Human remains have been interred in burial grounds since historic times. Although the re-use of graveyards differs from one country, region or time period to another, over time, graveyard soil may become contaminated or enriched with heavy metal elements. This paper presents heavy metal element soil analysis from two UK church graveyard study sites with contrasting necrosols, but similar burial densities and known burial ages dating back to the sixteenth century and some possibly older than 1,000 years. Portable X-ray fluorescence element laboratory-based analyses were undertaken on surface and near-surface soil pellets. Results show elevated levels of Fe, Pb, Mn, Cr, Cu, Zn and Ca in both necrosols when compared with background values. Element concentration anomalies remained consistently higher than background samples down to 2 m, but reduced with distance away from church buildings. Element concentration anomalies are higher in the clay-rich necrosol than in sandy necrosol. Study result implications suggest that long-used necrosols are likely to be more contaminated with heavy metal elements than similar soil outside graveyards with implications for burial grounds management, adjacent populations and where burial grounds have been deconsecrated and turned to residential dwellings.The online version contains supplementary material available at 10.1007/s11356-022-19676-z. Burial grounds are unique both in their natural environment, including soil type, parent material, vegetation, topography and climate, and in their anthropogenic burial numbers, styles, depths, body distributions and above-ground placement of memorials, buildings and installation of pathways and roads to access the site  is an analytical technique for determining total element concentrations in a wide variety of materials and is used in environmental applications. Traditional, laboratory-based, spectrometry including inductively coupled plasma mass spectrometry, atomic emission spectroscopy and optical emission spectrometry, among others, are widely used to determine trace and heavy metal elemental concentrations in soils  field surveys have been shown to be effective for rapid evaluation of heavy metal soil contamination  church necrosol graveyards with different soil textures. Study objectives were therefore to (1) use pressed powder soil pellets from topsoils from 2 contrasting soils from long-established church graveyards and analyse for heavy metals using pXRF, (2) assess the respective known burial population and areal extent to quantify the respective graveyard burial histories and (3) identify potential soil and water quality implications for those living adjacent to burial grounds or living on ex-burial grounds.Two Church of England graveyards were selected for this study, study site 1 at St. John’s Church in Keele Staffordshire, UK Fig. , and stuStudy site 1 is located in the rural Keele Village in Staffordshire, situated ~ 200 m above sea level and with an average rainfall of 806 mm/year. The site was a Knights Templar church built around 1160 CE, before being taken over by the Knights Hospitaller in 1324 CE. A new church was built by the local Sneyd family in the sixteenth century, before the present sandstone church was built in 1868–1870 CE Pevsner . A desktStudy site 2 is located in the rural Stockton village in south Norfolk, situated ~ 35 m above sea level, with an average rainfall of 620 mm/year. The church of St. Michael and All Angels is present on the study site, with a Saxo-Norman-style round tower, probably dating from the thirteenth to the fourteenth century, and with later medieval flintwork additions Knott . A desktFor site 1 at St. Johns, graveyard burial records showed there were 5,735 individuals from 1585 to 1970 CE and 1990 to 2018 CE see Fig. . The burFor site 2 at St. Michaels, graveyard burial records there were 669 individuals from 1561–2018 CE see Fig. . Average2 for study site 1 at St. John’s and 2,330 m2 for study site 2 at St. Michaels), burial densities of ~ 2.2 individuals/m2 at study site 1 and ~ 3 individuals/m2 at study site 2 were determined.When burial numbers were corrected for the areal extent of the graveyards  soil cores were taken using a handheld slimline hand auger at 32 and 31 sampling locations at study site 1 and study site 2 graveyards respectively. Cores were split into 0.25-m intervals   Fig . The resA grave digger was hand-digging a new grave in study site 1 whilst soil sampling was being conducted, so opportunistically the research team were able to collect deeper subsoil samples. Here, ~ 250 g of soil samples was collected every 0.25-m down to 2-m depth on the south end of the empty grave Fig . The resThe samples had routine soil characterisation analysis, including determination of electrical conductivity, pH and soil moisture content following standard methodologies. Study site 1 electrical conductivity (EC) of soils recorded an average of 47 μS/cm (28 SD), with study site 2 recording an average EC of 99 μS/cm (49 SD). Study site 1 pH of soils displayed an average of 6.3 (0.8 SD), with study site 2 recording an average of 7.8 (0.8 SD). Study site 1 soil moisture content ranged from 8 to 27% (average 14.1%), with study site 2 soil moisture content ranging from 13 to 27% (average 21%).Our results show clear differences in element concentrations in the soil samples measured, when compared to the background control samples, within each study site graveyard and between the two graveyards summarised in Tables At study site 1 with the sand-rich soil, surface soil pXRF measurements showed Pb element concentrations varying from 69 mg/kg up to a maximum of 742 mg/kg with an average of 188 mg/kg Table . ControlAt study site 2 with the clay-rich soil, surface soil pXRF measurements showed very high Pb concentrations, varying from 38 mg/kg up to a maximum of 2,310 mg/kg with an average of 317 mg/kg Table . ControlComparing element concentrations between graveyards, Pb concentrations were higher in the clay soil of study site 2 compared to the sandy soil of study site 1 which has been reported elsewhere  church necrosol graveyards with different soil types. The graveyard datasets acquired from surface soil (0–0.05 m), shallow soil depths (> 0.75 m) and single deep soil (> 2 m) (at study site 1 only) all showed heavy metal concentrations were not only higher than background control soil samples taken ~ 100 m away from both graveyards Tables . MeasureThere were also large variations of measured heavy metal element concentrations within each graveyard, with the highest concentrations being found generally in soil samples taken adjacent to church buildings. This would suggest building materials would be a major source of heavy metals found in the adjacent soils such as Pb flashings, particularly as element concentrations decreased with increasing spatial distance from the churches. Other elements also showed this trend such as Ca, which may suggest a higher burial density nearest the church and corresponding release of elements from coffins as Fiedler et al.  measured2), so element concentration differences may be due to the different soil type, with clay-rich soils having greater reactive surface areas which can bind metals and other mineral and organic substances in the environment  burial grounds, UK church graveyards in this case, whose necrosols are contaminated by heavy metals. Here we used pXRF to determine the spatial extent (distance and depth) of heavy metal contaminations, and highlight its use for rapid data collection across different environmental samples. In particular, we identified Pb concentrations were well in excess of current environmental guidelines, although these concentrations are not uniformly distributed, both in extent across the graveyards and in depth below ground level. The highest levels of contamination are in the top 0.25 m and adjacent to church structures, potentially due to high burial concentrations and/or due to relict church materials being incorporated into the soils. This will be important for burial ground management, those living adjacent to burial grounds, potential surface/groundwater contamination and where burial grounds have been deconsecrated and turned into residential dwellings.This paper is limited by only studying two UK graveyards, albeit long-used with different soil types, and by the numbers of soil samples collected, analysed and measured. However, the implications for other church graveyards to be similarly contaminated is clear. More accurate analytical equipment should be used to refine these initial results and obtain absolute element measurements to further validate the use of pXRF in determining heavy metal contamination as this could help speed up and reduce costs for soil testing in potentially contaminated sites. Here, we highlight graveyards are potential repositories for heavy metals but could also be possible stores for other emerging environmental contaminations too, such as pharmaceuticals which may persist in graveyard soils after decomposition. Further research is needed to explore other graveyards, cemeteries, green burials and other burial grounds with different burial ages, in other soil types, as well as collecting soil within and adjacent to graves in order to assess environmental contamination risks as well as future environmental sustainability.Supplementary file1 (DOCX 556 KB)Below is the link to the electronic supplementary material.


