assume the ice temperature at the contact to the bed to be at the pressure melting point and that the released latent heat from freeze-on is warming the basal water, maintaining it at the new pressure melting point, and thus allowing it to flow up the slope. Using Eq. (Gp becomes negative) and neglecting Gc, L = 3.06 × 108 J m−3 and cos(atan αb) transforms the result αb, to the z-coordinate. As αb in our calculations is so small (cos(atan αb) is nearly one) it can be omitted.  The geothermal heat flux Gg used ranges from 0.03 to 0.11 W m−2 and the heat of sliding Gs is calculated using the surface velocity us to describe the sliding velocity at the base of the ice sheet, thus overestimating Gs, so thatτb is the basal drag, the ice density ρi = 916 kg m−3 and the gravitational acceleration g = 9.8 m s−2.Following Alley et al.sing Eq.  on an adGw and Gp in Eq. (xH as (αs − αb), followed by replacing bed slope with αb = Sαs, we obtain for the water-flow heat sourceρw = 1000 kg m−3 is the water density, the volumetric heat capacity of water J = 4.2 × 106 J m−3 K−1, the Clapeyron constant C = −7.4 × 10−8 K Pa−1, and Expressing the heat terms p in Eq.  for planGp and Gw in Eq.  and ratios S > = −5, even for increased ice flow (i.e. 100 m a−1). For large water fluxes (i.e. Qw = 0.11 m3 s−1 m−1 width); however, the difference Gp − Gw is one to two orders of magnitude larger  and introducing Eqs. Gp and GwS, while the heat emitted by Comparing the non-dimensional heat terms αb and αs. However, the interpolation of the RES data to obtain the bed topography leads to large vertical errors in bed elevation exceeding 600 m in regions of sparse radar data . A minimum error of 200 m allows us to nearly continously visualise all used radar transects leading to a minimum transect width of ≈4 km in regions of sparse data. Increasing the minimum transect width to 5 and 10 km leads to a maximum error of 225 and 300 m, respectively. Note that Φ and the water path are consistent as the same data set for bed and surface is used, so that with each improved topography data set the Φ and water paths become more reliable.The calculation of the freeze-on index Φ depends highly on the bed and surface slope 41 with a 3 or 5 km step size and starting points (seeds) at a regular spacing (e.g. every 12 km) on a 1050 m gridded Greenland ice-sheet surface and bed topography26 .To visualise the direction in which water tends to flow in North Greenland, we use a simple approach by assuming that water is available everywhere and that it freely follows the steepest gradient of the hydrostatic potential. This is done by using the MATLAB streamline functionGg) ranging from 70 to 106 mW m−2 21. Within this zone, high basal melt rates have been inferred from internal layering along the ice ridge (around the NGRIP ice-core drill site), ranging between 0.005 and 0.02 m a−139, and towards the onset of NEGIS on local spots leading to rates exceeding 0.1 m a−1 42. Comparing our mapped plumes with a composition of areas with a temperate bed22, we find that most plumes are within areas of a ‘likely thawed’ or ‘uncertain’ bed, with water paths seeded from both areas reaching most plumes , transporting the subglacial water, have their source in the central region of the ice sheet Fig. , the mai3 km3). We assume an elevated basal melt rate ranging between 0.01 and 0.02 m a−1 over this catchment area, as it lies within the inferred zone of elevated geothermal heat flux21 and is affected by the high melt rates around NGRIP (west margin)39. To obtain the source area, we seed at 1050 m along the 6 km accretion length (observed in RES profile) and 15 km plume upstream along ice flow, and calculate streamlines along the reversed (upwards) hydraulic gradient using steps of 5 km  km a−1 = 0.1 km3 a−1 = 3.2 m3 s−1 and 0.2 km3 a−1 = 6.3 m3 s−1, respectively. This quantity does not include water flux from melt production (Gw and Gs) along the flow path.For the outlined catchment area and the relevant basal melt rates, we obtain a total water flux of 1043  from itF∕Q  passing at a flux of Qw = 0.11 m3 s−1 (water velocities of 0.11 m s−1). Observations inferred from RES data of Thwaites Glacier in Antarctica suggest that there are both channels and distributed conduits44. However, a distributed hydraulic system seems more likely, as on an adverse slope, steep enough for freeze-on to occur, a channelised system is not sustainable when modelling the subglacial hydrology of an overdeepening, and it therefore shuts down45.Finally, assuming a water body height of 1 m, we obtain maximum channel widths ranging between 29 and 58 m for the available basal meltwater flux  with the averaged results for 