E = 58.6 ± 4.6 kJ/mol, agreeing withthe proposed sublimation mechanism and at odds with surface diffusionor fluid flow or evaporation–condensation from a quasi-liquidlayer.We show that thesurface of ice is scratch healing: micrometer-deepscratches in the ice surface spontaneously disappear by thermal relaxationon the time scale of roughly an hour. Following the dynamics and comparingit to different mass transfer mechanisms, we find that sublimationfrom and condensation onto the ice surface is the dominant scratch-healingmechanism. The scratch-healing kinetics shows a strong temperaturedependence, following an Arrhenius behavior with an activation energyof Δ Already in 1860, Faraday observed that ice cubessinter together, and he concluded that there is always a liquid layerpresent on the ice surface, even at atmospheric temperatures far belowthe melting temperature.13 It took more than 100years until detailed measurements of the speed of sintering finallyruled out the idea that the flow of a liquid layer was at the originof the sintering dynamics.19 Explanations of the mass transfer phenomenon underlying ice sinteringincluded highly mobile surface molecules undergoing surface diffusion,16 bulk lattice motion,20 and condensation from the vapor phase.23 Although thesethree forms of thermal relaxation were studied thoroughly in the 1950sand 1960s, the mechanism that dominates the sintering process of icehas never been quantitatively established. Hence, Kingery’sexplanation by surface diffusion is frequently considered as beingcorrect, even though it yields an activation energy of more than twicethe latent heat of sublimation.Iceis one of the most actively studied solids,28 However, this one to two molecules thick layer cannotsimply be considered as a liquid since it exhibits viscoelastic properties.30 General crystal growth theory is insufficient to describe the diffusionlimited dynamics of ice crystals since it does not account for theambiguous disordered interface, cooperative intermolecular hydrogenbonding, and the degree of supercooling. Moreover, no model has beenproposed that completely describes the unique growth behavior of icethus far.35More recent research suggeststhat the outermost molecular layerof an ice crystal is disordered.36 The obtained activation energy corresponds to the known value forsublimation, which is significantly higher than the energy barrierfor liquid evaporation. This settles the long-standing debate on theorigin of the sintering dynamics of ice and explains why healing occursrelatively quickly in ice with its high vapor pressure.To shed new light on the complex molecular dynamics of thesurfaceof ice, we investigate the temporal evolution of a scratch made ina pristine surface of ice with submicrometer precision, under preciselycontrolled experimental conditions. We find that the scratch healsspontaneously over time, and that eventually the ice surface becomescompletely smooth again. By comparing the data quantitatively to modelsfor the different proposed healing mechanisms, we determine whichmechanism dominates the healing process. We conclude that scratchhealing of ice occurs by the detachment and reattachment of surfacemolecules. Since the transport of water molecules in the ambient phaseis limited by diffusion, this process is dominated by local sublimationfrom and condensation onto the surface.37 in a temperature and humiditycontrolled chamber. The humidity was regulated by an inflow of drynitrogen into the chamber and monitored by a thermal hygrometer . Furthermore, to reach low humidities,a controlled flow of liquid nitrogen  through a copper element acted as a cold trap to remove remainingwater vapor by condensation. Hence, a theoretical equilibrium betweenthe vapor pressure of the flat ice surface and the air could be achieved,which was calculated with the parameters of Murphy and Koop.38 When the measured humidity deviated from thetheoretical equilibrium by a larger value than the error margin ofthe hygrometer, the inflow of nitrogen was adjusted. Apart from thehealing of scratches, no sign of sublimation or condensation was observedon the horizontal surfaces, confirming the stability of the equilibriumin the chamber.Measurements were carried out with a confocal profilometer (KeyenceVK-X1100), with a lateral resolution of 212 nm and a vertical resolutionof 12 nm,39 The profile of the ice was regularly monitored by a 50×magnification Plan Apo objective  for the period of scratch healing. For a detailed 3D modelof the setup, see Figure S1.The ice layers were formed by cooling 3 mL ofultrapurified waterfrom a Milli-Q system on a copper plate (560 × 380 × 40mm). Cooling was done by a Peltier element in direct contact  with the copper plate to ensure an isothermal and homogeneouslayer of ice. The induced heat on the opposite side of the Peltierelement was extracted from the system by flow from a temperature bath.Ice temperatures were in the range from 243.0 to 272.6 K (measuredby a Voltcraft PL-125-T2USB VS temperature probe). Micrometer-sizedscratches were manually created with a sharp razor blade (Derby extraPaslanmaz Çelik) and positioned in such a way that the measuredarea of 212 × 283 μm contained only one defined crystalorientation, so grain-boundary dynamics could be excluded. Since differentgrains do not exhibit wide variations in molecular organization atthe surface, we were enabled to collect an ensemble of measurementsperformed under similar experimental conditions, as the effectivediffusion coefficients are expected to be similar.Our measurements provide highly detailed images of the ice scratchprofile as it slowly heals in time. As illustrated in 41 The Mullins model assumes that the attachment and detachment ofmolecules can occur everywhere on the surface, which is valid forice with its disordered interface. He derived that, in the case ofan initial sinusoidal profile with wavelength λ, only the overallamplitude of the profile changes with time, so the time-dependentdistance of the ice surface with respect to the unscratched surfaceis given by U = u(t) sin(2πx/λ), where x is the direction perpendicularto the scratch and t is time. The evolution equationfor the amplitude u(t) depends onthe mechanism and is given byCn(T) is atemperature-dependent prefactor and n is an integerdepending on the model: n = 1 for fluid flow, n = 2 for sublimation–condensation, n = 3 for volume diffusion, and n = 4 forsurface diffusion. Mullins also showed that the equations governingthe mass diffusion are linear in the sense that the sum of any twosolutions is again a solution.41 Hence,the evolution of an arbitrary initial profile can be obtained froma Fourier analysis, and this is how we calculate the time-dependentprofiles for each of the four models: we decompose the initial experimentalprofile as a Fourier sum  and propagate each component independently in time. Weapply a correction for a small overall slope of the initial profileif necessary. Our analysis involves the following simplifying assumptions:(i) the measurements are carried out in a closed system where thevapor pressures of the flat ice surface and air are in equilibrium,(ii) the mass transfer coefficients are not affected by the crystalorientation of the ice lattice, and (iii) the slope of the profilesis small enough to apply the small-slope approximation  as the only free parameter. We find that thesublimation–condensation process exhibits the lowest χ2 and thus best agreement with the measurements . In Figure S3. The differences among the four models are further illustrated byplotting the absolute maximum depth of the ice scratch profile developingin time for the best fitting parameters Cn(T) in We test each mass transfer modelby comparing the theoretical predictionwith the data of 30 independent measurements using χurements S2. In Fiurements S2. In Fiurements S2. In FiSupporting Information). This scratch-healingprocess is relatively fast in the first few minutes and slows downas the surface becomes less curved, as observed in In the sublimation–condensation driven healing process,the smoothening of the profile is driven by an increased vapor pressurefor curved surfaces  obtained from the fitfollow an Arrhenius temperature dependence with an activation energyof ΔE = 58.6 ± 4.6 kJ/mol, as shown in 38 The sublimation activationenergy of water was found to be in the range 53.1–57.3 kJ/mol,44 in good agreement with our result. Moreover, the energy barrierfor condensation is significantly lower: 43.35–45.1 kJ/mol.46 These results are strong indications that the scratch healing ofice is driven by local sublimation instead of local evaporation, beforecondensation occurs.To obtain more insightinto the healing mechanism, we investigatethe temperature dependence of the effective diffusion coefficient.To this end we perform similar least-squares fit analyses for 30 measurements(five scratches profiled at six different temperatures from 243.0to 272.6 K). The effective sublimation–condensation coefficients 49 However, none of the sintering experiments could be quantitativelyreproduced by the liquid layer model.3The above results indicate that the scratch healing of iceoccursthrough a sublimation–condensation mechanism. We now discussearlier experimental results that were previously interpreted in termsof the other proposed scratch-healing mechanisms. First, considerthe liquid layer interpretation of Faraday. This concept receivedwidespread acclaim by rather precarious comparisons between the physicalproperties of thin liquid layers and the topmost layer of ice.20 This inhibited the movement of the molecules along the surface and,more importantly, completely prevented mass diffusion via the vaporphase. Hence, this specific experiment on ice sintering is not generallyapplicable.Second, the research that concluded that volumetric bulk diffusionis responsible for the sintering of small ice beads was conductedin a liquid kerosene saturated environment.16 Remarkably, when we force the model for surfacediffusion onto the data of our scratch-healing experiments, we obtaina similar activation energy of ΔE = 100.0 ±11.1 kJ/mol. Weber et al. used friction experiments and moleculardynamic simulations on the topmost layer of solid water moleculesto demonstrate that the activation energy of surface diffusion isroughly 11.5 kJ/mol: 1 order of magnitude lower.50Third, the research on surface diffusion, done byKingery, matchedthe theoretical predictions of the neck growth between two touchingice spheres. However, the obtained activation energy of approximately115 kJ/mol is more than twice the latent heat of sublimation.To conclude, we find that the detachmentand reattachment of highlymobile water molecules on the ice surface causes scratches in theice surface to heal spontaneously. By quantitatively studying thescratch-healing behavior of micrometer-sized scratches, and comparingthe results with four models proposed for the transport of moleculeson the ice surface, we conclude that the main mechanism of transportis through sublimation and condensation. We propose that the efficientscratch healing of ice compared to other materials might be due tothe water molecules in ice being connected by hydrogen bonds: in contrastto the attractive interactions in the crystals of most other materials,hydrogen bonding is highly cooperative, meaning that breaking fourhydrogen bonds in the bulk requires much more than 2 times the energyrequired for breaking two hydrogen bonds at the interface. As a consequence,the water molecules at the surface can detach relatively easily, eventhough the bulk crystal phase is completely stable.