Cleavage of the severe respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) spike protein has been demonstrated to contribute to viral‐cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure‐based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline‐directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O‐linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS‐CoV‐2 spike protein cleavage sites. The S1 subunit contains the N‐terminal and C‐terminal receptor‐binding domains, which bind to host cell receptors to initiate viral entry. After receptor‐binding, host proteases can cleave the spike protein at various motifs, which subsequently exposes an internal hydrophobic fusion peptide in the S2 subunit that binds and fuses the host and viral membranes.Unlike other known coronaviruses, the SARS‐CoV‐2 spike protein contains an insertion of an RRAR furin cleavage motif at the S1/S2 site, which has, indeed, been proven experimentally to confer cleavage by furin.Recently, the SARS‐CoV‐2 Omicron strain has been found to have little ability to undergo cell surface membrane fusion and, as a result, primarily invades the host cell through endocytosis.22.1The amino acid sequences of the spike protein from the VOCs  and VOIs  were selected to represent the most impactful mutations at S1/S2 and S2' sites  among the three predicted sites  training data.Zhang et al.2.2.2Phosphorylation has been reported to occur at the SARS‐CoV‐2 spike protein S1/S2 site, which has also been shown to inhibit binding to proteases.As shown in Table Checking the expression levels of PKA, PKC, Cdk1, and Cdk5 revealed that PKA and Cdk5 are found at higher levels than PKC and Cdk1, respectively, throughout the respiratory tract, although all kinases were found to be expressed in the respiratory epithelium , Delta (P681R), and Omicron  sequences illustrate the variety of mutations at the S1/S2 site in all VOC/VOIs, they were selected for comparative structural analyses. Furin and TMPRSS2 were selected as host proteases for the protein−protein docking on account of their known involvement and available experimentally‐derived structures. As a result of the high flexibility of the S1/S2 site, no study utilizing cryo‐EM or X‐ray crystallography has been able to define a stable structure for this site. Thus, we used the full‐length spike model made by Woo et al.2.3.1Ensemble docking has been found to be useful for understanding protein−protein interactions in which one or more of the protein interfaces is flexible or found in different conformations.p = 1.27 × 10−8), Delta and Alpha (p = 2.53 × 10−3), Omicron and Wild‐type (p = 1.62 × 10−47), and Omicron and Alpha (p = 5.98 × 10−4)  but not between the Omicron and Wild‐type (p = 0.094) (Table p = 6.89 × 10−7)  Figure . A signi4) Table ,H. In co4) Table . Interes) Figure ,I. TheseNo significant differences were found between the predicted scores and number of residue 681‐interacting residues for ensemble docking of the Wild‐type, Alpha, Delta, and Omicron S1/S2 sites to TMPRSS2 , Alpha (S683), Delta , and Omicron (S683) spike structures Figure  and redo3Cleavage of the SARS‐CoV‐2 spike protein is an important virulence factor for viral cell entry and syncytia formation. While mutations in the Delta and Alpha strains have been shown to increase fusion capacity, the Omicron strain has been particularly shown to have lessened capacity for cleavage that allows for cell surface virus−host membrane fusion.All mutations were found to potentially increase affinity between the S1/S2 site and furin, whereas the mutations were not found to increase affinity to TMPRSS2 at the S1/S2 site. The P681R mutation in the Delta strain, in particular, was predicted to significantly contribute to the increased interactions with furin. The N676K mutation in the Omicron spike protein may increase the propensity for O‐linked glycosylation at the S1/S2 site. Although the P681H and N676K mutations in the Omicron strain were predicted to increase affinity to furin—indicating that more efficient cleavage may occur in comparison to the Wild‐type—structural analyses suggest that such glycosylation may abrogate recognition by host proteases at the S1/S2 site. Since cleavage of the spike S1/S2 site has been suggested to be necessary for subsequent exposure and cleavage of the S2' site at the cell surface, the predicted glycosylation at S683 in the Omicron strain may explain the exhibited decreased viral‐host plasma membrane fusion and syncytia formation. However, potential glycosylation at the Omicron spike S683 may depend on the expression of appropriate GALNTs; thus, the effect of glycosylation on cleavage capacity could change depending on the infected tissues . All mutations at the P681 residue were found to remove phosphorylation site at the S1/S2 site, which may prevent phosphorylation‐based inhibition of binding to proteases and, thus, increase fusion capacity for these variants. Experimental work is needed to confirm these results.Mutations in regions outside of the S1/S2 and S2' sites, such as those near the fusion peptide, may also have impacts on fusion capacity and kinetics. Additionally, mutations near other protease sites, such as those found for cathepsins, may reveal differences in cell entry.44.1Amino acid sequences of the Wild‐type (NCBI Accession: NC_045512), Alpha (MZ344997), Beta (MW598419), Gamma (MZ169911), Delta (MZ359841), Omicron (OL672836), Mu (OK005482), and Lambda (MW850639) strain spike proteins were obtained from the NCBI Viral Genomes Resource.4.2Although several glycosylation prediction tools were tested, such as SPRINT‐Gly and GPP, the known experimental results are shown to be most aligned with NetNGlyc 1.0 and NetOGlyc 4.0 for predicting N‐ and O‐linked glycosylation, respectively, and, thus, they were used in the analysis.4.3Proteomics, RNA‐seq, and microarray data for tissue‐specific expression data from healthy human cells were obtained from the Human Protein Atlas and EMBL‐EBI Expression Atlas.4.4The Alpha, Delta, and Omicron spike homology models were generated using MODELLER and were subsequently refined using a molecular dynamics with simulated annealing protocol.4.5Since the spike protein S1/S2 site is flexible and may be found at different conformational states before protease binding, we generated multiple conformations of the S1/S2 site for the Wild‐type, Alpha, Delta, and Omicron strains and docked them to furin (PDB: 6hzd) and TMPRSS2 (7meq). To generate representative conformers, the S1/S2 site of each spike protein was extracted (Wild‐type spike residues: C671‐Y695) from the structures and were submitted to the CABSFLEX 2.0 web sever, which utilizes molecular dynamics to measure flexibility and output structures at different conformations.4.6t‐tests were used to calculate statistical differences between groups . All data and structural models are available at the tlb‐lab github (https://github.com/tlb-lab).Statistical analyses and plots were generated using R version 3.6.3. Mean, standard deviation, and Student's Christopher A. Beaudoin, Arun P. Pandurangan, and So Yeon Kim performed sequence‐ and structure‐based analyses, summarized data, and wrote the paper. Samir W. Hamaia, Christopher L.‐H. Huang, Tom L. Blundell, Sundeep Chaitanya Vedithi, and Antony P. Jackson advised on methodologies, interpreted data, and wrote the paper.The authors declare no conflict of interest.Supporting information.Click here for additional data file.


