Despite the abundance of available software tools, optimal particle selection is still a vital issue in single-particle cryoelectron microscopy (cryo-EM). Regardless of the method used, most pickers struggle when ice thickness varies on a micrograph. IceBreaker allows users to estimate the relative ice gradient and flatten it by equalizing the local contrast. It allows the differentiation of particles from the background and improves overall particle picking performance. Furthermore, we introduce an additional parameter corresponding to local ice thickness for each particle. Particles with a defined ice thickness can be grouped and filtered based on this parameter during processing. These functionalities are especially valuable for on-the-fly processing to automatically pick as many particles as possible from each micrograph and to select optimal regions for data collection. Finally, estimated ice gradient distributions can be stored separately and used to inspect the quality of prepared samples.  •Develop a software tool for image segmentation based on estimated ice thickness•Present a method to detect and annotate ice contamination in the dataset•Show a procedure to equalize contrast on the micrographs with the non-uniform ice•Demonstrate a workflow to identify optimal ice for data collection/particle selection Olek et al. present a software tool, IceBreaker, for handling non-uniform ice thickness in cryo-EM micrographs. Ice thickness is believed to be a crucial factor that affects the quality of cryo-EM reconstructions. IceBreaker provides empirical estimation of the ice distribution and introduces an ice thickness parameter to the cryo-EM processing pipeline. Advancements in cryoelectron microscopy (cryo-EM) instrumentation, detector development, and data processing algorithms have allowed reconstructions to be obtained at atomic resolution . The finThe ideal setup for single-particle analysis would have the particles distributed in a thin, vitreous ice layer. The surface of the ice in the data collection areas should be flat and normal to the electron beam. Particles should occupy most of the grid holes, be oriented randomly, and not overlap with each other . Areas wOne main shortcoming common to most of the state-of-the-art automated tools is the fact that they do not take into consideration the fact that particles distributed in different ice thickness regions may have different quality and features. After the processing, most of that information, which could lead to the improvement of the final resolution, cannot be recovered. Currently there is no software tool that allows the user to easily connect the ice thickness parameter with the quality and state of the particles in different areas of the prepared sample.In this work, we present a software tool, IceBreaker, for the ice thickness estimation and digital ice gradient removal on the cryo-EM micrographs. The software allows the segmentation of the micrographs and grouping areas with similar ice features. It can be used for local image processing as filtering or contrast enhancement, as well as annotating and removal of the ice contamination and/or carbon film fringes. Importantly, it introduces the empirical ice thickness parameter that can be associated with each particle based on the picked coordinates. The described tool can be used as a stand-alone image processing software or as an external job in the integrated Relion workflow .The IceBreaker software allows segmentation of the cryo-EM micrographs based on the distribution of the pixel intensities recorded by the detector. The term “estimated ice thickness value” is introduced to describe and group the areas of the micrograph with similar pixel intensities. This information can be exploited during the later stages of the cryo-EM processing pipeline; e.g., particle picking, 2D classification, or 3D refinement. An overview of the workflow is presented in Input data: the required input is a set of motion-corrected cryo-EM micrographs. The IceBreaker can be run as an external job of the Relion project or as a stand-alone tool from the command line. It can be used as a part of the data collection pipeline or performed on historical data. Various tools for motion correction  can be iStep 1. Pre-processing: filtering and feature flattening: the 20 Å low-pass filter is applied to each micrograph to remove the high-frequency noise and reveal features such as particles, ice contamination, foil hole edges, and the ice gradient. Then, the micrograph is divided into a pre-defined number of patches: 40 in x and 40 in y direction, which is independent of the size of the micrograph. Within each patch, an average value of pixel intensities is calculated. This way local features are reduced to 1/1,600 of the micrograph area on top of the initial 20 Å filter. In our test cases, this was sufficient to reveal trends and low-frequency changes in the background, which represent the changes in the ice thickness. Additionally, the super-pixels represented by each patch can be used to reduce the size of the micrographs and improve the computation speed. Micrographs processed this way are used as input to the next stage of the processing.Step 2. K-means clustering: the K-means clustering algorithm is used to group together the areas of the rescaled, feature-flattened micrograph with similar values. By default, each micrograph is divided into 16 segments. Then, the segmented image is upscaled to match the original size of the micrograph. This results in a micrograph with 16 discrete regions with unique values of the intensities of the pixels. Each group populates the pixels that originally represented similar background features in a given neighborhood. The segmented micrographs are saved and can be used for further processing in two ways. First, for masking and local processing of the original micrographs, and second, as a reference to identify the micrograph quality in the neighborhood of the coordinates selected during the particle picking.Step 3. Local processing for contrast improvement or ice gradient estimation: the groups defined in the previous step allow the local processing of the original dataset. Each segment represents an area with similar background features and can be used as a local mask that can be applied to the original, motion-corrected micrographs. Within each mask, the image processing operations such as contrast improvement can be performed. The application of the contrast equalization in different areas of the micrograph separately results in the final image with a similar ratio between the particles and the background features. This also alleviates the problem of oversaturation of parts of the image when it is equalized as a whole. The resulting image has a similar ratio between the particles and the background, which can be beneficial for the particle picking tools based on the template matching algorithms.Another use of the presented approach allows estimating the average ice thickness in segmented micrographs. The defined local masks can be applied to the motion-corrected images. Within each mask, an average value of the pixel intensities can be calculated to estimate the ice thickness in the selected region. This way, a set of segmented micrographs with the estimated ice distribution is created and can be used to associate the picked particle coordinates with the background intensity in the area where they come from. The empirical ice thickness parameter describes whether the particle was picked from the area with high signal-to-noise ratio (which would correspond to the thin ice conditions) or low for the particles embedded in thicker ice. It also allows filtering and selecting subsets of particles of similar quality.The performance of the IceBreaker was tested using several datasets available from the Electron Microscopy Public Image Archive (EMPIAR) database. The presented results are focused on the main features of the software: (1) local contrast enhancement to improve the particle picking; (2) evaluation of the micrographs’ quality and identification of the ice contaminations and foil hole edges; and (3) the cryo-EM data processing with the newly introduced empirical ice thickness parameter.One of the main challenges when processing cryo-EM micrographs with non-uniform ice distribution is the fact that the contrast levels between the particles and the background features vary in different parts of the image. This can affect the performance of the automated particle pickers, especially those using a single value threshold to detect false-positives. In order to normalize the local contrast between the particles and the background across the whole micrograph, IceBreaker segments low-pass-filtered micrographs into areas of similar overall intensity. The procedure of local contrast enhancement is presented in The segmented micrographs can be used to evaluate the overall quality of the collected dataset, in addition to CTF estimation. The information about the distribution of the background pixel intensities can be associated with the coordinates of the particles picked using any available picking tool. With the IceBreaker, we introduce a new empirical particle parameter representing the estimated ice thickness based on the background features of the area where the particle is located. Users can check the overall distribution of the particles and their orientations with respect to their background quality. To further gauge the effect of ice thickness on 3D reconstruction, we regrouped the full dataset of picked particles into five groups based on the ice thickness parameter, as shown in The T20S proteasome has a D7 symmetry and may not be affected by the lower number of edge-on views in thin ice. We therefore selected another low-symmetry particle dataset, gamma-secretase (EMPIAR-10194), for the ice thickness-based refinement . The disThe non-uniform ice distribution on the cryo-EM micrographs affects the data processing and the quality of the final map. The thickness of ice in which the particles are embedded affects the local signal-to-noise ratio, particle quality, and behavior. The presented software, IceBreaker, aims to overcome the issues caused by the varying ice gradient. The tailored contrast enhancement can improve the micrographs’ interpretability and the performance of automated particle picking tools. At the same time, it allows the application of information about the ice distribution in the original micrograph for later stages of processing. To our knowledge, currently no other software offers this level of insight into the ice gradient in the micrograph.With the analysis of pixel intensity distribution in the segmented micrographs, users can get an insight into overall quality of the collected data. This helps the user to easily identify the micrographs with non-uniform ice distribution, ice contamination, and foil hole edges in the field of view. Based on the outlier analysis, a threshold can be applied to exclude areas of poor quality from further processing.Our software allows determining empirically and associating the ice thickness parameter with each particle. It allows us to select optimal particles and achieve the best possible resolution for collected cryo-EM datasets. It provides users with additional information about the dataset and the possibility to determine angular distribution of particles in different ice gradient regions. Users can filter and group the particles based on the estimated optical density of the micrographs, normally associated with amorphous ice thickness, ice contamination, or foil hole fringes. Presented results using the EMPIAR-10025 dataset as an example show improvement in the final resolution of the map with the particles picked from thinner ice. Because the T20S proteasome has high symmetry, the effect of missing orientations in thinner ice areas was less prominent. The fact that the non-symmetrical gamma-secretase dataset (EMPIAR-10194) has improved resolution of the map from thicker regions shows that the local ice conditions can affect the quality of the final map, and the thinnest ice sometimes has to be avoided. In this case, better results were obtained from thicker ice. This type of analysis can be done during the initial, small-scale data collection to determine the optimal setup for a given dataset and to target the best ice conditions, whether for the optimal angular orientation coverage or for a better signal-to-noise ratio.The IceBreaker can be run as an external job in an existing Relion3.1 project, as it has been integrated into Relion seamlessly, or run as a stand-alone software. Further integration with data collection pipelines, such as IspyB , can extpeijun.zhang@strubi.ox.ac.uk)Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prof. Peijun Zhang , p∈, there is a real number x that F(x) = p, therefore F-1(F(X)) = X  calculated for the histogram normalized between 0 and 1 has to be linearised to produce a new image with a flat histogram. The  describe(X)) = X . The tra(X)) = X : represents the 75th percentile. The size of the box is determined by the interquartile range (IQR) which is a distance between Q1 and Q3, IQR=Q3-Q1. The outliers are detected as observations outside the range:To evaluate the quality of the micrographs the box plots are used. They provide information about the data distribution based on the five-number summary . It inclThe deposited dataset was averaged, therefore no further motion correction was performed. The dataset was processed with Relion3.1 pipeline. The parameters of contrast transfer function were estimated with CTFFIND-4.1 . The mot5a63 (The dataset was processed with Relion3.1 pipeline. Motion correction was done using MotionCor2 with 5x5 patches and binning factor 2. CTFFIND-4.1 was used to estimate the parameters of contrast transfer function. 