Constraining the response time of the climate system to changes in North Atlantic Deep Water (NADW) formation is fundamental to improving climate and Atlantic Meridional Overturning Circulation predictability. Here we report a new synchronization of terrestrial, marine, and ice-core records, which allows the first quantitative determination of the response time of North Atlantic climate to changes in high-latitude NADW formation rate during the last deglaciation. Using a continuous record of deep water ventilation from the Nordic Seas, we identify a ∼400-year lead of changes in high-latitude NADW formation ahead of abrupt climate changes recorded in Greenland ice cores at the onset and end of the Younger Dryas stadial, which likely occurred in response to gradual changes in temperature- and wind-driven freshwater transport. We suggest that variations in Nordic Seas deep-water circulation are precursors to abrupt climate changes and that future model studies should address this phasing. The response time of North Atlantic climate to changes in high-latitude deep-water formation during the last deglaciation is still unclear. Here the authors show that gradual changes in Nordic Seas deep-water circulation systematically lead ahead of abrupt regional climate shifts by ~400 years. Through changes in high-latitude North Atlantic Deep Water (NADW) formation and export, AMOC exerts an important control on the global climate system by redistributing heat near the surface and regulating carbon storage at depth. In particular, the partitioning of carbon between the surface and deep ocean is thought to play a critical role in centennial-to-millennial-scale variations of atmospheric CO2 (refs 5). However, reconciling the deglacial history of changes in overturning circulation as recorded in marine records with North Atlantic climate and pCO2 as inferred by Greenlandic and Antarctic ice cores, respectively, remains challenging. First, highly resolved records from deep convection sites sensitive to NADW that monitor the descending branch of AMOC are still lacking. Secondly, large uncertainties in high-latitude marine reservoir ages6 limit the precision of marine 14C-based chronologies. Thirdly, direct alignment of marine records to far afield Greenland ice-core stratigraphies hinders testing hypotheses of synchronicity. Lastly, precise comparisons between marine and ice-core climate records are hampered by inconsistencies between the radiocarbon and ice-core timescales8.Precise reconstructions that resolve the relative timing of changes in North Atlantic Ocean circulation, climate, and carbon cycling are necessary to anticipate the mechanisms initiating and propagating abrupt global climate changes. During the last deglaciation , the climate system underwent numerous abrupt changes that have been attributed to variations in the strength of the Atlantic Meridional Overturning Circulation (AMOC)pCO2 records for the last deglaciation based on new marine and ice core data that allows us to conclude for the first time that changes in deep-water circulation in the Nordic Seas led rapid shifts in North Atlantic climate and changes in carbon cycling.Here we present a new synchronization of high-latitude NADW, climate, and 14C ventilation age from 14C measurements on planktic and benthic foraminifera (Methods) in sediment core MD99-2284  from the Norwegian Sea , is located at the gateway of the Faroe-Shetland Channel (FSC), where warm surface Atlantic water flows into the Nordic Seas and cold dense water overflows into the North Atlantic. Critically, this overflow water is one of two main NADW pathways flowing into the deep North Atlantic and a key constituent of the AMOC9. During the last glacial period and deglaciation, overflow through the FSC remained a continuous source of NADW11. Hence, because deep-water 14C activity reflects the circulation-driven exchange of carbon between the atmosphere and deep-ocean reservoir, bottom-surface water 14C age differences of the FSC directly inform past changes in Nordic Seas deep convection, NADW production, and its southward export13.Site MD99-2284, which is characterised by exceptionally high sedimentation rates  to calculate the marine 14C ventilation age. Our estimate was determined using a random walk model (RWM)  and WD2014 (ref. 16), and 14C timescales using previously published and new 10Be records from GRIP17 and WAIS Divide ice cores, respectively 20.Surface and bottom water mass reconstructions at our site are consistent with existing paleoceanographic records of water properties, transport and exchange between the Norwegian Sea and the northern North Atlantic  and iceberg rafting inferred from IRD , which corresponds to the Younger Dryas stadial (YD), the B-P age offset rapidly increased by ∼900 years starting at 13,250 years BP. Ahead of the termination of GS-1 (11577 ± 16 years BP), the B-P offset returned to modern values beginning at 11,890 years BP Figs. , relativIRD Fig. , and rel12  and during the early GI-1 (BA) is observed in data from both the northeast and equatorial Atlantic, whereas early changes prior to the start and end of GS-1 (YD) are evident in each of the records. We hypothesise that these parallel changes in radiocarbon across sites likely reflect downstream propagation of North Atlantic deep/intermediate water 14C signatures associated with high-latitude NADW ventilation rates. In addition, the timing and pattern of the reconstructed NADW changes are consistent with shifts in production-corrected atmospheric Δ14C (ref. 27)  Figs. , 4, whicpCO2 rise near the start of GS-1 (YD). Importantly, the latter finding substantiates the hypothesised31 lag between AMOC reduction and pCO2 rise during early deglaciation and is observed in climate simulations as a transient response of the global efficiency of the biological pump to AMOC slowdown .The lag between our high-latitude NADW records and Greenland temperatures across the transitions into and out of GS-1 (YD) requires further consideration. Cooling of sea-surface temperatures and sea-ice expansion in the Norwegian Sea preceding GS-1 (YD) have been documented in terrestrial temperature and isotope records38, and further support decreasing B-P offsets in MD99-2284 starting at 11,890 years BP. A lead of a few centuries of changes in high-latitude NADW formation ahead of Greenland temperatures is compatible with the timing of AMOC slowdown and recovery during deglaciation inferred from several Pa/Th circulation39 and other AMOC-sensitive proxies41. Despite uncertainties with the 14C chronologies and coarse temporal resolution, it has been demonstrated that a century-scale response of Pa/Th to AMOC changes should be additionally accounted for when interpreting this proxy as a function of changes in ocean circulation43. The existence of a significant lag time for Greenland temperature and Pa/Th records behind changes in NADW would represent an important observational constraint to account for, for example when attempting to infer the physical mechanisms of rapid climate and AMOC change from climate model simulations45. Equally abrupt and synchronous changes in the AMOC, Pa/Th and Greenland temperature  should not, therefore, be expected in numerical model simulations.On the other hand, ocean warming in the Nordic Seas, a northward diversion of the polar front, and NADW resumption during the second half of the GS-1 (YD) have been widely reported14C ventilation records, we propose that the lagged Greenland temperature response behind changes in Nordic Seas NADW formation can be understood as a threshold response to gradual changes in surface temperature-driven freshwater transport, which ultimately modulates the strength of the northward oceanic heat transport. Both empirical46 and climate modelling studies47 have proposed a nonlinear salt oscillator in the North Atlantic system, whereby meltwater production rates are greater  toward the end of warm (cold) episodes. This is consistent with evidence for southward (northward) migration of the polar front, more (less) extensive sea-ice cover, and increased (decreased) iceberg discharge at our coring site shortly before the termination of GI-1 (GS-1). We further hypothesise that shifts to higher (lower) rates of meltwater fluxes, although initially prompted by gradual climate warming (cooling), are amplified by concomitant changes in surface winds, which play a critical role in the rearrangement of water masses in the Nordic Atlantic. For instance, model studies suggest that sustained cold events like GS-1 (YD)48 and the Little Ice Age50 can be triggered by a positive feedback loop, whereby increasing export of meltwater to the subpolar gyre in response to warming leads to surface ocean cooling and consequent strengthening of atmospheric blocking over the North Atlantic–i.e. large-scale quasi-stationary anticyclonic circulation. This feedback can impede northward heat transport and suppress NADW formation for centuries. Moreover, reduced NADW can lead to an additional freshening through basin-wide subsurface warming52, which causes ice-shelf thinning and iceberg discharge52, further bolstering NADW formation weakening. However, we cannot rule out the potential amplifying role of a catastrophic drainage of freshwater from the Baltic Ice Lake into the Nordic Seas53, which occurred precisely at the start of GS-1 (YD), and could have further impacted regional NADW formation.On the basis of timing inferred from our 54) and glacial conditions55, the cold Fennoscandian Ice Sheet induces a strong southwesterly wind flow over the Norwegian Sea associated with blocking circulation over the ice sheet. This configuration is more conducive to reduced sea-ice cover through export of sea ice out of the Norwegian Sea and increased surface warming and salinity in response to northward oceanic transport of heat and salt, which in turn gradually promotes deep-water formation in the Norwegian Sea55. Our data and other existing records all lend support to the occurrence of this proposed mechanism midway through GS-1 (YD)  . New studies suggest that under full GS-1 (YD)  records from the ancient lake of Atteköp, southern Sweden63 66 run for 106 iterations. The approach involves nonlinear deformation of the SST time series onto the reference δD time series to deliver an optimal alignment accounting for uneven compaction and/or expansion of sediments over time, as well as for analytical errors associated with the proxy data , have been previously reported in core MD99-2284 (ref. 36). These horizons have been precisely and accurately radiocarbon dated in Lake Kråkenes, Norway70. The markers are here supplemented by the first reported marine occurrence of the Abernethy Tephra72  using an ARL SEMQ electron microprobe equipped with six wavelength-dispersive spectrometers and a Bruker 5030 SDD energy-dispersive spectrometer. Instrument conditions included a 14 kV accelerating voltage, 10 nA beam current, and beam size of 4 to 6 µm. Results were reported as non-normalized major oxide concentrations 72 and Abernethy Forest (AF555)71, in Scotland, were attributed to this eruption, which are both similar in stratigraphy and geochemistry to tephra that we have identified 70. Therefore we here assigned to the layer an age of 11,744 ± 50 years BP. Radiocarbon dating of this layer based on one terrestrial plant macrofossil71 from a sediment sequence in Abernethy Forest yields a calibrated age range of 12,380–11770 years BP, which is consistent with, or slightly underestimates our age assignment.Results from the analysis of tephra grains in samples 247.5 cm and 249.5 cm show a single geochemical population of rhyolitic composition similar to the widespread Vedde Ash erupted from the Katla volcano in Iceland during the middle of the YD. Recently, it has been shown that there was a second tephra-producing eruption of Katla within the YD, but closer to the YD-Holocene boundary named the Abernethy TephraNeogloboquadrina pachyderma (s) ), two samples of the benthic foraminifer Cibicidoides wuellerstorfi, one shell of Pyrgo murrhina, and a number of mixed benthic foraminifera were hand-picked from slices of core MD99-2284 for AMS 14C dating for a total of 88 measurements (41 planktic and 47 benthic), including 8 previously reported dates36  measurements, whereas large samples were additionally rinsed with dilute, organic-free HCl.Well-preserved monospecific shells of the planktic foraminifer 14C measurement procedures within the laboratories of Beta Analytic Miami (FL), USA, and ETH Zurich, Switzerland. All the small fraction samples were processed and analysed using a newly developed method77. The approach involves direct analysis of CO2 from ultra-small amounts of carbonate (∼0.5 mg) using a compact AMS facility equipped with a gas ion source at the Laboratory of Ion Beam Physics, ETH Zurich. To test for contamination by secondary carbonates on the small fraction samples, we performed leaching experiments on the sample material surface using HCl 0.02 M and following the procedure outlined in ref. 78. All radiocarbon ages are here reported according to the standard protocol of ref. 79. A subset of 7 samples were prepared at the University of Cambridge using methods detailed in ref. 80 and subsequently dated by AMS at the 14 Chrono Centre, University of Belfast.Large samples were converted to graphite and analysed using standard AMS 14C ages served as a basis to generate subsurface (∼100 m) and bottom water (1500 m) ventilation records employing the random walk model described below, and ultimately to reconstruct benthic-planktic (B-P) ventilation ages81. We identified seven outliers that were dismissed from our bottom water ventilation record . These were all ages based on benthic foraminifera, including one Pyrgo spp. age, which has been shown to yield old 14C ages12. The other benthic dates, which are all based on small shell fragments <1 mg in weight and all associated with the 63-150 μm fraction, were either significantly younger than the corresponding planktic ages (where available), or significantly younger than the contemporaneous atmosphere. Acid leaching tests indicate that four out of the seven outliers are likely affected by modern CO2 carbon contamination, as evidenced by the high 14C content in the respective leach fractions (Supplementary Data −1), we argue that complications associated with bioturbation and differential mixing effects are likely negligible83. Rather, we argue that the systematically younger ages measured in these samples may be the result of preferential modern-carbon contamination among the finer carbonate fraction through secondary growth of calcite within the sediment pore waters and exchange between calcite and the carbonate ions in pore waters83.The resulting R) and reservoir (B-Atm and P-Atm) estimates were inferred using a random walk model that incorporates both the uncertainty in the calendar age modelling and our 14C measurements ; and 14). We then infer the value of the absolute Norwegian Sea reservoir correction For each of our 14. At any calendar age We take a Bayesian approach for our estimation of 87) wherebyAdditionally, we require a prior model for the evolution of the Norwegian Sea’s reservoir age 14) then allows us to estimate the absolute Norwegian Sea reservoir correction The above random walk model is then combined with our prior on 10Be concentrations at UC Berkeley and at the Purdue University PRIME Lab  were analysed for 10Be and 14C in the upper atmosphere. The former are integrated in ice cores; the latter are incorporated in a number of absolutely dated records  that take up carbon directly from the atmosphere at the time of formation. Hence, matching of the globally synchronous common short-term 10Be and 14C variations as reconstructed in ice cores and IntCal13 records14, respectively, provides a unique tool for estimating timescale differences in a nearly continuous fashion8.Solar activity cycles modulate the amount of Earth bound cosmic rays that