Inhibition activity was induced when polymers at nearly fixed loci formed steady hydrogen bonding with the ice surface. However, the formation-and-dissociation dynamics of the interfacial hydrogen bonds, originating from and maintained by migrating polymers, resulted in an enhanced quasi-liquid layer that facilitated ice growth. Such ice growth activity is a unique property unseen in natural antifreeze proteins or their mimetic materials.In this study, a new phenomenon describing the Janus effect on ice growth by hyperbranched polyglycerols, which can align the surrounding water molecules, has been identified. Even with an identical polyglycerol, we not only induced to inhibit ice growth and recrystallization, but also to promote the growth rate of ice that is more than twice that of pure water. By investigating the polymer architecture and population, we found that the stark difference in the generation of quasi-structured H Regulation of ice growth and ice growth inhibition are critical and different materials mimicking anti freeze proteins or ice binding surfaces have been developed. Here, the authors combine both concepts in a hyperbranched polyglycerols to change the ice growth activity. Nature has evolved to produce antifreeze proteins (AFPs)2 to survive this freezing threat. Their specific amino acid sequence has been widely accepted to play a critical role in binding to ice9, which can result in antifreeze activity when the Kelvin effect is dominant at the ice interface. To date, various studies mimicking the unique antifreeze activity of AFPs have been reported by using glycoproteins10, carbohydrates12, polymers18, supramolecules19, carbon materials22, and gold nanoparticles23. On the contrary, ice-binding surfaces can also lead to heterogeneous ice nucleation when the appropriate chemical and dimensional aspects are satisfied. Ice nucleation proteins and their mimics possess a large ice-binding surface, which facilitates the organization of surrounding water molecules in an ice-like lattice that could promote ice nucleation30. Cryosurgery utilizing such promoted ice growth within cells has been suggested32. The effects of surface size, shape, and material type on antifreeze or ice nucleation were investigated through computational analyses36. Both phenomena, which require ice-binding characteristics in common, demand distinct design protocols, and thus active materials have been developed by tailoring them for respective purposes. In this work, we report a single type of hyperbranched polymeric agent that exhibits both promotion and inhibition activities. The polymer’s formation–dissociation dynamics of hydrogen bonds (H-bonds) with the ice interface regulated ice growth in opposite tendencies depending on its population.Water freezing is a commonly observed natural phenomenon; however, ice growth and recrystallization can critically damage living organismshbPG) to realize the Janus effect on ice growth and recrystallization. Owing to its unique three-dimensional architecture, which comprises a polyether backbone with several functional hydroxyl groups, along with its excellent biocompatibility and immunogenicity37, hbPG has gained significant attention in biological and biomedical applications39. Moreover, their facile synthetic nature and access to various architectures ) have prompted us to investigate the interaction of PGs with the ice surface to form dynamic H-bonds. Accordingly, we synthesized hbPG and linear polyglycerol (linPG) to study their effects on ice growth and recrystallization with respect to their architecture and concentration  and hbPG  for the one-directional freezing experiments. The ice/water interface in pure water , and 1.22 ± 0.06 μm/s at 1 mM (more than two times slower than pure water). Interestingly, the rate at 0.5 mM resembled those of more dilute solutions under 10 nM. In contrast, linPG exhibited a rather monotonic decrease in the ice-growth rate over the entire range of concentrations. PVA, a well-known polymer that exhibits some ice recrystallization inhibition (IRI) activity, exhibited a decreasing trend resembling that of linPG  of 1 mM hbPG was 81.5 ± 6.8  μm, indicating its ability to inhibit ice recrystallization. When its concentration decreased to 1 μM, the MLGS increased to 160.2 ± 12.1 μm, indicating that ice recrystallization was promoted compared to pure water, which is correlates with the enhanced ice-growth rate at 1 μM as described in Fig. linPG showed an inhibitory activity of recrystallization with an MLGS of 61.7 ± 7.3 μm at 1 mM, but gradually converged upon dilution to that observed with pure water. In the microscopic image  solution contained tiny ice grains and no noticeable change was observed over 30 min, while the 1 μM solution facilitated recrystallization significantly and the size of ice grains increased to more than twice that of pure water , hbPG inhibits the ice recrystallization by preventing the translocation of H2O molecules at grain boundary. Unlike linPG, which showed a consistent inhibitory effect  modeling  to investigate the cause of the Janus ice growth. As shown in Fig. map Fig. . When hb42. In the presence of hbPG, the amount of water molecules in the most immobile (MI) region increased by 28% compared to pure water and linPG  measurements, which indicated that the water molecules around the hbPG were more confined than those of linPG , were maintained in the range of concentrations in the present study nPG Fig. . Thus, h PG Fig. . Around 2O molecules and reverse-regenerated the fully-grown structure for 20 ns. In the presence of hbPG, a large portion of the H2O molecules were located between hbPG and the ice surface in the 20 ns past. In contrast, an identical analysis of linPG revealed that most of the water molecules were scattered throughout the bulk water. Based on the back-tracking results, we probed the molecular paths sequentially from liquid water to ice in terms of the following three characteristics  travel via PG, (2) travel via specific segments of PG, and (3) multiple H-bonding. In the left panels of Fig. 2O molecules are classified according to their travel path through the PG. In the case of hbPG, 23% of H2O molecules traveled via hbPG and were mostly localized around hbPG. Likewise, 25% traveled via linPG, but were randomly distributed. The center panels show that these H2O molecules traveled through four segments of PG: primary alcohol L13, secondary alcohol L14, terminal group T, and dendrimer D units. In hbPG, the H2O molecules that turned ice predominantly underwent H-bonding with the T and D units. In linPG, most of the H2O molecules traveled through L13. As for the multiplicity of H-bonding shown in the right panels, compared to linPG, hbPG