920,945 particles picked with crYOLO from 2,925 micrographs were used for reference-free 2D classification. The best 2D classes were selected manually. The initial 3D classification resulted with reported resolution 7.47 Å. 308,706 particles from the best 3D classes were used for the 3D refinement with C1 symmetry and resulted in 4.07 Å resolution based on the gold standard FSC = 0.143 criterion. The map was sharpened using a soft mask created from the atomic model PDB 5a63  and withThe methods of statistical analysis are provided in


Background: Most of the arthroplasty surgery failure due to prosthetic joint infections (PJI) is caused by biofilm-associated Staphylococcus aureus. In a recent experimental study, savirin has been used to prevent and treat S. aureus skin infections in animal models. We explored the application of savirin in a PJI mouse model to determine its utility as an adjunct therapy to prevent PJI.Materials and methods: The in-vitro antibacterial and antibiofilm activity of savirin, with or without antibiotics , against S. aureus were investigated using broth microdilution and crystal violet staining method, respectively. The effect of savirin treatment on the expression of the key biofilm-related genes  in S. aureus was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The in-vivo efficacy of savirin alone and with cefazolin to prevent S. aureus PJI was determined using a clinically relevant PJI mouse model. Mice were randomized into five groups (n = 8/group): 1) infected K-wire savirin treated group, 2) infected K-wire cefazolin treated group, 3) infected K-wire savirin plus cefazolin treated group, 4) infected K-wire PBS treated group, 5) sterile K-wire group. Savirin was administered subcutaneously immediately post-surgery and intravenous cefazolin was given on day seven.Results: Savirin inhibited planktonic and biofilm in-vitro growth of S. aureus, showed enhanced inhibitory activity when combined with antibiotics, and down-regulated the expression of key S. aureus biofilm-related genes . Savirin significantly reduced bacterial counts on joint implants in comparison with the PBS treated control, while savirin plus cefazolin reduced bacterial counts on both implants and peri-prosthetic tissues.Conclusion: Savirin adjuvant therapy may prevent biofilm formation and S. aureus PJI. This study gives baseline data for using savirin for the prevention as well as treatment of S. aureus PJI in future animal studies. S. aureus infection, is common (S. aureus growth mode that contributes to prosthetic joint infection (PJI) pathogenesis, is recalcitrant to antibiotic treatment , is a low molecular weight, lipophilic, synthetic molecule suitable for drug development  broth at 37°C for 48 h without shaking. Subculturing in 0.5% glucose containing LB (GLB) broth for 24 h induced ample biofilm production.A methicillin susceptible 5 cfu of S. aureus in 50 µL volume was added to eight two-fold serial dilutions of savirin (80 μg/ml to 0.62 μg/ml) in 50 µL volumes and incubated for 24 h at 37°C. This resulted in eight different savirin concentrations ranging from 40 μg/ml to 0.31 μg/ml. Antibacterial activity was determined by measuring the optical density (OD) of bacterial growth in microtiter plate wells at 600 nm. The minimum bactericidal concentration (MBC) of savirin was determined by plating the microtiter plate wells showing no bacterial growth for maximum 48 h. Microtiter plate biofilm assay procedures were adapted from a previous study , was used as a positive control and the sterile DMSO (0.08%) was used as a negative control.Broth microdilution and crystal violet staining methods were performed in triplicates using microtiter plates. The 10us study . PlanktoSavirin (26.67 μg/ml to 0.42 μg/ml) in combination with cefazolin (0.5 μg/ml to 0.007 μg/ml), or vancomycin (2.5 μg/ml to 0.03 μg/ml), or rifampicin (0.015 μg/ml to 0.0002 μg/ml) was used as described above, except with a total well volume of 150 µL . The combined effect was tested by checkerboard assay by determining fractional inhibitory concentration (FIC) index value. The interaction of savirin and antibiotics was categorized as synergy (FIC < 0.5), antagonism (FIC > 4), and additive effect (FIC = 0.5–4) . InhibitS. aureus culture treated with 10 μg/ml savirin (test sample) and 0.02% DMSO (control sample), using the Qiagen RNeasy mini kit. Ten µg/ml savirin was used, as this concentration reduced biofilm formation without inhibiting planktonic growth. The quality and quantity of RNA was determined using a Nanodrop 2000C spectrophotometer .RNA was extracted from log phase fema because the expression of this gene was not affected when S. aureus was treated with savirin. The reaction mixture (10 µL) consisted of 5 µL of 2 × iTaq universal SYBR green reaction mix, 0.125 µL iScript reverse transcriptase, 0.8 ng RNA template in 1 µL volume, 1 nmol of primer mix in 1 µL volume, and 2.9 µL nuclease free water. The cycling conditions used on the BioRad CFX96 Touch Real-Time PCR Detection System were: reverse transcription , polymerase activation and DNA denaturation  followed by 40 cycles of denaturation , and annealing/extension and plate read . The effect of savirin treatment on the expression of key S. aureus biofilm-related genes, icaA, icaD, eno, fib, ebps, agr was determined by the comparative Ct (ΔΔCt) method using BioRad CFX Manager software (Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) was performed in triplicate for each gene by using Bio-Rad iTaq universal SYBR green one-step kit . The refsoftware . The datEthics approval was obtained from the James Cook University Animal Ethics Committee (AEC2486). Six to 10 weeks old C57BL/6 female mice  were used. The mice were assigned to five experimental groups (8 mice/group): 1) infected K-wire savirin treated group, 2) infected K-wire cefazolin treated group, 3) infected K-wire savirin plus cefazolin treated group, 4) infected K-wire PBS treated group (positive control), and 5) sterile K-wire untreated group (negative control).S. aureus (TUH_MSSA_01) normal saline inoculum (500 cfu) was pipetted into the joint space. The kneecap was returned to its original position and the surgical site was closed with a 5–0 absorbable suture. A combination of subcutaneous (0.2 mg/kg) and oral (2.5 ml/160 ml drinking water) buprenorphine was administered for 72 h as post-surgical analgesia.Surgery was performed using previously described procedures . Mice wein-toto and peri-prosthetic tissues were collected aseptically for bacteriological culture.A single non-toxic subcutaneous dose of savirin (40 µg in 100 µL), as tested in the Vero cell line culture, was administered immediately after surgery to the infected K-wire savirin treated group. A single cefazolin  dose in 100 µL volume was administered on day 7 post-surgery to the infected K-wire cefazolin treated group. The infected K-wire savirin plus cefazolin treated group was administered a single dose of savirin  immediately after surgery followed by a single cefazolin dose  on day 7 post-surgery. Cefazolin was used in this manner not to sterilize the biofilm but to assess for any increased effect where it was used with savirin. Mice were weighed and animal well-being parameters, such as eating, drinking, mobility, interaction with other mice, and reaction to external stimuli were recorded daily. Mice were euthanized on day 14 post-surgery. The intramedullary K-wires were removed K-wires were collected in 5 ml of cold LB broth after washing three times with cold sterile PBS to remove planktonic cells. Sonication at 44 khz for 5 min using a waterbath sonicator was performed to thoroughly disrupt the attached biofilms. Similarly, tissues were collected in 800 µL of ice-cold PBS to minimize bacterial multiplication followed by homogenization using a Navy Lysis Kit . Bacterial quantification of sonication fluids and tissue homogenates was performed by the drop dilution method whereby they were serially diluted and cultured on LB agar and mannitol salt agar (MSA) at 37°C for 48 h.p-value < 0.05 indicated statistical significance.One-way ANOVA was performed using GraphPad version 8.2.0  followed by Tukey post-hoc test. Savirin’s minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) were 40 μg/ml and 20 μg/ml respectively. Savirin at 40 μg/ml, 20 μg/ml and 10 μg/ml inhibited biofilm formation significantly .S. aureus strain used were 0.5 μg/ml, 0.015 μg/ml, and 2.5 μg/ml respectively. Combined sub-inhibitory concentrations of savirin and antibiotics showed significant enhanced antibacterial and antibiofilm activity compared with that of each alone  in comparison with the untreated positive control .in-vitro activity when combined with savirin compared with other antibiotics tested. Additionally, this is the most common prophylactic antibiotic used in arthroplasty surgery. Therefore, cefazolin was chosen to use in the animal experiment over other antibiotics tested in-vitro. On animal well-being parameters testing, we did not report any adverse effects with the savirin concentration used in this study. Savirin significantly reduced bacterial counts on K-wires removed from the femur of mice with experimentally-induced prosthesis-associated septic arthritis in comparison with the PBS treated control  (p < 0.05). Similarly, savirin plus cefazolin reduced bacterial counts on both implants  and peri-prosthetic tissues  in comparison with the PBS treated control (p < 0.01)  . The absin-vitro antibacterial and antibiofilm activity, potentiated the in-vitro activity of selected antibiotics  against S. aureus, and downregulated all the biofilm-related genes tested. In the PJI mouse model, this molecule prevented S. aureus infection.Savirin showed the S. aureus to be 40 μg/ml and 20 μg/ml respectively. An earlier study also reported the direct in-vitro antibacterial activity of savirin (MIC = 36.8 μg/ml) (This study reported the MBC and MIC of savirin for 8 μg/ml) . Howeveragr quorum sensing system and a few other AgrA regulated genes, hla, psm alpha, pvl (lukS), have been reported previously  would have been expected to enhance biofilm formation  against the eviously . Howevereviously . Consequormation . However strains . The disnization , while ioduction . TherefoS. aureus skin and subcutaneous infections in mouse models (S. aureus mature biofilms (S. aureus infectious doses 2×107 to 4 × 107 cfu (S. aureus infective dose (500 cfu) than the previous study was used and confirmed that savirin prevented infection. The infective dose used in the PJI model was chosen to establish a chronic septic arthritis, while the savirin dose was chosen because of theoretical concerns of reduced penetration into joints or bones.There is only one other study that reported the influence of savirin on the prevention and treatment of e models . In the biofilms . Savirin 107 cfu . In thisSince the detailed animal toxicity profile, pharmacodynamics, and pharmacokinetics of savirin are not known, this study was unable to use higher savirin doses through different routes that might have sterilized the infection. Dose and toxicity finding studies including pharmacodynamics and pharmacokinetics studies are needed to allow for further animal model experiments. Testing of savirin in large animal prosthetic joint infection models that use materials and techniques used in a modern arthroplasty surgery is also recommended. These large animal models can better represent the real human infection pathogenesis compared with the simple mouse model used in this study.S. aureus did not develop resistance against low concentration of savirin (5 μg/ml) in comparison with antibiotics  resistance through selection of agr dysfunctional mutants or stimulation of drug efflux requiring higher savirin concentration is possible  and MRSA . Savirin was not active against P. aeruginosa, vancomycin resistant Enterococcus, and Klebsiella pneumoniae (detailed data not shown). These data indicate that savirin is, more broadly, an anti-staphylococcal agent with activity against both planktonic and biofilm growth forms. However, in the previous study no effect of savirin has been reported against S. epidermidis (This study also explored the dermidis . This diS. aureus PJI. This study lays a foundation for studying this molecule for the prevention and treatment of S. aureus PJI.From our results, it can be concluded that savirin should be considered for the development of an adjuvant therapy for the prevention of