High Arctic ecosystems and Indigenous livelihoods are tightly linked and exposed to climate change, yet assessing their sensitivity requires a long-term perspective. Here, we assess the vulnerability of the North Water polynya, a unique seaice ecosystem that sustains the world’s northernmost Inuit communities and several keystone Arctic species. We reconstruct mid-to-late Holocene changes in sea ice, marine primary production, and little auk colony dynamics through multi-proxy analysis of marine and lake sediment cores. Our results suggest a productive ecosystem by 4400–4200 cal yrs b2k coincident with the arrival of the first humans in Greenland. Climate forcing during the late Holocene, leading to periods of polynya instability and marine productivity decline, is strikingly coeval with the human abandonment of Greenland from c. 2200–1200 cal yrs b2k. Our long-term perspective highlights the future decline of the North Water ecosystem, due to climate warming and changing sea-ice conditions, as an important climate change risk. The North Water polynya is a unique but vulnerable ecosystem, home to Indigenous people and Arctic keystone species. New palaeoecological records from Greenland suggest human abandonment c. 2200–1200 cal yrs BP occurred during climate-forced polynya instability, foreshadowing future ecosystem declines. Diatoms, including open-water, marginal ice zone, and sea-ice (sympagic) taxa, are the main primary producers in the NOW (e.g. ref. 8) and although grazing pressure may vary, a significant fraction of the diatom production in the euphotic zone reaches the seafloor sediments either as intact cells and spores or empty frustules (after grazing or lysis)9. Following nutrient exhaustion, species belonging to the diatom genus Chaetoceros can produce large amounts of highly silicified, fast-sinking resting spores10 that generally preserve well in the sediments and reflect changes in productivity levels over time.The seasonal formation and physical conditions of the NOW rely primarily on the consolidation of an ice arch or bridge across the southern Kane Basin during winter, which blocks the inflow of multiyear sea ice from the Arctic Ocean Fig. . When th11. The little auk is tightly linked to the copepod Calanus hyperboreus, on which the chicks are raised, and >60 million birds depend on the unique availability of this prey item in the NOW12. By transporting vast quantities of marine-derived nutrients (MDN) from sea to land in the form of guano, little auks have transformed extensive parts of the NOW coastal landscapes into green oases14. At little auk colonies, temporal changes in the MDN flux in sediments can be used as a proxy for changes in bird numbers and, by inference, NOW productivity over time15.The NOW supports >80% of the global breeding population of little auk, which is the most abundant seabird in the North Atlantic16. The NOW is of critical significance to Inuit communities today17, and transformative shifts in the ecosystem, due to changing sea-ice conditions, represent an emerging hazard, in this context referring to a climate-related trend with negative impacts1. Ice-arch dynamics in Nares Strait have shown a tendency towards instability and earlier spring collapses, linked to changes in sea-ice regime and wind forcing20. This raises concerns as to whether the NOW ecosystem will persevere in a warming climate. Evaluating risks and defining climate adaptation measures for this unique ecosystem requires an understanding of past ecological and societal responses, but this knowledge is lacking due to a paucity of long-term records.Due to its resource richness in one of Earth’s most inhospitable environments, and its strategic location at the southern edge of the narrowest point between Canada and Greenland, the NOW region represents the gateway of prehistoric migrations into Greenland and has been the stage for cultural transitions since the first humans crossed the Nares Strait c. 4400 yrs b2k1). We applied a retrospective approach, reconstructing long-term trends in sea ice, primary (diatom) production and little auk colony dynamics based on two sediment core records (one marine and one lacustrine) spanning the last c. 4000 and 6000 years, respectively . As proxies of past sea-ice dynamics, we used IP25, a source-specific molecular biomarker produced by sympagic diatoms22, and its related compound HBI III (Triene), produced by certain pelagic diatoms thriving in the cold waters of the marginal ice zone23. Combined, these source-specific biomarkers track polynya activity and stability over time. To infer the presence and relative abundance of little auks, we analysed δ15N, cadmium to titanium ratios (Cd:Ti), the fluxes of cholesterol and ß-sitosterol, and changes in diatom assemblage composition in a sediment core from a lake within the catchment of a large little auk colony  and a box core (40 cm long) retrieved from a site centrally located in Smith Sound at 692 m water depth, south of the southernmost ice arch location  with glacial silty-clays and low organic content  was retrieved from a lake at Annikitsoq on the Cape York Peninsula at 34 m water depth and spans the last ca. 6000 years Figs. . As suchStaurosira construens and Stauroforma exiguiformis). Fragilarioids are common in Arctic lakes and ponds and are typically found in oligotrophic and circum-neutral to somewhat alkaline environments with prolonged ice cover and hence a short growing season25. After 4200 cal yrs b2k, there is a marked change in the lake diatom assemblages indicating a decrease in lake pH, which is seen as the dominance of acidophilous taxa (within the genera Psammothidium and Eunotia) and an overall decrease in planktic species, which are adversely affected by lower pH26 31  and the Arctic Oscillation (AO) are the two dominant (and closely related) modes of atmospheric variability for the mid-latitudes of the North Atlantic region and the entire Northern Hemisphere, respectively38. The NAO/AO have shown periodicities on multidecadal timescales that are linked with sea-surface temperature changes in the North Atlantic ), in turn influencing Arctic sea-ice variability40. Sea ice concentration and motion in the Arctic Ocean region are also highly impacted by the Dipole Anomaly (DA) pattern—the second mode of winter variability - which promotes sea ice transport from the western to the eastern Arctic Ocean and export towards the North Atlantic through Fram Strait, and appears to be particularly important during warmer periods43.Polynya dynamics in the North Water are strongly linked to sea ice conditions  and prevailing winds, and thus influenced by local and regional climate forcing. The inception of recurrent ice arches in the Nares Strait region is estimated to have occurred at the mid-to-late Holocene transition, following atmospheric cooling and extensive sea ice formation in the Canadian Arctic and Greenland45, Fig. 46. Ocean-atmosphere climate shifts after 2700 yrs b2k have been linked to a strengthening of the latitudinal temperature gradient and southward migration of the Polar Front47, coincident with cooling and freshening of bottom waters off the West Greenland coast from 2600 to 1900 cal yrs b2k48 The period of polynya instability spans two warm(er) intervals, the Roman Warm Period and the Medieval Climate Anomaly. Marine records from offshore Greenland indicate ocean warming, particularly during the Roman Warm Period , linked to an increased contribution of Atlantic-sourced waters to the West Greenland Current49.From c. 4400 to 2700 cal yrs b2k, the polynya remained relatively stable and productive, and paleo-records point towards predominantly weakly positive NAO and low magnitude variations in the AO during this period  are inferred for the period of human abandonment of Greenland c. 2200–1200 cal yrs b2k and coeval with our demonstrated decline in NOW productivity Figs.  and 6. Sata Fig. . While tOur long-term perspective on the evolution of the NOW underscores the tight coupling of this ecosystem to climate forcing and highlights its vulnerability to climate change. The concordance of past periods of decreased marine productivity with human abandonment of Greenland suggests that the future collapse of the NOW ecosystem is a legitimate climate change risk.19, which allows for increased late spring/summer ice drift and melt in the NOW region as ice is not held upstream of the arch 17.On our present climate trajectory, the NOW will likely cease to exist as a globally unique ice-bounded open-water ecosystem, and a winter refuge for keystone High Arctic speciesAmundsen during the ArcticNet 2015 Leg 4a expedition in 2015, in accordance with relevant permits and local laws. The CASQ corer recovered a sequence 543 cm long, while the box core was 40 cm long. Sediment material from these cores is stored at the Geological Survey of Denmark and Greenland and available upon reasonable request to the first and corresponding author (SRI).The Calypso Square gravity core AMD15-CASQ1  and accompanying box core  were retrieved aboard the CCGS Computed Tomography (CT) scanning of the core was performed using a Siemens SOMATOM Definition AS + 128 at the Institut National de la Recherche Scientifique (INRS), Quebec, Canada. The tomograms were converted into digital DICOM format using a standard Hounsfield scale  from −1024 to 3071, where −1024 corresponds to the density of air, 0 to the density of water and 2500 to the density of calcite.210Pb/137Cs measurements conducted on 20 samples at the Gamma Dating Center, Copenhagen University, Denmark. In the box core, the content of unsupported 210Pb showed a clear exponential decline with depth  values ranging from -40 years in the Inglefield Fjord to +320 years in Ellesmere Island (the latter consistent with the proposed 335 ± 85 years for the Canadian Arctic Archipelago56). However, these samples have been retrieved from shallow sites (<40 m water depth), which are unlikely to reflect influence from the West Greenland Current. Data from the only deeper site in the NOW region are based on measurements of the mollusc Astarte montagui and indicate an offset of 140 ± 60 years57. This value is consistent with measurements from the Disko Bay region, also under the influence of the West Greenland current58. We have therefore chosen to use an offset value of 140 ± 60 years. Ages are reported in calibrated years before 2000  and year CE  radiocarbon dates on mollusc shells  were powdered (<250 micron) and subjected to Rock-Eval type bulk flow pyrolysis using a HAWK instrument . Sets of one control-standard (in-house standard) and one blank were run every 10 samples to ensure instrument stability.Haslea and Pleurosigma genera are known to produce the sea ice biomarker IP25—a mono-unsaturated highly branched isoprenoid (HBI) alkene, biosynthesized in the sea-ice matrix22 and deposited in marine sediments following ice melt. Due to its source-specificity and