controls the production rates of cosmogenic radionuclides 10Be flux17  and new WAIS Divide 06A ice core (WDC) 10Be concentration data  to 14C from IntCal13 using a Bayesian wiggle match approach to estimate the likely time offset between the ice and radiocarbon timescales (Supplementary Data 10Be data are compared to the atmospheric 14C production rate (p14C) simulated from IntCal13 Δ14C data using a Box-Diffusion carbon-cycle model91 initialized with pre-industrial boundary conditions assuming a constant carbon cycle. Even though this assumption does not necessarily hold for climatically unstable period such as the YD, sensitivity experiments8 have shown that centennial-scale variations in atmospheric 14C  are largely unaffected by the dynamics of the carbon cycle and predominantly reflect solar cycle modulations. To estimate the uncertainty associated with carbon cycle effects on the simulated p14C, we performed four additional sensitivity experiments where the carbon cycle was perturbed by increasing or decreasing the air-sea gas exchange and the ocean diffusivity parameter by 50%, individually. These tests resulted in an uncertainty estimate of ± 2%, which was added to the nominal IntCal13 Δ14C error.Here we aligned relative changes in GRIP 10Be production rate variability and uncertainties accompanying longer-term climate and carbon cycle changes92. We also apply a 1-year lag for 10Be to account for the average delay between production and deposition, and ultimately scaled the 10Be time series by a factor of 0.7 to optimize the fit with the p14C data.Prior to wiggle matching, the cosmogenic nuclide records were bandpass filtered between cut-off frequencies of 1/100 and 1/500 years to focus on the common (likely solar-induced) secular changes in production. Low-pass filtering eliminates short-term weather noise and sampling inconsistencies between the data sets. On the other hand, high-pass filtering minimizes systematic errors associated with millennial 93 and methodology of ref. 8  were freeze-dried, homogenized, and lipids were extracted from the sediments via sonication with a 9:1 mixture (v/v) of dichloromethane:methanol for 20 min. The procedure was repeated three times and supernatants were combined. Total lipid extracts (TLE) were evaporated in a Biotage TurboVap under a stream of nitrogen gas. TLE were separated into constituent lipids by flash silica-gel chromatography (pre-cleaned 100% active silica gel) in hexane-saturated columns using as eluting solvents hexane (F1), dichloromethane (F2), 3:1 hexane:ethyl acetate (F3), and methanol (F4), separately.One hundred and sixty-nine sediment samples were collected from core MD99-2284 at 1-cm intervals. The samples 94, respectively, contained in the F1 fraction of 169 samples  and IP25 were identified and quantified on an Agilent 7890A gas chromatograph (GC) equipped with a mass selective detector (MSD). Samples were measured with an Agilent HP-5ms column . The GC oven was held for 1.5 min at 60 °C and ramped to 300 °C at 10 °C min-1. After 1 min it was ramped to 320 °C at 2 °C min−1 and held for 10 min. We used a PTV injector in splitless mode, with samples injected at 60 °C and the injector temperature immediately ramped to 320 °C at 900 °C min−1. All the compounds were identified in SIM mode based on mass spectra and retention times from the literature . A standard mixture of n-alkanes (C10-C40) was run after every sixth sample injection, and was used to evaluate instrument drift and correct for mass-dependent differences in the response factor of the MSD. Concentrations of individual n-alkanes were calculated based on the comparison of the adjusted peak areas relative to both C36 alkane and 5α-androstane used as an internal standard, and each added to samples before GC analysis. Similarly, IP25 was quantified based on comparison to the internal standard of 9-octyl-heptadec-8-ene (9-OHD)96 added before GC analysis. The measurement accuracy of both n-alkanes and IP25 was determined by monitoring the relative response factor for n-C36 and 5α-androstane, and for IP25 and 9-OHD (over a range of concentrations), respectively, every seven samples (1σ = ± 3.2%).To monitor changes in terrestrial meltwater discharge and paleo sea-ice conditions at the coring site, we quantified the abundance of long-chain les Fig. . The lat37 unsaturated alkenones97 contained in the F2 fraction of 154 samples  (1σ = ± 0.0072).To quantify changes in marine surface water phytoplankton productivity, we measured the concentration of C25 index (PIP25)99  the modelled data within its uncertainty, assuming a Gaussian error distribution. We then estimated the breakpoint structure of each Monte Carlo realization using the Segmented function run with 100 bootstrap iterations. The inferred likelihood distribution of each breakpoint allowed us quantifying the timing uncertainty for the start and end of the transitions under investigation (Fig. To estimate the timing of the transitions recorded in the proxy data, we used a Monte Carlo version of the function Segmented in the R package ‘Segmented’Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients’ blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization. It is characterized by the generation of allergen-specific immunoglobulin (Ig)E that binds to antigen-presenting cells and effector cells, i.e. mast cells and basophils, which following exposure to an allergen results in immediate allergic reactions3. The diagnosis of food allergy is determined by the oral food challenge (OFC), which arose in the 1950s when it was established that the elimination of allergenic foods resulted in a complete resolution of symptoms, while reintroduction of the allergen caused the return of those reactions5. Since then the procedure for the double-blind placebo-controlled food challenge was standardized and accepted as the “gold standard” for allergy diagnosis7. However, this procedure is burdensome for both patients and medical providers, as it takes multiple hours and carries inherent risks of severe allergic reactions. Over the years, a substantial effort has been made to refine the methods for allergy diagnosis that can complement and eventually replace OFCs.Food allergy is a type I immune hypersensitivity affecting 7.6% of children and 10.8% of adults in the United States10. Different in vivo subcutaneous tests, i.e. Skin-Prick or Prick-Prick, are still commonly used in clinic, as they can be interpreted immediately and allow testing of multiple allergens. However, they have been shown to lack specificity16, and must be interpreted only in conjunction with carefully collected medical history.The use of chemically extracted food fractions as a diagnostic tool for hypersensitivity dates back to 1912 when Schloss used proteins in the scratch skin testin vitro quantification of IgE specific to allergen extracts correlated with the likelihood of allergic reactions20, fueled the development of new diagnostic assays and the search for the ideal biomarker. The ability to refine proteins that drive IgE responses, e.g. Ara h 2 in peanut21, gave rise to component-resolved diagnostics (CRD)22. Further development of diagnostic methods occurred when researchers started mapping IgE-binding epitopes, short sequences of amino acids from allergenic proteins, using SPOT membranes24. This subsequently led to a more granular understanding of the immune mechanisms of food allergy. Studies of conformational and sequential epitopes uncovered different phenotypes of allergy, e.g. children who outgrew their milk or egg allergy had IgE to predominantly conformational epitopes while those with persistent allergy possessed IgE to sequential epitopes26. As the technology continued to evolve enabling the screening of large libraries of peptides using microarrays27, it was found that not only IgE binding to specific epitopes, but also antibody diversity – IgE recognition of greater numbers of different epitopes - was associated with the severity of allergic symptoms28.More