Floating ice shelves buttress inland ice and curtail grounded-ice discharge. Climate warming causes melting and ultimately breakup of ice shelves, which could escalate ocean-bound ice discharge and thereby sea-level rise. Should ice shelves collapse, it is unclear whether they could recover, even if we meet the goals of the Paris Agreement. Here, we use a numerical ice-sheet model to determine if Petermann Ice Shelf in northwest Greenland can recover from a future breakup. Our experiments suggest that post-breakup recovery of confined ice shelves like Petermann’s is unlikely, unless iceberg calving is greatly reduced. Ice discharge from Petermann Glacier also remains up to 40% higher than today, even if the ocean cools below present-day temperatures. If this behaviour is not unique for Petermann, continued near-future ocean warming may push the ice shelves protecting Earth’s polar ice sheets into a new retreated high-discharge state which may be exceedingly difficult to recover from. New experiments suggest that the Petermann Ice Shelf in northwest Greenland is unlikely to recover once a breakup occurs in the future. If this is not unique to this ice shelf, continued ocean warming may lead to high discharge from polar ice sheets. Atmospheric and ocean warming is known to have played a key role in this pattern13. Glacier retreat is projected to continue over the coming decades to centuries due to sustained climate warming (e.g. refs. 15). Nonetheless, the Paris Agreement aims to reduce emissions enough to limit future warming to 2 °C higher than in Earth’s pre-industrial climate. This could eventually permit a return to a colder climate, reminiscent of that of the pre-industrial period. How marine outlet glaciers and their ice shelves would respond to a cooling climate has, however, been difficult to study due to a pervasive lack of data from historical analogues of such climate transitions over the recent past. We have few modern observations of outlet-glacier growth, grounding-line advance and floating ice shelves that thicken and expand. Thus, the role this behaviour plays in a cooling climate lacks an observational underpinning and remains uncertain. Documented ice advances exist in paleo-records (e.g. refs. 17), but advancing glaciers generally leave fewer traces behind than those retreating because the geomorphological imprints are overridden by subsequent ice recession. Therefore, it remains unknown whether a return to the climate that prevailed in the pre-industrial period will permit ice sheets and glaciers to recover after decades to centuries of mass loss and retreat, and to what extent global sea-level rise from ice-sheet mass loss can be curtailed or even reversed. Specifically, it is unclear if a climatic reversal in a post-Paris world allows glaciers to re-advance, ice shelves to regrow and sea-level rise to be kept moderate.Since the late 1990s, ice shelves in Antarctica and Greenland have experienced thinning18). We examine the climate conditions required for Petermann to recover to its present-day (2008) state after an ice-shelf breakup and grounding-line retreat19. Petermann is the largest glacier by area in northern Greenland, drains about 4% of the entire Greenland Ice Sheet by area21 and has one of the northern hemisphere’s few remaining ice shelves. Note that Petermann’s ice shelf is confined in a fjord (sometimes referred to as an ice tongue), in contrast to many topographically unconfined ice shelves in Antarctica. Over the last decade, Petermann has lost ~40% of its ice shelf23 and concerns have been raised that further breakup would lead to grounded-ice speedup, retreat and accelerated mass loss26. Similar scenarios have been put forward for ice-shelf-glacier systems in West Antarctica in response to ongoing and future ocean warming (e.g. refs. 28).To tackle this global question, we investigate Petermann Glacier in northwest Greenland as an example Fig. a, using Here we show that once a confined ice shelf has been lost, the glacier feeding it may get locked into a new stable regime of sustained mass loss even if the climate cools again. The numerical experiments we present for Petermann Glacier demonstrate that a cooling well below present-day conditions, in conjunction with sea-ice growth, is required in order to escape this high-discharge state and thus avoid considerable sea-level rise.19. This corresponds to a ~3 °C ocean warming relative to pre-industrial times (before c. 1850). In a more complete future scenario, atmospheric warming would cause some additional mass loss, but this is ignored here for simplicity. Moreover, the ocean-induced melt is responsible for ~80% of Petermann’s current mass loss29. The applied ocean forcing is based on observations and results from the ocean model MITgcm (see Section “Ocean forcing”30).We start all our Petermann recovery simulations from a future retreated model state , thus lowering ice-shelf basal melt rates back to those associated with present-day ocean conditions .The dynamics of ice shelves depends on local processes, such as basal melt, but also on non-local processes such as the mass balance of the inland grounded iceDespite these more favourable conditions applied for glacier growth, Petermann Glacier Ice Shelf does not regrow to its original extent Figs. e and 3c.Thus far we have seen that a future reversal of ocean warming is not sufficient to recover Petermann’s ice shelf after a future breakup (Section “Ice-shelf breakup may cement pervasive sea-level rise”), neither is a more positive surface mass balance over the glacier (Section “Recovery in a post-Paris climate”). Cooling in a post-Paris world may, however, also have other impacts on the coupled ice-sheet-ocean-climate system, for example through changes to iceberg calving (Section “Sea-ice induced advance of ice shelves”). In a suite of simulations, we consider whether reduced calving rates can aid the recovery of Petermann Glacier Ice Shelf.35). We hypothesise that suppressed calving is linked to sea-ice growth and increased ice mélange in Petermann Fjord. The theoretical and empirical support for this link is discussed in detail in Section “Sea-ice induced advance of ice shelves” below.In the numerical model, we impose changes to the calving regime by increasing the stress threshold in the calving law .We find that reduced calving rates alone do not yield ice-shelf recovery Figs.  and S11.Clearly ocean cooling (Section “Ice-shelf breakup may cement pervasive sea-level rise”) and reduced calving are mutually dependent to trigger regrowth. Additional experiments confirm that this holds regardless of whether ocean cooling is rapid or sluggish, and whether the calving regime changes like a flip-switch or over several centuries Fig. .Note that ocean cooling and sea-ice growth do not necessarily vary in tandem in the real world. An ocean cooling in our experiments means that sub-ice shelf melt is reduced (cf. Section “Ocean forcing”). In reality, sub-shelf melt is mainly driven by available subsurface ocean heat which, in this case, is controlled by remote oceanographic conditions rather than local forcing. In contrast, local atmospheric conditions govern surface waters and sea-ice conditions. Indeed, the ocean—sea ice-— atmosphere is a coupled system, but changes are likely to be asynchronous and occur on different time scales.σmax , the ice shelf grows and attains the long equilibrium state; for smaller values of σmax, the ice shelf remains in the short state, regardless of the amount of ocean cooling. Provided sufficient ocean cooling, even stress changes of less than 1% near the critical σmax stress of around 440 kPa can trigger a feedback loop of thickening and advance, causing Petermann’s ice-shelf front to advance by 70 km and its grounding line by 20 km  a contemporary long ice-shelf state : for σm km Fig. . In contTo summarise, a transition from a short-shelf to a long-shelf regime can only occur if the shelf is preconditioned by sufficient ocean cooling. In this case, a relatively small increase in the calving stress threshold is required to trigger complete advance.38 to be influential. Upstream of the present-day grounding line there are local bedrock highs that pin the grounding line in the short-shelf regime  provide a mechanistic framework for the requirements to recover an ice shelf in the future. This framework may also help us to understand (re)growth of ice shelves in the past. This is relevant for the response to any climatic cooling throughout the geological history, not only in a future post-Paris climate scenario.40). This was followed by ice-shelf breakup c. 6900 yrs BP, according to sediment records taken below the present-day ice shelf16. The ice shelf was absent for the following several thousand years and started to grow again c. 2100 yrs BP, to reach its contemporary configuration ~600 years ago .Geological evidence suggests that Petermann’s grounding line retreated rapidly across a bathymetric sill and into Petermann Fjord during the early Holocene  Before 2100 yrs BP, a relatively warm ocean and vigorous calving regime prevented ice-shelf growth in Petermann Fjord; (1b) after 2100 yrs BP, an ocean cooling occurs, which in theory allows for an extensive ice shelf. Due to the difficulty to trigger recovery with an ocean cooling alone ; (3) a sustained weakened oceanic heat flux42 favours sea-ice growth, dampens calving and allows for regrowth of the ice shelf to present-day conditions at c. 600 years BP . In Section “Sea-ice induced advance of ice shelves” below we discuss the physical mechanisms involved between a cooling ocean, sea-ice expansion and reduced iceberg calving in more detail.Our simulations imply that a plausible explanation for the historic re-establishment of Petermann Glacier Ice Shelf was an ocean cooling combined with reduced calving. In light of empirical reconstructionsChanges to the calving regime are clearly necessary, yet on their own not sufficient, for the ice shelf to regrow in our simulations Fig. f and thuIce mélange is a dense mix of calved icebergs and sea ice that covers the inner part of many Greenland fjords  can be 49. In the cooling climates considered here, such as the late-Holocene and potentially a post-Paris world, the end effect is the same, but the mechanism is different. Landfast sea-ice growth would prolong the quiet calving season in winter as well as a favour a stronger mélange. This would reduce net annual mass loss from calving and thus allow for ice-shelf advance as seen in our Petermann experiments . In particular, they find