good preservation potential in marine sediments, IP25 constitutes direct evidence for seasonal sea ice. In marine settings, high sedimentary IP25 content generally reflects increasing seasonal sea ice concentrations, whereas the absence of IP25 can either indicate perennial sea-ice cover or open-water23. As such, downcore changes in sedimentary IP25 fluxes from a given location can be interpreted to reflect temporal fluctuations in sea ice conditions. A related lipid biomarker HBI III is produced by diatoms blooming in the often ice-loaded and relatively fresher and cooler surface waters typical of the marginal ice zone (e.g. ref. 59). It has recently been shown that the relative abundances of IP25 and other HBIs remain essentially unaltered in trophic food webs and faecal pellets60. These findings suggest that source HBI distributions remain unaltered following grazing, which implies that changes in grazing efficiency do not have a significant impact on the sedimentary signature of HBIs.A small number of common pan-Arctic diatoms belonging to the 2Cl2) and methanol (MeOH) . Extracts were pooled together, and the solvent was removed by evaporation under a slow stream of nitrogen. The total extract was subsequently resuspended in hexane and purified through open column chromatography (SiO2). Hydrocarbons (including IP25 and HBI III) were eluted using hexane (8 mL). Procedural blanks and standard sediments were analysed every 15 samples. Hydrocarbon fractions were analysed using an Agilent 7890 gas chromatograph (GC) fitted with 30 m fused silica Agilent J&C GC columns (0.25 mm i.d. and 0.25 µm phase thickness) and coupled to an Agilent 5975 C Series Mass Selective Detector (MSD). The following oven temperature programme was used: 40–300 °C at 10 °C min−1, followed by an isothermal interval at 300 °C for 10 min. The data were collected using Chemstation and analysed using the MassHunter quantification software. IP25 and HBI III were identified on the basis of retention time and comparison of mass spectra with authenticated standards. Abundances were obtained by comparison between individual GC–MS responses relative to those of the internal standard. Biomarker data presented here are reported as fluxes to account for changes in sedimentation rate.Prior to analytical treatment, an internal standard (7-hexylnonadecane) was added to 0.5 g of freeze-dried and homogenised sediment. Total lipids were ultrasonically extracted (×3) using a mixture of dichloromethane  and hydrochloric acid  to remove the organic material and carbonate, respectively. Residues were then rinsed several times with distilled water. A known volume of the final residue, homogenised in suspension, was added to a coverslip. Once the samples were completely dried, microscopy slides were mounted in Naphrax® for observation. Quantification of diatom valves was done using an optical microscope (Olympus BX43) with phase contrast optics at a magnification of 1000x. Concentrations were calculated based on the surface area of the slide that was analysed. Fluxes were calculated by combining diatom concentrations (ind. g−1) with mass accumulation rates (g cm−2 yr−1).Diatom fluxes were used to infer changes in marine primary production. For diatom quantification, sediment samples were treated with hydrogen peroxide  on July 30th 2015. The sediment core was recovered from 34 m water depth using a highly portable piston corer specially adapted for remote location use. The 177cm-long core was kept upright and drained of water, the sediment surface was secured by packing the core top with a rigid foam block (Oasis), and the core was kept cool and dark before and during transport from Greenland to Denmark. Fieldwork was conducted in accordance with local laws and permits. Sediment material is deposited at Aarhus University and available upon reasonable request from the last author (TAD).The lake was not stratified at the time of sampling (July 30–August 2 2015) and was partially ice-covered on the day of arrival. The surface waters were 4 °C and oxygen saturation was over 100% all the way to the lakebed.55. The age model was calculated using the R routine BACON54. Ages reported and used in the figures are median modelled ages converted to calibrated years before 2000 .The age control of NOW25c was attained by 10 accelerator mass spectrometry (AMS) radiocarbon dates at Aarhus AMS Centre (AARAMS), Aarhus University, 9 on terrestrial moss remains and 1 on humic extraction of a bulk sample  techniqueThe lake sediment core was split along its length then placed in an ITRAX core scanner to obtain high-resolution pictures and measure micro-XRF. The XRF scans were made at the Aarhus University core scanning facility with a molybdenum tube set at 30 kV and 30 mA with a dwell time of 4 s. Prior to analysis, the sediment surface was flattened and covered with a 4 µm ultralene film. A step size of 0.1 mm was selected to capture possible elemental variations even in small laminations. Count readings for less abundant elements, such as Ti, maybe too low with a 4 s dwell time, so counts were summed to at least 1 mm for analysis and presentation.15N (stable isotope of nitrogen), the ratio of Cadmium (Cd) and Titanium (Ti) concentrations in the lake sediments, concentrations and fluxes of cholesterol and β-sitosterol and diatom assemblage composition changes to assess the presence/absence and relative size of the adjacent little auk colony through time. δ15N differs markedly between marine and freshwater systems and has been shown to provide an unequivocal signal of marine-derived nutrients (MDN)13. Cd is also more concentrated in the marine system and thus can be used to trace seabird influence62. Cd is abundant in the seabird excrement, but it may also be present in the lake’s catchment and so the ratio Cd:Ti is used, as Ti is a proxy of catchment input to the lake. The development of soil, peat and permafrost in the catchment stores C, N and perhaps to a lesser extent Cd, reducing the quantities delivered to the lake and affecting isotope fractionation. The extremely high values of δ15N and cholesterol at the time of bird arrival likely reflect the absence of soil in the catchment and the unimpeded flow of bird-derived compounds into the lake. In the event that the inputs of C, N and Cd are reduced due to reduced supply as bird numbers fall, the catchment has the potential to act as a source. Thus, whilst the isotopes of N give a clear signal of bird arrival, they have the potential to be less reliable in tracking absence as a result of their storage and subsequent release. This may depend on the residence time of the lake and the degree of flushing by non-bird impacted waters. The combination of total cholesterol flux and the ratio of cholesterol to cholesterol plus β-sitosterol has been used to identify marine bird influence in freshwater systems in the Arctic (e.g. ref. 63).We used a combination of δ64. We found very high cholesterol concentrations (1497 µg g−1) and low β-sitosterol concentrations (15 µg g−1) in little auk excrement, contrasting with goose excrement collected in the same region (16 µg g−1 of cholesterol and 159 µg g−1 of β-sitosterol), a species which largely grazes on terrestrial vegetation. Thus, when seabirds are abundant, cholesterol values in the lake sediments can be expected to be high, and the ratio of cholesterol to cholesterol plus β-sitosterol should also be high. In the NOW region, the ratio is 10-fold higher in little auk excrement (0.99) compared with goose excrement (0.09). This ratio of cholesterol to cholesterol plus β-sitosterol has been previously used as an index of seabird influence, with a value of around 0.6 indicating that the majority of the cholesterol originates from seabirds64. This index was derived from studies of fulmar colonies in relatively oligotrophic systems, which is not the case at Annikitsoq, where the lake is situated in extremely eutrophic systems (due to the little auk colony) with lush vegetation in the catchment and freshwater algae thriving in the water column, both of which are sources of β-sitosterol. Here, the arrival of little auks, and with them large quantities of MDN, transformed nutrient availability, prompting a period of exceptionally high terrestrial and aquatic biological productivity. This is demonstrated by the fact that 2 m of peat accumulated in the catchment adjacent to the lake in just 1000 years, following little auk colonisation65, and the extremely high LOI values recorded in the lake (75% instead of <10% as expected for a High Arctic lake). Therefore, in addition to the large input of marine-derived cholesterol, there was also the input of cholesterol of terrestrial and freshwater origin, which has a higher proportion of β-sitosterol. Thus, a slight drop in bird input combined with increased terrestrial production, as nutrient levels remain sufficient, would result in a lower ratio, even when birds may still be present. Thus, whilst the ratio is still a useful indicator , we have not used a cut-off value signalling the dominance of seabird influence but instead simply present the index value over time. Bird absence is most likely when both cholesterol and the ratio of cholesterol to cholesterol plus β-sitosterol are low.Marine zooplankton, upon which the little auks feed, are rich in cholesterol but contain virtually no β-sitosterol. In contrast, terrestrial and freshwater primary producers contain a high proportion of β-sitosterol66. Specifically, Androstanol (0.1 mg mL−1) was added as an internal standard to each sample of approximately 0.5 g of dried, homogenised sediment. Lipid compounds were extracted with solvents  using Microwave-Assisted Extraction, saponified and separated into neutral and acid fractions using aminopropyl SPE columns. The neutral fraction of each sample was then separated using silica gel column chromatography. The sterol fraction was trimethylsilylated using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) (99:1 v/v) and heated at 70 °C overnight. Excess BSTFA-TMCS was removed by drying gently under nitrogen. Samples were dissolved in 50–100 µl of ethyl acetate prior to gas chromatography-flame ionisation detection (GC-FID) and gas chromatography–mass spectrometry (GC–MS) analysis. GC–MS analyses were performed on an Agilent 7890B GC injector (280 °C) linked to an Agilent 5977B MSD in full scan mode (50–600 amu s−1). Separation was performed on an Agilent fused silica capillary column  with Helium as a carrier gas. Sterol derivatives were analysed using the following temperature programme: 50 °C (held for 2 min) to 200 °C at 10 °C min−1, then to 300 °C at 4 °C min−1 and held for 20 min. GC–MS peaks were identified through comparisons with known mass spectra (NIST08) and standards where possible. Analytes were quantified based on internal standards. For 13 of the samples, lipids were extracted from the dry sediment using a chloroform:methanol 2:1 