recent work demonstrating that The granularity provided by the molecular-based diagnostic, i.e. peptide microarrays and SPOT membranes, gave hope of improving the accuracy of diagnostic methods. However, they came with drawbacks as SPOT membranes required large quantities of serum and did not allow clear quantification or multiple sample screening and, similarly to microarrays, required labor-intense protocols. To further advance the molecular allergy diagnostics and potentially bring it to the clinic, we have developed a Bead-Based Epitope Assay (BBEA) that allows high-throughput screening of samples and epitopes with significantly smaller volumes of serum or plasma compared to current molecular assays. BBEA provides greater sensitivity and reproducibility than microarrays and better resolution than component resolved diagnostics, making it a promising tool for the development of diagnostic and prognostic biomarkers.29. Here we present a validation of the BBEA to measure epitope-specific humoral immune profiles as potential biomarkers of food allergy. We show that allergenic epitopes detected with BBEA have high reliability within samples and reproducibility across different laboratories, large effect size across allergic and non-allergic patients, and inter-patient variability that allows discrimination between different disease phenotypes. Additionally, the high resolution provided by epitope analyses may enable further discoveries of the interplay between epitope-specific IgE and IgG4 antibodies in predicting food allergy thresholds as determined by the OFC.Ultimately the goal of the bioassay is to detect biomarkers that have low technical variability, low baseline variation within the individual, as well as high variation between individuals and a large effect size in response to perturbationThe bead-based epitope assay (BBEA) enables simultaneous quantification of antibodies binding to different sequential epitopes for multiple samples. Epitopes are first covalently coupled to unique fluorescent microspheres (Luminex beads). The epitope-loaded beads are then mixed together to form a multiplex master library and added to a 96-well microplate Fig. . To iden38 and consisted of 66 epitopes from the 5 most clinically relevant milk proteins, i.e. αS1-casein, αS2-casein, β-casein, β-lactoglobulin, and κ-casein; and 50 epitopes from the 3 major peanut allergens, i.e. Ara h 1, Ara h 2, and Ara h 3.The BBEA doesn’t limit epitope detection to any specific antigen and allows researchers to customize peptide libraries and antibodies. In this study we present a quantification characterization of IgE and IgG4 binding to milk and peanut epitopes using BBEA. The milk and peanut libraries were designed and created based on published experimentset al.39 previously showed that using at least 25 beads per antigen/peptide produces consistent MFIs, while using lower bead counts resulted in increased variability between identical samples. Samples with an average bead count less than 30 were thus excluded from the downstream analyses. The Cullen-Frey analysis41, which shows the parameters of theoretical distributions across 100 bootstrap simulations, indicated that the original distribution of the MFIs was generally skewed  incubating for a longer period of time (>1 hour) and consequently differences might be expected to occur between earlier and later rows. To investigate whether this is the case for BBEA, two plates of a peanut experiment had a completely randomized design  and principal variance component analysis (PVCA) in milk and peanut experiments49.High-throughput assays have well documented batch effects, where each experiment carries specific technical artifacts that can confound biological signalsIn the milk study, plate effect was seen on the PCA plots and estimated by the PVCA to account for 8% and 2% of total variability, for IgE Fig.  and IgG4PlateDesigner50, a web application specifically for the sample randomization across microplates. This design software may be applied to any study and was used for the design of the CoFAR2 peanut experiment.The plate estimation was quite straightforward since the experiment was designed ensuring random allocation of disease phenotypes and treatment groups across plates. As such, there was no confounding between experimental conditions and plates, and both factors could be adequately estimated. The milk study was relatively small and included only 4 plates for each antibody, which made it possible to empirically randomize the plates. However, for larger experiments, such ad hoc randomization may not always prove successful. As an added quality assurance measure, we have developed An important characteristic of any assay is that it is both reliable and reproducible. Reliability refers to the agreement between multiple measures assessed on the same sample under the same conditions; while reproducibility looks at the variation of the measurement on the same sample under varying conditions, i.e. different instruments or observers. We have assessed the reliability and reproducibility of the BBEA with a separate set of experiments where IgE and IgG4 binding to peanut epitopes was measured by 3 independent laboratories using Luminex-200 instruments.51, with the intra-class correlation coefficient (ICC) for agreement >0.9 for IgE . PCA plots and phylogenetic trees showed evident clustering of triplicates belonging to the same samples Figs.  and S3ABIgE Fig.  and >0.7IgE Fig. . Of noteIgE Fig.  had highIgE Fig. . This waIgE Fig. .Figure 3BBEA’s reproducibility across the three laboratories was evaluated using the average binding of the technical triplicates. PVCA analyses showed much more of the variance in the data attributed to the sample type than to the laboratory, with a weighted average proportion variance (WAPV) of 99% versus 0.5% for IgE Fig.  and 67% 52. However, MIA involves complex and lengthy protocols, and takes ~2 days from the start of the experiment to acquisition of the raw data. The goal of BBEA is to simplify and streamline the detection of epitope-specific antibody binding, thus shortening the procedure time while also increasing the sensitivity of the signal detection.The current method to study allergenic epitopes with microarray immunoassays (MIA) allows screening of large libraries of peptides simultaneouslyTo benchmark the performance of the BBEA to MIA, we’ve performed a separate set of experiments using milk peptides. For the MIA experiment, a positive pool (PP) and NC samples were run on two separate days, and the PP was done in duplicates. For the BBEA experiment, the PP and NC were repeated in replicates of 4 on two separate days Fig. . While MWe then compared the ability of BBEA and MIA to identify epitopes that differ between PP and NC. 100% (66/66) of the epitopes were detected by BBEA (detection threshold p < 0.05) and only 17% (11/66) by MIA, indicating that BBEA had superior sensitivity Fig. . The eff53. IgG4 is considered a blocking antibody, as it could potentially prevent allergic symptoms by out-competing IgE on effector cells for allergen. While the measurement of IgE and IgG4 to component proteins of an allergen, by microarray assays and ImmunoCAP, provides insights into the mechanism of food allergy, there are still no universally accepted set thresholds for their diagnostic utility. We sought to further investigate the relationship between IgE and IgG4 directed at allergenic epitopes in association with milk allergy severity and peanut allergy diagnosis.In IgE-mediated food allergy, protein recognition by IgE antibody is indicative of allergic sensitization; and it has been previously demonstrated that epitope-specific IgE binding is associated with different disease phenotypes55, we measured IgE and IgG4 binding to 66 milk epitopes. After OIT was discontinued for 8 weeks, patients underwent final OFC with 10 g of milk protein at month 32 of the trial. Epitope-specific IgE profiles were highly correlated with IgE specific to milk