The further evolution of the ice cream microstructure during storage and abuse was captured using ex situ tomography on a time scale of days. The morphology of the ice crystals and unfrozen matrix during these thermal cycles was quantified as an indicator for the texture and oral sensory perception. Our results reveal that the coarsening is due to both Ostwald ripening and physical agglomeration, enhancing our understanding of the microstructural evolution of ice cream during both manufacturing and storage. The microstructural evolution of this complex material was quantified, providing new insights into the behavior of soft-solids and semi-solids, including many foodstuffs, and invaluable data to both inform and validate models of their processing.Ice cream is a complex multi-phase colloidal soft-solid and its three-dimensional microstructure plays a critical role in determining the oral sensory experience or mouthfeel. Using in-line phase contrast synchrotron X-ray tomography, we capture the rapid evolution of the ice cream microstructure during heat shock conditions in situ and  Ice cream is a widely consumed dairy product whose complex microstructure determines its texture and oral sensory perception. The main constituents of ice cream are air cells, ice crystals, finely dispersed (emulsified) fat globules, and a continuous unfrozen aqueous solution phase (containing sugars and other additives) and, as shown in It is thus crucial to be able to observe how the complex three-dimensional structure of ice cream evolves over time with changes in temperature. The traditional method of examination using cryo-SEM of extracted samples b is capaIn the present paper, we will describe the application of 4D sCT to reveal the microstructural evolution in ice cream over a range of different timescales, namely that which takes place during the manufacturing process  and storage over long timescales (days/weeks). We apply in situ, time-resolved sCT to reveal the microstructure evolution mechanisms during manufacturing; whereas we use an ex situ sCT methodology to provide time-resolved data during long-term ice cream storage.The manufacturing process for ice cream introduces around 50 vol% of air cells so the typical make up of frozen ice cream, by volume, is 50% air cells, 30% ice crystals, 10–15% sugar solution matrix and the balance dispersed fat droplets and non-fat solids ,5,6,7,8.The texture and sensorial quality of ice cream are related to the size and connectivity of the air cells and ice crystals ,11,12. IWhilst significant progress has been made in elucidating the process-structure-quality relationships of ice cream, a major limitation is the complex procedures needed to perform microstructural observations by cryo-SEM or transmission electron microscopy (TEM). These are post-mortem techniques , and proIce cream falls into the category of soft solids  and has similar behavior and properties to many man-made and natural semi-solids. The microstructure of many of these materials evolves with time, changing material properties ranging from rheology to yield strength to plasticity, and, in the case of foodstuffs, mouthfeel. One way of quantifying this microstructural evolution is time-resolved radiography ,21 or toThe use of high brilliance synchrotron X-ray computed tomography (sCT), coupled with a precise cold stage, to study ice cream microstructures has recently been reported by the current authors. In this prior study, we obtained the three-dimensional quantification of microstructural changes arising from the thermal cycling at low heating and cooling rates over periods of weeks. This was undertaken to simulate storage and transport effects on the ice cream product . Howeveroperando, during the process. The second set of experiments involved monitoring the microstructural evolution over long timescales and so ex situ experiments could be used to capture the time-dependent evolution. Novel image-based quantification techniques were developed to precisely and robustly evaluate the structural characteristics in the ice cream samples. Variations in the size distribution of ice crystals and unfrozen matrix were followed during the thermal-induced microstructural evolution as these are recognized as indicators for changes in texture and oral sensory perception. In the present study, we used a bespoke cold stage (capable of controlling the sample temperature precisely between 233 K and 293 K with 0.1 K accuracy) and employed high resolution  sCT to conduct 4D (3D plus time) studies on the microstructural evolution of ice cream samples undergoing different types of thermal histories. In the first set of experiments, the ice cream samples underwent fast heating and cooling cycles to simulate aspects of manufacture and were continuously scanned, in situ and 500 mL blocks of fresh ice cream containing 5% fat and 40% ice were prepared . Prior to the in situ tomographic experiments, a block of fresh ice cream was left at room temperature to melt and partially de-aerate. Kapton tubes with 3 mm inner diameters and 67 μm wall thickness  were filled with this liquid dairy mixture using a 5 mL syringe, followed by mounting them onto a specially designed cold stage which will be described in the next section.For the ex situ samples, Kapton tubes were inserted into the central region of individual ice cream blocks and the dairy mixture was first subjected to blast freezing at 238 K and then stored at 248 K. Immediately before observation by synchrotron imaging, the Kapton tubes filled with ice cream were cut from the bulk samples on a dry ice bed (maintained at around 193 K) and then inserted into the specially designed cold stage. The cold stage assembly is shown schematically in In order to investigate processes relevant to the manufacture of ice cream, the in situ thermal cycling experiment was performed following the thermal cycles as described. A Kapton tube containing melted ice cream mixture was firstly mounted into the cold stage at the temperature of 270 K i.e., just below the melting temperature of the ice cream formulation. It was held at 270 K for 15 min and then its temperature was rapidly reduced to 250 K with a fast rate of cooling of 5 K/min (referred to as FC) and held at this temperature for 10 min. After that, the sample was heated to 267 K, at a heating rate of 5 K/min and held there for 10 min. Subsequently, it was cooled down from this temperature to 250 K at a slower rate of 0.05 K/min (referred to as SC). The overall thermal cycle for in-situ experiments is illustrated in the temperature versus time plot of Blocks of ex situ ice cream samples were cycled between 258 K and 268 K multiple times, as shown in Concurrently, with the thermal cycling of the sample, a series of X-ray tomographic images were acquired using sCT on I13-2 beamline at Diamond Light Source . This has a high flux undulator, producing a pink beam, with peak modes of narrow bandwidth (ca. 300 eV), with high and low bandwidth filters removing modes outside the energy ranging from 15 to 30 keV. This combined with the ca. 250 m beamline length provides excellent in-line phase contrast. A 2560 × 2160-pixel PCO Edge 5.5 CMOS camera optically coupled to a single crystal CdWO4 scintillator was used to record the projection images. The distance between the sample and the scintillator was optimized to be ~35 mm to achieve an optimum imaging quality. The final pixel size obtained was 0.81 µm for the scans.During the in situ experiments, each tomographic run includes collecting 720 projections evenly spaced over a 180° rotation with the exposure time of 0.1 s. For FC, the scans were not recorded continuously due to the limitation of sampling rate relative to the fast cooling rate (5 K/min) and two images were acquired at the start and end points of the process, i.e., 270 K and 250 K, respectively, indicated as pentagons in The collected 2D radiographs/projections were stacked and converted into sinograms and in which any apparent continuous lines were removed by interpolation to reduce ring artifacts due to imperfections from the detector/camera. The sinograms were then used to reconstruct the volume slices using a filtered back projection (FBP) based algorithm. Because the ice cream samples were relatively low attenuating to the incident X-ray beam, the reconstructed volumes exhibited a relatively high level of noise. Therefore, the 3D volumes need to be filtered before any feature segmentation and quantification can be applied. Due to the microstructural differences in ex situ and in situ experiments, the data processing methods are slightly different.For the ex situ experiments, a novel image processing strategy and reconstruction algorithm were used to achieve better quality images from the collected data; for details, see References ,7. A morIn the in situ study, an image analysis based quantification methodology of the ice crystal size in the ice cream samples was developed which is similar to the techniques for porous structure characterization for biomedical and geological samples . DependiA 3D distance map was first created by a Euclidean distance transform from the binarized image.For the current samples, 13 sampling spheres with a diameter equally spaced between 5 µm and 40 µm were chosen to balance the quantification accuracy and computational expenses.The radius of each sampling sphere (in voxels) was compared to the voxel intensities in the distance map, determining the centers of the spheres of a radius that can be completely placed within the ice phase.A dilation algorithm was then applied to the voxels correlated to the sphere centers, using a spherical kernel of the same radius. The volume after dilation is the volume correlated to the sampling sphere.Step 3–4 were repeated for all the sampling sphere dimensions chosen in step 2, providing a range of reachable volumes corresponding to increasing sphere diameters.The volume fraction was calculated by dividing each volume by the total volume of the ice phase. Then an ice crystal size distribution can be calculated as the differential of the area under the cumulative volume percentage curve.The variation in accessible volume was used to estimate the ice crystal (or any segmented phase) size distribution, as follows:For the ex situ study, the reconstructed volumes from the data were cropped into a smaller volume, followed by the microstructural quantification in 3D. The three-dimensional rendering of the features, as well as the quantification of the ice crystals, was performed by manually using Avizo 9.4 . For more details, see References ,7.First, a tomographic scan was collected at 270 K prior to the onset of fast cooling. Except for air cells, it was not possible to resolve structural features in the material at this temperature. This indicates that it was above the freezing point of the ice cream composition under investigation and additionally significant supercooling is required before ice crystal nucleation occurs.s) which is given by the interfacial area between ice (solid) and unfrozen matrix (liquid), denoted as A, divided by the total enclosed volume of ice (solid), denoted as Vs, i.e., fication . There ification d.The 3D rendering of ice crystals and un-frozen matrix from representative regions of the same size are shown in The size distributions of ice crystals and the unfrozen matrix, obtained from the 3D accessible volume method, are shown in In a complementary manner, the effects of the thermal history on the unfrozen matrix were also quantified. These qualitative observations are supported by the numerical data. The unfrozen matrix at 267 K (20.2 µm) after heating is much greater in thickness than that measured at 250 K (8.2 µm), which supports the idea that small crystals were melting into the unfrozen matrix as the temperature increased. During the SC regime, a trend of decreasing size of the unfrozen matrix is observed when the temperature of the sample was slowly cooled down to 250 K. This is presumably due to the growth increasing volume fraction of ice crystals, and is consistent with our quantification for the overall increased size of the ice crystal.s) as revealed in mm−1 when cooled down to 250 K.The morphological changes of ice crystals are accompanied by changes in the total ice fraction and specific interface area (Ss) of ice crystals decreased during heating and holding, which supports the observation of the partial melting and coarsening of ice crystals. During this period, the needle-shaped ice crystals at 250 K became more spherical after the heating regime . The fraction of ice decreased significantly from 38% at 250 K to 28% 267 K, which is consistent with the melting phenomenon while heating. In the SC regime, we noted that the increase of the ice volume fraction with freezing is not completely monotonic. One possible reason for this is the relative movement of the entire volume during cooling, which might change the features that are measured.s of ice crystals first decreased dramatically from 599 to 301 mm−1 during the fast heating and holding period. However, the morphology of the ice crystals showed only small changes during the SC regime, as indicated by the average Ss decreasing from 301 mm−1 at 267 K to 247 mm−1 at 263 K, and then continuously decreased to 193 mm−1 when cooled down to 250 K.The SApart from the dynamic evolution mechanism of the individual features in ice cream which were elucidated in the in situ study, an examination of the microstructural evolution over long timescales (days/weeks) was also performed. The results are summarized in Compared with the ex situ study, the size distribution obtained following the FC stage of the in situ work has a much smaller modal value of 8.2 µm. Such a fine scale microstructure is possible due to the fast cooling from the ice cream mix (liquid), which is different from the ex situ coarsened ice cream sample which was being “thermally abused” for a number of cycles between 258 and 268 K, i.e., the maximum temperature was well below the temperature of zero ice fraction a.operando time-resolved synchrotron tomography in a bespoke cold stage to quantify the fast evolution of the different microstructural phases in ice cream during processing, including ice crystals and the unfrozen matrix. To capture the long-term microstructural evolution during storage, we used an ex situ sCT methodology.In the present study, we applied in situ and The in situ experimental results in this study reveal that the coarsening of ice crystals was due to both Ostwald ripening and physical agglomeration during heating and cooling. This change in the ice crystals size and morphology strongly influences our sensory perception of ice cream’s taste. During the subsequent storage, we demonstrate that fluctuations in storage temperature can cause a partial-melting and recrystallization process, increasing the rate of coarsening. These processes were quantified, providing valuable data to both inform and validate models of the behavior of soft-solids.