that the sea-ice regime in Petermann Fjord was discontinuous during the warm mid-Holocene, with prolonged summers with open water, consistent with a weak ice mélange and the absence of an ice shelf. This was followed by a shift towards more extensive, near-perennial sea ice from 3,900 yrs BP onwards, and a shift from a northern to a southern ice arch in the Nares Strait. This precedes the reconstructed ice-shelf recovery at 2100 yrs BP16. These changes may have been accompanied by an ocean cooling similar to what we impose in our experiments. This connection between ocean and sea ice is seen also under modern conditions, as sea ice modifies the ocean circulation in Nares Strait42. Specifically, landfast sea ice weakens the inflow of warm Atlantic Water into Petermann Fjord42, while more mobile sea ice is associated with increased warm-water inflow. Detlef et al.41 also suggest that an earlier-than-usual breakup of landfast sea ice in spring preceded the major calving events at Petermann in 2010 and 2012.For Petermann, our findings suggest that in a cooling climate, ice-mélange-induced suppression of calving can facilitate ice-shelf recovery, an effect which in our simulations is promoted by a higher calving stress threshold . We postulate that shorter calving seasons and a stronger ice mélange, both promoted by sea-ice growth, are key physical mechanisms for ice-shelf recovery that has occurred in the past and may occur in the future. This hypothesis is supported by reconstructions of sea-ice and ocean conditions over the Holocene. Based on marine sediment cores and multiple sea-ice proxies, Detlef et al.50.Increased sea ice offshore of Petermann Fjord may also have prevented flushing of the ice mélange, otherwise done efficiently by prevailing katabatic winds. This sea-ice damming effect is currently visible in Sherard Osborn Fjord, located north of Petermann Fjord, where Ryder Glacier drains. The thick sea ice in Lincoln Sea offshore of Ryder is pushed towards the fjord entrance and thereby efficiently prevents iceberg export from Ryder Glacier51). Still, the calving law we employ has been compared against several others for a range of Greenland glaciers52, and was found to give the most realistic behaviour. Similarly, the choice of friction law influences glacier flow, grounding-line behaviour and the response to external forcing56. Nevertheless, we find that post-breakup recovery of Petermann is similarly difficult using a Schoof friction law (Section 58).The difficulty in recovering Petermann Glacier after the breakup of its ice shelf is robust over multiple time scales and forcings, as discussed above. There are nonetheless several aspects of our study that must be considered when evaluating the results. We have parameterised some key physical processes, including iceberg calving  and basal friction . The ty60) and atmospheric forcing than what we use (Sections “Ocean forcing” and “atmospheric forcing”). The atmospheric forcing is relatively simple; to impose a climate cooling (Section “Recovery in a post-Paris climate”), we shifted the present-day surface mass balance field in a positive direction (Section “Atmospheric forcing”). An alternative would be to exploit surface mass balance products from model intercomparisons such as ISMIP614, combined with a regional surface mass balance model. Nevertheless, recovery triggered from the atmospheric side appears very difficult; even with stronger positive shifts to the surface mass balance than what is presented in Figs. Future studies could consider more elaborate parameterisations of the ocean . Supraglacial lakes cover ~2.8% of the surface of Petermann’s contemporary ice shelf62, but most of these lakes drain or are evacuated through supraglacial rivers every summer (cf. ref. 63). In the present climate, Petermann Glacier Ice Shelf is therefore not thought to be susceptible to collapse due to large-scale hydrofracture. In theory this could change with the increasing availability of supraglacial water in a warmer climate62. In the case of the cooling climate considered here, the reduced availability of surface water may reduce fracture development. This would mean a higher effective viscosity of the ice shelf and thus increased buttressing (cf. ref. 64), in turn potentially reducing calving rates, providing another link between the atmosphere and ice-shelf advance.In a warmer climate, surface ablation will strengthen and runoff will thus increase. This supraglacial water can accumulate as surface lakes at an ice-shelf surface. These lakes can drain rapidly to the ice-shelf base through large-scale 65.We postulate that sea-ice growth and a stronger ice mélange is a viable escape route from sustained post-breakup sea-level rise. Empirical glacier reconstructions support this idea , and our calving parameterisation implicitly accounts for the effect of ice mélange through the calving stress threshold . Still, this hypothesis is yet to be tested in a model framework with an explicit parameterisation of ice mélange66. However, the presence of sea ice may increase the effective viscosity of an ice shelf, allowing the shelf to buttress ~10% of the extensional driving stress64. Similarly, reduced sea ice offshore of Antarctic ice shelves, as well as loss of protective landfast ice, has been shown to coincide with shelf disintegration67. Conversely, heavy pack ice (ice mélange) has been suggested to protect ice-shelf fronts from flexure by ocean swells, reducing calving rates and preventing collapse67, and even limiting the effects of a potential marine ice-cliff instability65. While intriguing and physically sound, a direct causal connection between sea-ice loss and shelf breakup is yet to be established, or similarly, between the sea-ice expansion and shelf advance postulated here. Deciphering these aspects remain a priority for future work.Finally, we have illustrated that a major climatic cooling would be needed to regrow Petermann Glacier Ice Shelf after its breakup. These findings may be transferable to topographically less confined Antarctic ice shelves, with implications for the future Antarctic contribution to sea-level rise. The general notion is that the laterally extensive, unconfined ice shelves typical for present-day Antarctica provide little buttressing to upstream iceWe have used a numerical ice-sheet model to study the recovery of Petermann Glacier in northwest Greenland, after potential future ice-shelf breakup. Our model simulations show that post-breakup recovery of the ice shelf is difficult and requires major climatic cooling to occur. For Petermann, a mere reversal of future ocean warming back to contemporary conditions is insufficient for recovery, and dynamic ice discharge remains 40% higher than the present. Instead, we highlight sea-ice and ice mélange-induced suppression of calving, potentially accompanying colder ocean temperatures, as a viable escape route from sustained mass loss and associated sea-level rise. While the suppressive effect of sea ice on calving is supported by empirical evidence, it is not explicitly modelled and only implicitly accounted for in our calving parameterisation. Given the difficulty to regrow ice shelves once they have collapsed, the rationale to avoid ice-shelf breakup in the first place should be clearer than ever. Future research needs to pin down the exact mechanisms and thresholds of ice-shelf breakup, and to what extent ice shelves in Antarctica exhibit the same behaviour as Petermann Glacier Ice Shelf in Greenland.69 on a finite-element mesh comprising 42,000 elements. The mesh resolution varies from 0.5 to 10 km based on the steepness of the bed topography. In addition, the mesh is refined to 0.5 km where observed velocities exceed 500 m/a.Ice dynamics is modelled using the two-dimensional Shelfy-Stream Approximation71. The domain extends ~70 km offshore of the present-day calving front, to facilitate glacier advance during the recovery experiments outlined in Sections “Ice-shelf breakup may cement pervasive sea-level rise”, “Recovery in a post-Paris climate”, “Reduced calving as an escape route from pervasive mass loss”. Bedrock topography is taken from BedMachine v372 and we use ice-sheet geometry from the Greenland Ice Sheet Mapping Project75.The model domain .Grounding-line migration is modelled with subelement resolution and is based on a flotation criterion77, which calculates basal drag τb asα is a friction parameter, ub is basal velocity and N the effective pressure. We assume a perfect hydrological connection between the subglacial drainage system and the ocean, defining N as the difference between ice overburden and hydrostatic pressure: N = ρigH + ρwgzb, where ρi, g, H, ρw, and zb are ice density, gravitational acceleration, ice thickness, seawater density and bed elevation, respectively. While this parameterisation of the effective pressure is not realistic far from the coast, it allows for a smooth transition of basal stress in the grounding zone, which has been shown to significantly improve the fit with remote sensing data56. The basal friction parameter α is inverted for using an adjoint method and remotely sensed ice velocities71.Basal friction is modelled using a linear viscous Budd law19 for details). Ice viscosity for grounded ice is assumed to be uniform and corresponds to an ice temperature of −12 °C based on Greenland-wide ISMIP6 experiments14.Similarly, ice-shelf viscosity is estimated using an adjoint method minimising the absolute misfit between modelled and observed velocities , and σmax is a stress threshold. We use σmax = 1 MPa for grounded ice, and calibrate σmax to 300 kPa for floating ice by reproducing the present-day ice-shelf margin after a 50-year transient relaxation, where the grounding line and calving front can evolve freely (see ref. 19 for details). Floating-ice thickness smaller than 100 m is not allowed, to avoid unrealistic calving model behaviour along the fjord walls.A von Mises law34. This surface mass balance is derived from monthly means for each year.Present-day atmospheric forcing is imposed using a climatic surface mass balance (1979–2014) from the regional climate model MAR 3.5.278. This means that coastal areas would thicken more than would the interior ice sheet, which results in a flatter ice-surface profile. For example, model studies and empirical evidence suggest that a divide higher than ~3500 m a.s.l. is unlikely to have occurred during past cold periods, such as the Last Glacial Maximum and the Younger Dryas81. In comparison, the elevation of the present-day divide is only a few hundred meters lower74. In simulations where positive shifts of the surface mass balance are imposed (ocean cooling + increased SMB), see Section “Recovery in a post-Paris climate” and Table In a colder climate, decreased ablation is not expected to be compensated by an equivalent increase in accumulationzdivide is the elevation at the ice divide (upper boundary of domain) and z0 = 0 is the elevation at sea level. The anomalies at sea level  casts from the Petermann 2015 Expedition83. In the model, we apply annual oceanic melt rates of the ice-shelf base. Melt rates vary linearly from zero at depths shallower than 200 m to 30 m/a at depths deeper than 600 m  at the calving front is only applied if the calving front is grounded. However, Petermann’s ice shelf never disappears completely in our simulations, so grounded-front melt is never applied in practice.Ocean forcing is parameterised based on observations of Petermann’s ice shelf and modelling using the ocean model MITgcm30. Associated depth—melt rate profiles for a given temperature are given in Fig. In experiments where the oceanic melt is altered, oceanic melt rates are based on modelling experiments bySupplementary InformationPeer Review File