formed more multiple H-bonds with H2O molecules , indicating the three ice layers were promoted to grow in 130 ns in our simulation. In contrast, no stepwise growth occurred in linPG: it grew to 0.19 nm in 130 ns and then to 1.10 nm in 250 ns. It is interesting to note that the stepwise growth of ice in the presence of hbPG is evidently related to the formation of H-bonds with the ice surface  as an internal standard in the deuterated solvents. Gel permeation chromatography (GPC) measurements (Agilent 1200 series) were performed using DMF as an eluent at 40 °C with a flow rate of 1.0 mL min−1 using a refractive index detector. GPC instrument equipped with PLgel 5 µm guard and two PLgel 5 µm mixed-D columns (Agilent). Standard poly(ethylene oxide) (PEO) samples were used for calibration to determine the number- and weight-averaged molecular weight (Mn and Mw). All solvents and reagents were purchased from commercial sources . Ethoxyethyl glycidyl ether (EEGE) was prepared. N-methyl-2-pyrrolidone (NMP), EEGE, and glycidol were purified by vacuum distillation over calcium hydride (CaH2) prior to use.t-BuP4  in n-hexane was added to the solution of benzyl alcohol  in NMP under an N2 atmosphere. Then, 10 mmol of glycidol, EEGE, or glycidol/EEGE mixture solution (monomer: NMP ratio = 1:1 (v/v), the total volume of NMP was set to 2.4 mL) was added dropwise to the initiator solution for 24 h and then stirred for 6 h. After polymerization was completed, the reaction mixture was quenched with excess methanol. The polymer in methanol solution was passed through an Amberlite IR-120(H) ion exchange resin twice to remove the phosphazene base. The polymer solution was then vacuum distilled to remove the remaining NMP to obtain pale yellow transparent polymers of P(G-co-EEGE) s. Successful polymerization of P(G-co-EEGE) was confirmed by 1H NMR and GPC. 1H NMR  δ 4.70 , 4.53 , 3.97–3.26 , 1.24–1.29 .  N3 initiated PGs were polymerized from 6-azido-1-hexanol.All glassware was washed and flame-dried before polymerization. A 0.11 mL solution of hbP(G-co-EEGE) solution in a 1:1(v/v) mixture of 1 M HCl and MeOH was stirred at 40 °C for 24 h. After removing the solvent, the polymer was dissolved in MeOH and precipitated twice in cold diethyl ether. The polymer solution was concentrated in vacuo and further dried at 60 °C for 24 h to obtain transparent pale yellow hbPGs (yield: 28–40%). The successful hydrolysis of P(G-co-EEGE) was confirmed by various characterization techniques, including 1H and inverse-gated 13C NMR, GPC. 1H NMR  δ 7.39 , 4.55 , 3.98–3.42 , 13C NMR  δ 79.61, 78.23, 72.16, 70.63, 69.01, 62.66, 60.83. A 10 wt.% hbPGs were characterized by inverse-gated 13C NMR spectroscopy . Each Peltier module was controlled using a PID Controller  that was operated with LabVIEW software. The temperature measurements of the two stages was acquired independently, and the temperature precision/stability was <0.1 °C. The sample holder, a Hele-Shaw cell, consisted of a sandwich of two glass slides separated by an 80 µm gap. The sides of the sample holder were sealed with carbon tape, and the exterior surface was fluorinated to prevent heat loss and allow heat to flow from the hot to the cold stage. Polarized optical and fluorescence microscopes were used to observe the movement of the ice/water interface over time. The experiments proceeded with the sample holder located at the fixed position in the middle of the temperature-controlled stage. Upon nucleation on the cold side, ice-growth proceeded so that the ice/water interface formed a straight line and moved towards the hot stage throughout the growth experiments in this study. Maintaining flat ice interface in our set-up was enabled in the operation window having cold-block temperature between −5 °C and −15 °C and hot-block temperature between 5 °C and 15 °C. In this study, the temperature gradient was 3.33°C/mm by setting cold-block at −5 °C and hot-block at 5°C. The ice-growth rates obtained by using this method, operated at the above temperature gradient, were in good agreement with those by the conventional moving-stage method. The present set-up afforded a simple way to observe the Janus regulation phenomenon of ice growth. Each experiment was repeated three times or more, and the ice growth rate was calculated using the following equation Fig. :2\documeFor RI activity, a splat-freezing method was used. The 10 µL sample droplets were rapidly frozen by dropping on a precooled stage, which was cooled using liquid nitrogen . The frozen samples were subsequently annealed at −6 °C on a temperature-controlled microscope stage and the transition through recrystallization observed for 30 min. After 30 min, the average size of the 10 largest ice domains was estimated, and the MLGS was evaluated by comparing it with the size obtained with pure water. The MLGS presented in the main text were calculated using four observations.45. The PG solution mixed with D2O (volume ratio of 1:99) was filled into the NMR tube. All the T2 relaxation behaviors for the water proton were fitted with a biexponential decay curve.The proton spin–spin relaxation time (T2) measurements of water in PGs samples were carried out on a 400 MHz NMR spectrometer  using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. A tau interval of 1.2 ms between the 90° and 180° pulse was used, and the spin-lattice relaxation time between successive scans was set sufficiently (at least 10 times) to fully recover the magnetization between acquisitionsc) by using the Bloembergen Purcell and Pound equation asC is a constant for water of 5.33 × 109 s−2 and ω0 is the Larmor frequency46.The dynamic of water can be represented by the correlation time for the motion of water (τhbPG and linPG) were separately prepared at a concentration of 1.0 mM in D2O. For diffusion measurements, pulsed-field-gradient DOSY NMR experiments were performed with a maximum gradient of 52.9 mT m−1. The pulse sequences contained a 2.0 ms delay to stabilize the gradients. The magnetic field gradient amplitudes were gradually increased from 2.10 × 10−2 up to 5.2 × 10−1 T m−1 for the maximum gradient strength in a linear ramp. For each gradient amplitude, 16 transients of 16384 complex data points were obtained for a total experimental time of 16 min. The diffusion gradient length (δ) of 2 ms was selected for the diffusion time, with a diffusion delay of 20 ms. The NMR data were processed, and the diffusion coefficients (D) were determined using the DOSY Toolbox software package. Errors of approximately 1.8% were obtained for the diffusion experiments. The main source of error in the diffusion experiments was the reproducibility of data acquisition. The signal decay caused by gradients was obtained by DOSY