We find that glacial discharge - sourced primarily from ice shelf and iceberg melting along the eastern Antarctic Peninsula – remained largely stable between ~6,250 to 1,620 cal. yr BP, with a slight increase in variability until ~720 cal. yr. BP. An increasing trend in glacial discharge occurs after 550 cal. yr BP (A.D. 1400), reaching levels unprecedented during the past 6,250 years after 244 cal. yr BP (A.D. 1706). A marked acceleration in the rate of glacial discharge is also observed in the early part of twentieth century (after A.D. 1912). Enhanced glacial discharge, particularly after the 1700s is linked to a positive Southern Annular Mode (SAM). We argue that a positive SAM drove stronger westerly winds, atmospheric warming and surface ablation on the eastern Antarctic Peninsula whilst simultaneously entraining more warm water into the Weddell Gyre, potentially increasing melting on the undersides of ice shelves. A possible implication of our data is that ice shelves in this region have been thinning for at least ~300 years, potentially predisposing them to collapse under intensified anthropogenic warming.The Antarctic Peninsula Ice Sheet is currently experiencing sustained and accelerating loss of ice. Determining when these changes were initiated and identifying the main drivers is hampered by the short instrumental record (1992 to present). Here we present a 6,250 year record of glacial discharge based on the oxygen isotope composition of diatoms (δ Some of the most dramatic changes have been observed in the Antarctic Peninsula (AP) region, where up to 87% of glaciers are retreating5 and several ice shelves have collapsed7. At least some of the increased melting relates to rapid atmospheric warming associated with a more positive phase of the Southern Annular Mode (SAM)8, the leading mode of climatic variability in the Southern Hemisphere11. This has resulted in a strengthening of warmer westerly winds12 and increased surface melting on the eastern side of the AP13, which has been linked to the collapse of the Larsen B ice shelf14. Stronger westerlies also drive warm Circumpolar Deep Water (CDW) onto the western AP continental shelf, leading to enhanced transfer of heat to the underside of ice shelves15, and further melting. On the eastern AP, inter-annual fluctuations in bottom-water properties are governed by the wind-driven Weddell Sea Gyre, which is also sensitive to SAM17.The Antarctic Ice Sheet (AIS) is losing mass at an accelerating rate13, which has been linked to atmospheric warming driven by a positive trend in SAM18. Although the relationship between atmospheric temperatures and melting is well-quantified for this high altitude  ice core site, there is little corresponding data on how the combined atmospheric and ocean temperature changes have driven retreat of glaciers and ice shelves at or close to sea level, where most of the recent mass loss has occurred19. One attempt to understand the thinning history of marine-terminating glaciers on the western AP analyzed the oxygen isotope composition of marine diatoms (δ18Odiatom) in a sediment core from the near-coastal Palmer Deep20  of change are uncertain due to insufficient resolution of this record, which is limited to four data points covering the past 500 years20. In addition to this, there is also a suggestion that the climate of the western and eastern AP was characterised by opposing temperature anomalies during the late Holocene23, further highlighting the need for additional constraints on glacier melting from a number of locations, covering a range of time-scales. Acquiring the data necessary to bridge the gap between the short instrumental record of mass loss (1992 to present1), estimates of surface melting in ice cores13 and existing marine records of glacial discharge from the western AP20 is the motivation of this study.While the rates and drivers of modern warming are now well documented, the interplay between SAM and its impacts on ice sheet mass loss on longer, centennial to millennial timescales is poorly understood. This is principally a result of short instrumental records and a paucity of proxy data linking climate variability to glacier melting. Analysis of an ice core from James Ross Island (JRI) off the north-eastern tip of the AP Fig.  shows anp20 Fig. . In this18Odiatom in marine core PS67/182-1 from the South Orkney shelf, at the northeastern tip of the AP . The restriction of melt to this specific geographic corridor of the ocean is steered by the strong cyclonic circulation of the Weddell Gyre  supports a small terrestrial ice cap, ~620 km2 in size. Onshore lacustrine sedimentation at nearby Signy Island  had occurred by the early to mid-Holocene. Whilst moderate variations in glacier size have occurred since this time30, the cumulative flow of ice shelf and iceberg-derived meltwater to the region, as derived from a numerical model, is up to ~3000 mm/yr31 and thus nearly one order of magnitude greater than local meltwater input or precipitation (~400 mm/yr32). Our record thus provides a unique opportunity to assess the past sensitivity of ice shelves and glaciers in the NW Weddell Sea to known changes in climate.Here we present a high-resolution proxy record of glacier mass loss during the Holocene by measuring δ AP Fig. 24–27 - ryre Fig. , which cyre Fig. 27,28, buent Fig. 25,26. Inand Fig.  began ov210Pb and 14C methods, with age-depth modelling performed in CLAM v2.233  between 1,617 and 720 cal. yr BP followed by a decline  counts (>2 mm) and/or sand fraction  show centennial to millennial-scale variability . The sediment sequence was dated using both  BP Fig. . The firine Fig. . Since cine Fig. . A furthine Fig.  shows a ion Fig.  particulene Fig.  but therity Fig.  that are18Odiatom values prior to c. 1,617 cal. yr BP are indicative of a marine-water dominated signal with a reduced influence from glacier and iceberg melting relative to modern-day values  shows centennial-scale warm events that are superimposed on a gradual warming trend (0.3 °C) from ~7,000 to 500 cal. yr BP 39 were also observed on the western AP at this time, suggesting coherent temperature trends during the mid-Holocene  occurred at this time, reducing the entrainment of warm CDW into the Weddell Gyre resulting in sea-ice growth and reduced ice-shelf melting. The apparent increase in δ18Odiatom variability between c. 3,200 and 1,900 cal. yr BP is broadly coincident with a mid-Holocene climate optimum inferred from multiple indicators of increased biological production in lakes on nearby Beak Island 40 and on Signy Island 41. , where warmer SST on the western AP coincides with cooler atmospheric temperatures on the eastern side. Opposing temperatures during this period have been related to the establishment of Antarctic dipole-like conditions, driven by opposite temperature and sea-ice anomalies in the Weddell and Amundsen-Bellingshausen seas. The development of dipole-like conditions during the late Holocene, which has been linked to ENSO, might explain why the long-term trend to increasing glacial discharge on the western AP, starting around ~2,500 cal. yr BP20  peaking around ~1,100 cal. yr BP before declining Fig. . This va44 Fig. . Along t44 Fig.  have beeies Fig.  throughoP20 Fig. , is not P20 Fig. 38. Recen23 and the Beak Island lake record40 on the eastern AP show a warming trend from 550 and 543 cal. yrs BP respectively 20  and 244 cal. yr BP (A.D. 1706) bracket pronounced changes in discharge at site PS67/182-1, which first declines until ~550 cal. yr BP (A.D. 1400) before displaying a pronounced increasing trend until the modern day (A.D. 2005). At the same time, both the JRI ice coreely Fig. , while Sely Fig. 38. Glaci)20 Fig. . Warmer  40 Fig. . We note 40 Fig. . After 20 and 244 40 Fig. . We ther8 and, during a more positive SAM, the belt of westerlies intensifies and contracts poleward, bringing stronger, warmer westerlies to the AP region. Analysis of contemporary temperature data (spanning 1979–2015) reveals that SAM impacts the west and eastern sides of the AP in different ways12. On the eastern AP, inter-annual variability of temperatures is most sensitive to zonal wind (west-east) anomalies crossing the Peninsula and resultant leeside adiabatic warming (Föhn winds) rather than to meridional wind anomalies , which is closely tied to variability in the zonal portion of the SAM pattern12. More frequent Föhn events drive enhanced surface melting that has been linked to collapse of the Larsen B Ice Shelf in 2002 through hydrofracture46. The oceanic response to the shifting SAM – on the eastern side of the AP - is mostly through Ekman transport16. Positive SAM is associated with increased wind stress and reduced sea-ice48 and, over longer time periods, stronger westerlies promote greater entrainment of warm CDW water into the Weddell Gyre16 with the potential to increase melting of fringing ice shelves38.SAM denotes the pressure gradient between mid and high latitudes49  have undergone enhanced mass loss during the past ~300–500 years. In the case of the eastern AP, the most recent changes (since A.D. 1706) are unprecedented during the past 6,250 years.We hypothesise that a progressive shift to positive SAM since the mid-fifteenth century, and particularly since the early 1700s promoted stronger westerlies that triggered increases in glacial discharge, via the mechanism that we see today; that is, atmospheric and ocean-driven thinning and retreat of ice shelves. Whilst there is a dearth of wind-proxies for the AP covering this time-interval, sea salt aerosol (ssNa+) concentrations in EPICA Dome C ice core49 Figs  and S2 i 50 Fig.  and soutean Fig. . At the ean Fig. 23,38. Th000 Fig.  has beenast Figs  and 3e f18. Abram et al.18 also note a strong co-dependence between ENSO and SAM during the last 1000 years, suggesting the maximum in Niño3.4 SST during the fifteenth century might have contributed to the SAM minimum   which is thought to reflect the dominant influence of rising greenhouse gas levels and ozone depletion. However, despite evidence for a clear co-dependence between SAM and ENSO during the late Holocene, questions exists about the relative impacts on AP temperatures, and how this might vary on the west and east sides of the Peninsula.The mechanisms driving SAM trends during the last millennia are only partially understood. Coupled climate simulations indicate that increasing solar irradiance after the Spörer Minimum (~A.D. 1415–1534) resulted in a small positive forcing on SAMmum Fig. . Instrumño) Fig.  would be7 - most notably the Larsen A-Prince Gustav Channel in 1995 and Larsen B in 2002  to variations in the westerlies, it is likely that mass loss will continue to accelerate through the mid-late twenty first-century, raising global sea-level1, impacting marine ecosystems3 and potentially leading to meltwater-induced sub-surface ocean warming which has the potential to drive even greater melting underneath ice shelves4.Models predict further poleward intensification of the westerliesPolarstern. Core PS67/182-1 penetrated a thick (>60 m) package of acoustically laminated sediments  PS67/182-1  and corresponding trigger core (TC) were recovered from 364 m water depth within the eastern tributary of Signy Trough, ~15 km south of Coronation Island, South Orkney Islands (SOI) Fig. , during nts Fig. , interprmud Fig.  with isomud Fig. . X-ray f57. Water flows westwards in the southern limb (Weddell Sea embayment) and eastwards in the northern limb of the gyre . Seasonal fluctuations of WSBW properties are governed by the seasonal cycle of the winds over the western margin of the Weddell Sea. Inter-annual fluctuations are linked to the variability of the wind-driven Weddell Sea gyre and hence to large-scale climate phenomena such as the SAM and El Niño/Southern Oscillation16.The South Orkney Islands (SOI) are located at the north-western limb of the cyclonic flowing Weddell gyre Fig. , which iyre Fig. . Due to ies Fig. . Warm Ci3 volume before and after freeze drying. Grain-size composition (gravel-sand-mud) was analysed on samples of ~60 cm3 volume from 1-cm intervals by wet- and dry sieving over >63 μm and 2 mm, respectively. The mud fraction (<63 μm) was retained and used to analyse sortable silt (SS) following McCave et al.60. Biogenic opal was first digested using a 2 molar Sodium Hydroxide (NaOH) solution, heated at 90 °C for an hour. The NaOH solution was removed with three deionised water rinses. In addition, organic material was digested using a 30% H2O2 solution, shaken for 24 hours at room temperature. Samples were placed on a vertical carousel for 24 hours prior to analysis and the sonicator was used for 30 seconds before each measurement. Analyses was performed on a Coulter Counter (Multisizer-3) at the Earth Sciences Department, University of Cambridge and mean SS calculated using the equations in McCave et al.60. Carbonate was not removed as it forms a very small component of the samples. Ice-rafted debris  was counted at 2 cm intervals in PS67/182-1 using high-resolution X-radiographs61. Total organic carbon (TOC) was measured every 10 cm with an Elementar Vario EL at the AWI. TOC contents were determined after removal of the total inorganic carbon  with HCl using an ELTRA CS-2000. Biogenic opal  was analysed at a sampling interval of 10 cm using an automated leaching technique62. The archive halves of the sediment cores were scanned with an Avaatech XRF-core scanner63 at AWI with a sampling interval of 1 cm and spot size of 1 cm2.Volume specific magnetic susceptibility (MS) and wet-bulk density (WBD) were analysed at 1 cm intervals on whole cores using a GEOTEK multi-sensor core logger  at the Alfred Wegener Institute for Polar and Marine Research (AWI) . MS was also measured on split cores at 1 cm intervals (using a Bartington MS2 susceptibility meter with MS2F sensor). Lithology, colour and sedimentary structures were described visually on the split cores, and refined using X-radiographs of 1 cm-thick sediment slabs taken parallel to the core axis. Water contents were determined by weighing samples of 10 cm14C) dating of calcareous micro-fossils and the humic acid in the organic fraction of the sediments where no microfossils were present , Beta Analytic, Florida (BETA), University of Kiel (KIA) and Queen’s University Belfast (UBA) for accelerator mass spectrometry (AMS) radiocarbon dating. The 210Pb and 137Cs activities were measured on 1–2 cm thick sediment slices by gamma spectrometry using a Canberra ultra-low background Ge-detector. Specifically, 210Pb was measured by way of its gamma peak at 46.5 keV, 226Ra by way of its granddaughter 214Pb (peaks at 295 keV and 352 keV), and 137Cs by way of its peak at 661 keV. The TC showed surface contents of unsupported 210Pb of 392.73 Bq kg−1 with a tendency for exponential decline with depth in the upper ~20 cm  model with errors calculated on the basis of error propagation64. All 14C samples were calibrated using the Marine13 calibration curve65 within CLAM v2.233. Because the marine reservoir is poorly constrained in this region66, we follow the approach used by the RAISED Consortium35 and apply uniform marine reservoir of 1,300 years (ΔR of 900 years). All ages are reported as calibrated years before present , where ‘present’ corresponds to A.D. 1950. The calibrated ages exhibit a near linear increase with depth (r2 = 0.9891 with linear fit) with no age reversals . Age-depth modelling of the 210Pb and 14C age-data was undertaken with CLAM v2.233 using linear interpolation between neighboring levels  datasets, which is most suitable for identifying changes throughout the 20th Century and Holocene, respectively. Significant CPs were identified at 38 cal. yr BP, 244 cal. yr BP; 720 cal. yr BP and 1,617 cal yr BP  is missed in the 100-yr smoothing  analysisset Fig. . Analysiset Fig.  and 100 set Fig.  LOESS-fiing Fig. .et al.68. ~0.5 g samples were digested using a 30% Hydrogen Peroxide (H2O2) solution, heated to ~70 °C for up to two weeks. The peroxide solution was washed off with three deionised water rinses in the centrifuge. Carbonate was removed using a 5% Hydrochloric (HCl) acid solution, left at room temperature for at least 24 hours. The HCl solution was also washed off with three deionised water rinses. The diatoms were isolated from the terrigenous material using the heavy liquid Sodium Polytungstate (SPT), made up to an initial specific gravity of 2.25. Once the cleaned diatom residues were obtained, they were sieved through a 38 μm sieve, and filtered through a 3 μm membrane to further remove contaminants . This step removes the vast majority of whole spicules although occasional spines may pass through the sieve. Sample purity was assessed on a sub-set of samples prior to analysis using X-Ray Fluorescence (XRF)  between repeat measurements of δ18Odiatom ranged from 0.05 to 0.4‰, with average of 0.23‰ (n = 9). Crucially, the magnitude of variations in δ18Odiatom are far greater than the analytical precision (Table Samples were prepared following the methods outlined in Swann F) Table ; an alumty Table . Sampleson Table .Supplementary Information


Modeling the evolution of in situ 14C confirms that Holocene exposure is unlikely at all but one of the sites. Viewed in the context of temperature records from Greenland ice cores, our results suggest that summer warmth of the past century exceeds now any century in ~115,000 years.Arctic temperatures are increasing faster than the Northern Hemisphere average due to strong positive feedbacks unique to polar regions. However, the degree to which recent Arctic warming is unprecedented remains debated. Ages of entombed plants in growth position preserved by now receding ice caps in Arctic Canada help to address this issue by placing recent conditions in a multi-millennial context. Here we show that pre-Holocene radiocarbon dates on plants collected at the margins of 30 ice caps in Arctic Canada suggest those locations were continuously ice covered for > 40 kyr, but are now ice-free. We use in situ  14C ages of plants and cosmogenic 14C concentrations from these landscapes indicate that recently exposed landscapes have been continuously ice covered for > 40,000 years.Shrinking Arctic Canada ice caps are revealing preserved landscapes containing a record of past glacier activity. Here the authors show that  The large influence of summer temperature on glacier mass balance also suggests that past changes in glacier dimensions can be used to place contemporary warming in a multi-millennial perspective6. Here we exploit the high potential for thin, cold-based, non-erosive ice caps to preserve the landscapes over which they advance, including delicate tundra plants and un-eroded rocks. Under these conditions, radiocarbon dates of plants newly exposed by recent melting (hereafter plant 14C) constrain the time of plant death due to snowline lowering and glacier expansion9. Recently reported plant 14C dates > 40 ka from four ice caps on Baffin Island, Arctic Canada, further suggest that ice cover at those locations may have persisted through the warmest part of the Holocene7. However, given the small number of sites, it is possible that local conditions may have favored anomalous preservation of plants following melt out and exposure, allowing for subsequent reburial. Here we expand the number of ice caps with plant 14C ages > 40 ka from 4 to 30, greatly reducing the likelihood that the earlier dates reflected anomalous conditions. We also provide in situ cosmogenic 14C inventories from rock surfaces  adjacent to plant collections with radiocarbon ages > 40 ka to test the assertion that such sites were continuously covered by ice throughout the Holocene. The combination of 30 retreating ice caps revealing plants with pre-Holocene radiocarbon ages and measured in situ 14C inventories provides strong evidence for continuous ice coverage of these locations for >40 ka. When viewed in context of temperature records from Greenland ice cores, these results suggest that the past century of warming is likely greater than any preceding century in the past ~115,000 years.Summer warming in the Arctic has significantly outpaced global average warming over the past several decades14C analysis; with radiocarbon ages of 40 to >50 ka, close to or beyond the range of 14C dating (Table 2), and all lie within a region of 170 × 70 km , but subsequent analyses of the same samples following rigorous pretreatment yielded 14C ages > 40 ka , suggests that plants collected within 1 m of the ice margin were likely first exposed the year they were collected. In rare cases, preservation of dead plants exposed by ice retreat has been noted up to 200 m beyond current ice margins, indicating that they have survived on the landscape for several decades following exposure12. Given the possibility that some plants may have been exposed and then re-entombed, we independently evaluate the exposure history of the sites using in situ 14C in associated rock samples.Plant 14C inventory in the surface of rocks now emerging from beneath receding cold-based ice reflects the cumulative 14C production during exposure when the rocks were ice-free, and attenuated production when rocks were covered by thin ice, as well as losses due to the continuous decay of 14C  and erosion of the rock surface. Unlike plant 14C, which records the timing of plant death by snow and/or ice cover, in situ 14C records the cumulative exposure and burial history of a rock surface over the lifespan of the isotope . Two mechanisms dominate in situ 14C production: (1) energetic spallation reactions between a high-energy nucleon and a target atom, and (2) low-energy (slow) muogenic reactions that occur when a negatively charged muon falls into the electron shell of an atom and is captured by the nucleus14. Various higher-energy (fast) muogenic reactions are less important for in situ 14C production17 but cannot be neglected. Although in situ 14C production is dominated by spallation in unshielded rock surfaces17, these reactions are negligible beneath ~10 m of ice18. Muogenic production in unshielded rock surfaces is much lower than production by spallation, but decreases more slowly with ice thickness, not reaching negligible amounts until ice thickness exceeds 40 m. Since any in situ 14C produced prior to ~40 ka will have decayed below detection, and all sites are expected to have been covered by at least 40 m of ice during the last glacial maximum (LGM), in situ 14C inventories21 at all our sites must have been negligible by the onset of deglaciation. Thus, measured in situ 14C inventories reflect primarily the local variation of ice thickness history during and after regional deglaciation.The in situ 14C concentrations in rock surfaces adjacent to nine of the study sites with plant 14C ages > 40 ka  from a location that has been ice-free since local deglaciation ~13 ka22. Several rocks on the central Baffin Island plateau repeatedly exposed and buried by local ice caps during the Holocene have in situ 14C concentrations of ~85,000 at g−123. The new inventories reported here are generally significantly less than reported elsewhere on Baffin Island, despite site-specific production rates at the new, higher elevation sites that are on average ~60% higher than at these other locations on Baffin Island  to 70 m during the LGM . This leads to inventories that are near zero by the onset of deglaciation, a conclusion that is largely insensitive to the functional form of the assumed preceding thickness history , 1900 CE, before thinning to zero at present. We test for possible ice-free intervals by allowing for exposure in increments of 10 years prior to 4.5 ka for all sample locations. All solutions that are consistent with measured in situ 14C inventories and their measurement uncertainties are considered for each site, and expressed in Table We use a numerical simulation to estimate in situ 14C simulations do not encompass all possible scenarios, they demonstrate that significant Holocene exposure at all but one of the sample sites is unlikely, and is not possible under any realistic scenario for most sample study locations.Under these conditions, only the highest inventory, measured at site 23, permits exposure during the Holocene Table . While c28. We propose two possible explanations for the range of implied ice cover histories. First, due to differences in the patterns of accumulation and ablation, ice caps rarely expand and recede symmetrically, so that upon retreat, landscapes are not necessarily exposed in uniform reverse chronological order. Second, some ice caps behave as threshold systems29. All but 7 of the 30 sites with plant 14C > 40 ka are from high elevations, 