Angiotensin-converting enzyme (ACE) regulates several biological functions besides its vital role in immune functions. ACE is elevated in immune cells in inflammatory diseases including atherosclerosis, granuloma, chronic kidney disease, and also autoimmune diseases, like multiple sclerosis, rheumatoid arthritis, and type I diabetes. No significant information prevails in the literature regarding the isolation, identification, and profiling of potential ACE inhibitory peptides. In the present study, indigenous crop varieties like seeds  were used to evaluate their ACE inhibition activity. Variables including hydrolysis time, enzyme-to-substrate ratio (E/S), pH, and temperature were standardized to acquire the most suitable and optimum ACE inhibition activity. Seeds of cotton, chickpea, and peanuts displayed remarkably maximum ACE inhibition activity than other plants. The study disclosed that maximum ACE inhibitory activity (86%) was evaluated from cottonseed at pH 8.0, temperature of 45°C, hydrolysis time of 2 hrs, and enzyme to the substrate (E/S) ratio of 1 : 5 followed by peanuts (76%) and chickpea (55%). SDS-PAGE confirmed that vicilin protein is present in cottonseed and peanut seed while cruciferin and napin proteins are present in chickpeas. LC-MS/MS analysis disclosed potential novel peptides in hydrolyzed cottonseed that can be ascribed as potential ACE inhibitors which have never been reported and studied earlier. The current study further showed that cottonseed peptides due to their promising ACE inhibitory activity can be a valuable source in the field of ACE inhibitor development. Angiotensin-converting enzyme (ACE) is involved in inflammation and in the stimulation of T-cells by certain antigenic peptides. In autoimmune disorders, ACE levels are increased. Certain immune functions are suppressed and inflammatory or autoimmune diseases are inhibited by ACE inhibitors . AutoimmPeptides and peptidomimetics can act as immunomodulators by either blocking or stimulating the immune response to generate tolerance. The study of peptide synthesis and modified amino acid side chains has led to a new class of autoimmune therapeutic drugs . PeptideThe discovery of plant primary metabolites such as proteins and peptides for disease management has ushered in a new era for the development of plant-based peptides as therapeutic candidates . BioactiTo the best of our knowledge and information available, no significant literature prevails regarding the screening and development of inhibitory peptides and their role in autoimmune diseases. Therefore, the aim of the present research was to evaluate the ACE inhibition potential of indigenous plants for the production of inhibitory peptides. Our study focused on the screening of plants for their ACE inhibition activity. This study also is aimed at development of potential ACE peptide inhibitors from plants and protein profiling using the liquid chromatography mass spectrometry (LC-MS/MS) approach. These identified potential ACE inhibitory peptides from plants can be used as a source in the development of antihypertensive drugs in the pharmaceutical field in the future to cure autoimmune diseases/disorders.The seeds of local varieties used in this study were obtained from the Seed section of Ayyub Agriculture Research Institute (AARI), Faisalabad, Punjab, Pakistan. A fine powdered seed of each plant was mixed with distilled water thoroughly for 2 hrs until the mixture was homogenized. The extract was filtered through Whatman No. 1 filter paper, and the solvent was evaporated with a rotary evaporator. Extracts were stored at -20°C for further analysis.ACE extracted from rabbit lung powder was used as reported earlier with minor modifications . FreshlyμL of each sample solution, 60 μL of Hippuryl-L-histidyl-L-leucine (5 mM) and 100 mM of sodium borate buffer (pH 8.3) having NaCl (300 mM) were mixed at 40°C for 15 minutes. The reaction was initiated by adding 10 μL of ACE, and the mixture was further incubated later at 40°C for 35 min. The incubated reaction was terminated using HCl (1 M). 0.5 mL of ethyl acetate was added and centrifuged at 3000 rpm for 15 min at 4°C to extract hippuric acid (HA). Upper ethyl acetate layer (0.2 mL) was separated and was evaporated at room temperature. The resulted HA was dissolved in 1.0 mL deionized distilled water, and the optical density of this solution was calculated at 228 wavelength using a UV spectrophotometer. The ACE inhibition activity was determined by Zheng et al.'s method [The ACE inhibition activity of plants seeds was determined using the reported method . To prepA − B) − (C − D)]/(A − B)).(1)ACE inhibition (%) = 100 ×  were prepared using NaOH (0.09 M) at room temperature. The extracts were centrifuged at 3000 rpm for 25 minutes. The higher film was standing apart, set to pH 5.5 using HCl (2 M), and centrifuged at 3000 rpm for 25 minutes. Samples were washed twice using pure water and quantified. Around 0.2 mL solution containing alcalase (0.05%) and inhibitor solution (protein) was incubated with potassium phosphate buffer (0.1 M) having pH 8.0 for 6 hours at 37°C. To stop the hydrolysis, samples were boiled for 6-7 min and centrifuged at room temp for 10-15 minutes + in the mass spectrum  is a powerful analytical technique, used for the identification of unknown compounds, and structure elucidation to find the chemical properties of different compounds/peptides. It is highly sensitive and selective for the analysis of compounds present in trace amounts , 38. In spectrum  was idenn plants , 40. Ther [M+H]+ . Pattern[M+H-81] .m/z 253 [M + H]+ corresponds to the His-Gly-Gly tri-peptide. The product peak at m/z 137.92 due to the [M + H-Gly-Gly] and at m/z 109.83 due to [His-NH=CH], and m/z 120 due to the removal of [His-H2O]. A molecular ion peak at m/z 280 [M+H]+ was identified as Phe-Met dipeptide (m/z 148.83 due to the phenylalanine amino acid, at m/z 166.92 due to the addition of water molecule [Phe+H2O], at m/z 133.92 due to the methionine amino acid, and at m/z 115.92 due to dehydration [Met-H2O] (m/z 312 [M+H]+ was detected as Val-Pro-Gly-Gly tetra-peptide. Spectra produced at m/z 255.83 and m/z 197.00 resulted due to the elimination of [M+H-Gly-Gly], m/z 213.02 due to the [M+H-Pro], and m/z 100.83 due to the Val peptide (m/z 413 [M+H]+ was due to Lys-Val-Trp tripeptide (m/z 314.18 due to [M+H-Val], m/z 369.1 due to [M+H-43], and m/z 338.6 due to [M+H-73], and 226.00 is ascribed due to [M+H-Trp]. Pentapeptide Glu-Gly-Ala-Ser-Asp produced ion spectrum band at m/z 459 [M+H]+, spectra at m/z 360.33 due to [M+H-Val], m/z 441.16 due to dehydration [M+H-H2O], m/z 429.34 due to [M+H-31], m/z 401.00 due to [M+H-58], and m/z at 344.17 in the spectra can be attributed due to [M+H-Asp] (The peak at ipeptide  which prMet-H2O] . The fra peptide . A molecipeptide , which pM+H-Asp] . PreviouAccording to the best of our information available, this was the first performed study regarding the protein profiling of cottonseed using LC-MS/MS technique, and the peptides obtained are explored for the first time. From the current findings, it is clear that cottonseed, peanut, and chickpea have the potential against hypertension. However, among all the studied plants, cottonseed contains important amino acids which are essential for antihypertensive activity. Hence, it can be concluded that antihypertensive proteins from cottonseed are valuable alternatives because of their cost-effectiveness and no side effect.There must be detailed studies on the specific structural unit of the peptides by MALDI-TOF analysis, which is very costly and time taking, followed by docking analysis and protein-protein interactions to analyze the best fit model of peptides in autoimmune disorders/diseases.


We have recently illustrated that nebivolol can inhibit angiotensin II (Ang II)-mediated signaling in cardiomyoblasts; however, to date, the detailed mechanism for the beneficial effects of nebivolol has not been studied. Here, we investigated whether the inhibition of NO bioavailability by blocking eNOS  using L-NG-nitroarginine methyl ester (L-NAME) would attenuate nebivolol-mediated favorable effects on Ang II-evoked signaling in H9c2 cardiomyoblasts. Our data reveal that the nebivolol-mediated antagonistic effects on Ang II-induced oxidative stress were retreated by concurrent pretreatment with L-NAME and nebivolol. Similarly, the expressions of pro-inflammatory markers TNF-α and iNOS stimulated by Ang II were not decreased with the combination of nebivolol plus L-NAME. In contrast, the nebivolol-induced reduction in the Ang II-triggered mTORC1 pathway and the mRNA levels of hypertrophic markers ANP, BNP, and β-MHC were not reversed with the addition of L-NAME to nebivolol. In compliance with these data, the inhibition of eNOS by L-N⁵-(1-Iminoethyl) ornithine (LNIO) and its upstream regulator AMP-activated kinase (AMPK) with compound C in the presence of nebivolol showed effects similar to those of the L-NAME plus nebivolol combination on Ang II-mediated signaling. Pretreatment with either compound C plus nebivolol or LNIO plus nebivolol showed similar effects to those of the L-NAME plus nebivolol combination on Ang II-mediated signaling. In conclusion, our data indicate that the rise in NO bioavailability caused by nebivolol via the stimulation of AMPK/eNOS signaling is key for its anti-inflammatory and antioxidant properties but not for its antihypertrophic response upon Ang II stimulation. The stimulation of the renin–angiotensin–aldosterone system (RAAS) enhances the activation of angiotensin II (Ang II), a peptide linked to cardiovascular diseases (CVDs) ,3,4,5,6.The activation of the β3-adrenergic receptor induces negative inotropic effects opposite to those of the β1-adrenergic receptor and the β2-adrenergic receptor by stimulating the NOS pathway . The agoAng II-induced amplification in ROS and diminution in NO bioavailability activate redox-sensitive genes, which augment inflammatory and growth factors . We haveAng II and nebivolol were obtained from Sigma-Aldrich , and L-NAME was obtained from Calbiochem . Antibodies for phospho-mTOR (pSer2448), mTOR, phospho-p70S6K (pThr389), p70S6K, phospho-RPS6 (pSer235/236), and RPS6 were obtained from Cell Signaling Technology Inc. . ECL substrates for high-sensitivity Western blot detection, Pageruler plus prestained ladder, and SuperScript™ III First-Strand Synthesis System were obtained from Thermo Fisher Scientific . TRIzol™ Reagent was obtained from Invitrogen , and PowerUp SYBR Green master mix was obtained from Applied Biosystems .H9c2 cardiomyoblasts derived from rat embryonic cardiomyocytes were obtained from the American Type Culture Collection (ATCC) . Cultures were grown on Dulbecco’s Modified Eagle Medium , supplemented with fetal bovine serum (FBS) (10%)  and penicillin/streptomycin (100 U/mL: 100 mg/mL). Cells were cultured in an incubator with a 37 °C temperature and 5% CO2-air. The cultures were passaged after 70–80% confluence was achieved. For Western blotting, cells were collected using trypsinization after treatment. Lysis buffer was added, and samples were lysed on ice as demonstrated previously [Binding of antibody to membrane was detected using ECL substrates, and images were captured using G: BOX . Protein band density was quantified using Gene Tools image analysis software . The data are presented as the normalized protein band density in arbitrary units.