10,000 randomly chosen locations. For the reversed streamlines calculated on a 1050 m grid, as used in this paper, we find the best match between plumes and Φ areas for the large plumes, followed by the small plumes and the random set, which are reduced by up to 8% and a further 9%, respectively . Consequently, we argue that the plume distribution is related to the pattern in Φ, since if in the observational data set the plumes are randomly distributed we would expect a similar mismatch between the data sets.To quantify the relation between plumes and areas with freeze-on index, we follow streamlines along the reversed (surface) ice-flow gradient starting at each plume, and determine if they cross areas of freeze-on index Φ. Further, we compare the results between the mapped plume sets . Larger RES mapping errors for small plumes (see Methods section ‘RES data’) partly explains the better result for plumes >H/3.Calculating reverse streamlines on a 150 m gridSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen. Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections.Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is  Acinetobacter baumannii. Importantly, differences in ribosome structure among prokaryotes can potentially be exploited to develop therapies that selectively target pathogens and leave protective normal flora intact, thus avoiding subsequent therapy-associated colonization with resistant pathogens and morbid diseases such as Clostridium difficile colitis.The ribosome represents a major target for antimicrobial agents as it is vital for protein synthesis and is one of the largest molecular machines in the cell . AlthougBacterial 70S ribosomes consist of two subunits, which are the 50S and 30S , 4, 5. TAcinetobacter baumannii 70S ribosome. A. baumannii has emerged as one of the most problematic and highly antibiotic-resistant superbugs  structures of three different conformational states of the A. baumannii 70S ribosome. Extensive classification gave rise to five different classes within this single data set  in each 50S subunit of these 70S ribosomes. We also resolved the small 30S subunit of these three 70S ribosome structures. Based on the high-resolution cryo-EM maps, we identified the 16S rRNA and 20 r-proteins in each 30S subunit.We used cryo-electron microscopy (cryo-EM) to determine high-resolution structures of the data set . The firdata set . These tdata set . Like otThe fourth ribosomal class contains only the disassembled 50S subunit  and 2. TE. coli 70S ribosome structure. Thus, an fMet molecule was included at the aminoacyl end of the P-site-bound tRNA  Expanded region showing density corresponding to an amino acid (modeled as fMet) at the aminoacyl end of the tRNA near the peptidyl transferase center (PTC) and exit tunnel. Download FIG S1, PDF file, 0.5 MB.Aminoacylated P-site tRNA. (A) P-site occupied structure of the Copyright © 2020 Morgan et al.2020Morgan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In comparison with the P-site-occupied, E-site-occupied, and empty structures, there is only a subtle difference in the overall conformation of individual rRNAs and r-proteins. A pairwise superimposition of the 23S rRNAs from these three ribosome structures gives rise to a range of root mean square deviations (RMSD) between 2.0 and 2.6 Å. However, the relative orientations of the 50S and 30S subunits are quite distinct among these structures, along with the rotation of the 30S head between P-site-occupied, E-site-occupied, and empty structures . As an aUpon examining the 70S structures, it is noticeable that there are significant differences in intersubunit interactions between the three conformations. Most notably, changes are seen in contacts between the A-site finger (H38 of the 23S rRNA) and r-protein S13 (30S subunit) alongside interactions between r-protein L5 (50S subunit) and r-protein S13 . These iEscherichia coli  in cryoSPARC, version 2 . In eachThe A-site finger (H38) of the 23S rRNA and r-protein L5 from the 50S subunit have been previously shown to form an important bridge with the 30S subunit . In the Studying the 3D variability analysis results of the E-site-occupied particle set showed that the interaction is indeed transient due to the extensive motions of the 30S head. The ratcheting and rolling motions of the 30S move the interaction sites of the A-finger helix and r-protein L5 with S13 up to 18 Å away . This isLast, the ability of this technique to identify partially populated regions of the final EM map was examined  Final map of the E-site populated ribosome. (B) Ribosomal protein S2 (cyan) refined to a notably lower resolution than the rest of the structure. This