component proteins and with the OFC outcome. The overall z-score for all 66 IgE epitopes had a Pearson’s correlation of 0.81 for milk, 0.87 for casein, and 0.78 for beta-lactoglobulin , while for individual epitopes these correlations ranged from 0.34 to 0.85. On the other hand, IgE binding to 7 epitopes most correlated with OFC, showed a dose-dependent relationship with the OFC outcome (p < 0.001), while IgG4 (p = 0.128) or the ratio of the two antibodies  had no clear pattern  that underwent milk oral immunotherapy (OIT) ± omalizumab in a prospective placebo-controlled randomized clinical trialern Fig. . We obseern Fig. . A simplern Fig. .Figure 557 were analyzed. Allergic diagnosis was based on either OFC, or IgE to peanut ≥14kUA/L, or IgE to peanut ≥0.35kUA/L and Skin-Prick test ≥3 mm plus a suggestive history. IgE specific to 22 epitopes had significant binding differences (fold change (FCH)≥1.5 and FDR <0.05), while only 4 IgG4 epitopes and 14 IgG4-IgE epitopes showed significant differences  recruited for the NIH-sponsored CoFAR2 natural history cohortces Fig. . All theWe have developed a Bead-Based Epitope Assay to measure antibody responses directed at allergenic epitopes. The assay enables multiplexing of epitopes, allowing multiple results from a single specimen, and is done in a 96-well plate, streamlining multiple sample processing within one experiment Fig. . The BBEHigh-throughput assays are known to have technical artifacts that may confound biological signals. We have considered both well and plate effects as potential sources of bias and showed that neither well position nor reading order influenced binding values Fig. . This al59 or ComBat60; but in order for them to work, there should be no confounding between the batch and experimental variables. For example, if samples from all allergic patients are processed on one plate and non-allergic individuals on another, it is not possible to reliably estimate how much of the result is attributable to either plate effect or allergy diagnosis alone. To avoid such scenarios, samples must be randomized across and within plates. To achieve this, large experiments with peanut epitopes were designed with PlateDesigner50 and the original plate effect that accounted for > 10% of the variability was successfully eliminated.We have identified a batch effect that is indicative of individual microplate runs, and as shown in Figs. Epitope-specific antibody binding detected by BBEA is reliable and reproducible Figs. , with hiBiomarkers for allergic sensitization include allergen recognition by IgE antibodies, which are the least abundant isotype in serum, making it the most challenging to detect. We showed that the BBEA provides greater sensitivity for detection of allergenic epitopes compared to the microarray immunoassay Fig. , capableet al.61 have shown using BBEA that antibody binding to milk epitopes discriminates between different phenotypes of milk allergy, with highest epitope-specific IgE in children reactive to baked-milk, followed by fermented milk, and then whole milk. Suarez-Farinas et al.55 developed prognostic models that were able to identify subjects that would achieve sustained unresponsiveness (remission) to milk OIT. The best model consisted of IgE epitopes and had better performance than the epitope-specific IgG4, CRDs, or combinations of those biomarkers. IgG4 antibody is considered to play a protective (blocking) role in allergy, as it competes with IgE for allergen binding68, and a higher ratio of IgG4 to IgE could be indicative of better outcomes and less severe allergic phenotypes74. Some groups have found either no significant association between IgG4 to IgE ratios of milk proteins and allergic phenotypes36, or possible effect modification by IgG4 rather than a linear relationship76. Using IgE and IgG4 binding profiles to milk epitopes, we showed that the relationship between the two antibodies is not necessarily linear  were conjugated to the LumAvidin xMAP microspheres  and stored in PBS-TBN buffer . The peptide-coupled beads are stable for up to 1 year at 4–8 C, when prepared with 0.05% Sodium Azide in the storage buffer. A master mix of microspheres was then made in PBS-TBN buffer and 100 μL/well of mix was added to 96-well filter plates. After washing the microspheres with PBS-TBN, 100 μL of plasma samples diluted 1:50 for milk- and 1:10 for peanut-allergic patients was added to the wells in triplicates. Plates were incubated on a shaker for 2 hours at 300 rpm at room temperature. Excess plasma was then removed and the plate was washed. 50 μL/well of R-phycoerythrin (PE) labeled mouse anti-human IgE-PE  or IgG4-PE  secondary antibody was added and plates were incubated for 30 minutes at room temperature. After a final wash, 100 μL of PBS-TBN was added to each well to re-suspend the microspheres, which were then transferred to fixed-bottom 96-well reading plates and quantified with the xPONENT® software on Luminex200 instrument .ij was defined as:NSB represents non-specific binding  wells. This quantitative outcome denotes epitope-specific antibody binding.Median Fluorescence Intensity (MFI) was obtained directly from the Luminex xPONENT® software. For the non-specific signal detection for every peptide, each plate included 3 wells with only PBS-TBN buffer and no plasma sample. For each sample i, and epitope j, the binding measurement B77. The milk library consisted of 66 peptides from αS1-casein (n = 18), αS2-casein (n = 13), β-casein (n = 14), β-lactoglobulin (n = 8) and κ-casein (n = 13) proteins. The peanut library had 50 peptides from Ara h 1 (n = 27), Ara h 2 (n=13), and Ara h 3 (n=10).The libraries of 20-mer milk and 15-mer peanut peptides  were designed based on the meta-analyses of BBEA and previously published SPOTs membrane and peptide microarray studies55 and (ii) peanut CoFAR256 cohorts for the quantification and detection of technical artifacts, as well as the analysis of IgE and IgG4 in relation to allergy severity and diagnosis, (iii) pooled samples for the reliability and reproducibility study, and (iv) an allergic pool and a negative control for the comparison of BBEA and MIA. The assay for milk OIT cohort used 4 IgE and 4 IgG4 plates; while peanut CoFAR2 cohort was run on 22 IgE and 28 IgG4 plates. The first 2 IgE plates had all triplicates randomly assigned to wells, in order to stimate an effect of well position. After ascertaining the presence of a plate effect in milk OIT, the intraplate controls  were included in triplicates on all plates of the CoFAR2 experiment. The reliability and reproducibility experiment consisted of 8 different samples, each was a pool of patients with different peanut reactivity, and measured IgE and IgG4 to peanut epitopes. This experiment was repeated by 3 independent laboratories, located in the Icahn School of Medicine at Mount Sinai , AllerGenis , and Carrera Bioscience . With the exception of the MIA study, all samples were processed in technical triplicates. BBEA versus MIA experiment measured IgE to milk epitopes and consisted of the replication of positive  pool and one negative control, and was repeated on two separate days  milk OITays Fig. .CoFAR2 and milk OIT studies were funded by the NIH, AI-66738 and AI-44236. Milk OIT + omalizumab clinical trial was registered on the ClinicalTrials.gov, NCT01157117. All protocols and consent/assent forms were reviewed and approved by the Program for the Protection of Human Subjects (PPHS) at the Icahn School of Medicine at Mount Sinai and the Internal Review Boards of participating institutions: milk OIT – Johns Hopkins University; CoFAR2 – Johns Hopkins University, Arkansas Children’s Hospital, University of North Carolina and National Jewish Health. Informed consents/assents were obtained for all subjects. All research methods were performed in accordance with relevant guidelines and regulations.37. In brief, a library of 235 peptides consisting of 20 amino acids overlapping by 17 (3 offset), corresponding to the primary