fitting of the Stejskal–Tanner equation:S is the signal amplitude as a function of gradient strength g, S0 is the signal amplitude at g = 0, D is the diffusion coefficient, γ is the proton gyromagnetic ratio, δ is the gradient pulse duration, and Δ is the diffusion time.Two samples (47 package (version 5.1.4) and the CHARMM general force field (CGenFF)48 for all-atomic (AA) modeling. The TIP4P-ice water model, which can simulate the water-ice phase transition well, was used49. To control the temperature, a V-rescale50 was used as the thermostat for equilibrium. The pressure was maintained at 1 bar using a Berendsen51 and Parrinello-Rahman52 barostat for the equilibrium and production runs, respectively. Neighbor lists were built using the Verlet cutoff scheme with a cutoff radius of 1.2 nm. The linear constraint solver (LINCS)54 algorithm was used to constrain the bond lengths. All simulations were performed using a leapfrog integrator with time steps of 2 fs during 150,000,000 steps  for ice-growth simulation. Electrostatic interactions were calculated using particle mesh Ewald (PME)55 with a cutoff of 1.2 nm in AA-MD.All simulations were performed using the GROMACShbPG, a molecular model with a different distribution of segments was used in the repeat calculations . In the high-concentration simulation, a system was constructed by inserting two molecules of five models for a total of 10 PG molecules.PG molecules were parameterized based on the CHARMM36 force field (version mar-2019) parameters. Molecular structures were designed based on experimental NMR results  were recorded.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


The response of the East Antarctic Ice Sheet to past intervals of oceanic and atmospheric warming is still not well constrained but is critical for understanding both past and future sea-level change. Furthermore, the ice sheet in the Wilkes Subglacial Basin appears to have undergone thinning and ice discharge events during recent decades. Here we combine glaciological evidence on ice sheet elevation from the TALDICE ice core with offshore sedimentological records and ice sheet modelling experiments to reconstruct the ice dynamics in the Wilkes Subglacial Basin over the past 350,000 years. Our results indicate that the Wilkes Subglacial Basin experienced an extensive retreat 330,000 years ago and a more limited retreat 125,000 years ago. These changes coincide with warmer Southern Ocean temperatures and elevated global mean sea level during those interglacial periods, confirming the sensitivity of the Wilkes Subglacial Basin ice sheet to ocean warming and its potential role in sea-level change. Crotti et al. reconstructed the dynamics of the Wilkes Subglacial Basin (Antarctica) during the past 350,000 years. Their study reveals that a portion of the East Antarctic ice sheet experienced an extensive retreat 330,000 years ago. Over the last decade, it has been observed that excess basal melting in Antarctica, arising from ocean heat supply, has increased the dynamic mass loss of grounded ice shelves bordering the Southern Ocean (SO)2. This observation has implications for future ice sheet stability, and also suggests that ocean warming may have played a role in controlling past ice sheet dynamics in Antarctica.The growth and decay of polar ice sheets exert important controls on regional and global climate, while their future behaviour is a key uncertainty in predicting sea-level rise during and beyond this century3, is well known for its past and future vulnerability to a warming climate5. In contrast, past stability of the much larger East Antarctic Ice Sheet (EAIS), which is characterized by a total potential contribution to Global Mean Sea Level (GMSL) of 53 m6, of which around one-third is marine-based ice, is still under debate10. The Wilkes Subglacial Basin, which contains 3 to 4 m sea-level equivalent3, is characterized by a reverse-sloping bed with an elevation below sea level 14, following the near-complete loss of the Cook West Ice Shelf between 1973 and 1989 that resulted from intense oceanic warming during the middle of the 20th century16. Model simulations suggest a modest sensitivity of the Wilkes Subglacial Basin ice sheet to oceanic warming17 and margin retreat controlled by the presence of a coastal ice plug18. However, projected atmospheric and oceanic warming could soon lead to the crossing of tipping points in Antarctica, and hence to the destabilization of marine-based sectors of the ice sheet20. In light of this potential vulnerability, future predictions of the Wilkes Subglacial Basin ice dynamics should be refined by studying previous occurrences of instabilities during past warm climatic periods when temperatures were comparable to, or warmer than, modern conditions.Satellite altimetry and images reveal that the Cook Glacier, which drains a large proportion of the Wilkes Subglacial Basin, has experienced thinning of 33 ± 12 cm/a (year) over the past 25 years21.Here we explore the past ice dynamics of the Wilkes Subglacial Basin during the recent interglacial Marine Isotopic Stages (MIS) 5.5, 7.5, and 9.3 of the last 350 ka (thousand years ago). These warm periods can be considered similar, in terms of atmospheric warming and GMSL increases, to a range of near-future climate projections22. The recent extension of the TALDICE ice core chronology back to 343 ka (TALDICE-deep1) and the observation of a unique behaviour of the δD record during the interglacial periods23 may therefore come helpful for deciphering the late Pleistocene dynamics of the peripheral EAIS.The Talos Dome Ice Core (TALDICE) , which was drilled in a peripheral area of the East Antarctic Plateau  from the TALDICE ice core at Talos Dome, in comparison to the EDC ice core record at Dome C, which is representative of East Antarctic Plateau conditions under the influence of the Southern Indian Ocean  Hole U1361A, offshore Wilkes Subglacial Basin7  periods, TALDICE and the plateau ice cores  share common isotopic maxima (δ18O and δD), between 12 and 9 ka in the Holocene and at 128 ka during the LIG30. The LIG isotopic maxima indicate Antarctic atmospheric temperatures 2–4.5 °C warmer than the Holocene33, probably arising from the operation of the bipolar seesaw mechanism during deglaciation of the Northern Hemisphere ice sheets35. However, during the late stage of MIS 5.5, the TALDICE isotopic record shows a δ18O peak at 117 ka followed by an abrupt decrease towards the glacial inception, and such features are not recorded in the cores from the plateau30.The EDC and TALDICE water isotopic records are both influenced by precipitation originating mainly from the Southern Indian Ocean18O data (measured at 5 cm resolution) from MIS 5.530, MIS 