1380–1600 m asl, and most of these are from the margins of pedestal ice caps , flat-topped summits, surrounded by steep slopes where maximum ice thicknesses are limited by ice rheology to <70 m30. Once the equilibrium line altitude (ELA) drops below the pedestal surface, ice will grow quickly to a physically determined thickness, but no thicker, even for continued ELA descent. In contrast, lower elevation ice caps can form and expand on less constrained terrain. Consequently, such ice caps became thicker and covered larger altitudinal ranges than pedestal ice caps that formed much earlier, but with dimensions constrained by topography. The result is that the thickness of pedestal ice caps is insensitive to ELA changes below the pedestal base, but is highly sensitive to ELA change as it rises above that level  and much older (>40 ka) landscapes  were sonicated in deionized water and treated with an acid-base-acid36 wash before being converted to graphite by the Laboratory for AMS Radiocarbon Preparation and Research . Graphite targets were measured at the W.M. Keck Carbon Cycle Accelerator Mass Spectrometry Laboratory at the University of California Irvine.Plant samples were collected late in August, to capitalize on the accessibility of plants exposed by ice margin retreat during the summer melt season. Plants (mostly bryophytes), still in growth position, were collected within 1 m of the modern ice margin at most sites14C is produced in situ in quartz from rock surfaces by spallation reactions between incoming secondary nucleons (neutrons + protons) and Si and O. More than ~6 m of ice cover eliminates most in situ 14C production by spallation. However, muons are only weakly attenuated with depth, allowing low rates of in situ 14C production via muogenic reactions beneath >6 m of overlying ice. Rocks continuously beneath >6 m of ice since the LIG will have low concentrations of in situ 14C that reflect muogenic production and decay. Any exposure during the Holocene will greatly increase 14C inventories by spallation reactions.14C collection sites were sampled for in situ 14C to a maximum depth of 4 c using a hammer and chisel. Quartz was isolated and purified from whole rock at the University of Colorado Boulder Cosmogenic Isotope Laboratory using standard techniques37. Samples for in situ 14C concentration were prepared at the Purdue Rare Isotope Measurement Laboratory (PRIME Lab) using current methods21. The automated extraction process involves combusting ~5–10 g of quartz at 600 °C for 1 h to remove atmospheric contaminants, followed by dissolution in a previously degassed LiBO2 flux at 1100 °C for 3 h. Both steps take place in an atmosphere of ca. 50 torr of Research Purity O2. Any carbon species released during the high-temperature step are oxidized to CO2, which is then purified, measured quantitatively, diluted with 14C-free CO2, and converted to graphite for 14C measurement by AMS at PRIME Lab21. In situ 14C concentrations were calculated from measured AMS isotope ratios38, less representative sample-specific procedural blanks.Stable granitic rock surfaces located near nine of the plant 14C atoms to 154,200 ± 30,900 14C atoms21  from site elevation, latitude 41, while accounting for attenuation of spallogenic42 and muogenic43 production due to ice thickness at each timestep. The in situ 14C concentrations are iteratively calculated at annual time steps using the following equation49:No is the existing in situ 14C concentration (at g−1) at the beginning of each timestep, λ is the decay constant of 14C (yr–1), and dt is the annual timestep. Differences in production arising from changes in atmospheric density at each site due to sea level rise are negligible at these elevations44. Rock erosion is assumed to be negligible given the field evidence that ice cover was cold-based.For each realization of ice thickness history for each sample, we determine the production rate at the rock surface (Supplementary InformationPeer Review File


Surgery for adult spinal deformity requires optimal patient-specific spino-pelvic-lower extremity alignment. Distal fixation in thoracolumbar spinal deformity surgery is crucial when arthrodesis to the sacrum is indicated. Although we had performed sacro-pelvic fixation with bilateral S1 and bilateral single iliac screws previously, iliac screw loosening and/or S1 screw loosening occurred frequently. So, the authors attempted to fuse spino-pelvic lesions with the dual iliac screws and S1 pedicle screws.Twenty-seven consecutive adult spinal deformity patients underwent thoracolumbar-pelvic correction surgery with bilateral double iliac screws between May 2014 and September 2015. Sagittal vertical axis, lumbar lordosis, pelvic tilt, sacral slope, T1 pelvic angle, and global tilt were assessed radiographically and by computed tomography both preoperatively and 24 months postoperatively. Iliac screw loosening, S1 pedicle screw loosening, and screw penetration of the ilium were evaluated 2 years postoperatively.Only two patients (7.4%) at 1 year and three patients (11.1%) at 2 years presented with iliac screw loosening postoperatively. Loosening of the S1 screw occurred in three cases (11.1%) 2 years postoperatively. Displacement of the iliac screw occurred in eight cases (25%). Internal and external perforation of the ilium by the iliac screw occurred in six (22.2%) and three (11.1%) cases respectively. One reoperation was performed due to back-out of the iliac screw and rod breakage.Bilateral dual iliac screws and an S1 pedicle screw system achieve longer stability for spinal and pelvic fusion in adult spinal deformity patients, with few severe complications. The pathology of adult spinal deformity (ASD) includes various components such as malalignment of the sagittal plane, lower angle of lumbar lordosis, and pelvic retroversion , 2. The All patients were considered candidates for thoracolumbar correction if fusion was indicated because of ASD and if a full course of conservative care had been exhausted. The inclusion criteria were symptoms including postural imbalance , low back pain, and/or gastro esophageal reflux disease, and a radiographic diagnosis of ASD defined by at least one of the following parameters: a coronal Cobb angle > 30°; a C7 sagittal vertical axis (SVA), which is the distance between the C7 plumb line and the posterosuperior edge of S1, > 5 cm; and/or pelvic tilt (PT), which is the orientation of the pelvis with respect to the femurs and the rest of the body, > 30°. Patients were excluded if they had rigid severe kyphosis, ankylosing spondylitis, a rounded back because of Parkinson’s disease, or if they had not been followed up for at least 1 year. Twenty-seven ASD patients were consecutively enrolled and underwent surgery between May 2014 and September 2015. Iliac screw loosening, pedicle screw loosening of S1, and misplacement causing screw penetration of the iliac table were assessed using computed tomography (CT) 1 and 2 years postoperatively. Screw loosening was defined as a lucent zone around the screw. Concurrence of at least two of the observers was mandatory to diagnose screw loosening . The accIn addition, posterior-anterior and lateral radiographs of the entire spine in the standing position were investigated preoperatively, 12 months postoperatively. The SVA, lumbar lordosis (LL), PT, sacral slope (SS), T1 pelvic angle (TPA), and global tile (GT) were measured. All radiological analysis was performed two times by three independent doctors in a blinded fashion, and the measured values were averaged. This study was approved by our institutional review board (No. 1101).An anterior approach was selected to perform lateral interbody fusion (LIF) or posterior lumbar interbody fusion (PLIF) from the L1–2 or L2–3 to the L4–5 disc level to obtain adequate coronal and sagittal spine alignment in the ASD patients. Then, the patient position was changed to the prone position. The Ponte osteotomy and PLIF at the L5–S1 disc level were performed, and spinal kyphosis was corrected through cantilever force using bilateral S1 screws and bilateral dual iliac screws. In the case of loss in flexibility of spinal motion, a Ponte osteotomy, pedicle subtraction osteotomy, or vertebral column osteotomy was added.To place the screws, the paravertebral muscles including the multifidus muscles around S1 and S2 spinous processes first were removed to visualize the lamina and spinous processes. Muscle fascia on the ilium was incised to provide access to the iliac screws Fig. , resultiThe size of the iliac resection must be large enough to set the dual iliac screws so that the heads of the screws are not prominent. The surrounder was directed to the line from the anterior superior iliac spine to the greater trochanter in a manner similar to the ball-tip method Fig.  12]. An. An12]. The length between the iliac screws and the rod of the pedicle screw was measured. Then, the pedicle screws were connected to the rod of the iliac screws with an offset connector through a hole inside the paravertebral muscles from the ilium to the sacrum Fig. .Rods of adequate length extending from the cranial to caudal surgical level were prepared. The lateral connectors bind the iliac screws to a rod which was concatenated with the S1 pedicle screw Fig. . The spiA 72-year-old female patient had adult spinal kyphosis and showed a postural imbalance such as leaning forward or to the left side when walking and standing, resulting in claudication within 2 min. She underwent surgery including LIF at L2–3, 3–4, and 4–5 disc levels and posterior lumbar interbody fusion at L5–S1, and posterior corrective fusion from T10 to the ilium with bilateral S1 pedicle screws and bilateral dual iliac screws. It took 7 h and 18 min in surgical time and 179 ml of blood given intraoperatively Fig. .Fig. 6CaThe patients’ age at surgery, gender, bone mineral density (BMD), number of osteoporotic vertebral fractures (OVF), operation time, bleeding, and location of upper instrumented vertebra are presented in Table Surgical complications presented by iliac screw loosening occurred in two cases 1 year and three cases 2 years postoperatively. Loosening of the S1 screw occurred in three cases. Displacement of the iliac screw occurred in eight cases 2 years postoperatively. Internal perforation of the ilium by the iliac screw occurred in six cases, whereas external perforation of the ilium by the iliac screw occurred 2 years postoperatively in three cases. One reoperation was performed due to back-out of the iliac screw and breakage of the rod.Surgery decreased the SVA from 86.7 to 14.9 mm and increased the LL from 15.1 to 52.4°. Surgery decreased the PT from 31.8 to 20.6° and increased the SS from 16.5 to 30.3°.We have demonstrated how to perform dual iliac screw fixation as an iliac anchor to stabilize the spino-pelvic junction in ASD patients with osteoporosis. When considering a surgical strategy for ASD, the surgeon’s plan should consider not only global spinal balance but also pelvic alignment. Therefore, both global tilt and T1 pelvic angle are important to evaluate global alignment of the spine and the lower extremity through the pelvis. Proper alignment allows stability when standing , 14. PelAlternatively, S2-alar iliac pelvic fixation has been developed . PreviouTo settle these concerns, we have developed bilateral dual iliac screws and S1 pedicle screw fixation to strengthen the support of the pelvis. The initial idea for this procedure came from cases in which dual iliac screw fixation accompanied by total sacrectomy and tumor excision was used for lumbosacral tumors , 19. TheHowever, this study has some limitations. First, it is a relatively small study with a limited number of patients. Second, the follow-up observation period of 1 year was short. Third, this study did not evaluate clinical outcomes. Fourth, this study did not compare surgical outcomes with bilateral dual iliac screws and a single iliac screw, or S2-alar iliac pelvic fixation. Future study is required to address these points.The bilateral dual iliac screw and S1 pedicle screw system provide 6 points as pelvic anchors and promise improved stability for long spinal and pelvic fusion for ASD. It is accompanied by few complications and may allow a superior correction over single iliac fixation to obtain ideal spino-pelvic alignment.