sequences of αS1-, αS2-, β-, and κ-caseins and β-lactoglobulin was printed in 2 sets of triplicates (6 spots per peptide) onto Arrayit SuperEpoxy glass slides . The slides were blocked with 400 μL of 1% human serum albumin (HSA) in PBS containing 0.05% Tween 20 (PBS-T) for 1 hour at room temperature, then incubated with 250 μL of patient serum diluted 1:5 in PBS-T/HSA at 4 °C overnight. Slides were then washed with PBS-T and incubated for 1 hour at room temperature with a mixture of 3 monoclonal mouse anti-human IgE and IgG4 antibodies 36. Slides were then incubated for 3 hours at 31 °C with a cocktail of Anti-BiotinDendrimerOyster 550 (350) and Anti-fluorescein isothiocyanate Dendrimer Oyster 650 (350) in Dendrimer buffer , both at 0.6 μg/mL with the addition of 0.02 μg/mL of salmon sperm DNA (Invitrogen), followed by a wash with PBS-T, 15 mM Tris, 0.1× PBS, and 0.05× PBS. Slides were centrifuge dried and then scanned by using a ScanArrayGx . Images were saved as TIF files.Six plasma samples from milk allergic patients were mixed 1:1 to generate a positive control pool (PP); and a plasma sample from one non-allergic participant was used as the negative control (NC). MIA was performed as previously described with some modifications52 following the following formula:The fluorescence signal of each spot was digitized with the Scan Array Express Microarray Analysis System (PerkinElmer), exported as comma-delimited files, and quantified as Z-scores49.All data processing, quality control, and analyses were performed in R v3.5.1. Unless otherwise noted, results were considered statistically significant if the two-sided nominal p-value was less than the 0.05. After the MFI was converted to binding scores, batch effect was quantified using PVCA, with the principal component threshold set to 0.8ij with j = 1, …, k; i = 1, …, n) can be written as:xP-1 matrix where each column p  is Zi = , β is a P-1 dimensional vector containing the plate effect with respect to the reference plate (Plate1), X is the design matrix representing known experimental factors which effect on binding we wish to study, and the residuals εij contain any other unknown sources of variation and random fluctuations. Under this model, random allocation of samples to plates will preclude collinearity between Z and X and the P-1 coefficients associated to the plate effect γ, can be unbiasedly estimated (as γ*) using classical linear regression or linear mixed effect models. As such, the binding can be adjusted as:Under the assumption that samples are randomly allocated to plates, the binding for epitope j and sample i , good (0.60–0.74), fair (0.40–0.59), and poor (<0.40). Unsupervised clustering of the samples was performed using spearman correlation as a distance metric and “average” agglomeration algorithm.For the reliability analysis a two-way ICC for agreement was used to assess reliability across triplicates, and an ICC for consistency when assessing reproducibility across the laboratories. According to the ICC and Kappa guidelinesFor the BBEA versus MIA comparisons sample reliability across days was evaluated with the two-way ICC for agreement. Epitope detection was carried out by comparing the differences (𝛿) in binding between positive and negative pool using a moderated paired t-test. Effect size was estimated as 𝛿/SD(𝛿) and epitopes were considered detected if p < 0.05, with no adjustment for multiple hypotheses testing, given the small sample size. As a sensitivity analysis, the same comparisons were made after adjusting BBEA and MIA data for batch effect, i.e. experimental date.tree79 R package using IgE and IgG4 as predictors of the binary OFC outcome (10 g versus <10 g).To evaluate the relationship between epitope-specific antibody responses and milk OFC dose, we focused on seven milk IgE-specific epitopes with Pearson correlation less than −0.50 with the OFC dose. The association between the average binding of the 7 peptides for either IgE or IgG4 and the OFC dose  was evaluated using an ordinal logistic regression. A classification tree with 4 terminal nodes was constructed with the limma framework, and epitopes with FCH≥1.5 and FDR <0.05 were considered statistically significant. To be consistent with the literature, the IgG4-IgE is referred to as a “ratio”; however, since the binding was quantified in the logarithmic scale, the difference between the two antibodies rather than their quotient was calculated.IgE, IgG4, and IgG4-IgE binding to all 50 peanut epitopes was modeled for peanut allergic and non-allergic patients using linear models in the Supplementary Figures and Tables


The results showed that TGR5 was significantly up-regulated in H9C2 (rat cardiomyocyte cells) and human cardiomyocytes (HCMs) after H/R. Overexpression of TGR5 significantly improved cell proliferation, alleviated apoptosis rate, the activities of caspase-3, cleaved caspases-3 and Bax protein expression levels, and increased Bcl-2 level. Overexpression of TGR5 significantly up-regulated ROS generation, stabilized the mitochondrial membrane potential (MMP), and reduced the concentration of intracellular Ca2+ as well as cytosolic translocation of mitochondrial cytochrome c (cyto-c). Meanwhile, overexpressed TGR5 also enhanced the mRNA and protein levels of interleukin (IL)-10, and decreased the mRNA and protein levels of IL-6 and tumor necrosis factor α (TNF-α). The shTGR5+H/R group followed opposite trends. In addition, overexpressed TGR5 induced an increase in the levels of p-AKT and p-GSK-3β. The protective effects of TGR5 were partially reversed by AKT inhibitor MK-2206. Taken together, these results suggest that TGR5 attenuates I/R-induced mitochondrial dysfunction and cell apoptosis as well as inflammation, and these protections may through AKT/GSK-3β pathway.Ischemia/reperfusion (I/R) injury reduces cell proliferation, triggers inflammation, promotes cell apoptosis and necrosis, which are the leading reasons of morbidity and mortality in patients with cardiac disease. TGR5 is shown to express in hearts, but its functional role in I/R-induced myocardial injury is unclear. In the present study, we aimed to explore the underlying molecular mechanism of TGR5 in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury  The m pathway . HoweverG protein-coupled receptor TGR5, also named as GPBAR1, BG37 or M-BAR, belonged to G-protein-coupled receptors (GPCRs) family . TGR5 paThe present study examined the putative role of TGR5 in the pathophysiology of the TGR5 or shTGR5 cardiac warm hypoxia/reoxygenation (H/R) injury model. Our results demonstrated that TGR5 can ameliorate mitochondrial dysfunction, cell apoptosis and inflammation due to H/R, suggesting the protective effects of TGR5 in the maintenance of myocardial homeostasis via the activation of the AKT/GSK-3β signaling pathway.2 at 37°C. To stimulate hypoxia, cells were incubated in a hypoxic incubator containing 95% N2 and 5% CO2 for 4 h at 37°C. Subsequently, reoxygenation  was performed for another 4, 6, and 12 h. Cells were collected after the ending reoxygenation. The control cells were cultured under normoxic conditions.H9C2 and human cardiomyocyte (HCM) cells were cultured in Dulbecco’s Modified Eagle’s Medium , which is composed of 10% fetal bovine serum and 100 U/ml penicillin. Then, the culture was placed in an incubator containing 5% COOverexpression plasmid of TGR5 was constructed and purchased from Bio‐transduction Lab . Lentivirus‐mediated short hairpin RNA (shRNA) targeting TGR5 mRNA was synthesized by Shanghai GeneChem . Overexpression plasmid of TGR5 and TGR5 shRNA (shTGR5) were transfected into cells using Lipofectamine 2000 transfection reagent , according to the instructions.Total RNA was extracted using RNAiso Plus Reagent  and NanoDrop 1000 Spectrophotometer  was used to check the quality and concentration of RNA. The first strand complementary DNA (cDNA) were synthesized by using PrimeScript® RT Reagent Kit . The expression of TGR5, interleukin (IL) 10 (IL-10), IL-6 and tumor