7.5, and MIS 9.3 with the published EDC δ18O record37, which is representative of the Antarctic Plateau conditions, in order to investigate TALDICE isotopic patterns during previous late Pleistocene interglacials (Methods). Interestingly, the TALDICE δ18O record from MIS 9.3 exhibits a double-peak shape that is similar to the one observed for MIS 5.5, which is not seen in the EDC record  differences in moisture sources, (ii) changes in sea-ice extent in the Ross Sea, and (iii) a local decrease of elevation at Talos Dome.d-excess = δD − 8·δ18O) thanks to the new TALDICE d-excess profile (5 cm resolution) for MIS 5.5, 7.5, and 9.3. This second-order parameter is an indicator of climate conditions in the vapour source regions, and is therefore sensitive to changes in the provenance areas or changes in air mass trajectories towards the sites40. Our comparison shows no clear differences between the TALDICE and EDC41d-excess profiles, which exhibit consistent patterns during interglacial stages  fluxes in TALDICE for MIS 128 and MIS 5.544, and new data for MIS 7.5 and MIS 9.3, as a proxy for sea-ice coverage in the western sector of the Ross Sea and in the southern Indian Ocean facing the Wilkes Subglacial Basin28. The high-resolution (7-8 cm) TALDICE ssNa+ flux record for MIS 5.544, MIS 7.5, and MIS 9.3 agrees well with the EDC record37 and no site-specific differences are identifiable  and LIG47. To estimate the imprint of elevation changes at Talos Dome during the LIG, Sutter et al.9 applied a δ18O-elevation relationship of −0.53‰/100 m, based on multiplying the present day lapse rate of −0.8 °C/100 m by the local δ18O isotope-temperature relationship of 0.66‰/°C estimated from the atmospheric general circulation model ECHAM5-wiso equipped with an isotope module47. In contrast, Goursaud et al.29 obtained a relationship of −0.93‰/100 m for the LIG, based on the simulated isotopic response to idealised changes in Antarctic ice sheet elevation in the isotope-enabled coupled ocean-atmosphere-sea-ice general circulation model HadCM348. As a third approach, we directly compute the isotope-elevation relationship from present-day snow-pit δ18O data collected along the traverse from GV7 site to Talos Dome49, which takes into account the provenance of air masses reaching Talos Dome (see details in “Methods”). This method leads to an estimate of the modern isotopic lapse rate for Talos Dome of −1.35 ‰/100 m.The relationship between δ18O lapse rate estimates for TALDICE to calculate the elevation changes required to explain the observed anomalous increase of the isotopic signal (Δδ18O) (see “Methods”) for MIS 5.5 (+1.68‰), MIS 7.5 (+0.68‰), and MIS 9.3 (+1.42‰)  for those intervals. Hence, while subject to uncertainty from the choice of lapse rate, we conclude from the TALDICE isotopic record that the Talos Dome site was subjected to an elevation decrease on the order of −100 to −300 m during MIS 5.5 and MIS 9.3  temperatures. The NADW is produced by deep convection of dense waters at high northern latitudes, then flows southward in the deep Atlantic Ocean, and upwells in the SO. Due to the lack of a suitable SO proxy record for sub-surface oceanic temperatures spanning the last 400 ka, this estimate of upwelled NADW temperature is a reasonable indicator for the oceanic heat available in Antarctic coastal regions that affects the sub-shelf melting rate at the grounding line. We have also performed additional sensitivity tests using two other estimates of the sub-surface oceanic conditions in the SO  and doubling the original sub-shelf melting24, starting from the Interglacial Start (IS) represented by the present-day Antarctic ice sheet state. The DS state is considered more likely to reproduce Antarctic ice sheet conditions during MIS 11 (~400 ka) in comparison to the IS state. Simulations have also been performed applying a IS state and those results are presented in Supplementary Table 24 and derived from the ODP 980 benthic record50, (ii) the signal defined by Quiquet et al.24 increased by 5%, and (iii) the signal defined by Quiquet et al.24 increased by 10% over the past 400 ka  the initial state of the Antarctic ice sheet at 400 ka, and (ii) the sub-shelf melting rate. The GRISLI experiments labelled DS (Deglaciated Start) adopt an initial Antarctic interglacial ice sheet state, while the GS  experiments use a Last Glacial Maximum 21 ka) initial state ka initiThe ice thickness results from the GS and DS sensitivity experiments are compared to ice thickness variations estimated from the isotopic record at Talos Dome Table , with siThe main grounding line retreat during MIS 5.5 is seen earlier in the model 128 ka) than is suggested by the TALDICE isotopic signal (117–127 ka) 54, recovered from the continental rise adjacent to the Wilkes Subglacial Basin  discharge can indicate dynamic ice loss, while Nd isotopes measured on detrital sediments (εNd) provide a provenance indicator for changes in subglacial erosion and transport that could reflect ice sheet retreat56  of the ice sheet in the Wilkes Subglacial Basin during the warmest late Pleistocene interglacials. Notably, our simulated Talos Dome elevation changes during MIS 5.5 are consistent with recent studies10, while the GRISLI GS-5 simulation depicts a larger retreat at the margin of the Wilkes Subglacial Basin which is in closer accordance with the U1361A record7.By combining the TALDICE δ57. In addition, the deglacial weakening of the Atlantic Meridional Overturning Circulation may have led to warmer Southern Ocean temperatures through the bipolar seesaw mechanism, with early interglacial atmospheric warming also attributable to CO2 escape from deep waters59. The more southerly and warmer Antarctic Circumpolar Current could have induced a dynamic response in the EAIS during MIS 5.561 and a comparable scenario can also be anticipated for MIS 9.3. The distinctive late interglacial δ18O peaks in the TALDICE record during MIS 5.5 and MIS 9.3 occurred ~9–11 ka after the interglacial isotopic maxima and are synchronous with the local summer insolation maxima at 65°S  during MIS 118.Interestingly, a new late Pleistocene Subantarctic sea surface temperature reconstruction from the Indian sector of the SO (core DCR-1PC) shows that both MIS 5.5 and MIS 9.3 were characterized by a double warming phasen58 Fig. . Over ti64. For MIS 9, a stacked GMSL reconstruction based on various proxies indicates a sea-level highstand of ~9 m above present65. The GS-5 experiment indicates that the grounding line in the Wilkes Subglacial Basin may have retreated by several hundred kilometres during those interglacials, leading to contributions of approximately +0.5 m and +0.9 m to the GMSL increases during MIS 5.5 and MIS 