mixture and then sonicated for 10 min, after which 0.75 mL of distilled water was added. Lipids were fractionated into neutral lipids , glycolipids, and phospholipids (PLs) using a Bond Elut (0.5 mg) silica cartridge. First, the resin of the cartridges was conditioned using 5 mL of chloroform. Subsequently, the total lipids (1 mL) were applied to the resin, rinsed using chloroform, and then the NLs (including sterols) were collected under vacuum using 10 mL of chloroform. Sterols from the NL fraction were silylated with BSTFA, TMCS, and pyridine at 70 °C for 1 h. Trimethylsilyl (TMS) derivatives of sterols were analysed with GC–MS (Shimadzu) and GC-FID (Shimadzu) equipped with a Phenomenex (USA) ZB-5 Guardian column (30 m × 0.25 mm × 0.25 μm). Cholesterol and β-sitosterol were identified using characteristic ions of GC–MS runs64 and quantified with GC-FID using authentic standard solutions of plant sterol mixture from Larodan , and cholesterol from Sigma-Aldrich. The recovery percentage of the sterol samples was calculated using 5-α-cholestane (Sigma-Aldrich) as an internal standard.Sterol analysis followed standard protocols13C) and nitrogen (15N) isotope analyses were conducted using an elemental analyser and a continuous flow isotope ratio mass spectrometer (IRMS). Specifically, an Elementar Vario EL Cube  interfaced with an Isoprime VisION IRMS . Samples were combusted at 1080 °C in a reactor packed with chromium oxide and silvered copper oxide. After combustion, a reduction reactor trap removed oxides and a helium carrier then flowed through a water trap (magnesium perchlorate and phosphorous pentoxide). CO2 was held in an adsorption trap until the N2 peak was analysed; after which the CO2 was released by heating to the IRMS. Reference materials included: IAEA-600, USGS-40, USGS-41, USGS-42, USGS-43, USGS-61, USGS-64, and USGS-65. A sample’s isotope ratio is expressed relative to a reference gas peak analysed with each sample. These provisional values are finalised by correcting the values for the entire batch based on the known values of the included laboratory reference materials. The long-term standard deviation was 0.2 per mil for 13C and 0.3 per mil for 15N. The delta values are expressed relative to international standards VPDB (Vienna Pee Dee Belemnite) and Air for carbon and nitrogen, respectively.For stable isotope analysis, samples were taken at 2 cm intervals, freeze-dried for 48 h and ground into fine powder. The samples were packed into tin cups and analysed at UC Davis Stable Isotope Facilities, California, USA. Here, carbon  in two regions of interest—NOW and Ice Arch Fig. . We usedChaetoceros spores flux, and HBI III) were assumed to have a measurement uncertainty of ±7.5% (5–10%) and the depth-dependent dating uncertainty shown in Fig. 15N, and 0.001 mg cm−2 yr for fractional sterol, with depth-dependent dating uncertainty , while HBI III loads negatively (PC1 loading of −0.47). We interpret this as suggesting that the first marine principal component is positively correlated with primary production. In the lake record, all-time series  load positively on the first principal component  approach, which optimises predictive accuracy and allows for heteroscedasticity in the data. After the model was fitted , and took a flexible approach to the degree of smoothing employed in modelling the response of both the marine and lake record proxies. Where possible, the degree of smoothing was estimated using restricted maximum likelihood (REML), and the model contained a continuous-time first-order autoregressive process (CAR(1)) to account for temporal autocorrelation. This solution was optimal for the marine record proxies Diatoms, Supplementary InfoPeer Review File


The replacement of the stripped screw resulted in a POS of 158.4 ± 64 N for the screw of larger diameter, while the screws of the same diameter but increased length or those with extended thread length yielded POS values of 138.4 ± 42 and 185.7 ± 48 N, respectively. Screw stripping is a frequent intraoperative complication that, according to our findings, cannot be addressed by leaving the screw in place. The holding capacity of a stripped screw implanted in cancellous bone can successfully be restored with a different screw of either larger diameter, longer length, or extended thread length.Screw stripping during bone fixation is a common occurrence during operations that results in decreased holding capacity and bone healing. We aimed to evaluate the rescue of the stripped screw site using screws of different dimensions. Five screw configurations were tested on cadaveric specimens for pull-out strength (POS). The configurations included a control screw tightened without stripping, a configuration voluntarily stripped and left in place, and three more configurations in which the stripped screws were replaced by a different screw with either increased overall length, diameter, or thread length. Each configuration was tested five times, with each screw tested once. The POS of the control screw, measured to be 153.6 ± 27 N, was higher than the POS measured after stripping and leaving the screw in place (57.1 ± 18 N,  A common problem encountered in orthopaedic surgical procedures is screw stripping due to overtightening, because a stripped screw offers poor fixation . FletcheThe existing literature offers several strategies that can be employed in the case of screw stripping, such as plating , augmentWith the aim of exploring rescue approaches for cancellous bone that are less invasive than bone cement, in this study, we compare the holding capacity of the primary screw with three rescue strategies that do not require alternative holes or supplemental fixation. We hypothesized that despite the bone damage due to stripping, pull-out strength can be recovered in the same location of the stripped screw using a screw of the same type, but of different dimensions.The study was performed according to the rules of the institution ethical committee.control configuration. A second configuration was constituted by the screw voluntarily stripped at time of insertion and left in place. The three rescue configurations were recreated by intentionally stripping the screws and replacing them with screws of either larger diameter, longer length, or with an extended thread. All tests were performed on human cadaveric specimens harvested from donors with mean age of 65.2 ± 8 years old. The proximal tibial metaphysis of 25 human cadaveric specimens were used as models of human cancellous bone [In this experiment, the screw pull-out strength (POS) was used to judge fixation quality  and streous bone . CT scanlarger screw had the same length and thread as the control screw but a diameter of 8 mm, the longer screw had the same diameter and thread of the control screw but a length of 120 mm, and the extended thread screw had same length and diameter of the control screw but a thread extension of 32 mm.Titanium cannulated screws  were chosen with dimensions associated with each configuration see . The conAn experienced orthopaedic surgeon implanted each of the screws by hand 10 mm below the articular surface of the tibia with a gap of at least 5 mm from the medial cortex. Each screw was tightened in correspondence of the tibial coronal plane along the lateral to medial direction. An adjustable spacer was used to ensure consistency in screw placement see . Followicontrol configuration constituted by the screw inserted without stripping was obtained by screwing up to about 50% of the peak torque that was observed in the beginning of the compression phase  in accordance with the Standard Specification and Test Methods for Metallic Medical Bone Screws (ASTM F543). The peak force values recorded during the extraction provided the POS of the screws see .t-test. The significance level of all the analyses was set at p < 0.05 (two-tailed). All statistics, descriptive and analytic, were performed using SPSS .Differences among configurations were analysed with ANOVA, while specific differences between groups were identified using Student’s p > 0.05). None of the 25 used screws were damaged during testing. The stripped screws resulted in a peak torque of 0.74 ± 0.3 Nm, which was higher than the control screw torque of 0.36 ± 0.1 Nm (p = 0.018). The rescue screws were inserted with a torque of 0.31 ± 0.01 Nm, 0.19 ± 0.1 Nm and 0.24 ± 0.1 Nm, respectively, for the larger screw, longer screw, and extended thread screw. The POS of the control screw, measured to be 153.6 ± 27 N, was higher than the POS measured after stripping and leaving the screw in place . All three rescue strategies were able to recover the POS to the level of the control (p > 0.05). The extended thread screw yielded the highest POS , while the POS for the longer screw and larger screw were limited to values of 158.4 ± 64 N (p = 0.011) and 138.4 ± 42 N (p = 0.016), respectively (see p > 0.05).CT analyses did not yield bony defects or hardware in any of the specimens. The mean attenuation coefficient was 206.3 ± 48 HU (Hounsfield Units) and no differences in density were reported among groups (vely see . No statp = 0.001) for the stripped screw. Comparably, Collinge et al. and Marmor et al. reported a strength reduction ranging from 76% to 82% when stripping occurred while testing screws of 3.5 mm and 4 mm in a bone substitute [Poor stability and low compression in the site of fracture have been related to a higher rate of late consolidation, non-union, and chronic pain in multiple scenarios ,21. Suffbstitute ,24. ThesThe main finding of this study is that the holding capacity of a stripped screw in cancellous bone can be restored by replacing it with a screw either longer in length, larger in diameter, or with an extended thread, thereby salvaging the original screw site. The larger implanted screws replacing the stripped screws had values of POS of 158.4 ± 64 N. Those of same diameter but longer or those with extended thread yielded POS values of 138.4 ± 42 and 185.7 ± 48 N, respectively. In general, our findings show that having more depth available to the stripped screw would allow the implantation of a longer screw, while a larger segment under fixation would allow the use of a screw larger in diameter. The cannulated screw type used in our study can be used in femoral neck fractures, which have a similar composition to the proximal tibia in that both are composed of cancellous tissue . ConsideIn conclusion, we found that screw stripping is a frequent intraoperative complication that cannot be addressed by leaving the screw in place. The holding capacity of a stripped screw implanted in cancellous bone can successfully be restored with a different screw of either larger diameter, longer length, or extended thread length. The specific choice among these three rescue screw configurations should be made by considering the morphometry of the specific anatomical site and object of instrumentation.


Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders. In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga,  Polysiphonia morrowii Harvey (P. morrowii), a red alga, produces several bioactive substances such as 5-bromo-3,4-dihydroxybenzaldehyde (BDB), 3,4-dihydroxybenzaldehyde (DHB), 3-bromo-5-(ethoxymethyl)-1,2-benzenediol, 3-bromo-4,5-dihydroxybenzyl methyl ether, 3-bromo-4,5-dihydroxybenzaldehyde, and bis  ether [P. morrwii possess antiallergic effects in IgE-induced mast cell activation and passive cutaneous anaphylactic (PCA) reaction in mice model [The rapidly increasing incidence of allergic diseases, including asthma, allergic rhinitis, and atopic eczema, has become a significant public health problem ,2. Amongl) ether ,17,18,19l) ether ,17,18,19ce model . Additioce model . HoweverTo identify the cytotoxicity of DHB on BMCMCs, the MTT assay was performed. At all of the studied concentrations, DHB did not show any cytotoxicity in the BMCMCs compared to the untreated control cells A. On theTo identify whether DHB inhibits the expression of FcεRI and the FcεRI-IgE binding on the surface of BMCMCs, we performed flow cytometric analysis. DHB dose-dependently reduced the expression of FcεRI compared to that in the untreated control cells A. TypicaTo further elucidate the effect of DHB on the secretion of allergic cytokines in IgE/BSA-stimulated BMCMCs. DHB treatment dose-dependently decreased the secretion of allergic cytokines such as IL-4, IL-5, IL-6, IL-13, and TNF-α at concentrations of 31.3 and 62.5 μg/mL . InteresThe suppression of various cytokines expression via NF-κB inhibition is a key signaling pathway in the antiallergic effect . TherefoTo confirm the effect of DHB on the IgE/BSA stimulation-induced PCA reaction in mice, we monitored the amount of Evans blue dye extracted from the mice ear tissues. As shown in Prunus mume seed has antiallergy effect by suppressing the β-hexosaminidase release in antigen-stimulated RBL-2H3 cells and BMCMCs [P. morrowii, a red alga, in IgE/BSA-stimulated BMCMCs and a PCA mouse model and evaluated its biological mechanism.DHB, known to be a protocatechuic aldehyde and a water-soluble compound, is a phenolic aldehyde. Generally, phenolic compounds represent the main type of secondary metabolite in plants and show multiple biological effects . Therefod BMCMCs . Howeverd BMCMCs . In the P. mume seed inhibited β-hexosaminidase release upon antigen-induced degranulation of RBL-2H3 cells at the half maximal inhibitory concentration (IC50) values 0.6 mM, and it was similar to the effects of DHB on the degranulation of BMCMCs [The annual increase in allergic diseases is caused by the inappropriate response of immune cells to allergens characterized by the excessive production of IgE ,28. IgE-f BMCMCs . Generally, mast cells express FcεRI on their membranes, which are important for the proinflammatory allergic response, and when an IgE-antigen binds with FcεRI, the receptor is activated and complex biological reactions occur, causing allergic reactions, including inflammatory disorders . InhibitFurthermore, the activation of mast cells leads to a potential source of many cytokines and a chemokine that might affect IgE-mediated allergic inflammation. These cytokines and chemokines include IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-13, GM-CSF, TGF-β, TNF-α, IFN-γ, TARC, and several others . IL-4 anThe expression of cytokines is regulated by several intracellular signaling pathways, especially NF-κB, a transcription factor that plays a central role in the induction of gene expression and is considered as a promising target for the treatment of disease ,35. NF-κFinally, we examined the anti allergic effects of DHB in a localized IgE-mediated allergic response based in vivo study. PCA reaction, which evaluate by increased vascular permeability and ear swelling, is that responses through binding of the IgE and FcεRI in mast cells ,20. TherMinimum Essential Medium α (α-MEM), penicillin-streptomycin, fetal bovine serum (FBS), and dimethylsulfoxide (DMSO) were purchased from Thermo Fisher Scientific . Pokeweed mitogen (PWM), 2-mercaptoethanol (2-ME), 3--2,5-diphenyltetrazolium bromide (MTT), Evans blue reagent, monoclonal anti-dinitrophenyl (DNP)-IgE (clone SPE-7), and β-actin antibody were purchased from Sigma-Aldrich . DNP-BSA was purchased from LSL Japan Inc. . Primary and secondary antibodies were purchased from Cell Signaling Technology Inc. .P. morrowii harvested from the coast of Jeju Island, according to the method described by Ko et al. (2019) [P. morrowii dry powder was suspended in water and fractionated into chloroform. The chloroform fraction was further separated on a silica open column via stepwise elution of chloroform and methanol mixtures of different ratios. The fractions with the highest bioactivity were pooled and further separated on a Sephadex LH-20 column with 100% methanol elution followed by reversed-phase (C18) HPLC purification . The identity of the purified compound was confirmed using NMR. The purified DHB was diluted using PBS (2000-fold) for biological experiments (DHB was purified from . (2019) . Brieflyeriments .C57BL/6 male mice (8-week-old), reared under specific pathogen-free conditions, were purchased from Orient Bio . BMCMCs were obtained from 8-week-old male C57BL/6 mice according to a previous method . The cel4/well) were treated with different concentrations of DHB (31.3 and 62.5 µg/mL) and incubated at 37 °C for 24 h. Subsequently, 15 μL of 5 mg/mL MTT solution was added, and the cells were further incubated for 4 h. The formazan crystals formed within the cells were dissolved using DMSO, and the absorbance was measured at 540 nm using a microplate reader .The effect of DHB treatment on cell viability was evaluated using an MTT assay ,10. Acco5/well) were first incubated for 1 h with different DHB concentrations and sensitized using monoclonal anti-dinitrophenyl (DNP)-IgE (clone SPE-7) (1 μg/mL) for 4 h [To assess the inhibitory effect of DHB on mast cell degranulation, BMCMCs  was carried out using total RNA isolated from BMCMCs using Trizol reagent . The cDN6/well) were incubated for 1 h with different DHB concentrations and then sensitized with anti-DNP-IgE (1 μg/mL) for 4 h. Next, the sensitized BMCMCs were incubated in DNP-BSA at 37 °C for 24 h with a CO2 humidified atmosphere. The cell suspension supernatants were analyzed using mouse cytokine ELISA kits  according to the manufacturer’s instructions.The BMCMCs (2 × 105/well) were incubated with DHB for 6 h at 37 °C. To measure the surface expression levels of IgE- FcεRI binding, the cells (4 × 105/well) were reacted with DHB for 2 h and sensitized using DNP-IgE (1 μg/mL) for 4 h at 37 °C. The prepared cells were blocked using anti-CD16/CD32 monoclonal antibodies  for 20 min to prevent nonspecific binding of the antibodies. To detect the expression of FcεRI, the cells were incubated with FITC-conjugated anti-FcεRI antibodies ; to detect the expression of IgE-bound FcεRI, the cells were incubated with FITC-conjugated anti-mouse IgE monoclonal antibodies . The fluorescently stained cells were measured using flow cytometry . The total BMCMCs were gated on a forward scatter (FSC-A)/side scatter (SSC-A) plot to omit outliers.To prepare the cells for the measurement of the surface expression levels of FcεRI, the BMCMCs (4 × 105/well) were incubated with DHB for 1 h before anti-DNP-IgE (1 μg/mL) sensitization, which was carried out for 4 h. Indomethacin (I.M) was used as a positive control. Then, the cells were treated with DNP-BSA (100 ng/mL) for 30 min. Cytosolic and nuclear proteins were separately obtained from the cells using a NE-PER® nuclear and cytoplasmic extraction kit . The protein concentrations of the cell lysates were measured using a BCA protein assay kit  according to the manufacturer’s instructions. IκBα, phospho (p)- IκBα, p65, and p-p65  and β-actin  were used as the primary antibodies, and the secondary antibodies used were HRP-conjugated anti-mouse IgG and anti-rabbit IgG . The densitometry analysis of the Western blot results was performed using the ImageJ software to quantify the protein levels relative to corresponding housekeeping proteins [The BMCMCs 2 × 105/well wehttp://www.pdb.org, accessed on 5 July 2017). The ligand (DHB) structure was optimized to a stable geometry via Gaussian semiempirical PM6 calculations using Gaussian 09 software. Ligand charge and torsional bonds were analyzed, and hydrogen atoms were added to both proteins and ligand before docking simulations. Flexible ligand docking and docking refinements were carried out using the CDOCKER plugin (default settings) in Accelrys Discovery Studio 3.5 , which utilizes a molecular dynamics simulated-annealing-based algorithm. Potential binding sites for DHB were identified in the known binding pockets of the proteins.The binding energies and types of intermolecular interactions between DHB with the active sites of IgE  and the IgE-FcεRI complex (PDB ID: 1F6A) were analyzed via molecular docking. The crystal structures of the respective proteins were obtained from the Protein Data Bank , reared under specific pathogen-free conditions, were purchased from Orient Bio . The in vivo effects of DHB were analyzed in a PCA mouse model induced using DNP-IgE together with DNP-BSA. First, anti-DNP-IgE (500 ng) was intradermally injected into the dorsal skin of both ears. Then, using a 1 mL syringe, DHB was topical applicated to the mice 2 h before the induction of anaphylaxis. Subsequently, the mice were intravenously treated with 30 μL of DNP-BSA (10 mg) saline solution containing 4% Evans blue dye. The control group was treated with saline instead of DHB. After 30 min, the mice were euthanized by anesthetizing them with isoflurane followed by cervical dislocation, and skin tissues collected from the dorsal ear were soaked in 1 mL of formamide and left overnight (24 h) at 64 °C. The absorbance intensity at 620 nm was taken as a measure of the amount of extravagated dye. Histological analysis was conducted according to the method described previously . The coup values < 0.05 were considered significant.The data were compared one-way ANOVA and Duncan’s multiple range test using the SPSS Statistics V20 software package . The values are presented as the means ± standard error (SE), and P. morrowii may be used as an agent for functional foods and pharmaceuticals to suppress allergic reactions.In conclusion, the present findings demonstrated that DHB attenuated the antiallergic response through the inhibition of the mast cell degranulation, FcεRI expression, the crosslinking of IgE to FcεRI, and allergic cytokine secretion via the NF-κB activation in IgE/BSA-stimulated BMCMCs, and suppressed the allergic reactions in IgE/BSA-stimulation induced PCA model. DHB isolated from


Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization. Atypical chemokine receptors (ACKRs) are part of the superfamily of seven transmembrane receptors known as G-protein-coupled receptors (GPCRs). These receptors have been coined atypical, since, unlike classical GPCRs, they do not signal via G proteins upon activation ,2. ACKRsThe atypical chemokine receptor 3  is a GPCR sharing endogenous ligands with chemokine receptors CXCR3 CXCL11)  and CXCR  and CXCIn this study, we show that upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles, via G-protein-coupled receptor kinase (GRK) 2 and 3. ACKR3 internalizes and thereafter, recycles back to the cell surface. We demonstrate that β-arrestin recruitment to the receptor is potentially determined by a single phosphorylation-cluster on the C-tail, and that single residues serve as key phosphorylation sites for β-arrestin1 recruitment. Although phosphorylation of the C-tail appears essential for ligand-induced internalization and important for β-arrestin recruitment, agonist-induced internalization of ACKR3 can also occur in the absence of β-arrestins.CXCL12 was obtained from Almac. CMPD101 was obtained from Tocris Bioscience. Hanks’ balanced salt solution, Dulbecco’s Modified Eagle medium and penicillin/streptomycin were obtained from Gibco. Fetal bovine serum was obtained from Bodinco . Coelenterazine-h was obtained from Promega .2 in Dulbecco’s Modified Eagle medium  supplemented with 10% fetal bovine serum (Bodinco), 50 IU/mL penicillin, and 50 mg/mL streptomycin (Gibco).Human Embryonic Kidney (HEK)293T cells (ATCC) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) HEK293 β-arrestin1/β-arrestin2 KO cells ,33 were 3 plasmid already containing the RLuc8 gene with an in frame NotI restriction site at the 5′ end. SNAP-tag ACKR3 mutant constructs were generated from a pcDNA3.1 SNAP-tag ACKR3 plasmid kindly provided by Thierry Durroux  by cloning the promoter, the SNAP-tag and the N-terminus of ACKR3 into the pcDEF3 HA-tag ACKR3 construct using internal MluI and BstXI restriction sites. Following, the mutant C-tails were introduced using internal BstXI and NotI restriction sites from the HA-tag constructs.N-terminus HA-tag and alanine substitution of all serine and threonine residues in the C-tail were introduced by DNA synthesis followed by PCR-based site-directed mutagenesis. The PCR fragments were cloned using internal BamHI and NotI restriction sites into a pcDEF3-hACKR3-RLuc [3-hACKR3mut-RLuc and 1.6 µg of the completing BRET pair  plas3.1-SNAP-hACKR3-RLuc combined with 198 ng pcDEF3 plasmid) was diluted in optiMEM (ThermoFisher) to have a total volume of 25 µL. To this, 0.5 µL lipofectamine2000 mixed with 24.5 µL optiMEM was added. The transfection mixture was then incubated for 15 min at room temperature. Fifty µL of this mix was added to each well and 100 µL of medium containing 25,000 HEK293T cells was added on top of the transfection mixture. Two days after transfection, plates were washed twice with 100 µL TagLite® buffer  and incubated with 75 µL 100 nM SNAP-Lumi4-Tb (Cisbio) at 16 °C to block constitutive internalization for at least 1 h 30. Plates were then washed four times with 100 µL TagLite®. Ninety µL 25 µM fluorescein (Cisbio) (diluted in TagLite®) was added per well and 10 µL buffer or CXCL12 solution (ten times concentrated) were added per well. Internalization was followed as a ratio of 620/520 nm while exciting Lumi4-Tb at 340 nm and measured on the Infinite F500 plate reader . 620/520 ratios were normalized to values between 0 and 1. Data was analyzed using Prism 7 (GraphPad).HEK293T cells were transfected in the 96-well plate  using lipofectamine2000  agent according to manufacturer’s protocol. Ninety-six-well plates were coated with poly-ornithine (Sigma). For each well, a total of 200 ng of DNA (2 ng pcDNAv/v) NP40, 0.5% Na-deoxycholate supplemented with 1 mM NaF, 1 mM NaVO4 and cOmplete protease inhibitor cocktail , sonicated and centrifuged to remove insoluble cell debris. Protein content of the lysates was determined using a bicinchoninic acid assay (BCA) protein estimation assay according to manufacturer’s instructions . Western blot was performed according to standard procedure and blotted against: β-arrestin1/2 , β-actin . The western blot images were acquired by the use of a ChemiDoc imager  and quantified by densitometry using Image Lab software (Bio-Rad).Western blots were performed as previously published . Briefly50 values are also similar for the two isoforms (8.1 vs. 8.2)  on the C-terminal tail (ACKR3-RLuc) as well as β-arrestin1 tagged with the eYFP fluorescent protein (βarrestin1-eYFP), or β-arrestin2 tagged with the mVenus fluorescent protein (βarrestin2-mVenus), creating a Bioluminescent Resonance Energy Transfer (BRET) pair sensor  in HEK29vs. 8.2) .50 values for recruitment of each of the GRKs are in a similar range as the pEC50 values for recruitment of the β-arrestins  of ACKR3, we generated a set of mutant receptors by mutating potential phosphorylation sites, Serine (S) and Threonine (T) residues, to an Alanine (A) residue. We evaluated these mutants in the BRET-based β-arrestin recruitment assay. A decrease in BRET signal may indicate a decrease in protein recruitment and/or a decrease in BRET efficiency (donor energy transfer to acceptor) , due to The S/A 355 mutant showed impaired β-arrestin1 BRET signal. Yet, the T/A 352 and S/A 355 mutant were still able to recruit β-arrestin2 upon binding of CXCL12, with only slightly reduced BRET signal compared to WT ACKR3 for T/A 352 d. The inSince phosphorylation of the C-tail of ACKR3 seems to be playing an important role in β-arrestin recruitment, we studied its effect on CXCL12-mediated ACKR3 internalization. To monitor the internalization of the WT and mutant receptors, we used the BRET-based Rab5a sensor a, and a To enable the use of this technology with ACKR3, we fused a SNAP tag onto the N-terminus of WT and mutant receptors, to allow labeling by a fluorophore (Lumi4-Tb) and transfected HEK293T with these constructs. Labeling was performed at 16 °C to prevent protein trafficking in the cells ensuring that Lumi4-Tb will only label receptors initially present at the cell membrane, allowing detection of internalization events when exposing the cells to 37 °C, by a decrease of TR-FRET between the Lumi4-Tb and the non-cell penetrating fluorophore in solution. Upon stimulation by CXCL12 , the int-arrestin1/2 recruitment still showed internalization, we hypothesized that the internalization process of ACKR3 is not fully dependent on β-arrestin  appears crucial for both β-arrestin1 and 2 recruitment, and much less for 335SAKTGLT341 (cluster 1) despite having some effect on β-arrestin1 interactions. There are clear differences of the contribution of phosphorylation sites within cluster 2, which could lead to differential recruitment of one β-arrestin isoform over another. It should be noted, however, that there is no evidence thus far of region-specific phosphorylation in different cell types upon agonist exposure. The ST/A 350–355 mutant serves as an ACKR3 β-arrestin1 and 2-deficient mutant, defective in β-arrestin1 and 2 recruitment, which may be of interest to dissect the importance of β-arrestin recruitment for ACKR3 function. Recently, the structure of β-arrestin2 in complex with the phosphopeptide of the ACKR3 C-tail 335SAKTGLT341 (cluster 1) has been resolved [350SETEYS355 cluster is responsible for the differential regulation of the β-arrestins, and the C-tail in its entirety regulating internalization of the receptor. It is also noted that β-arrestin2 has been shown to be more flexible than β-arrestin1 [Phosphorylation of residues within the resolved . Bindingrrestin1 ,66, whicIn summary, we provide evidence of β-arrestin1/2 and GRK2/3/5 interactions with ACKR3 upon activation by its cognate ligand CXCL12 . C-tail


Our hypothesis is that, contrary to the safety claims of the world’s largest commercial developer, Coldwell Banker Real Estate/Trammell Crow (CBRE/TCC), the authors’ screening assessment, especially its lab-certified, toxic-site, indoor-air tests, show violations of all three prominent government, cancer-safety benchmarks. If so, these facilities require additional testing/remediation, likely put site renters at risk, and may reveal problems with privatized hazardous cleanup. To our knowledge, we provide the first independent tests of privatized, toxic-site assessments before cancer reports occur. Our screening assessment of this hypothesis tests indoor air in rental units on a prominent former weapons-testing site  that is subject to carcinogenic vapor intrusion by volatile organic compounds, VOCs), then compares test results to the redeveloper’s site-safety claims, made to government officials and citizens through traditional and social media. Although NOTSPA toxic soil-gas concentrations are up to nearly a million times above allowed levels, and indoor air was never tested until now, both the regulator and the remediator (CBRE/TCC) have repeatedly claimed on social media that “the site is safe at this time.” We used mainly Method TO-17 and two-week sampling with passive, sorbent tubes to assess indoor-air VOCs. Our results show that VOC levels at every location sampled—all in occupied site-rental units—violate all three government-mandated safety benchmarks: environmental screening levels (ESLs), No Significant Risk Levels (NSRLs), and inhalation risks based on the Inhalation Unit Risk (IUR); some violations are two orders of magnitude above multiple safety benchmarks. These results support our hypothesis and suggest a need for independent assessment of privatized cleanups and media-enhanced safety claims about them. If our results can be replicated at other sites, then preventing health misinformation and toxic-facility safety threats may require new strategies, one of which we outline.Health misinformation can cause harm if regulators or private remediators falsely claim that a hazardous facility is safe. This misinformation especially threatens the health of children, minorities, and poor people, disproportionate numbers of whom live near toxic facilities. Yet, perhaps because of financial incentives, private remediators may use safety misinformation to justify reduced cleanup. Such incentives exist in nations like the United States, where most toxic-site testing/remediation is semi-privatized or voluntary, conducted by private parties, commercial redevelopers, who can increase profits by underestimating health harm, thus decreasing required testing/remediation. Our  In the United States and many other nations, most hazardous-facility testing and remediation is semi-privatized or “voluntary”; that is, private parties  conduct sampling and cleanup under varying degrees of government oversight ,2,3. In Of course, as with any health-affecting activity that is at least partly motivated by private profits, semi-privatized toxic-site testing/remediation is the subject of controversy. Privatization proponents claim their assessments/cleanups are cheaper, faster, and safe, but critics say such testing and remediation is risky, typically driven by purely private-profit motives, and frequently lacks adequate government oversight, e.g., .Who is right about toxic-site health and safety? Do private, commercial hazardous-site redevelopers sometimes conduct flawed assessments and cleanup, misrepresent site health and safety, then use traditional and social media to publicize their misinformation? This analysis begins to answer these questions and to address the data gap regarding the adequacy of semi-privatized toxic-site assessment, remediation, and its possible misrepresentation.Until human-health problems have appeared, to our knowledge, no one has conducted proactive, independent , sampling to test whether semi-privatized toxic-site testing/cleanup actually satisfies the health-and-safety claims that private interests sometimes make on its behalf. New Jersey and Massachusetts, for instance, have two of the best-established, semi-privatized assessment/remediation programs; yet their results suggest safety problems, despite the limited evaluations government gives them. Only about 2% of Massachusetts’ semi-privatized projects receives minimal, post-cleanup government evaluation. Although 10% of New Jersey toxic sites receives annual, post-cleanup audits, these typically involve neither sampling nor site visits, and none of these audits are truly independent. Yet, Massachusetts data show that an average of only 29% of “completed” privatized cleanups pass this evaluation and have no major safety problems; 71% of audited sites fails to pass this safety test, and these results presumably are applicable to other, non-audited, hazardous sites—that are never investigated .One obstacle to independent post-testing or cleanup sampling is how to fund it. An additional obstacle is how scientists can secure permission for it, given that toxic-site owners know they could face personal-injury, trespass, and endangerment lawsuits because of testing results. Later paragraphs show this is a problem we face in our toxic-site sampling.objective is to begin to determine whether or not interested parties misrepresent health harm (at hazardous facilities that they test/remediate/redevelop) when they use traditional and social media to claim that these sites are safe. To begin to remedy the data gap regarding the adequacy of semi-privatized testing/cleanup and the veracity of safety claims about it, we sample a toxic site, claimed to be safe by its private remediators. We provide a preliminary assessment   site is safe at this time. Contamination is below paved surfaces and confined” .To perform a preliminary test of this hypothesis  per day.This assessment likewise is preliminary in focusing only on residential safety benchmarks for the former-NOTSPA toxic site. For three reasons, we spend little time assessing NOTSPA with respect to commercial benchmarks. First, the California regulator, DTSC, says that it “recommends that a residential scenario be assumed for site screening at all [toxic] facilities, both active and closing/closed.” Part of its reasoning is “that reuse of hazardous-waste sites could result in a change of ownership and land use, including potential residential reuse of the property.”  