+/NADPH Assay Kit  as per manufacturer specifications. After assay, as per kit instructions, the absorbances of NADP+ and NADPH were measured at OD450 nm. The concentrations of NADP+ and NADPH were calculated as per the given protocol.The intracellular levels of NADP+ and NADPH were determined by employing colorimetric assay using NADP−ΔΔCt method [TRIzol reagent was used for the extraction of RNA from H9c2 cells as per the manufacturer guide. RNA quantity was determined using Nano drop (Thermo Scientific™ Nano Drop 2000c). Reverse transcription was used to synthesize cDNA using 400 ng of RNA per 20 µL reaction using SuperScript™ III First-Strand Synthesis System. Real-time-PCR was carried out by using RT-PCR detection System  using SYBR Green PCR master mix . Target mRNA levels were normalized to control levels, and relative levels of mRNA were calculated by using the 2t method . The expThe redox-sensitive dye 2,7-dichlorodihydrofluorescein diacetate  was employed to measure generation of ROS in cells . H9c2 caAfter treatment with Ang II and inhibitors for 48 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were then stained with 1% Crystal violet (Sigma) for 10 min. Six to eight images were acquired randomly from each group using Zeiss AxioCam MRc5 color camera attached to ZEISS Axio Observer inverse microscope . The cell surface area of nearly 100 cells was measured using Zen 2 lite software .t-test. Two-group analysis was performed using Student’s t-test (paired or unpaired as appropriate). A value of p < 0.05 is considered significant.Data represent means ± SEM of at least three to six separate experiments. Differences in the multiple groups were compared using analysis of variance (ANOVA) followed by Student’s Increases in NO bioavailability via the nebivolol-mediated activation of eNOS reduce oxidative stress . To eval+/NADPH ratio. Previous studies have demonstrated NADPH oxidase as a dominant site for superoxide synthesis in response to Ang II stimulation in H9c2 cells [+ and NADPH, and then we measured the NADP+/NADPH ratio. Compared to the untreated control, Ang II elevated the NADP+/NADPH ratio in H9c2 cardiomyoblasts. Consistent with the above observation, pretreatment with the L-NAME plus nebivolol combination had no influence on Ang II-stimulated NADPH oxidase activity , B-type natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) A–C. AddiInterestingly, in contrast to our above observations, pretreatment with the L-NAME plus nebivolol combination noticeably diminished the gene expression levels of ANP, BNP, and β-MHC and significantly reduced the cell surface area in H9c2 cardiomyoblasts A–E. ThesAng II-induced cardiac hypertrophic growth is known to increase by the deregulation of mTORC1 signaling. Thus, to further demonstrate the influence of L-NAME and nebivolol co-treatment on Ang II signaling, we compared the impact of nebivolol and the nebivolol plus L-NAME combination on Ang II mediated on the mTORC1 pathway by determining mTOR, S6K1, and RPS6 phosphorylation levels. Similarly, to nebivolol, the nebivolol plus L-NAME combination exhibited a substantial reduction in the phosphorylation of mTOR, S6K1, and RPS6 A–C, suggThe activation of pro-inflammatory cytokines by Ang II plays a marked role in the advancement of CVD . To furtTo determine whether the inhibition of AMPK, an upstream kinase regulator of eNOS would produce effects similar to those of eNOS inhibitors on Ang II signaling, we evaluated the effects of the AMPK inhibitor, the compound C plus nebivolol combination, on Ang II-provoked ROS formation and the transcription of inflammatory and hypertrophy genes in H9c2 cardiomyoblasts. Pretreatment of H9c2 cardiomyoblasts with compound C plus nebivolol did not reduce ROS or the gene expressions of TNF-α and iNOS induced by Ang II A–D. HoweTo further confirm the above observation, we next evaluated the effects of a different inhibitor of eNOS, the LNIO plus nebivolol combination, on Ang II-induced signaling. In accordance with our above findings, pretreatment of H9c2 cardiomyoblasts with LNIO plus nebivolol did not alter Ang II-induced ROS generation in H9c2 cardiomyoblasts . SimilarHere, we demonstrate that the inhibition of Ang II-mediated signaling by nebivolol is partially dependent on its bioavailable NO production in H9c2 cardiomyoblasts. We also demonstrate that NO bioavailability caused by nebivolol is not vital for the inhibition of hypertrophy but that it is compulsory for the suppression of ROS and the proinflammatory actions triggered by Ang II. Nebivolol is a third-generation β1-adrenergic receptor antagonist, and its vasodilator properties have been demonstrated to have a hemodynamic profile, which provides beneficial effects in its approved use in hypertension and heart failure ,22,24. T+/NADPH ratio were not suppressed by treatment with either L-NAME or nebivolol plus L-NAME. Thus, it is conceivable that nebivolol-mediated inhibitory actions in Ang II-mediated specific effects are regulated by eNOS activation.The growing evidence supports the notion that the over-activation of RAAS triggers a redox imbalance, thereby eliciting ROS and decreasing NO production. NO reduces the expression of AT1R, and the eNOS inhibitor promotes AT1R expression, indicating that NO might act as an inhibitory regulator of AT1R expression . FurtherDespite such compelling evidence, Ang II-mediated mTOR and the phosphorylation of its downstream signaling molecules RPS6 and S6K1 were decreased almost to the same extent in both pretreatments, nebivolol alone and nebivolol plus L-NAME. The deregulation of mTORC1 signaling is anticipated to induce myocardial hypertrophy , which iOur results were further validated by using an inhibitor of AMPK, compound C, and a selective pharmacological inhibitor of eNOS, LNIO, in combination with nebivolol on Ang II-provoked signaling. AMPK is an upstream serine/threonine kinase known to phosphorylate eNOS at Ser-1177 and stimulate NO synthesis . The actThe mounting evidence demonstrates that Ang II modulates the expressions of pro-inflammatory mediators in the myocardium, which is attributed to hypertension and CVDs ,36. In pOur data suggest that the inhibition of NO bioavailability caused by antagonizing eNOS or its activator AMPK alleviates the defensive effects of nebivolol on the Ang II-stimulated increase in ROS formation. Additionally, our data reveal that the nebivolol-promoted increase in NO bioavailability is vital for its anti-inflammatory effects on Ang II stimulation. The findings from this study reveal for the first time that eNOS suppression, by either pharmacological inhibitors eNOS or AMPK, does not downregulate the cardioprotective effects of nebivolol on Ang II-triggered hypertrophy . These f


The evolution and driving mechanism of the South Asian summer monsoon (SASM) are still poorly understood. We here present a 12-Myr long SASM record by analyzing the strontium and neodymium isotopic composition of detrital components at IODP Exp. 359 Site U1467 from the northern Indian Ocean. The provenance investigation demonstrates that more dust enriched in εNd from northeastern Africa and the Arabian Peninsula was transported to the study site by monsoonal and Shamal winds during the summer monsoon season. A two-step weakening of the SASM wind since ~12 Ma is proposed based on the εNd record. This observational phenomenon is supported by climate modeling results, demonstrating that the SASM evolution was mainly controlled by variations in the gradient between the Mascarene High and the Indian Low, associated with meridional shifts of the Hadley Cell and the Intertropical Convergence Zone, which were caused by interhemispheric ice-sheet growth since the Middle Miocene. The εNd record from the IODP Exp. 359 Site U1467 in the northern Indian Ocean, along with climate modeling, reveals a two-step weakening of the South Asian summer monsoon (SASM) wind since 12 Ma. The SASM evolution was mainly caused by interhemispheric ice-sheet growth since the Middle Miocene. Consequently, its long-term evolution and the underlying causes have attracted much attention from the paleoclimate research community7. Increasing evidence indicates that the Asian monsoon was initiated no later than the Early Miocene7; however, the mechanisms driving its subsequent evolution are highly debated. Suggested mechanisms include the development of high topographic relief linked to the uplift of the Tibetan-Himalayan system9, global cooling as a consequence of polar ice-sheet development11, the retreat of the Tethys Sea12, and variations in pCO213. Among these, much attention has been paid to the global cooling associated with the expansion of polar ice-sheets, which is evidenced by both sedimentary records11 and climate modeling14. However, details of exactly how global cooling controlled the Asian monsoon remain unclear. In contrast to the long-term evolution of the East Asian monsoon (EAM), which is recorded by many eolian sequences in northern China15, SAM records extending to the Middle Miocene are scarce, especially from within the marine realm of the SAM-influenced region, with few exceptions16.The South Asian monsoon (SAM), an important component of the global climate systemo 51ʹ N, 73o 17ʹ E; ~12 Myr7), recovered from the Maldives Inner Sea in the southeastern Arabian Sea, from a water depth of ~487 m 32. The average εNd value of marine sediments and rocks from the margins of the northeastern African and the southern Arabian Peninsula too are highly radiogenic (average –4.6)33. These more radiogenic Nd values observed in the northeastern Africa and the Arabian Peninsula are consistent with the Kenya Rift basalts 34 and the Arabian-Nubian shield (εNd: 1 to 6)35. By contrast, the Sr and Nd isotopic composition of the Indian Thar deserts showed much less radiogenic Nd values 36.The Arabian desert dust reveals a more radiogenic εNd of –6, and 34, and from the Thar desert, with less radiogenic εNd values36  has potentially influenced the intermediate water of the northern Indian Ocean42, but the εNd of AAIW is generally fluctuated between –8.4 and –6 for the glacial and interglacial periods44, which is far more radiogenic than those of our lithogenic fraction sediment data  from the Indus Fan, indicate a strong SASM during ~16–10 Ma interval, followed by a gradual weakening during ~10–3 Ma48 , interrupted by a further decrease at ~5–7 Ma50 , possibly from the southern Indian continent0 to –18,57  over the Asian continent and the Mascarene High (MH) in the southern Indian Ocean57 Fig. . The MH g60 Fig. . Specifi 61 Fig. , and oves62 Fig. . We suggThe proposed linkage between the SASM evolution and the growth of polar ice sheets over the last ~12 Myr was examined using numerical simulations. Sensitivity results reveal relatively consistent variations in the MH-IL pressure gradient, 850-hPa wind speed, and precipitation during the summer season  in the study area Fig. , which a63. There was a major expansion of the East Antarctic ice sheet from ~13.9 Ma onwards and a further expansion of the West Antarctic ice sheet until ~8 Ma65  is associated with the ascending branch of the Hadley Cell, its position is closely coupled with the movement of the Hadley Cell, as well as the MH and IL. A previous study suggests that the ITCZ, and thus the Hadley Cell, was displaced toward the warmer hemispherea65 Fig.  leading  62 Fig. . This ch 62 Fig. ; Fig. 6ca67 Fig. , causing 62 Fig. . This pu 62 Fig.  and 6d, 8, closure of the Tethys Sea12, and changes in pCO213 may also have contributed to the evolution of the SASM. However, the continental collision between India and Eurasia is thought to have commenced no later than 40 Ma74. The Himalayan-Tibetan region may have reached its present height by the Early-Middle Miocene75 and there was no significant tectonic uplift thereafter. Similarly, the final closure of the Tethys Sea was much earlier (by ~15 Ma)76 which precludes its potential impact on changes in the SASM. There is uncertainty regarding the role of pCO2 in affecting monsoon climate77. However, a recent study excluded it as a controlling factor in the evolution of the East Asian monsoon78. Considering that pCO2 and climate are coupled throughout the Cenozoic79, we suggest that CO2 might not directly drive the evolution of the SASM, but might act through its modulation of glaciations of the polar regions80. In summary, our proxy records combined with numerical modeling, demonstrate for the first time a linkage between the SASM and interhemispheric ice-sheet growth via the modulation of the meridional shifts of the Hadley Cell and its associated MH-IL pressure gradient since the Middle Miocene. Our study thus provides a more comprehensive interpretation of how interhemispheric ice-sheet development and associated global atmospheric circulation regulated SASM evolution on geological timescales.In addition to global cooling, the uplift of the Himalayan-Tibetan regiono 51ʹ N, 73o 17ʹ E, water depth 487 m) in the Maldives Inner Sea was cored through a sequence of carbonate drift deposits  and 146Nd/144Nd (0.7219), respectively. Repeated analyses of the NBS987 standard yielded 87Sr/86Sr = 0.71032 ± 0.000006 , and the JNdi-1 standard yielded 143Nd/144Nd = 0.512116 ± 0.000005 . Nd results are expressed as εNd = [(143Nd/144Nd(measured)/143Nd/144Nd(CHUR)) − 1] × 104, using the CHUR value of 0.512638 given by ref. 84.The strontium and neodymium isotopes of 29 samples from Site U1467 were analyzed using a High-Resolution Multi-Collector Inductively Coupled Plasma Mass Spectrometer (HR-MC-ICPMS). Pretreatment methods followed ref. 85. Early and the latest version of the model (CESM1.2) are often used to simulate past climate change86, as well as present and future climate assessments87 (IPCC AR5). In this study, the horizontal resolutions of the CAM and CLM are approximately 0.9° × 1.25° and the vertical dimensions contain 26 and 15 levels, respectively. The POP and CICE components have a horizontal resolution of 1° × 1° and depth (z) coordinates of 40 levels.Sensitivity experiments were performed using the Community Earth System Model (CESM1.2) released by the National Center for Atmospheric Research (NCAR) in 2014. CESM1.2 is a fully coupled global climate model that consists of five components: atmosphere , land , ocean , sea ice  and the Coupler67. S1 and S2 represent the ice cover accumulated in the East Antarctica and the whole Antarctica, respectively  for all the sensitivity experiments are based on the pre-industrial revolution scenario integrated from the model. The orbital parameters for 195088 are applied in the model simulation. To ensure that the output climate field of the numerical experiments has reached an equilibrium state, all of the numerical experiments are continuously integrated for 500 model years, and the average field for the last 50 years is used for analysis. Precipitation and wind speed are averaged for the region influenced by the SASM . The MH  and IL  cells are calculated from the atmospheric pressure at sea level averaged over the respective fixed areas. The MH-IL pressure gradient is calculated as the mean sea-level pressure over the Mascarene High and Indian Low pressure cell regions.The response of the Mascarene High (MH), Indian Low (IL), MH-IL pressure gradient, summer  precipitation, and 850-hPa wind speed were examined under three different scenarios (labeled S1, S2 and S3; Supplementary Fig. Supplementary InformationPeer Review File