9.3, respectively. These instabilities of the Wilkes Subglacial Basin ice sheet appear to have been driven mainly by increases in SO temperatures and/or CDW intrusion during past interglacials.Finally, sea-level reconstructions for MIS 5.5 and MIS 9.3 are also consistent with our reconstruction of the Wilkes Subglacial Basin ice dynamics during the late Pleistocene. Data from corals and other sea-level proxies indicate a late peak in GMSL during MIS 5.5 of ~6–9 m above present at ~119 ka17, in order to improve our understanding of EAIS dynamics during past interglacial intervals and on a future warming Earth.Despite the qualitative agreement between the ice core data, marine sediment core proxies, and the ice sheet modelling results, it will be essential to better assess the quantitative differences between the data and models, as well as the relative roles of atmospheric and oceanic warming in triggering ice sheet instabilities18O and δD) and d-excess (d = δD − 8 ×  δ18O) of new and published profiles measured at high resolution (5 cm) in the TALDICE ice core during interglacials. Here we present the published δ18O bag resolution profile (1 m) for the Holocene34, the published profile for MIS 5.530, and new 5 cm resolution profiles for MIS 7.5 and MIS 9.3. For the d-excess, we show the published bag resolution signal (1 m) for MIS 128 and the new 5 cm resolution signal for the oldest interglacials. We plot data from MIS 1 (0–20 ka) and MIS 5.5 (~115–132 ka) on the AICC2012 age scale66. The new data for δ18O and d-excess during MIS 7.5 (~240–246 ka) and MIS 9.3 (~324–339 ka) are plotted on the TALDICE-deep1 age scale23.In this study we focus on the oxygen and hydrogen isotopic composition  and France (LSCE) using the Cavity Ring Down Spectroscopy (CRDS) technique. Analyses were performed using a Picarro isotope water analyser (L2130-i version for both laboratories). The data were calibrated using a three-point linear calibration with three lab-standards that were themselves calibrated versus Standard Mean Ocean Water (SMOW). Intercomparisons between the two laboratories have been performed over the analysis period. The average precision for the δ18O and d-excess records from the TALDICE and EDC cores41, which are characterized by different temporal resolution, mostly due to differences in the sampling interval, snow accumulation, and thinning function at different depths (Table In this work we compare the δhs Table .Table 2Cfind change points with a maximum of 3 or 4 change points. The software identifies points with changes in slope (derivative) and intercept  were then calculated as the differences between values in the resampled record between the two change points  and resampled them at the same time step of 200 year. We then searched for changes using the MATLAB task ept Fig. . Isotopi25. In contrast, at EDC the modern precipitation originates mainly from the western Southern Indian Ocean (85%), with a small amount coming from the east via the Ross Sea and Transantarctic Mountains (15%).In the modern day, Talos Dome receives 50% of its total precipitation from the west (Southern Indian Ocean), 30% from the east (Ross Sea and Southern Pacific Ocean), and ~15% from the interior18O) in permil (‰) for every 100 m change in altitude. Here we calculate the isotopic lapse rate for TALDICE using the ITASE traverse dataset49. We take into account the δ18O values of snow between the GV7 and Talos Dome sites sampled every 5 km and the altitude profile from the GV7 site to Talos Dome. This approach seems reasonable, given that the air masses travelling to Talos Dome mainly originate from the Southern Indian Ocean at 60°S and likely follow a similar path to the traverse once they reach Antarctica25. The calculated isotopic lapse rate is −1.35‰/100 m.The isotopic lapse rate expresses the variation in the oxygen isotopic composition (δ+) flux measured in the TALDICE ice core during MIS 1 (1 m resolution)28, MIS 5.5 (1 cm resolution)44, and new data for MIS 7.5 and MIS 9.3 (8 cm resolution). The concentrations of ssNa+ were measured by classical ion chromatography on discrete samples collected using a melting device connected to an auto-sampler for the MIS 1, MIS 7.5, and MIS 9.3 samples, whereas Continuous Flow Analysis (CFA) was applied for the MIS 5.5 samples44.In this study we present the sea-salt sodium , while for the deepest part (1438–1578 m) the accumulation rates are obtained from the TALDICE-deep1 age scale23.The total deposition ssNa67. The TALDICE chronology is based on the AICC2012 age scale until ~150 ka66, built with a multi-site approach including both Greenland and Antarctic ice cores, while for the older part it is based on TALDICE-deep1 chronology23.To compare the sedimentological and geochemical records from core U1361A with the TALDICE ice core isotopic records, we need to define a common chronostratigraphy. The original chronology for the U1361 composite splice (U1361A and U1361B) is based on biostratigraphic and magnetostratigraphic data and one radiocarbon date66 as a reference curve in order to compare the late Pleistocene sediment core data from U1361A to the TALDICE ice core record. Specifically, we refine the existing U1361A age model through the alignment of barium/aluminium (Ba/Al) ratios from XRF-scanning7 with the EDC δD record on the AICC2012 age scale. Glacial-interglacial cycles are clearly expressed in the U1361A Ba/Al record, with higher ratios reflecting warmer conditions and reduced sea-ice extent during interglacials7. As such, and similar to the EDC δD record, the Ba/Al record reflects a combination of local forcing and global climate boundary conditions. We apply a conservative tuning strategy to align the two records, using tie points  only at the mid-points of the major glacial terminations I-V, where large and rapid signals are recorded in both the U1361A Ba/Al and EDC δD records. Such transitions are likely to be synchronous in the two records, at least at the multi-ka sample resolution of the U1361A marine record, whereas there is greater uncertainty in the exact timing and progression of productivity changes during the steps from interglacial to glacial periods. The U1361A Ba/Al record on its original chronology and on its refined age model is presented in the Supplementary Information , the new age model for core U1361 differs by only 0–6 ka compared to the previous age model in which the sedimentation rate was assumed to be constant24 to model changes of the Antarctic ice sheet between 100 and 400 ka, since it is mostly designed for multi-millennial integrations. The model combines an inland ice model with an ice shelf model, extended to the case of ice streams considered as dragging ice shelves. The latest release includes