Second, 3 above their current residential ESLs , ev, ev28], Thus, in summary, as the authors’ 3/day = 10−5 risk), the highest and lowest ranges of PCE average residential exposures, respectively, are (31.0–20.8 µg/m3) (20) or 620–416 µg/m3/day for highest exposures and (3.3–2.2 µg/m3) (20) or 66–44 µg/m3/day for lowest exposures. That is, 620 µg/m3/day and 44 µg/m3/day, respectively, at NOTSPA-sampling-locations B and H, represent the highest and lowest ends of the range of generally calibrated PCE exposures, respectively. This OEHHA PCE residential-exposure range corresponds to a risk range between 4.4 (10−4) µg/day and 3.1 (10−5) µg/day. Indeed, even the uncalibrated risks of the highest PCE samples are 19 times higher than this second or NSRL safety benchmark, and the corresponding generally calibrated samples are more than double that uncalibrated risk.As the authors’ 3/day = 10−5 risk) and the commercial (10-h/day) NSRL, the NOTSPA generally calibrated PCE exposures reveal a risk up to 22 times above the commercial NSRL.Likewise, as already mentioned , the Staaverage workplace exposures per day are 310 µg/m3 and 22.0 µg/m3, respectively, at NOTSPA-sampling-locations B and H. This commercial/workplace-exposure range corresponds to a risk between 2.2 (10−4) and 1.6 (10−5). Thus samples, at all locations tested, violated even the more lenient commercial NSREL safety benchmarkThat is, the highest to lowest PCE 3 and the more realistic or generally calibrated CT results of 1.1–1.6 µg/m3 violate both the commercial and the residential NSRL of 5 µg/m3 per day (=10−5 risk). The highest to lowest range of generally calibrated CT average residential exposures are (1.6–1.1 µg/m3) (20) or 32.0–22 µg/m3 per day. This CT residential-exposure range corresponds to a risk range between 6.4 (10−5) µg/day and 4.4 (10−5) µg/day. Even the risk from the lower or uncalibrated CT residential (13.6 µg/m3 per day) concentration at NOTSPA is roughly three times higher than the NSRL for CT. Similarly, as the author’s −5 risk, CT cancer-inhalation risks from NOTSPA are as high 67 times above the allowed level. The highest and lowest CT average inhalation risks, respectively, are (6.7) 10−5 and (4.6) 10−5, given Beacon’s general calibration. Finally, as −5] than the 10−6 NOTSPA safety benchmark. The inhalation cancer risk of corresponding generally calibrated PCE sample is roughly 190 times higher than this benchmark. Thus, as the authors’ For PCE, Of course, the preceding results and interpretations are based on two main assumptions. The first assumption is that the renter has lifetime exposure at the level indicated. The second main assumption is that it is reasonable to apply Beacon’s own general calibration interpretation, given the absence of site-specific calibration. However, because the sorbent-tube results are preliminary  reduction of VOC exposures in situations where TCE concentrations, for instance, are as low as 0.5 µg/m3  will not allow any development to occur on the site unless it’s cleaned up to the highest residential standards” . AlthougFirst, one problem is that site renters deserve to know what risks they face, to know whether they are being protected, as well as what protections government guidance and regulations require, and to be protected by government at those required levels. Yet, as previous paragraphs show, site renters have been deprived of both information and protection. Although the responsible parties  have known about high site risks since CBRE/TCC’s 2007 site testing, no one has conducted the required indoor-air, groundwater, or additional soil-gas testing. Another question in response to our results  is why CBRE/TCC should test and clean up the former NOTSPA, to protect current renters, when it says it will do so after it purchases the site? While this question appears reasonable, there are several difficulties. Second, in addition, site renters especially deserve this information and protection for another reason. This that, although both the regulators and the responsible parties have said that the NOTSPA is now safe, our sampling results suggests the opposite. In particular, current site renters may not be safe.Third, site renters deserve risk information and protection now because otherwise, they may never get it. CBRE/TCC has not yet purchased the site; it has been in escrow to buy the site for decades, and many prior contracts to purchase the site have failed, once prospective buyers discovered NOTSPA toxicity. Hence, the CBRE/TCC purchase may never be accomplished. If there is no purchase, then at a minimum, the site should made safe for current renters and future people. One cannot merely assume that protection will come in the future, including because, as already noted, CBRE/TCC will leave “in place” most site VOCs, at levels above those at which the state requires remediation.Fourth, site renters especially deserve immediate protection because they appear not to have been protected in the past. Instead, regulators and the responsible parties failed to act in response to the already-mentioned 2004 Imminent and Substantial Endangerment Order. Because our 2021 sampling appears at least partly to confirm the very risks that the 2004 Order was meant to investigate and mitigate, arguably the regulators and the responsible parties may have failed because since 2007, they have neither given adequate risk information to site renters nor tested site indoor air. As result, they likely failed to provide adequate protection to NOTSPA renters.−6 for cancer risk…. For any exceedance of the points of departure for risk or hazard, based on soil-gas data, proceed to Step 3 for an indoor air investigation at current buildings” …10ildings”  p. 17)..−6 for cOur results suggest that a trend-setting, major commercial redeveloper of toxic sites, CBRE/TCC, appears to have spread misinformation about both the current safety and the future risks associated with the dangerous, inadequately tested, former NOTSPA—most of whose contaminants CBRE/TCC will leave onsite. Yet as already mentioned, CBRE/TCC will redevelop the site for 550 small residential apartments, disproportionate numbers of which are reserved for affordable, low-income, and moderate-income families. Given these facts, future research might attempt to replicate our results. That is, scientists might attempt to discover the prevalence of flawed or incomplete toxic-site testing and cleanup, as well as the prevalence of assessors’/remediators’ spreading apparent misinformation about hazardous-facility risks, testing, cleanup, and the degree to which some toxic-site redevelopments put people at risk. If our results can be replicated, then as the recent Hunter’s Point, California, residential redevelopment of another prominent former US military site suggests ,54,55, aIf our results can be replicated, they also suggest that government leaders may need to re-assess the reliability and performance of toxic-site regulators, regulatory-oversight programs, and semi-privatized testing/assessment. After all, in 2019 California banned private prisons, saying they are “driven to maximize shareholder profits” and “lack proper oversight” . Howeverhypothesis is that, contrary to the safety claims of the world’s largest commercial developer, and a major redeveloper of hazardous-waste facilities (CBRE/TCC), the authors’ screening assessment, especially its lab-certified, toxic-site, indoor-air samples show violations of all three prominent government, cancer-safety benchmarks—at every NOTSPA location tested. The results of this hypothesis are important because these findings suggest that toxic facilities may put site renters at risk, require additional testing/remediation, and reveal both scientific, public-health, and environmental-justice problems with privatized hazardous cleanup.In the United States and several other nations of the world, most toxic-site testing/remediation/redevelopment is conducted by private parties, usually redevelopers seeking commercial profits. However, because few studies have analyzed the effectiveness of such semi-privatized hazardous-site assessment and remediation programs, this analysis conducted a preliminary assessment of an important hypothesis. The methods to sample, then analyze two weeks of continuous, indoor-air testing with passive sorbent tubes; these results provide a preliminary assessment . Moreover, although state regulatory-guidance documents require indoor-air testing whenever soil-gas-VOC levels indicate a lifetime risk higher than 10−6, neither CBRE/TCC nor the regulator ever tested site indoor air. As a result of the preceding facts, the misleading safety claims of CBRE/TCC, and our indoor-air sampling results, this study draws the preliminary conclusion , the authors’ lab-certified, toxic-site, indoor-air samples show violations of all three prominent government, cancer-safety benchmarks.On one hand, the prominent site assessor/remediator/redeveloper has repeatedly used traditional and social media to claim that the former NOTSPA toxic site is “safe at this time.” On the other hand, despite such safety claims, CBRE/TCC’s own studies show NOTSPA soil-gas-VOC levels pose a lifetime risk as high as 7.4 (10If these CBRE/TCC results can be replicated at other brownfields-redevelopment sites, they suggest that government may need to reconsider the public-health consequences of semi-privatized toxic-site testing/cleanup and the adequacy of government oversight of such facilities. Scientists and policymakers also may need to assess whether the financial incentives for reduced testing/cleanup, reduced public safety, and increased private profits may jeopardize the economic benefits of semi-privatized toxic-site assessment/remediation. The warning of social reformer and author Upton Sinclair seems appropriate to the CBRE/TCC situation. He said “It is difficult to get a man to understand something [such as the health threats caused by health misinformation about poor toxic-site testing/cleanup], when his salary depends on his not understanding it.” .