Climate-forced, offline ice-sheet model simulations have been used extensively in assessing how much ice-sheets can contribute to future global sea-level rise. Typically, these model projections do not account for the two-way interactions between ice-sheets and climate. To quantify the impact of ice-ocean-atmosphere feedbacks, here we conduct greenhouse warming simulations with a coupled global climate-ice-sheet model of intermediate complexity. Following the Shared Socioeconomic Pathway (SSP) 1-1.9, 2-4.5, 5-8.5 emission scenarios, the model simulations ice-sheet contributions to global sea-level rise by 2150 of 0.2 ± 0.01, 0.5 ± 0.01 and 1.4 ± 0.1 m, respectively. Antarctic ocean-ice-sheet-ice-shelf interactions enhance future subsurface basal melting, while freshwater-induced atmospheric cooling reduces surface melting and iceberg calving. The combined effect is likely to decelerate global sea-level rise contributions from Antarctica relative to the uncoupled climate-forced ice-sheet model configuration. Our results demonstrate that estimates of future sea-level rise fundamentally depend on the complex interactions between ice-sheets, icebergs, ocean and the atmosphere. Most global sea level projections ignore the active role of ice-sheet-climate coupling. Including this effect, a new modeling system simulates irreversible Antarctic ice-sheet loss and accelerating sea level rise for global mean temperatures >1.8oC. This trend is likely to accelerate in response to increasing atmospheric greenhouse gas concentrations and anthropogenic warming. With a considerable fraction of the world’s population living near coastlines, it is crucial to provide accurate projections of global and regional future SL trends and constrain remaining uncertainties. The largest uncertainty originates from the response of the Antarctic ice-sheet (AIS) to greenhouse warming. Recent model-based estimates of the 21st century AIS contribution to future SL rise for a Representative Concentration Pathway (RCP) 8.5 scenario range from 0 to 1.4 m8.Global mean sea-level (SL) has risen over the past century by about 20 cm, in part due to the thermal expansion of seawater, glacier and ice-sheet melt and changes in groundwater storage9, (ii) several processes such as iceberg calving or basal melting are not well constrained and represented; also different ice-sheet models show varying sensitivities to warming scenarios11, and (iii) the impact of climate-ice-sheet coupling is not included in offline ice-sheet model simulations, even though it may play an important role in Southern Hemisphere climate change13. Here, we present a new suite of coupled future earth system model simulations, which captures important interactions between atmosphere, ocean, ice-sheets, ice-shelves, and icebergs in both hemispheres.These estimates were obtained with offline ice-sheet models that use atmospheric and oceanic forcings from future climate model projections. Accordingly, several issues need to be considered: (i) the transient and equilibrium climate sensitivities of the current generation of earth system models still have remaining uncertainties of 1.5–2.2 K and 2.6–4.1 K, respectively15. This in turn can increase Southern Ocean (SO) stratification and subsurface warming due to reduced vertical heat exchange. Subsurface Southern Ocean (SSO) warming enhances sub-shelf melting19 which can lead to a reduction of the buttressing effect of ice-shelf on grounded ice21. As a consequence the flow of ice streams can accelerate toward the ocean22, which would translate to SL rise. These processes can be further amplified by other feedbacks, including the Marine Ice-Sheet Instability (MISI) along retrograde slopes23, hydrofracturing and the Marine Ice-Cliff Instability (MICI)5—all of which remain poorly constrained10.Observational data shows that AIS meltwater discharge has increased over the past decades25, which capture the complex interactions between climate components and ice-sheet, ice-shelf and iceberg dynamics and thermodynamics. Our study focuses on the AIS and Greenland ice-sheet (GrIS) contributions to future SL projections using an ensemble of simulations conducted with the coupled three-dimensional climate-ice-sheet-iceberg modeling system26 LOVECLIP 29. Biases of surface air temperature, precipitation, and SSO are corrected from LOVECLIM to PSUIM26. PSUIM is forced by surface air temperature, precipitation, evaporation, solar radiation, and annual mean subsurface ocean temperature. Surface air temperature and precipitation are downscaled vertically to PSUIM grid with applied lapse rate corrections28.To quantify the effect of these interactions on future SL projections, one needs to employ coupled global climate-ice-sheet models2 concentrations following the Shared Socioeconomic Pathway (SSP) 1–1.9, 2–4.5 and 5–8.5 scenarios30 until 2150 CE. To further elucidate the effect of AIS meltwater flux on polar climate, the stability of the ice-sheets and SL we performed an additional idealized sensitivity experiment (Supplementary Table −2 and VCLIF = 0 km/year) with and without meltwater flux coupling (experiments SSP5-8.5_HFCMOFF and SSP5-8.5_MWHFCMOFF). Furthermore, to estimate the sensitivity of Antarctic ice-shelf mass loss to SSO warming, we doubled the sub-shelf SSO temperature anomaly (relative to 1850 CE) in the SSP5-8.5 scenario with (Re_SSP5-8.5_2xSOTA) and without meltwater fluxes (Re_SSP5-8.5_2xSOTA_MWOFF).The simulations include an 8000-years-long coupled pre-industrial spin-up run for initialization and 10 member ensemble of simulations forced by increasing CO32. Quantifying the natural and anthropogenic contributions to this trend remains difficult because of our limited understanding of naturally occurring low-frequency ice-sheet dynamics and the relatively short observational period. Here we compare the observed 19-year trend of ice mass balance from the Gravity Recovery and Climate Experiment (GRACE)33 for the period 2002–2020 CE to the corresponding values in forced experiments as well as a 5000-year-long pre-industrial control run (CTR) . Consistent with the GRACE measurements, changes in the mass balance are calculated only from the grounded parts of the ice-sheets for LOVECLIP. Interannual variability of the mass balance recorded by GRACE and simulated by the forced LOVECLIP experiments during 2002–2020 CE fall within the range of natural variability exhibited by the CTR  relative to the pre-industrial levels (1850–1900 CE) amounts to 1.4 ± 0.17 °C (1.2 ± 0.14 °C), 2.4 ± 0.15 °C (2.7 ± 0.16 °C) and 4.0 ± 0.15 °C (5.3 ± 0.09 °C) for the SSP1-1.9, SSP2-4.5, and SSP5-8.5 scenarios, respectively Fig. . The unc38 and Antarctica40. One factor impacting the LOVECLIP ice-sheet response is the relatively weak temperature sensitivity to greenhouse forcing compared to most CMIP6 models  around 2100 CE for SSP2-4.5 and SSP5-8.5 , dominate the negative mass balance and global SL contribution. The accelerated mass loss over the AIS is related to a combination of surface melting, basal melting and grounding line retreat which contributes to the massive ice calving fluxes . Increased AIS meltwater fluxes in the fully coupled model experiment (experiment SSP5-8.5) reduce surface ocean salinity in the SO relative to SSP5-8.5_MWOFF .When hydrofracturing and ice-cliff failure parameterizations are turned off in the additional model experiments  near the Antarctic ice-shelves during 650 years without greenhouse forcing. SSO temperatures in the Antarctic ice model ure Fig.  increaseure Fig.  relativeure Fig. . This isure Fig.  and de fure Fig. . Ice calure Fig. . Howeverure Fig. .Fig. 6TrThe result of increased Antarctic meltwater fluxes by enhanced SSO warming concurs with our previous discussion of a global warming slowdown by 0.4 °C, relative to a simulation without such coupling Fig. . Not uneHere we used the coupled three-dimensional climate-ice-sheet model LOVECLIP to better understand the impact of ice-sheet/ice-shelf/ocean/atmosphere coupling processes on the future evolution of GrIS and AIS, and to estimate their respective contributions to SL rise.st century global surface temperature rise to 2 °C above the pre-industrial level47 would be insufficient to slowdown the rate of global SL rise over the next 130 years42. Only the more aggressive low greenhouse gas emission scenario (SSP1-1.9), with temperatures leveling off below 1.5 °C 29. Here we review its main components, the coupling algorithm, experimental setup, and highlight modeling differences to previous studies.The coupled climate-ice-sheet-ice-shelf-iceberg model LOVECLIP27 used here, LOVECLIM model (version 1.3), includes ocean, atmosphere, sea-ice, iceberg components as well as a vegetation model. The atmospheric model ECBILT52 uses a T21 spectral truncation  and the prognostic quasi-geostrophic atmospheric equations are solved on three vertical levels. The model includes parameterizations of ageostrophic terms53 to better capture tropical dynamics. The free-surface primitive equation ocean model, CLIO56, which is also coupled to a thermodynamic-dynamic sea-ice model, adopts a 3° × 3° horizontal resolution and 20 vertical levels. The coupling between atmosphere and ocean is expressed in terms of freshwater, momentum and heat flux exchanges. An iceberg model integrates iceberg trajectories, melting and freshwater release along individual simulated iceberg trajectories58. VECODE59, the terrestrial vegetation model of LOVECLIP calculates the temporal evolution of annual mean desert, tree and grass fractions in each land grid cell. LOVECLIM has been used extensively to study the earth system response to a variety of boundary conditions63. Here the model is configured with a present-day land mask and an open Bering Strait.The earth system model of intermediate complexity29 is used here in a bi-hemispheric configuration. By adopting shallow ice and shallow shelf approximations, the model retains the ability to simulate streaming and stretching flow and to capture ice streams and floating ice-shelves. Floating ice-shelves, grounding line migration, and basal ice fluxes are parameterized21. PSUIM estimates the surface energy and ice mass balance by accounting for contributions from changes in temperature and radiation65. Similar to previous versions of the model29, we include parameterizations for enhanced calving caused by rainwater-driven hydrofracturing and surface melting, as well as a representation of marine ice-cliff failure. Through calculating changes of ice calving, floating-ice, grounding line migration and pinning by bathymetric bedrock perturbations, the SL is estimated. A horizontal resolution of 1° latitude and 0.5° longitude is used in the Northern Hemisphere and a stereographic grid is adopted for Antarctica with a resolution of 20 km.The ice-sheet/shelf model PSUIM5 in that our spatial resolution is lower over Antarctica. Moreover, different parameters were used, namely those characterizing sub-ice-shelf ocean melting (OCFAC), the coefficient in the parameterization of hydrofracturing due to surface liquid (CREVLIQ), and the maximum rate of horizontal wastage due to ice-cliff failure (VCLIF). The default parameter values are OCFAC = 1.0, CREVLIQ = 100 m per (m/year)−2 and VCLIF = 3 km/year5. Here we use a value of OCFAC = 1.5, which were chosen such that the AIS has a realistic extent under pre-industrial conditions and for the corresponding LOVECLIM climate forcing. Additionally, LOVECLIP realistically simulates the Antarctic ice velocity and shape of ice-shelves compared to the mean of 1996–2016 Antarctic ice velocity obtained from the satellite data67  2 concentrations in LOVECLIP follow the historical from the year 1850 to 2014, and SSP1-1.9, SSP2-4.5 and SSP5-8.5 from the year 2015 to 2150 4. The SSP1-1.9 and SSP2-4.5 simulations were further extended until 2500 CE , in which CO−2 and VCLIF = 0 km/year) with and without meltwater flux coupling (experiments SSP5-8.5_HFCMOFF and SSP5-8.5_MWHFCMOFF)  Fig. . To explTIM are calculated using Eq.  to the Antarctic ice shelves in the SSP5-8.5 scenario with/without Antarctic meltwater flux (experiments Re_SSP5-8.5_2xSOTA and Re_SSP5-8.5_2xSOTA_MWOFF). Specifically, SSO temperatures in the Antarctic ice model Supplementary InformationPeer Review File