was revealed to be due to partial population through 3DVA. (C) Focused classification of this protein in Relion and reconstruction allowed for a complete density to be achieved. (D) The protein refined to a threshold and resolution similar to levels for the rest of the ribosome map. Download Copyright © 2020 Morgan et al.2020Morgan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. baumannii ribosome have allowed us to identify several unique static and dynamic subunit and substituent interactions. Taken together, these observations contributed to our understanding of shared and unique attributes of A. baumannii ribosomes that might be exploited for future development of ribosome-based therapeutics.In summary, our cryo-EM structures of the A. baumannii ribosomes were purified directly from A. baumannii strain AB0057. The cells were reconstituted in fresh ribosome lysis buffer  and lysed using a French pressure cell. To pellet insoluble material, the crude lysate was centrifuged at 30,000 × g for 30 min. The resulting supernatant was then carefully layered on top of a sucrose cushion buffer (lysis buffer with 1.1 M sucrose) and spun for 16 h at 100,000 × g. This resulted in a ribosome-containing pellet that was stored at –80°C until further use.s buffer  (20 mM T4Cl) and examined using negative stain to test for purity. While the sample was largely pure, there was a significant population of 50S and 30S ribosomal particles alongside the full 70S particles. Therefore, it was determined that a second purification step was favorable to remove some of the smaller subunits. This was accomplished through the use of sucrose gradient purification. Two sucrose buffers were prepared using ribosome buffer  with 10% and 40% sucrose and mixed using a gradient maker. After the solution was cooled for >2 h, a portion of the crude ribosomal pellet was resuspended using ribosome buffer and layered onto the gradient and then centrifuged for 16 h at 100,000 × g. The resulting solution was fractionated by pushing a 60% sucrose gradient from the bottom of the tube, and fractions were analyzed using a plate reader. Fractions containing 70S ribosomal particles (largest peaks) were collected and exchanged into buffer containing 20 mM HEPES-KOH, 10 mM MgOAc, and 100 mM KCl using 100,000-molecular weight-cutoff Amicon centrifugal filters, flash frozen in liquid nitrogen, and stored at –80°C until further use.A small portion of sample was reconstituted in ribosome resuspension buffer  and standard protocols . Once sa2  with a GIF energy filter (20 eV). Data were collected in superresolution mode at defocus values between −1.0 and −2.5. Microscope nominal magnification was ×130,000, providing a physical pixel size of 1.064 Å/pixel (0.532 Å/pixel in superresolution). This provided 1,528 micrographs for data analysis.The NCI’s cryo-EM service was utilized for high-resolution data collection, providing 24 h of collection time in a Titan Krios (300 keV) equipped with a K2 Summit camera. Data were collected over 40 frames for 9 s at a nominal dose of 40 e/Åab initio reconstruction (5 classes), separating individual subunits  from the assembled ribosomal particle  and junk (1 class).All data were processed using a combination of cryoSPARC, version 2 , and Relab initio classification using 8 classes to inspect the particles for any differences. Along with motions between the 50S and 30S ribosomal subunits as expected, three different tRNA populations were discovered in the single data set, P site occupied, E site occupied, and empty. To focus classification based on tRNA population, all particles were recombined and underwent ab initio reconstruction, followed by homogeneous refinement. At this stage, clear density for E-site tRNA was present while there was no clear density for P-site tRNA as the E-site tRNA population was found to be the most populated. Therefore, a mask of the E-site tRNA was created. The particle set then underwent 3D focused classification without particle alignment in Relion. Csparc2star.py from pyEM was used to create the .star file from .cs files. Separating populated and unpopulated particles, the unpopulated particles underwent another round of ab initio reconstruction followed by homogeneous refinement in cryoSPARC, showing clear density for P-site tRNA. The 3D focused classification process was repeated with this P-site tRNA, resulting in a populated and unpopulated class. This entire process resulted in the 3 classes solved, P-site-occupied, E-site-occupied, and empty ribosomal particles.The three 70S classes were recombined and subjected to another round of The particles were reextracted to full resolution and refined using