Insulin resistance (IR) is considered as a risk factor for atrial fibrillation (AF) even before diabetes develops. The pathophysiology and underlying mechanism are largely unclear.We investigated the corresponding mechanism in two IR models of rats fed 15-week high-fat (HFa) and high-fructose/cholesterol (HFr) diets. AF was evaluated and induced by burst atrial pacing. Isolated atrial myocytes were used for whole-cell patch clamp and calcium assessment. Ex vivo whole heart was used for optical mapping. Western blot and immunofluorescence were used for quantitative protein evaluation.Both HFa and HFr rat atria were vulnerable to AF evaluated by burst atrial pacing. Isolated atrial myocytes from HFa and HFr rats revealed significantly increased sarcoplasmic reticulum calcium content and diastolic calcium sparks. Whole-heart mapping showed prolonged calcium transient duration, conduction velocity reduction, and repetitive ectopic focal discharge in HFa and HFr atria. Protein analysis revealed increased TGF-β1 and collagen expression; increased superoxide production; abnormal upregulation of calcium-homeostasis-related proteins, including oxidized CaMKIIδ, phosphorylated-phospholamban, phosphorylated-RyR-2, and sodium-calcium exchanger; and increased Rac1 activity in both HFa and HFr atria. We observed that inhibition of CaMKII suppressed AF in both HF and HFr diet-fed rats. In vitro palmitate-induced IR neonatal cardiomyocytes and atrial fibroblasts expressed significantly more TGF-β1 than did controls, suggesting paracrine and autocrine effects on both myocytes and fibroblasts.IR engenders both atrial structural remodeling and abnormal intracellular calcium homeostasis, contributing to increased AF susceptibility. The inhibition of CaMKII may be a potential therapeutic target for AF in insulin resistance. Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical settings, affecting ~ 2% of patients worldwide and carrying a significantly increased risk of ischemic stroke and mortality . DiabeteMale Sprague–Dawley rats weighing 150–170 g were assigned to three groups fed one of the following three diets for 15 weeks as previously described:  (1) a re3, 0.9 mM NaH2PO4, 1.8 mM CaCl2, 0.5 mM MgSO4, and 5.5 mM glucose. Subsequently, the heart was connected to a Langendorff apparatus and perfused with oxygenated Tyrode’s solution heated to 38.0 °C. The composition of Tyrode’s solution was 134 mM NaCl, 4.5 mM KCl, 0.5 mM MgCl2, 2 mM NaH2PO4, 23 mM NaHCO3, 1.8 mM CaCl2, and 5.5 mM glucose, equilibrated with 95% O2 and 5% CO2 to maintain a pH of 7.4 [The heart was harvested immediately and cannulated through the aorta after the rats were killed. Blood was flushed out by injection of a 30-mL cardioplegic solution composed of 134 mM NaCl, 15 mM KCl, 20 mM NaHCO2/pixel for 4 s [The perfusate was maintained at 38.0 °C with a flow rate of approximately 10 mL/min. After 30 min of stabilization, 3.0 mM calcium-sensitive dye  and 10 mM voltage-sensitive dye  were added to the perfusate. The heart was washed for 30 min, followed by the addition of 20 mM excitation–contraction uncoupler . A laser light at 532 nm was used to excite the heart, and the fluorescence signal was collected by two fast-speed charge-coupled device cameras. One camera acquired voltage fluorescence through a 710-nm long-pass filter, whereas the other camera simultaneously recorded calcium fluorescence through a 585- to 620-nm bandpass filter. Fluorescence images were captured at a frame rate of 2 ms/frame and 100 × 100 pixels as well as a spatial resolution of 0.35 × 0.35 mm for 4 s .2+ transients were elicited by field stimulation through a pair of platinum electrodes, with a 2-ms suprathreshold square wave voltage pulse delivered by the electrical stimulator at 1 Hz for 30 s to allow a steady-state condition to be reached. A Leica SP8 scanning system  was used for confocal imaging of Ca2+ fluorescence. Fluo-4 was excited by the 488-nm line of an argon laser, and emission signals over 505 nm were collected. For recording of Ca2+ transients/sparks, a line scan mode was normally used. After sequential scanning, a two-dimensional image of 512 × 1000 lines was generated and stored for offline analysis [The isolated atrial cardiomyocytes were harvested and stored in oxygenated Tyrode’s solution. For calcium indicator staining, 10 μL of Fluo-4 AM stock solution was added to 1 mL of cell suspension . The cardiomyocytes were placed at room temperature for 20 min to allow for complete de-esterification of AM esters. Caanalysis .Whole-cell patch-clamping was applied for electrophysiological recordings as described previously . The act2+/calmodulin-dependent protein kinase II (t-CaMKIIδ), sodium–calcium exchanger (NCX1), sarco/endoplasmic reticulum Ca2+-ATPase II (SERCA II) , oxidized CaMKII (ox-CaMKII), phosphorylated phospholamban (p-PLB), total phospholamban (t-PLB) , phosphorylated ryanodine receptor type 2 (p-RyR2), total RyR2 (t-RyR2), , phosphorylated AKT (p-AKT), total AKT (t-AKT) , phosphorylated CaMKIIδ (p-CaMKIIδ) , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , followed by antirabbit and antimouse immunoglobulin G -conjugated secondary antibodies. Signals were detected through an electrochemiluminescence assay and quantified using densitometry. Immunoreactive signal bands were in the linear range and expressed relative to GAPDH. All the Western blot data were normalized to the expression of GADPH. The phosphrylation sites of proteins were Ser16 on PLB, Ser2814 on RyR and Thr286 on CaMKIIδ. The oxidation site was Met281/282 on CaMKII.Proteins were extracted and processed as described elsewhere . BrieflyImmunohistochemistry were performed as previously described , 20. ImmTransvenous atrial stimulation was performed as described previously, with modification . BrieflyS-transferase beads. Lysates were then incubated with p21-activated protein kinase 1 p21-binding domain agarose for 1 h at 4 °C. Beads were washed three times in MLB, and samples were prepared for electrophoresis by adding 1 × SDS loading dye. Samples were boiled for 5 min and resolved by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and guanosine-triphosphate-bound Rac1 was identified using anti-Rac1 antibody.Rac1 activity was determined using a Rac1 activation assay kit . Briefly, tissues were lysed in magnesium lysis buffer (MLB) supplemented with a protease inhibitor mixture. Tissue lysates were precleared for 10 min with glutathione Fibroblasts were obtained from the atria of adult male Wistar rats  by using collagenase and trypsin digestion and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS. Only cells from early passages (1–3) at 80%–90% confluence were used. The medium was replaced with serum-free DMEM for 24 h and subsequently treated with or without glucose (25 mM), insulin (200 nM), and palmitate (50 mM) for an additional 24 h in DMEM with 10% FBS. Neonatal cardiomyocytes were obtained from neonatal mouse heart, and transfer trypsin (50 µg/mL) was placed in a Petri dish and mixed completely, followed by mixture with transfer trypsin inhibitor  in a 37 °C water bath. Collagenase and Leibovitz L-15 media were added and mixed in the tube using a shaking instrument (2–4 rpm) at 37 °C and incubated for 30 to 45 min. The mixture was then triturated 10 times to release cells with a plastic serological pipette. The medium was treated with or without glucose (25 mM), insulin (200 nM), and palmitate (50 mM) for an additional 24 h in L-15 medium with 10% FBS.t test for two groups and one-way ANOVA with post hoc Tukey’s tests for multiple comparisons were applied. Two-way repeated measures ANOVA models followed by post hoc Bonferroni test were applied with factors including APD80, coefficient of variation (COV) of APD80 and CV versus PCL. A value of P < 0.05 was considered to be significant.All values were expressed as the mean ± SE. Unpaired Student’s P < 0.001) than did HFr and control rats. The heart-to-body-weight ratio was significantly lower in the HFa rats than it was in the control rats. As shown in Additional file Table None of the control, HFa, or HFr rats had a spontaneous AF attack at baseline. To evaluate whether the HFa and HFr rats were more vulnerable to atrial tachyarrhythmia, we performed burst atrial pacing using a transvenous approach. As illustrated in Fig. 20), 50% repolarization (APD50), and 90% repolarization (APD90) , suggesting increased diastolic calcium leaks in the HFr atria   studies in atrial cardiomyocytes obtained from the HFa and HFr rats. The whole-cell patch-clamp study showed that the control, HFa, and HFr atrial myocytes had similar resting membrane potential and action potential duration values at 20% repolarization (APD80) compared with the control rats at each PCL. At 200-ms PCL, the APD80 