Osteoporosis-related proximal femur fractures continue to increase significantly due to demographic change. This study was designed to evaluate the biomechanical stability of two different fixation methods (cerclage vs. screw) for refixation of a trochanter minor fragment in the pertrochanteric fractures in cadaveric bones.n = 14) and human bones (n = 16) were treated with a DHS and the trochanter minor fragment was reduced by cerclage wiring or direct screw fixation. After preloading the simulated iliopsoas with 10 N, a tensile test was performed, ending with either a 70% loss of strength or avulsion of the fragment. The mean values of the avulsion force and the surface strain were recorded.Artificial bones (All tensile tests showed no significant differences between refixation using a direct screw or wire cerclage, for both artificial bones and human specimens. Absolute values showed higher avulsion forces after direct screw fixation than refixation with a wire cerclage. The surface tension of specimens treated with direct screw fixation was lower than that of specimens treated with wire cerclage. An opposite effect was seen in artificial bones. Both effects were not statistically significant.Based on the equal stability after lag screw placement compared to cerclage wiring, we promote the placement of a lag screw into the lesser trochanter fragment in pertrochanteric femur fractures when using a dynamic hip screw.Level III. Fractures of the proximal femur are among the most frequent injuries in orthopedic trauma surgery. Those fractures often occur during low-energy-trauma, such as a tripping fall, which is common for elderly people , 2. The In AO type 31.A1.3 as well as A2 proximal femur fractures according to AO (Arbeitsgemeinschaft für Osteosynthesefragen), the lesser trochanter fractured. Furthermore, fractures on the lesser trochanter can also occur isolated (AO type 31.A1.1o) or during periprosthetic fractures (Vancouver AL). If the lesser trochanter is affected during proximal femur fractures, the fracture is generally assumed to have a lack of medial support and therefore instability . PreviouSurgical treatment of proximal femur fractures is usually performed using either a dynamic hip screw  or a proximal femoral nail with a hip component. The current research data does not prove clearly which of the two procedures offers a better clinical outcome –21, wherSo far in case of intraoperative refixation of the lesser trochanteric fragment, the standard procedure is open reduction and implantation of a wire cerclage around the lesser trochanter. However, this procedure causes an extended time of surgery and results in increased blood loss and soft tissue damage, which combined lead to an increased surgical risk for the patient , 23. A rThe aim of this study is to provide the biomechanical basis for decision-making on the optimal choice of the type of refixation of the lesser trochanter which is performed after implantation of a dynamic hip screw (DHS) once by means of a wire cerclage and once by means of a screw refixation of the lesser trochanter by orthogeriatric patients.Our study included two biomechanical tests. The initial testing started with artificial bones, while the final testing was performed on a human cadaver. After obtaining approval from our institutional review board, a total of 14 biomechanical artificial bones  were used for the first part of this study. They were split up in two groups, each containing seven artificial bones. For the second part, 16 fresh frozen human femora were commercially obtained from Science Care . The 16 human femora were split up into two groups, each consisting of 8 specimens. The femora were frozen at − 24 °C and thawed at room temperature 24 h before experimental testing. To increase comparability and reduce bias, only matched pairs of femora were used.The surgical technique of DHS implementation was performed according to the manufacturer´s instructions . First, anteversion of the femoral neck by inserting a new Kirschner wire anterior to the femoral neck was determined. Then, a new DHS guide wire with the appropriate aiming device at the desired angle was inserted. The guide wire should be placed at the center of the femoral head and extend into the subchondral bone. After radiological confirmation of the correct guide wire position, the length of the DHS blade was measured with the measuring rod directly on the guide wire. When the guide wire was inserted into the subchondral bone, 10 mm from the reading was substracted. The three-step method was used to drill the length of the selected implant. Under fluoroscopy, the guide wire was checked for any migration during drilling. Then, the desired DHS plate and DHS screw were inserted over the guide wire to the desired depth. Using the centering sleeve, the DHS plate was placed flush onto the bone. 4.5 mm self-tapping cortical screws were used to fix the plate onto the bone.The osteotomy of the lesser trochanter was already described elsewhere  and is sThe specimen was mounted in a material testing machine  and filmed with a camera system with a resolution of 1936 × 1216 pixel  Fig.  was usedAfter the biomechanical testing, the data were analyzed using the testXpert software , GOM Correlate , SPSS  and Excel .t test for unpaired samples was used. For non-homogeneous variances, the Welch test was used. Differences with p ≤ 0.05 were considered statistically significant.For data collection and analysis, SPSS  and Excel  were used. All charts were created with Prism 9 . The Shapiro–Wilk test was used to evaluate the data for normal distribution. Homogeneity of variances was evaluated using the Levene test. For homogenous variances, a p = 0.069), the mean average tension force for screw osteosynthesis was 34% higher compared to wire cerclage.The average force needed to cause avulsion of the lesser trochanter fragment in artificial bones treated with screw osteosynthesis was higher (664.61 N ± 115.00 N) than in specimens treated with a wire cerclage (438.24 N ± 51.45 N) .Correspondingly, the average surface strain was lower in specimens using a wire cerclage (0.17% p = 0.454). The average surface strain around the fragment was higher in the group treated with wire cerclage (0.75% p = 0.137) between both groups.Similarly to the tensile test with artificial specimen, the average force needed to cause avulsion of the lesser trochanter fragment in human specimen after screw osteosynthesis was higher  after fixation of pertrochanteric fractures. He was able to demonstrate that patients with reduction of medial cortical integrity had the least loss in neck–shaft angle and neck length, were walking much earlier, and displayed good functional outcomes and less hip–thigh pain presence than patients with negative reduction .These results were confirmed by studies demonstrating a correlation of hip flexion strength and dislocation of the lesser trochanter  as well Controversial data were published by Liu et al. in a retrospective analysis of 85 intertrochanteric, intramedullary stabilized fractures . RegardiDiscussing a possible benefit for the lesser trochanter fragment with orthopedic trauma surgeons always raises doubts and questions about the relationship for the additional surgical trauma involved in this procedure possibly outweighing the biomechanical benefits of a refixation. Possible complications of a surgical lesser trochanter refixation, such as increased risk of bleeding, infections and incarceration of vessels/ nerves, have to be taken into account when considering this kind of surgical technique.While it must be admitted that surgical incision as well as dissection and consecutively risk for complications will increase in cases with intramedullary nail osteosynthesis, no need for additional dissection is necessary when using a dynamic hip screw. Therefore, the risk for possible complications should be significantly decreased as well. The dynamic hip screw has the additional possible benefit of potentially using a screw hole opposite to the lesser trochanter fragment for placement of a lag screw for refixation of the lesser trochanter.Although nowadays many surgeons prefer intramedullary devices due to its minimally invasive, time-sparing approach as well as theoretically better biomechanical intramedullary load distribution , the dynTherefore, increase of care quality in this collective might show great effects in respect to patient care and socio-economic impact.If we now compare our result of equivalent stability after lag screw osteosynthesis with that of cerclage wires with reduced risk profile for refixation of the lesser trochanter when using the dynamic hip screw, then this should be the reason to reconsider one's own surgical procedure.Possible limitations of the study arise from the clinical transferability of our biomechanical results. Even after successful refixation of the lesser trochanter, traction of the iliopsoas muscle would repeatedly be applied at the lesser trochanter fragment, which could ultimately lead to dislocation of the fragment after loosening of the cerclage wire. Additionally, repeated gait cycles could also result in loosening of the cerclage wires with the positive effect of lesser trochanter refixation only being transient. The fact that an explicit DXA measurement of the bone donors is missing is a possible limitation, but the donors were selected according to orthogeriatric criteria . These criteria best reflect the orthogeriatric reality and daily routine in emergency departments. The strengths of the study are the reproducible fracture patterns and the standardized measurement of the dislocation. After the preliminary tests on artificial bones, this study was carried out exclusively on human femora, which represents a much more realistic test setup than numerous published studies that were only carried out on artificial bones.Based on the equal stability after lag screw placement compared to cerclage wiring, we promote the placement of a lag screw into the lesser trochanter fragment in pertrochanteric femur fractures when using a dynamic hip screw in the orthogeriatric population. By application of this technique, the surgeon combines the biomechanical advantages of lesser trochanter refixation with increase of primary osteosynthesis stability without disadvantages, such as additional surgical dissection and increased risk of bleeding, when using a dynamic hip screw compared to an intramedullary implant.