necrosis factor α (TNF-α) was quantified using SYBR Premix Ex Taq  and β-actin was used as the internal control. The primers for TGR5, IL-10, IL-6, TNF-α and β-actin are as follows:TGR5 F: 5′-CAGTCTTGGCCTATGAGCGT-3′;TGR5 R: 5′-CTGCCCAATGAGATGAGCGA-3′;IL10 F: 5′-ACTGCACCCACTTCCCAGT-3′;IL10 R: 5′-TGTCCAGCTGGTCCTTTGTT-3′;IL6 F: 5′-GCTACCAAACTGGATATAATCAGG A-3′;IL6 R: 5′-CCAGGTAGCTATGGTACTCCAGAA-3′;TNF-α F: 5′-GCCTCTTCTCATTCCTGCTTGT-3′;TNF-α R: 5′-TTG AGA TCC ATG CCGTTG-3′;β-actin F: 5′-CTAAGGCCAACCGTGAAAAG-3′;β-actin R: 5′-GCCTGGATGGCTACGTACA-3′.CT−ΔΔ method.RT-PCR was performed on the ABI 7300 Sequence Detection System . The relative gene expression was calculated by the 25 cells/well). Cell proliferation was evaluated by the Cell Counting Kit-8 (CCK8)  according to the instructions.H9C2 and HCM cells were distributed into a 12-well plate  according to the instructions.H9C2 and HCM cells were planted into 12-well plates at a density of 1 × 10The intracellular calcium concentration was detected by Fluo-3 AM , following the instructions.Mitochondrial membrane potential (MMP) was detected using a mitochondrial membrane potential assay kit with JC-1  according to the manufacturer’s protocol.c subjected to Western blotting.Cell Mitochondria Isolation Kit  was used to isolate mitochondria and cytosol, according to the instructions. Sample protein of the cyto-Cell apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime), according to the instructions .g, 10 min) at low temperature and then using BCA Protein Assay Kit  to ensure the concentration. Proteins were separated on 10–12% SDS/PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% non-fat milk with 1 h, primary antibody against TGR5, cyto-c, Bax, Bcl-2, cleaved caspases-3, p65, Lamin B1, p-IκB-α, IκB-α, p-AKT, p-GSK-3β and β-actin  were incubated overnight at 4°C. Finally, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. β-actin was used as an internal control for the correction of protein expression. Finally, the protein bands were detected using an enhanced chemiluminescence detection system  and the relative band densities were quantified using the Multi-Analyst software package (Bio-Rad).The cells were collected and lysed using RIPA buffer . The protein was obtained by centrifuging , according to the instructions. And serum expression of IL-10, IL-6 and TNF-α were examined using commercial kits according to protocols recommended by the manufacturers .t test or one-way ANOVA. The data were represented as the mean ± SD. All experiments have three biological repetitions where each one has three technological repetitions. P<0.05 was considered with statistically significant.SPSS 22.0 was used to analyze the experimental data. The significance was analyzed by Student’s P<0.05), with a peak at 6 h and then falling rapidly (P<0.05)  C and D. P<0.05, P<0.05)  D–F. Cons P<0.05) F. These 2+ and the relative expression of cyto-c were down-regulated in TGR5+ H/R group and up-regulated in shTGR5+ H/R group, compared with H/R group in H9C2 and HCM cells (P<0.05). However, MMP was improved in TGR5+ H/R group and alleviated in shTGR5+ H/R group, compared with H/R group in H9C2 and HCM cells . However, shTGR5+ H/R group significantly down-regulates level of IL-10 and improves levels of TNF-α and IL-6, compared with H/R groups in H9C2 and HCM cells (P<0.05). Similar results were found in serum levels of IL-10, IL-6 and TNF-α (P<0.05) (Anti-inflammatory cytokine (IL-10) exhibits a protective role in I/R-stressed cardiomyocytes. However, inflammatory cytokines (TNF-α and IL-6) display pro-inflammatory effect in ischemia cardiomyocytes after reperfusion. To further ensure the cardioprotective effects of TGR5, the transcriptional levels of IL-10, IL-6 and TNF-α were determined A and B. (P<0.05) C and D. P<0.05) (P<0.05) (P<0.05) . These data suggested that overexpressed TGR5 alleviates mitochondrial pathway and inflammation via the activation of AKT/GSK-3β pathway.To gain a comprehensive understanding the mechanism of TGR5 in regulating myocardial H/R-induced apoptosis, mitochondrial pathway and inflammation, we further examined the AKT/GSK-3β pathways. Western blot analysis showed that levels of phosphorylated AKT and GSK-3β were significantly increased by overexpression TGR5 (P<0.05) A. The spP<0.05) B–G. Trea(P<0.05) B–D. Cons(P<0.05) E–G. ThesHere, we studied the function and mechanism of TGR5 in H/R-induced injury in H9C2 and HCM cells. We found that the levels of TGR5 mRNA and protein were significantly increased and peaked after 6 h of reperfusion following Hypoxia for 4 h, which is similar to the studies of Yang et al.  and Zhua2+ overload, the depolarization of MMP and the releases of the proapoptotic protein, cyto-c, from the mitochondria [c from the mitochondria to cytoplasm caused by the change of mitochondrial membrane permeability is the key link of apoptosis induced by intracellular mitochondrial pathway [Previous studies showed that myocardial I/R induces mitochondrial dysfunction including the enhancement of ROS generation, mitochondrial Cachondria . The rel pathway . Therefo pathway . Velazqu pathway  reported pathway . Here, w−/− compared with Tgr5+/+ mice upon LPS stimulation, and LPS-INT-777 treatment significantly decreases the mRNA levels of IL-6 and TNF-α in primary macrophages. The activation of NF-κB is one of the critical cellular responses to inflammations [Inflammatory response plays a pathogenic role in myocardial I/R injury, especially innate immune responses involved in cytokines, including IL-10, IL-6 and TNF-α, and so on . In our mmations . Previoummations . In our 216 and decreased by Ser9 phosphorylation [9 protects the heart during I/R [9 phosphorylation, thus decreaseing myocardial I/R injury [473 in aortic endothelial cells or macrophage [. In addition, and the specific inhibitor of AKT (MK-2206) partially blocked the effects of TGR5 on H/R-induced mitochondria dysfunction and apoptosis as well as inflammation. Together, these results support mechanistic involvement of Akt/GSK-3β signaling pathway in TGR5-mediated anti-apoptotic and anti-inflammation effects.GSK-3β is a serine/threonine kinase, which is involved in many cellular functions including gene expression, hypertrophy and apoptosis in the heart ,31. GSK-rylation ,33. An iring I/R . In addiR injury ,36. AKT R injury . Kida etR injury  reportedcrophage . In agreIn summary, our results demonstrated that TGR5 offers cardioprotection against myocardial H/R injury by reducing inflammation and inhibiting mitochondria-mediated apoptosis. Activation of Akt/GSK-3β signaling is involved in the cardioprotective effect of TGR5 . These fClick here for additional data file.


Incomplete lateral compression fractures (including AO Type B2.1) are among the most common pelvic ring injuries. Although the treatment of choice remains controversial, sacroiliac (SI) screws are commonly used for the operative treatment of incomplete lateral compression fractures of the pelvic ring. However, the disadvantages of SI screws include the risk of nerve root or blood vessel injury. Recently, tape sutures have been found useful as stabilizing material for the treatment of injuries of the syndesmosis, the rotator cuff and knee ligaments. In this current study, we aimed to test the biomechanical feasibility of tape sutures to stabilize the pelvis in the setting of AO Type B2.1 injury.Six human cadaveric pelvises underwent cyclic loading to compare the biomechanical stability of different osteosynthesis methods in a B2.1 fracture model. The methods tested in this experiment were a FiberTape® suture and the currently established SI screw. A 3D ultrasound tracking system was used to measure fracture fragment motion. Linear regression was used to model displacement and stiffness at the posterior and anterior pelvic ring.P >  0.999; stiffness 50.8 ± 13.0 N/mm, P > 0.999).At the posterior fracture site, the FiberTape® demonstrated similar displacement (2.2 ± 0.8 mm) and stiffness (52.2 ± 18.0 N/mm) compared to