High-strength screws represent one of the main joining or fastening components which are commonly used in the process of installation of frame constructions for information boards or signposts, relating to the traffic roads. The control of the production process may not always be a sufficient method for ensuring road safety. The backward investigation and control of the screw material processing seems to be the one of the most important procedures when there is the occurrence of any failure during the operation of the screw. This paper is mainly focused on the analysis of the failure of the high-strength screw of 10.9 grade with M diameter of 27 × 3 and a shank length of 64 mm. The mentioned and investigated screw was used as a fastener in a highway frame construction. In the paper, there is mainly the analysis of the material for a broken screw in terms of the material micropurity, the material microstructure, the surface treatment as well as chemical composition. The evaluation was based on investigation by optical microscopy, scanning electron microscopy and energy dispersive spectroscopy. Important knowledge and results were also obtained due to information on micromorphology and material contrast of the fracture surface resulting from fractographic analysis, using the method of scanning electron microscopy. In the case of the production of the high-strength screws, the tempering stands for the decisive or crucial process of heat treatment because the given process can ensure a decrease in hardness, while the required ductile properties of the material are kept and this is also reflected in the increase of strength and micromorphology of the fracture surface. From the aspect of micropurity, inclusions of critical size or distribution were not identified in the material, referring to Czech standard ČSN ISO 4967 (420471). The microstructure corresponds to tempered martensite, but the fracture surface of the broken screw was based on an intercrystalline micromechanism, which is undesirable for the given type of component. Combined with the measurement of the HV1 (Vickers hardness at a load of 1 kg) from the edge to the central area of the screw, the analysis revealed the significant drawbacks in the heat treatment of the high-strength screw. Highway steel frame constructions have the function of a supporting structural part for information and signaling boards or signposts in relation to the traffic roads. Particular attention is paid to the keeping of all relevant or appropriate requirements, from installation to maintenance ,2. In reMaterials for production of screws undergo various production technologies to improve their properties but the inadequate selection of material as well as inappropriate manufacturing procedure can lead to the damage or rupture as well as subsequent fracture or failure of the material . This tyHigh-strength steels are particularly susceptible to brittleness of the material, while the given embrittlement is seriously dangerous. Depending on the chemical composition of the steel and the tempering temperatures or the course of the thermal process, there can be the occurrence of the changes in the microstructure with a negative effect if the material reaches the region of the tempering embrittlement ,2. BasedProper tempering temperature has a significant effect on the tempering embrittlement. The given tempering temperature represents a decisive or crucial factor in the extent of structural changes and changes related to the mechanical properties of stainless steels. The values of notch and fracture toughness are decreased in the steel with the occurrence of tempering embrittlement and, moreover, there is a higher susceptibility to corrosion cracking or hydrogen embrittlement ,15,16. NDuring slow cooling from the tempering temperature, embrittlement can occur due to the development of the so-called anisothermal constituent part. The susceptibility of steels to tempering embrittlement is based on the chemical composition of steels, especially on the content of the surface-active elements, such as P, S, Sn, As, Sb and others ,20. Low High-temperature tempering embrittlement and the maximum decrease in toughness occur at a relatively high tempering temperature, which is around 500 °C. The high-temperature tempering can be carried out under the isothermal or anisothermal conditions of the heat treatment ,23. The The main objective of this paper is to obtain knowledge from individual applied analyses and to summarize the results in order to gain the information on the reasons of failure of a high-strength screw which was mounted in a highway frame construction. The resulting information can help to identify the individual procedures in manufacturing process because inappropriate manufacturing procedures can lead to defects in the microstructure.Relating to the mechanical joints of steel constructions, the fasteners, including screws, have to fulfill the requirements which are prescribed in the standards. The material of the screws and screw joints has to meet the requirements of STN EN 1993-1-1 and STN EN 1993-1-8 and STN EN 1090-2 + A1. Non-prestressed screw joints, which are made of carbon steels, alloyed steels and austenitic stainless steels have to meet the requirements according to STN EN 15048-1. High-strength prestressed screw joints have to meet the requirements according to STN EN 14399-1 .The investigated broken M 27 × 3 screw with a shank length of 64 mm  was usedTo be able to evaluate the micropurity, the microstructure and the surface treatment of the broken screw, a sample from the longitudinal section was taken from the area close to the fracture surface. The gaining of the given information was connected with the preparation of the samples in a metallographic way. Analysis of micropurity, which was performed by optical microscopy, showed that high-purity steel with local occurrence of subcritical oxides was used for the production of the high-strength screw of 10.9 strength grade. Oxide inclusions  chrome steel of 10.9 grade with M 27 × 3 dimensions and shank length of 64 mm. Using the method of optical microscopy in combination with the standard series based on standard ČSN ISO 4967 (420471), the analysis of the material micropurity showed that the screw was made of high-purity steel. After etching, oxide inclusions on the basis of Al, Si as well as complex sulfoxides were detected. The given inclusions were subcritical in size but they were distributed close to grain boundaries and it can be dangerous from the point of view of cohesion properties in relation to inappropriate thermal treatment.According to the technical standard mentioned above, there was the large number of manganese sulfides up to grade 1 in the central area and the given sulfides were distributed in parallel lines. In relation to the observation of the microstructure, the heat treatment of material was based on quenching and a subsequent hardening process. In addition, the hardness value of 695 HV1 was reached in the central area. The micromorphology and hardness correspond to the structure of the lower bainite. Using the methods of scanning electron microscopy and energy-dispersive spectroscopy, the surface Zn layer was identified on the surface of the screw and it represented the anticorrosion protection. On the other side, from the results of the performed investigations, it is important to point out that the given protective layer exhibited a large number of defective areas. According to the thickness of the Zn surface layer, it is possible to assume that it was formed in an electrochemical way by the electroplating process. In the polished state, microcracks were visible in the steel and the propagation of these microcracks was observed from the surface of the threaded part. In addition, the microspace of the microcracks was filled with corrosive products and Zn. In relation to the mentioned microcracks, it is possible to assume that the initiation and subsequent propagation results from the screw production process before the final surface treatment.The presence of the surface microcracks can have a negative effect on the cohesive strength of the steel metal matrix. From the measured values and the obtained results as well as knowledge, it can be concluded that the main reason for the screw failure was based on the inappropriate heat treatment of steel. In relation to the application of the screw, the required strength of this type of screw can be reached by quenching and tempering up to the upper bainite structure. The mentioned bainitic structure can only be reached by higher tempering temperatures and it results in the required fracture and notch toughness of the material. The chrome steel used to produce the high-strength screw also corresponds to this mentioned process of heat treatment. In addition, the fracture surface of the high-tempered martensite exhibits noticeable pit micromorphology, which stands for high-energy rupture of the material. However, the microstructure of the investigated broken screw was formed by lower bainite and this type of structure represents the increase in hardness values, but it also represents the decrease in toughness values of the material. During the tempering process, the tempering temperature was selected inaccurately and, moreover, the processed material was in the region of low-temperature tempering embrittlement during the given heat treatment. The introduced type of the material embrittlement commonly occurs at temperatures from 350 to 450 °C. Apparently, when a longer tempering time was used, the carbides were dissolved in the bainitic ferrite and subsequently, they were precipitated towards the boundaries of the original austenitic grains. The surface-active elements in the steel can be segregated towards the grain boundaries and it leads to the significant reduction of the cohesive properties in these areas. The occurrence and size of the carbide phase in the bainitic ferrite plates reached higher values and it could cause the increase in the hardness as well as brittleness of this microstructure.These facts are also reflected in the character of the fracture surface which was based on low-energy intercrystalline rupture with pit micromorphology in the areas of revealed grain boundaries. An interesting finding is also connected with the results of the comparative method which was based on the comparison of the fracture surfaces of the broken screw under shear operational loading and tensile loading. The given comparative method gives information on the weakness of grain boundaries. The occurrence of weakness was not observed in the whole volume of the screw but it was only in the area of the operational fracture. The rest of the screw volume exhibited the transcrystalline high-energy fracture with the significant pit micromorphology. This character of the fracture surface corresponds to the structure of the lower bainite with higher values of surface hardness. Moreover, the course of HV1 hardness measurements from the edge to the central area indicates an impropriate tempering of screw in overall cross-section because the hardness, which was measured at the edge, was 891 HV1. The given measured hardness value corresponds to the martensitic structure. It can be assumed that there was no optimal loosening over the entire cross-section of the screw. Based on the mentioned facts, it can be concluded that a neither optimum nor effective tempering process was used.Metallographic and fractographic analysis of broken high-strength screws can contribute to improvement of the measures for the production process and it can lead to the increase in the safety of the surroundings in which the given components are mounted. High-strength screws are used in the assembly of detachable joints of steel structures, which are subject to high strength requirements. These components are used, for example, in construction and steel structures in the transport industry. In the case of a similar fracture of the screw, it would be possible to proceed in the manner as shown in this article. Important information provides knowledge about the microstructure and micromorphology of the fracture surface. It is appropriate if microscopic methods are supplemented by information on the basic mechanical properties of the material. Of course, it is always necessary to look for the cause of the fracture, for whether the structure was not loaded more than the permissible load limit, and for whether the prescribed conditions for tightening the torsions were not violated, etc.