homogeneous refinement in cryoSPARC. While the particle set was nominally high resolution, significant blurring of the 30S was seen due to interparticle dynamics between the 50S and 30S in the assembled particle, as expected . TherefoE. coli ribosome (PDB accession number 5AFI)   as a stare, MDFF  from there, MDFF  simulatire, MDFF .E. coli ribosome structure (PDB accession number 5AFI)   as a moder 5AFI) . In regiA. baumannii Met-tRNA was modeled in its place. The P-site tRNA and E-site tRNA molecules from 5AFI were used as starting models and mutated to the A. baumannii fMet-tRNA sequence. Moreover, three residues from an mRNA were clearly present to pair with the anticodon of tRNA, and therefore this density was modeled as 5′-AUG-3′ to complement the Met-tRNA.While not manually added during creation of the sample, tRNA was clearly present in the final densities and was therefore modeled. Since it is not possible to determine the exact tRNA that is present in the structure and also unlikely that there is only one tRNA since it came along from the cellular mixture, the 2+ ions were manually added in Coot using the unmodeled blobs function at a threshold of 1.8 RMSD and the locally filtered maps to avoid placing atoms in noisy regions. After addition of Mg2+, water molecules were added using the “find waters” function in Coot. Two additional ions, Na+ and Zn2+, were also added.MgOnce modeled, structures were refined in Phenix . A singlDocking experiments were performed with AutoDock Vina  using ni24–To examine the overall flexibility and dynamics of the assembled ribosome, the three different structures, P-site occupied, E-site occupied, and empty, were studied using 3D variability analysis in cryoSPARC, version 2 . To saveA. baumannii P-site-occupied, E-site-occupied, empty, 50S, and 30S ribosome structures have been deposited in the RCSB Protein Data Bank under accession codes 6V39, 6V3A, 6V3B, 6V3D and 6V3E and in EMDB under accession numbers EMD-21030, EMD-21031, EMD-21032, EMD-21033, and EMD-21034, respectively.Atomic coordinates for the


However, the rest of compounds among the targeted series exhibited a low to moderate enzyme inhibition potential. To better understand the compounds’ underlying mechanisms of the inhibitory effect (3a and 4a) and their most active metal complexes (3b and 4b), we performed an enzymatic kinetic analysis using the Lineweaver–Burk plot in the presence of different concentrations of inhibitors to represent the non-competitive inhibition nature of the compounds, 3a, 4a, and 4b, while mixed type inhibition was represented by the compound, 3b. Moreover, molecular docking confirmed the binding interactive behavior of 3a within the active site of the target protein.The present study focuses on the design and synthesis of a cage-like organic skeleton containing two triazole rings jointed via imine linkage. These molecules can act as urease inhibitors. The in-vitro urease inhibition screening results showed that the combination of the two triazole skeleton in the cage-like morphology exhibited comparable urease inhibition activity to that of the reference thiourea while the metallic complexation, especially with copper, nickel, and palladium, showed excellent activity results with IC The development of competitive and non-competitive enzyme inhibitors based on a metalloskeleton has attracted wide interest of the scientific community because metal ions are found in the active sites of a large number of metalloproteins, such as hemocyanin, and also in metalloenzymes, like ureases, tyrosinase, laccase, and ascorbate oxidase ,5,6,7,8.−  assay. The results showed no toxicity for L-929 cells at 50 μM ligand concentrations. The safe profile of the ligand towards the living cells might open the ligands’ possibility as bioimaging probes. The safe nature of the triazole skeleton warrants further characterization of the compound. 3a-f and 4a-f, for their inhibitory effects on urease. Besides current urease inhibitors . Tw. Tw17]. 