the sacroiliac screw .Considering the anterior fracture site, the FiberTape® again demonstrated similar displacement (3.8 ± 1.3 mm) and stiffness (29.5 ± 9.0 N/mm) compared to the sacroiliac screw (displacement 2.9 ± 0.8 mm, The newly presented osteosynthesis, the FiberTape®, shows promising results for the stabilization of the posterior pelvic ring in AO Type B2.1 lateral compression fractures compared to a sacroiliac screw osteosynthesis based on its minimal-invasiveness and the statistically similar biomechanical properties. Fractures of the pelvic ring are frequently seen, especially in polytrauma patients or geriatric patients with poor bone quality –3. IncomRecently, tape sutures have proven to be a stabilizing method for syndesmotic injuries in the ankle joint, for injuries to the rotator cuff of the shoulder or for ligamental knee injuries –16. SimiThe disadvantage of the transiliac internal fixator lays in the possible disturbance of the soft tissue structures dorsal of the posterior superior iliac spine . By usinIn this study, we assume that a tape suture will perform the semi-rigid transfixation of both sacroiliac joints. In addition, when inserting a tape suture in the posterior superior iliac spines, no relevant nerve structure is endangered.For this reason, we performed a biomechanical analysis on 6 fresh frozen cadaver pelvises to analyze the stability of the tape suture applied to the posterior pelvic ring compared to a sacroiliac screw.A total of 6 human cadaver pelvises collected between January 2016 and April 2017 were used in this study with the approval of our institution’s ethics committee and the approval of the donors’ relatives given prior to the experiments. Only intact pelvises without any preexisting damage to the musculoskeletal structure, a tumor, or tuberculosis disease was included in this study. The characteristics of the pelvises are displayed in Table Prior to any processing, the bone density of all pelvises was measured with a qCT scan using the fourth and fifth lumbar vertebrae Table .The pelvises were unfrozen 1 day prior to their experiment. On the day of the experiment, each pelvis was heated in a water bath at roughly 35 °C for 30 min in order to approximate body temperature.Once heated, the tissue covering the spots needed to install the experiments equipment Fig. a was disAfter the removal of the tissue, each pelvis was mounted on the testing machine and the 5-step protocol Table  was applNext, the fixation methods were applied with the either tape suture  spanning between both posterior superior iliac spines Fig. b or the For the insertion of the tape suture (FiberTape®), two osseous channels were drilled through both posterior superior iliac spines, beginning from the fracture site using a high speed drill. The drill went through the contralateral posterior superior iliac spine from medial to lateral to achieve an osseous channel in the same direction as on the ipsilateral site. The FiberTape® was then threaded through the bony holes and a metal washer  first on the contralateral site. Both ends were pulled back through the osseous channel with the tape consequently spanning between both iliac spines. The ipsilateral ends were then threaded through a second DogBone®-Button and manually surgically tied. Consequently, both metal washers were lying on the lateral sides of the osseous channels on each iliac spine Fig. b.For the S1 screw, the following technique was used. The S1 partially threaded screw was inserted under fluoroscopic control on the fractured site using the S1-corridor above the first neuroforamen, with at first drilling of a Kirschner-wire and secondly inserting the screw using the previously drilled K-wire.Finally, the pelvises were set up in the testing machine.In this study, we used an all-electric testing machine  and a 3D-ultrasound measuring system  for the recording of all data.The ultrasound system consisted of 3 sensors that were placed onto the pelvis as seen in Fig. For the conduction of the experiment, a 5-step protocol was applied according to McDonald et al.  Table 22.Every 30 ms, the position of all sensors was recorded, allowing the calculation of the relative distances between the sensors at any time. These distances were then used for the statistical analysis.t tests. The Bonferroni method was used to adjust the P values for multiple comparisons. For all tests, α was set to 0.05. Descriptive statistics are presented as mean ± standard deviation (SD) wherever appropriate.We used linear regression to model the magnitude of displacement between the fracture fragments representing the functionality of the stabilizing techniques used: native pelvis (reference), SI screw, and FiberTape®. Clustered standard errors were calculated using the Huber-White method. Pairwise comparisons were carried out using P >  0.999; stiffness 50.8 ± 13.0 N/mm, P >  0.999)  and stiffness (52.2 ± 18.0 N/mm) compared to the sacroiliac screw   and stiffness (29.5 ± 9.0 N/mm) compared to the sacroiliac screw  can be performed by various conservative and surgical procedures –23. HoweMinimally invasive posterior tension banding with suture tape through the posterior superior iliac spines is a newly introduced method for stabilizing the posterior pelvic ring. This procedure is a minimally invasive fixation method with a sufficient posterior fracture stabilization without implanting any soft tissue disturbing components such as pedicle screw constructs. In addition, the extra-sacral position avoids an injury to the sacral nerve roots. Compared to SI screw fixation, tension banding does not compromise the intact SI joint. We assume that this semi-rigid fixation method also allows physiological mobility in the SI joint, so that removal of osteosynthesis implants is not necessary and there is no danger of stiffening of the joint. Positioning of the drill holes in the posterior superior iliac spine is usually easy to determine from the anatomical landmarks or to visualize in X-rays. This means that no special equipment such as 3D x-ray or navigation system is required to ensure correct positioning of the implants. We think that the radiation exposure and the surgical demands are also lower in this minimally invasive procedure compared to standard sacroiliac screw fixation.Our biomechanical results showed no significant difference between screw fixation and minimally invasive posterior pelvic tension banding considering the dislocation of both the anterior and the posterior fracture gap under cyclic loading. Using the FiberTape®, the dislocation of the posterior pelvic ring was 0.2 mm higher (3.2 mm ± 0.9) than with the sacroiliac screw (3.0 mm ± 0.5), however not significantly. Considering the anterior pelvic fracture, the dislocation using the FiberTape® was 0.9 mm higher than the sacroiliac screw fixation, but again not significantly. With a resolution of the ultrasound measuring system of 0.1 mm, we showed that the stabilization of the posterior pelvic ring with a minimally invasive tension banding achieves a comparable stability as a sacroiliac screw. The effect of a higher dislocation of the anterior pelvic fracture as seen in the FiberTape® group could also be seen in conservatively treated pelvic fractures type B 2.1 without any problems in bone healing of the anterior pelvic ring in patients under 65 years . We therIn the results presented here, we showed that the stabilization of the posterior pelvic ring was sufficiently achieved by minimally invasive dorsal tension using tape suture.The newly presented osteosynthesis of the posterior superior iliac spine using FiberTape® shows promising results for the stabilization of the posterior pelvic ring in AO Type B2.1 lateral compression fractures of the pelvis when compared to sacroiliac screw osteosynthesis. Further advantages of the technique presented here are the lack of the necessity of implant removal, lower surgical demands, and lower risk of injury to relevant neural or vascular structures of the patient.