values for the HFa, HFr, and control RAAs were 59.6 ± 9.8 (n = 5), 68.0 ± 9.1 (n = 5), and 66.4 ± 6.5 ms (n = 5), respectively. At 80-ms PCL, the values were 38.4 ± 7.4, 42.8 ± 5.6, and 39.2 ± 5.9 ms for the HFa, HFr, and control RAAs, respectively. In contrast to the small discrepancy of APD80, both the HFa and HFr RAAs showed a significantly longer CaTD at 80% repolarization (CaTD80) than did the control rats. At 200-ms PCL, the CaTD80 values for the HFa, HFr, and control RAAs were 78.8 ± 1.8, 82.4 ± 4.9, and 69.2 ± 5.0 ms, respectively . At 80-ms PCL, these values were 66.4 ± 4.8, 65.6 ± 5.5, and 51.6 ± 3.3 ms for the HFa, HFr, and control RAAs, respectively . In general, the coefficient of variation for either APD80 or CaTD80 was similar between the HFa, HFr, and control RAAs, suggesting a similar degree of heterogeneity  and 100 ms  . Figure P = 0.045 for HFa vs. control rats; P = 0.114 for HFr vs. control rats). We also attempted AF induction by providing a single premature stimulus (S2) after a train of regular pacing in some atria (S1). The interval between S1 and S2 started at 200 ms and decreased stepwise by 10 ms until AF or an effective refractory period was observed. As presented in Fig. P = 0.06 for HFa vs. control rats; P = 0.02 for HFr vs. control rats). We observed two mechanisms associated with the initiation and maintenance of AF in HFa and HFr atria. The first mechanism is the reentry. A representative recording of optical mapping in one atrium revealed that S1 pacing captured the tissue near the pacing site. However, the impulse was blocked halfway through the atria, leading to reentry and nonsustained AF episodes .As illustrated in Fig. We also tested whether the inhibition of CaMKII can reduce the inducibility and maintenance of AF in HFa and HFr atria. KN-93 (1 µmol/L), a specific CaMKII inhibitor, was applied in five HFa and five HFr atria. We observed that three of the five HFr and five of the five HFa rats exhibited elimination or increased threshold of AF inducibility after KN-93 infusion Fig. . By contTGF-β1 is an important mediator in AF-related structural remodeling . To evalTo reveal the potential underlying mechanisms of abnormal triggers and increased diastolic calcium leak in IR atria, we evaluated the expression of calcium-homeostasis-related proteins in the HFa and HFr atria. As presented in Fig. To evaluate whether the cardiomyocytes and fibroblasts of IR hearts present factors favoring AF-related pathophysiology, we established in vitro IR models by treating neonatal mouse cardiomyocytes and rat atrial fibroblasts with palmitate, and we evaluated the expression of TGF-β1. As shown in Fig. We provide several lines of evidence demonstrating calcium handling–related and structural remodeling in the rat atria of IR models, which were established by feeding them a 15-week HFa or HFr diet. We showed that the HFa and HFr rat atria were more vulnerable than the controls to AF induced by rapid pacing. We showed abnormal diastolic calcium leak and repetitive ectopic focal discharge in isolated atrial myocytes and whole atria of HFa and HFr rats, suggesting calcium-related triggered activity as the mechanism of AF. The HFa and HFr rat atria presented increased oxidative stress, NADPH oxidase activity, and CaMKII oxidization as well as both calcium handling–related and structural remodeling processes, including increased phosphorylation of RyR and phospholamban and upregulation of collagen and TGF-β1. Upon phosphorylation, the inhibitory effect of PLB on the function of SERCAII is relieved and SR calcium uptake is increased. In our study we showed the SR calcium content was significantly increased both HFa and HFr atria Fig. c, which People with diabetes may have diabetic cardiomyopathy and progress to heart failure independent of coronary artery disease, hypertension, or chronic kidney disease. The pathogenesis of diabetic cardiomyopathy includes impaired myocardial insulin signaling, mitochondrial dysfunction, endoplasmic reticulum stress, impaired calcium homeostasis, abnormal coronary microcirculation, inappropriate neurohumoral activation, and maladaptive immune response . The preRecently, Maria et al. reported that in long-term high-fat diet–induced IR rats, insulin-stimulated translocation of glucose transporters 4 and 8 as well as total protein expression was downregulated in the atria , which sWe also showed that TGF-β1 was upregulated in the atria of IR rats. The in vitro study suggested that palmitate-induced IR may have a direct effect in TGF-β1 upregulation independent of hyperglycemia or dyslipidemia. TGF-β1 is among the major profibrotic mediators in the atria. The findings are consistent with those of a previous study that pioglitazone, acting as an insulin sensitizer, attenuated atrial fibrosis in a rat model of AF through the suppression of TGF-β1 .In this study, we showed that IR was associated with various aspects of remodeling in the atria, including increased oxidative stress, hyperphosphorylated calcium handling–related proteins, and increased interstitial fibrosis , 28. RemAn animal study demonstrated that the diabetic rat atrium induced by streptozotocin had increased interstitial fibrosis, reduced connexin 40 expression, and decreased CV, resulting in APD prolongation and dispersion and increased incidence of APD alternans , 11. AnoIn contrast to the presentation of the streptozotocin-induced diabetic rats with overt hyperglycemia and severe body weight loss, which favorably mimic the effects of uncontrolled hyperglycemia and type 1 diabetes rather than IR, the HFa and HFr rats presented with ~ 30% elevated blood sugar and 0%–20% increased body weight after 15-week HFa and HFr feeding, which were set for the models of IR and prediabetes. Our findings more favorably reflect the changes in the atria of patients with prediabetes and metabolic syndrome.2+-activated K+ current in atria from patients with AF [We observed increased NADPH oxidase activity in IR rat heart, contributing to increased oxidative stress and upregulated ox-CaMKII. Oxidative stress can induce arrhythmogenic hyperphosphorylation of calcium-handling proteins through the activation of CaMKII. CaMKII hyperactivity has been demonstrated to be causatively associated with AF and ventricular arrhythmias in heart failure through modulating calcium handling–related protein activities , 32. Rec with AF . A recen with AF . Exposur with AF . In the  with AF .Some antidiabetic medications, including metformin, thiazolidinediones, dipeptidyl peptidase 4 inhibitors, and sodium/glucose cotransporter 2 inhibitor, have been shown to improve myocardial glucose uptake and contractile dysfunction in humans  and in aWe could not separately evaluate the effect of IR without conditions of elevated blood pressure, blood sugar, lipids, and cholesterol in these rats, all of which also contribute to atrial remodeling. Rats and humans may have different expression levels of calcium-handling proteins, which may lead to different mechanisms of regulation in calcium homeostasis.We conclude that IR promotes factors leading to interstitial fibrosis and abnormal calcium homeostasis in atria, which alter the CV and increase ectopic activities in the atria, contributing to AF genesis. IR may have a direct effect on the expression of TGF-β1 in myocytes and fibroblasts, contributing to atrial fibrosis. These results suggest that upstream therapy targeting CaMKII and TGF-β1 as well as reducing oxidative stress is a potentially effective strategy for preventing AF caused by a diet high in fat, sugar, and cholesterol.Additional file 1: Figure S1. Expression of phosphorylated and total AKT in insulin-treated high-fat (HFa) and high-fructose/cholesterol (HFr) diet-fed rat atria. Representative examples (upper panel) and mean ± SE of the relative expression levels of each protein quantified by densitometry and normalized to the control level, which was set at 1.0 (lower panel). Each value represents the mean ± SE of three independent experiments.Additional file 2: Table S1. Cellular electrophysiology in HFa and HFr rats.Additional file 3: Figure S2. Effects of KN-92 in atrial fibrillation (AF) induced by short pacing cycle length (PCL) in high-fat (HFa) and high-fructose/cholesterol (HFr) diet-fed rats. The figure shows representative optical membrane voltage (AP) and calcium transient (CaT), action potential duration at 80% repolarization (APD80), and CaT duration at 80% repolarization (CaTD80) maps of right atrial appendages (RAAs) in (A) one HFr diet-fed rat and (B) one HFa diet-fed rat at short PCLs before and after application of KN-92 (1 µmol/L), respectively. None of the HFr or HFa rat atria (n = 5 for each group) exhibited decrease or increase in AF duration after KN-92 infusion.