3a for binding conformation inside the active site of jack bean urease. Docking and fitting (3a) calculated a binding energy value of −10.40 kcal/mol. The 3a-docked complex showed that compound 3a was enclosed in the active site of the jack bean urease. Compound 3a formed four active hydrogen bonds with the protein active site. The carbonyl oxygen atom on the triazole ring was H-bonds with Arg439 residue with bond lengths of 2.20 and 2.46 Å, respectively. Similarly, the triazole N2 hydrogen likely interacted with Ala636 through hydrogen bonding, having a bond length of 2.19 Å. Moreover, the carbonyl oxygen formed another hydrogen bond with Arg609 with a bond length of 2.19 Å , H2SO4, acetic acid, and HCl  were used in this experiment. Urease from jack bean (EC 3.5.1.5), Thio-urea, sodium nitroprusside, and active chloride were purchased from Sigma . Stock solutions of the reducing substrates were prepared in phosphate buffer . The 4-methoxybenzoic acid, pyridine-2,6-dicarbaldehyde, phosphorous oxychloride, carbohydrazide, isocyanate and isothiocyanates, Hydrazine hydrate (80%), TEA, CSf values were determined with pre-coated silica gel aluminum plates, Kieselgel 60 F254 from Merck . TLC plates were visualized under a UV lamp . The melting points were determined on a Fisher Scientific  melting point apparatus. The FT-IR spectra were recorded in KBr pellets on a Shimadzu FTIR–8400S spectrometer . Proton and carbon nuclear magnetic resonance (1H-NMR and 13C-NMR) spectra were recorded on a Bruker Avance 400 MHz spectrometer with TMS as an internal standard. The chemical shifts are reported as δ values (ppm) downfield from the internal tetramethylsilane of the indicated organic solution. Peak multiplicities are expressed as follows: S, singlet; d, doublet; and m, multiplet. Mass spectra were recorded on the AB SCIEX Co. 4000 QTRAP LC/MS/MS System. The UV-visible absorption measurements were carried out using [SCINCO] UV-Vis Spectrophotometer “S-3100” . Abbreviations are as follows: CD3OD, deuterated methanol; DMSO–d6, dimethyl sulfoxide-d6, and FT-IR spectroscopy, Fourier transform infrared spectroscopy. The reaction progress was monitored by thin layer chromatography (TLC), and the R2 was prepared according to our previously reported procedure  solution. Following 2 h of incubation at 37 °C, growth medium was removed gently from the plate and 200 μL/well dimethyl sulfoxide was added to solubilize the produced purple formazan crystals. Later, the absorbance for each well was measured at 570 nm using microplate spectrophotometer systems  and results were calculated in percentage with respect to the untreated sample called the control. The same procedure was adopted for all the compounds and their metal complexes.MTT assay was done following the reported procedure . Briefly2HPO4, 1 mM EDTA, and 0.01 M LiCl2, pH 8.2), were incubated for 30 min at 37 °C in a 96-well plate. Briefly, 50 μL each of phenol reagents  and 50 μL of alkali reagent  were added to each well. The absorbance at 625 nm was measured after 10 min, using a microplate reader . All reactions were performed in triplicate. The urease inhibition activities were calculated according to the following formula:control and ODsample represents the optical densities in the absence and presence of the sample, respectively. Thiourea was used as the standard inhibitor for urease.The urease activity was determined by measuring the amount of ammonia produced with the indophenols method previously described ,27. The C. ensiformis) was accessed from the Protein Data Bank (PDB) (www.rcsb.org) with PDBID 4H9M. The selected protein structure was minimized by employing UCSF Chimera 1.10.1 [The three-dimensional protein structure of jack bean urease  was sketched in the ACD/ChemSketch tool and accessed in the mol format. Furthermore, the UCSF Chimera 1.10.1 tool was employed for energy minimization of 3a having default parameters. The steepest descent steps were adjusted to 100 with a step size of 0.02 (Å), conjugate gradient steps also fixed to 100 with a step size of 0.02 (Å), and the update interval was retrained at 10. Finally, Gasteiger charges were added using Dock Prep in the ligand structure to obtain the good structure conformation. The molecular docking experiment was run through the PyRx virtual screening tool with the AutoDock VINA Wizard approach [3a-docked complex were accomplished by Chimera 1.10.1.Based on the in vitro results, approach ,33. The 3a, 4a, and 4b, while mixed type inhibition by the compound, 3b. Further, molecular docking studies showed that 3a interacted within the active region of jack bean urease and exhibited a good binding energy value of −10.40 kcal/mol. These combined results showed that synthesized molecules can serve as structural templates in the design of novel drugs against the urease enzyme.In this study, we joined two triazole rings to form a series of Schiff base derivatives into a cage-like structure. Then, we evaluated their urease inhibition activity as well as their underlying mechanism for inhibition with enzyme kinetics studies. Among the tested series, copper, nickel, and palladium complexes displayed appreciable enzyme inhibition results, while the rest showed low to moderate enzyme inhibition potential. The kinetic analysis by the Lineweaver–Burk plot suggested the non-competitive inhibition nature of the compounds,