The patho-mechanism leading to airway wall remodeling in allergic asthma is not well understood and remodeling is resistant to therapies. This study assessed the effect of immunoglobulin E (IgE) in the absence of allergens on human primary airway smooth muscle cell (ASMC) remodeling in vitro. ASMCs were obtained from five allergic asthma patients and five controls. Proliferation was determined by direct cell counts, mitochondrial activity by expression of cytochrome c, protein expression by immunoblotting and immuno-fluorescence, cell migration by microscopy imaging, and collagen deposition by cell based ELISA and RNA expression by real time PCR. Non-immune IgE activated two signaling pathways: (i) signal transducer and activator of transcription 3 (STAT3)→miR-21-5p→downregulating phosphatase and tensin homolog (PTEN) expression, and (ii) phosphatidylinositol 3-kinases (PI3K)→protein kinase B (Akt)→mammalian target of rapamycin (mTOR)→ribosomal protein S6 kinase beta-1 (p70s6k)→peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α)→peroxisome proliferator-activated receptor-γ (PPAR-γ)→cyclooxygenase-2 (COX-2)→mitochondrial activity, proliferation, migration, and extracellular matrix deposition. Reduced PTEN expression correlated with enhanced PI3K signaling, which upregulated ASMC remodeling. The inhibition of microRNA-21-5p increased PTEN and reduced mTOR signaling and remodeling. Mimics of microRNA-21-5p had opposing effects. IgE induced ASMC remodeling was significantly reduced by inhibition of mTOR or STAT3. In conclusion, non-immune IgE alone is sufficient for stimulated ASMC remodeling by upregulating microRNA-21-5p. Our findings suggest that the suppression of micoRNA-21-5p may present a therapeutic target to reduce airway wall remodeling. Asthma is a worldwide increasing health problem and approximately 60% of all patients suffer from allergic asthma . DespiteAirway wall remodeling in asthma is characterized by deranged epithelium, thickened sub-epithelial basement membrane, accumulated sub-epithelial extracellular matrix (ECM), and hyperplasia/ hypertrophy of ASMC . Airway Altered mitochondria activity has been linked to the pathogenesis of various airway diseases including asthma, and presents a potential target for therapies for airway remodeling ,14. In ARecently, the role of non-coding microRNAs contributing to airway wall remodeling in asthma has been described . DeregulThis investigation assessed the hypothesis that non-immune IgE in the absence of allergens directly affects ASMC remodeling by activating two independent signaling pathways, which enhance each other. We hypothesize that: IgE activates PI3K→Akt→mTOR, which enhances the synthesis of pro-inflammatory ECM components and increases mitochondria mass and activity. In parallel, IgE activates signal transducer and activator of transcription 3 (STAT3) and upregulates microRNA-21-5p. The latter inhibits phosphatase and tensin homolog (PTEN) expression, and, thereby, removes a potent inhibitor of Akt signaling, as shown in the p < 0.01), but not of FcεR-II  were expressed by ASMC from asthma patients and controls A. Compar FcεR-II A. The in FcεR-II B. Regarding the increased deposition of the extracellular matrix during airway wall remodeling, we confirmed the previously reported effect of non-immune IgE on the deposition of collagen type-I, and fibronectin by ASMC of asthma patients. IgE (1 μg/mL) significantly stimulated the deposition of collagen type-I and fibronectin by ASMC over 24 and 48 h, as determined by cell based ELISA C. IgE-inp < 0.01) when compared to unstimulated ASMC , Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ), and Peroxisome Proliferator-Activated Receptor γ Coactivator-1α (PGC-1α) in ASMC was determined on the pre-transcriptional and post-transcriptional level in ASMC obtained from asthma patients and controls. p < 0.05) and reached a 4.5-fold increase (p < 0.01) after 24 h, as compared to unstimulated cells (p < 0.05) and this effect remained significant up to 48 h (p < 0.001) with a 20-fold increase (p < 0.05) and continued to increase until 48 h (p < 0.05) after IgE stimulation , IgE stimulated COX-2 mRNA expression, which increased significantly after 3 h (ed cells A. Additiincrease A. The gemulation A. p < 0.05), and remained at a high level up to 48 h (p < 0.05), as shown by a representative Western blot from ASMC of an asthma patient (p < 0.05), which peaked at 6 h (p < 0.01) and reduced thereafter (p < 0.05), which steadily increased over 48 h (p < 0.05), and further increased over the next 48 h , peaking after 1 h, and declining to baseline levels thereafter (p < 0.05) and further increased over 48 h (p < 0.05) and the protein remained phosphorylated until 48 h  was determined by Western blotting and the optical density of the protein bands was determined by imageJ in five blots. A representative Western blot and the kinetic of the proteins as the line chart is shown in ereafter A. This ever 48 h A. Similatil 48 h A.Immunofluorescence microscopy confirmed the IgE-induced phosphorylation of PI3K in sub-membranous protrusion sites, intracellular areas, and in the nuclei, as shown for 30 min and 1 h in p < 0.05) and was maintained over 48 h (p < 0.05) after IgE stimulation, as shown by the representative Western blot and subsequent image analysis  as compared to ASMC of non-asthma controls (n = 5) (p < 0.01) and declined thereafter  E. Stimulereafter F.p < 0.05, In In order to determine the contribution of the signaling cascade described above in ASMC controlled airway wall remodeling, the deposition of collagen type-I and fibronectin was determined in the presence and absence of IgE and the signaling inhibitors, as shown in IgE-induced migration of ASMC was inhibited by all three signal inhibitors, as shown in Next, we assessed the effect of the signaling inhibitors on IgE-induced mitochondria activity by immunofluorescence microscopy D. As depRegardless of the diagnosis of the cell donors and in the absence of IgE, the overexpression of microRNA-21-5p in ASMC increased collagen type-I and fibronectin deposition, while anti-sense microRNA-21-5p reduced both A. SimilaThe overexpression of microRNA-21-5p in ASMC decreased baseline PTEN expression in a dose-dependent manner C. In conMitochondrial activity was monitored by cytochrome c expression, which was significantly upregulated in ASMC after overexpression of microRNA-21-5p E. In conThe ASMC driven airway wall remodeling in allergic asthma is a major irreversible pathology that was assumed to be the result of chronic inflammation . HoweverIncreased serum IgE levels is one of the characteristic pathology of allergic asthma, which persists for several weeks after the clearance of allergens from the blood ,26,27,28Earlier studies showed that ASMC express the high affinity IgE-receptor FcεR-I, and responded to extracellular IgE by increased proliferation and ECM deposition ,9,11. ThIn a mouse model, prolonged antigen exposure induced airway wall remodeling, but did not increase inflammation further . In humaIt has been shown that activated PPAR-γ regulated glucose/lipid metabolism as well as the inflammation response and may act through NF-κB, p38-MAPK, or Erk1/2-MAPK signaling . PGC-1α Abnormal mitochondrial function in asthmatic ASMC has been previously reported ,14,29. BActivated mTOR signaling participated in IgE-activated mast cells and lymphocytes in asthma, while no such data was reported for ASMC remodeling . Akt → mCirculating microRNA-21-5p was suggested as a biomarker for allergic inflammatory disease during childhood . IncreasThe results of this study showed that microRNA-21-5p is essential for IgE-dependent ASMC remodeling and, thus, presents a target for therapies of airway wall remodeling in asthma. These findings are in line with earlier studies, which suggests that microRNA-21-5p plays a role in asthma ,20,40,41Human bronchial biopsies were obtained from patients with asthma or from non-asthma/non-COPD (controls) that underwent bronchoscopy for diagnostic reasons at the Clinic of Pneumology, University Hospital Basel, Switzerland. Each patient gave a written consent for using one additional biopsy sample for scientific investigations. The local Ethics Board  approved the protocol. The clinical parameters of all tissue donors are presented in The primary ASMCs were isolated from the biopsies (obtained from five patients with asthma and five controls) after removing the epithelium and cultivated in a specific medium  . PositivASMC from patients with asthma were seeded into 6-well plates and allowed adherence for 24 h, which was followed by overnight serum deprivation in DMEM containing 0.1% serum (control medium). ASMCs were further incubated in the absence (control) or in the presence of human non-immune IgE  for up to 48 h.Cell signaling pathways were confirmed by selective chemical inhibitors, which were added to ASMC 1 h before IgE stimulation: (a) STAT3 inhibitor at 20 μM , (b) PI3K inhibitor at 1 μM , and c) mTOR inhibitor Rapamycin at 10 nM . For microRNA functional evaluation, ASMCs were seeded into 6-well plates and transiently transfected using HiPerfect Reagent Kit . Synthetic miR-21-5p mimic and inhibitor were from Qiagen .Ct−ΔΔ algorithms. Total RNA was purified from ASMC by using the Quick-RNA Mini Prep kit  and was followed with cDNA synthesis by High-Capacity cDNA Reverse Transcription Kit . To detect miR-21-5p, the miRNA-specific stem loop reverse transcription (RT) reaction was used to synthesize cDNA by Mir-XTM miRNA First-Strand Synthesis kit . Real-time quantitative PCR (RT-qPCR) was performed by the Applied Biosystems 7500 Real-time system with FastStart Universial SYBR Green Master Rox  for quantification. The mRNA expression levels were normalized to GAPDH, while miR-21-5p expression was normalized to U6 snRNA. Purity of PCR products was confirmed by melting curve analysis, and the final data was analyzed using the 2Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α ; Peroxisome Proliferator-Activated Receptor-gamma ; and Cytochrome c Oxidase Subunit 2 . Primer sequence for miR-21-5p was: 5′-TAG CTT ATC AGA CTG ATG TTG A-3′. All primers were synthesized and purchased from Microsynth, Basel, Switzerland). The conditions for real-time PCR was: 95 °C and 30 s for template denature, 60 °C as the annealing and extension temperature for mRNA, and 55 °C for microRNA with 40 cycles to complete.ASMCs were washed once with cold DPBS  and lysed by RIPA buffer . Cell lysates were centrifuged at 12,000 rpm (10 min) and the protein concentration of the supernatant was measured by using the BCA protein assay kit . Equal amounts of denatured proteins (20 μg) were separated in 4%–12% SDS–PAGE , and, subsequently, transferred onto a nitrocellulose membrane . Proteins of interest were detected by specific antibodies, including total- (t-)PI3K (#4292), and phosphorylated- (p-)PI3K (#GTX132597), t-AKT (#4691) and p-AKT (#4060), t-p70S6 kinase (#2708) and p-p70S6 kinase (#9205), t-STAT3 (#9139) and p-STAT3 (#9145), PTEN , PGC-1α , t-PPARγ , and p-PPARγ . GAPDH  was used as an endogenous control for a semi-quantification measure. Protein bands were visualized by Luminata Forte Western HRP substrate  after binding specie-specific secondary HRP conjugated antibodies .For immunofluorescence analysis, ASMC cells were seeded on 10 mm sterile glass slides. Following experimental protocols, cells were fixed (2 × 5 min) by 4% paraformaldehyde (in DPBS). Immuno-fluorescence staining was performed by p-mTOR , p-PI3K , and PTEN . Antibodies were diluted, according to the manufacturer’s instructions in PBS containing 2% bovine serum albumin and incubated at 4 °C overnight, followed by three washes and subsequent staining with Alexa488 goat anti-rabbit IgG . Cytoskeleton counter staining was performed by TRIC-Phalloidin , while nuclei were stained by DAPI . Images were acquired by the OLYMPUS BX63 upright fluorescence microscope under the 20x objective. The combined 3-channel images in the figures are generated by FIJI.ASMCs were seeded onto coverslips in 24-well plates and incubated under various conditions for up to 48 h. Afterwards, ASMC were fixed by 4% formaldehyde , washed once with DPBS, permeabilized by 0.15% Triton , and blocked with 5% bovine serum albumin (BSA) in DPBS for 1 h. The primary mouse anti-Cytochrome c  antibody or FITC anti-human FcεR-Iα  was stained overnight at 4 °C. Alexa488-labeled anti-mouse secondary antibody  was used to visualize Cytochrome c. F-actin is stained with TRIC-Phalloidin. Nuclei were stained with DAPI. Coverslips were mounted with Vectashield Antifade Mounting Medium . Images were acquired by using the Nikon Confocal A1 microscope with a 40× 1.3NA FI oil objective of 0.225 μm Z-stack step. The printed images are presented as Z-stack projection generated by imaging software FIJI. Single cell (50 cells/group) cytochrome c expression was calculated by FIJI with the “3D objects counter” plugin and finally normalized to the control group after 6 h. The cellular FcεR-Iα expression level was measured by the “Analyze Particle” feature of FIJI, quantified by an average of 10 cells/cell line, five cell lines/group, and analyzed by one-way ANOVA.4 cells/well) were seeded into 96-well plates and then treated with different conditions before fixation in 4% formaldehyde (15 min). Antigen-Retrieval was induced by the citric acid method  and un-specific binding was blocked by 3% BSA (1 h). Collagen type-I deposition was detected by anti-collagen-1A1  and fibronectin was detected by anti-fibronectin antibody . After overnight incubation (4 °C), cells were washed three times with T-PBS (0.05% Tween20 in PBS), and incubated for 1 h with a secondary HRP-conjugated antibody . After another three washes with PBS, TMB substrate  was added . The reaction was terminated by adding 1M HCl and the absorption was determined at 450 nm . All experiments were performed in quadruplicate.Collagen-I and fibronectin deposition was determined by cell-based ELISA, as described previously . ASMC  after mechanical wounding on a confluent cell layer, under selective designed experimental culture conditions at 0, 12, 24, and 36 h. The cell migration comparison was achieved by measuring the wound closure rate at a time point of 24 h. t-test. p-values < 0.05 were considered as statistically significant. Statistical analysis was performed by software GraphPad-Prism7.The primary null-hypothesis was that IgE had no effect on any parameter of ASMC remodeling. A second null-hypothesis is that neither signaling inhibition nor microRNA modification modified IgE induced ASMC remodeling. All data are expressed as mean ± SEM, and were analyzed by unpaired one-way ANOVA and a subsequent Student’s Non-immune IgE stimulated ASMC remodeling by upregulating microRNA-21-5p, which, thereby, downregulated PTEN and, thus, supported mTOR signaling. The data suggests that microRNA-21-5p is a potential target for novel therapies that aim to reduce airway wall remodeling in asthma.


Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s).The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed.We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell β-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes.We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment. Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) are major sources of indoor inhaled allergens underlying immunoglobulin E (IgE)-mediated anaphylactic reactions [Allergic diseases constitute the most widespread type of immune disorder in the world . The houeactions , which ceactions . HDM alleactions , are impeactions . The rateactions . The preeactions  and up teactions . Thus, Hhttp://www.allergen.org). The most clinically important HDM allergens identified thus far have been group 1, group 2, and group 23 allergens [The identification of novel allergens benefits allergic disease diagnosis and treatment. Of the 38 HDM allergen groups identified, allergens from 32 groups and 23 groups have been identified for Der f and Der p, respectively, and catalogued in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) allergen database  in vitro methods as well as in vivo mouse immunization and human skin prick tests (SPTs), and then compared IgE binding activity across Der p groups’ IgE epitopes.Serum samples from 50 HDM allergic patients  and 20 non-allergic control group individuals  were provided by the First Affiliated Hospital of Guangzhou Medical College. The HDM allergic group consisted of patients who had experienced HDM-triggered anaphylaxis or IgE levels higher than 3 when measured by the ImmunoCAP allergen detection system . A subsample of 10 allergic patients were recruited separately for Der p extract and recombinant (r)Der p 24 skin prick testing  trypsin/1 mM EDTA (Thermo Fisher Scientific).Rat basophilic leukemia cell lines (RBL-2H3 cells) were purchased from the Chinese Academy of Sciences Cell Bank. The cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% The Basic Local Alignment Search Tool (BLAST) was used to find a UQCRB protein homolog in Der p based on aa sequence. The Der p UQCRB protein was expressed as inclusion bodies, solubilized with guanidine hydrochloride, purified with Ni-NTA resin, and analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specific truncated forms of Der p 24 were used to screen for IgE epitopes. Der p 24 open reading frame cDNA was amplified by polymerase chain reaction (PCR). The sequences of the primers used in PCRs and expression subcloning are shown in Table Escherichia coli BL21 (DE3) plysS for recombinant protein expression. The expression and purification of recombinant proteins was performed as described previously [PCR products were confirmed by DNA sequencing, cloned into pET-His vectors, and transformed into eviously . Followi81EYIQHNGVVQESYY94 and 101QSCRRPNAQRF111) and for Der p 2 (65VPGIDPNACHYMK78) were used as positive IgE binding controls. All of these polypeptides were synthesized by Shanghai Qiang Yao Biotech  and coupled with BSA at the N-terminus or C-terminus with a purity > 95%; the sequences were confirmed by matrix-assisted laser desorption ionization time-of-flight MS analysis.Overlapping polypeptides of Der p 24 were designed based on the N-terminal 32 residues region of Der p 24, as shown in Fig. Purified proteins were separated by 12% SDS-PAGE and then transferred to polyvinylidene-fluoride membranes  for western blotting. For dot blotting, 2-µg aliquots of polypeptides were applied serially onto nitrocellulose membrane ; BSA was used as a control. The membranes were blocked with 5% Difco™ skim milk  diluted in Tris buffered saline containing 0.05% Tween 20 (TBST) at 4 °C overnight. Serum samples were incubated for 2.5 h at 37 °C, and then incubated with horse radish peroxidase-conjugated mouse anti-human IgE antibody (1:2000 in TBST with 1% DSM) for 1 h at 37 °C. Antigen–antibody complexes on membranes were visualized with a Pierce™ 3′-diaminobenzidine substrate kit.2SO4. Absorbance was measured at 450 nm by a microplate reader . IgE-ELISA results with a positive/negative value > 2.1 were considered positive . All tests were performed in triplicate.Microtiter plates were coated with 100 μl of 2 μg/ml polypeptides  at 4 °C overnight. The plates were blocked with 5% (w/v) DSM in phosphate-buffered saline containing 0.05% Tween 20 for 3 h at 37 °C. After washing, the plates were incubated with serum from HDM-allergic patients (1:10) for 2.5 h at 37 °C, followed by mouse anti-human IgE-horse radish peroxidase conjugated (1:2000) for 1 h at 37 °C. The signal was detected by adding 3,3′,5,5′-tetramethylbenzidine substrate and the reaction was stopped with 2 M HD. pteronyssinus extracts  in 10 dust mite-allergic patients with allergic rhinitis and/or asthma and 12 healthy controls (Table The Skin prick test (SPT) was performed as previously described . SpecifiOnline SWISS-MODEL software was used to model the three-dimensional structure of Der p 24 based on its aa sequence. The 32 N-terminal residues were labelled with a red mark.Balb/c female mice (6–8 weeks old), purchased from Guangzhou Experimental Animal Center , were maintained under specific pathogen free conditions. Mice were sensitized by intraperitoneal injection of 50 μg of OVA or OVA-Der p24(1–32aa) antigen with 2 mg of Adjuvant  on experimental days 0, 7, and 14. They received three OVA or OVA-1–32aa antigen boosters by subcutanoeous injection on experimental days 25, 26, and 27. On day 28, serum levels of IgE and IgG specific to BSA-Der p24(1–32aa) were measured by ELISA as described previously [2, and 0.1% BSA, pH 7.4), cells were exposed to sera of sensitized mice for 2 h and then stimulated with 10 μg/ml BSA-1–32aa conjugate for 45 min. Degranulation was assessed by measurement of ß-hexosaminidase release into the supernatant and of unreleased enzyme in the respective cell lysate. The percentage release of total ß-hexosaminidase content was calculated from these values and reported.Degranulation assays were performed as previously reported with slight modifications . BrieflyLung tissues were removed under aseptic conditions for hematoxylin and eosin (H&E) staining. The tissues were fixed in 4% paraformaldehyde for 72 h, embedded in paraffin. The paraffinized samples were sliced into 5-μm sections, stained with H&E, and examined under 100 × magnification. Peribronchial and perivascular inflammation were assessed.t test for multiple comparisons. P values < 0.05 were considered significant.Data are presented as means with standard deviations (SDs). Statistical analyses were conducted in GraphPad Prism7 . Differences between groups were determined by analysis of one-way variance (ANOVA) followed by Dunnett’s BLAST analysis revealed a UQCRB protein homolog in Der p Fig. A. The cohttp://www.allergen.org/viewallergen.php?aid=855).IgE western blot analysis showed that the rDer p UQCRB protein reacted with serum IgEs from individual HDM allergic patients Fig. D. In SPTTruncated forms of Der p 24 used to screen IgE binding epitopes were expressed in a pET expression system, purified with Ni-NTA resins, and analyzed by SDS-PAGE , BSA-Der p 24(1–20aa), BSA-Der p 24(10–23aa), BSA-Der p 24(13–26aa), BSA-Der p 24(16–29aa), and BSA-Der p 24(19–32aa) -conjugate, OVA, or PBS (negative control) in accordance with the experimental design shown in Fig. IgE-ELISAs with separate serum samples from 30 individual HDM allergic patients showed strong IgE binding activity of the 32-residue N-terminal region of Der p 24, exceeding that seen for the main linear IgE epitopes of Der p 1 or Der p 2 Fig. a. IgE-doBased on the presently reported in vitro and in vivo IgE binding activity data, the UQCRB protein homolog identified in Der p was named Der p 24 and catalogued as such in the WHO/IUIS allergen database. The N-terminal 32-residue region of Der p 24 was bound by specific IgEs from HDM allergic sera in vitro and produced specific IgE antibodies in vivo, indicating that this region is an immunodominant IgE epitope of Der p 24 allergen.Although the allergenic components of the globally important HDM species Der f and Der p  are highThe epitopes of most HDM allergens have yet to be clearly resolved. The identification and analysis of immunodominant IgE epitopes of allergens, especially the major allergens, can help to elucidate allergen-induced sensitization mechanisms and provide information that can be used improving immunotherapy outcomes . Prior tGenetic recombinant hybrid proteins have become a favored immunotherapy approach . Der p 1The ongoing discovery of HDM allergens may ultimately lead to a complete HDM allergen panel that can enable component-resolved diagnosis and patient-tailored therapies . ComponeSensitization mechanisms have been elucidated for very few of the 38 recognized groups HDM allergens. Hence, questions remain regarding the allergenic mechanisms of HDM proteins, with potential mechanisms including native protein function effects and immunogenicity of unique sequences that form IgE B cell epitopes. The clinically important group 1 and 2 HDM allergens—which exhibit IgE-binding activity with sera from most patients with mite allergies  while DeThe presently reported experiments led to the identification of Der p 24 as a major HDM allergen with robust IgE binding activity. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced high titers of specific IgE antibodies in vivo. The present demonstration that the Der p 24 N-terminal epitope exhibited stronger IgE binding activity than linear IgE epitopes of Der p 1 or Der p 2 suggests that it may be useful as a component resolved diagnostic maker for HDM allergic diseases. These findings have theoretical implications for the development of diagnostic and therapeutic reagents for HDM allergy.


Czarnecka K, Girek M, Maciejewska K, et al. New cyclopentaquinoline hybrids with multifunctional capacities for the treatment of Alzheimer’s disease. J Enzyme Inhib Med Chem 2018;33:158-170.“Kinetic characterisation of AChE inhibition” and “Kinetic characterisation of BuChE inhibition” on page 161 have to be replaced by the following paragraph.In the “Materials and methods” section the two paragraphs Characterisation of AChE and BuChE inhibition3b against AChE and BuChE was further characterized in kinetic measurements using the Ellman’s method,3b, and with BuChE (4 U/ml in assay) at inhibitor concentrations of 0.2 µM, 1.1 µM and 2.2 µM. The reciprocal activities were plotted as function of the reciprocal substrate concentrations at the indicated inhibitor concentrations in form of Lineweaver-Burk plots.The inhibitory potency of compound Tables 2 (page 164) and 3 (page 165) in the “Results and discussion” section have to deleted.Furthermore, The mechanism of EeAChE inhibition …) and page 164  has to be replaced by the following paragraph:The text between page 163  its potency was further investigated. The obtained Lineweaver–Burk plots for the inhibition of AChE  and PAS , which leads to a mixed-type of inhibitionDue to the relatively strong inhibitory potency of compound  of AChE  and BuCh of AChE  by compo