a better representation of grounding line migration and a sub-glacial hydrology model. The model uses finite differences on a Cartesian grid at 5–40 km resolution depending on the application. Here we use a 40 km grid to take advantage of the model calibration performed in Quiquet et al.24. Given its low numerical cost at this resolution, Quiquet et al.24 perform an ensemble of 600 simulations to calibrate the mechanical parameters. We use the ensemble member of Tsai et al.68 for the formulation of the flux at the grounding line, since it best reproduces the glacial-interglacial transitions. This ensemble member is labelled AN40T21324. Note that, due to the relatively coarse resolution (40 km) applied here, fine-scale structures such as individual ice streams might not be properly represented. The model setup used to perform the transient paleo ice sheet simulations is identical to that used by Quiquet et al.24. The GRISLI simulations results are presented in the Supplementary Information.The GRISLI (Grenoble ice sheet and land ice) model is a large-scale three-dimensional thermomechanical ice sheet model. In this work, we use the GRISLI version 2.0Supplementary InformationPeer Review File


Mitophagy, a type of selective autophagy, specifically targets damaged mitochondria. The ULK complex regulates Parkin-mediated mitophagy, but the mechanism through which the ULK complex initiates mitophagosome formation remains unknown. The Rab7 GTPase (herein referring to Rab7a) is a key initiator of mitophagosome formation, and Ser-72 phosphorylation of Rab7 is important for this process. We have previously identified LRRK1 as a protein kinase responsible for Rab7 Ser-72 phosphorylation. In this study, we investigated the role of LRRK1 in mitophagy. We showed that LRRK1 functions downstream of ULK1 and ULK2 in Parkin-mediated mitophagy. Furthermore, we demonstrated that ectopic targeting of active LRRK1 to mitochondria is sufficient to induce the Ser-72 phosphorylation of Rab7, circumventing the requirement for ATG13, a component of the ULK complex. Thus, the ULK complex recruits LRRK1 to mitochondria by interacting with ATG13 to initiate mitophagosome formation. This study highlights the crucial role of the ULK complex–LRRK1 axis in the regulation of Parkin-mediated mitophagy. Summary: LRRK1 is recruited to damaged mitochondria by interacting with ATG13, a component of the ULK complex, during Parkin-mediated mitophagy and initiates mitophagosome formation via Rab7 Ser-72 phosphorylation. Autophagy is an evolutionarily conserved cellular process that removes unnecessary or dysfunctional cellular components . MitophaThe molecular mechanisms of mitophagy have been extensively studied, resulting in the discovery of several important signaling pathways. The most well-understood form of mitophagy is orchestrated by the mitochondrial kinase PTEN-induced kinase 1 (PINK1) and the E3-Ub ligase Parkin, which are encoded by two genes that are mutated in familial PD . Geneticin vitro. Ser-72 is located in the switch-II domain, which is involved in both GDP–GTP exchange and interactions with other proteins (Recent studies have identified Rab7a guanosine triphosphatase (GTPase) (hereafter referred to as Rab7) as a key component in the initiation of mitophagosome formation in Parkin-mediated mitophagy . The Rabproteins . Residueproteins , 2017. Fproteins .We recently identified LRRK1 as a kinase responsible for the phosphorylation of Rab7 at Ser-72 . The LRRParkin is specifically recruited to the mitochondria in response to mitochondrial depolarization induced by the proton uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) . ThroughTo investigate whether LRRK1 kinase activity is required for the removal of depolarized mitochondria, we conducted rescue experiments using an LRRK1 kinase-negative mutant [LRRK1(KM)], in which Lys-1243 in the LRRK1 kinase domain was replaced with methionine . In LRRKRab7 plays a significant role in the early stages of Parkin-mediated mitophagy, especially in mitophagosome formation, by targeting ATG9-containing vesicles around damaged mitochondria . Ser-72 We recently reported that LRRK1 phosphorylates Ser-72 in GTP-bound Rab7 at the endosomal membrane . Then, wWe next examined whether LRRK1 kinase is activated in Parkin-mediated mitophagy. We monitored the LRRK1 kinase activity in cell extracts by assessing pS72-Rab7 in HEK293 cells expressing endogenous Parkin. Briefly, the cells were co-transfected with Flag–LRRK1 and Flag–Rab7, treated with CCCP for 3 h, and immunoblotted with the anti-pS72-Rab7 antibody. We found that co-transfection with Flag–LRRK1, but not with LRRK1(KM), induced Rab7 phosphorylation after CCCP treatment E. Thus, The ULK kinase in the ULK multiprotein complex is an upstream-acting component of the macroautophagy machinery . A recenTo elucidate the relationship between ULK1, ULK2 and LRRK1, we examined the effect of the constitutively active LRRK1 mutant LRRK1(Y944F)  on the pNext, we examined the relationship between LRRK1 and the ULK complex in Parkin-mediated mitophagy. Considering that the ULK complex is composed of the ULK1 or ULK2 kinase, FIP200, ATG13 and ATG101 , we examWe next investigated whether ATG101 and ATG13 are required for Rab7 Ser-72 phosphorylation on depolarized mitochondria. ATG101 knockdown with siRNA in Flag–Parkin-expressing U2OS cells did not affect the Ser-72 phosphorylation of Rab7 induced by CCCP . In contWhy does LRRK1(Y944F) suppress ULK1 and ULK2 depletion but not ATG13 knockdown? The ULK complex is recruited to the damaged mitochondria, leading to ULK kinase activation . TherefoThe above-mentioned results suggest that ATG13 interacts with and recruits LRRK1 to the mitochondria during Parkin-mediated mitophagy. We also found that overexpressed LRRK1 interacted with endogenous ATG13 in a CCCP or O/A stimulation-dependent manner . SimilarNext, we investigated whether the role of ATG13 for LRRK1 in Parkin-dependent mitophagy is only to recruit LRRK1 to the mitochondria. Thus, we examined whether ectopic recruitment of ATG13 to the mitochondria by CID could induce Rab7 Ser-72 phosphorylation. We found that the addition of rapalog to U2OS cells expressing FKBP–GFP–ATG13 and FRB–Fis1 failed to elicit Rab7 Ser-72 phosphorylation on the mitochondria decorated with GFP–ATG13 C,D. Thusin vitro kinase assays using purified recombinant glutathione S-transferase (GST)-tagged ULK1. Immunopurified kinase-negative GFP–LRRK1(KM) was incubated with GST–ULK1 in vitro. The kinase-negative form of LRRK1 was used as a substrate to prevent autophosphorylation of LRRK1. We found that GST–ULK1 phosphorylated both itself and GFP–LRRK1(KM) (How does the ULK complex activate LRRK1 in Parkin-mediated mitophagy? ULK kinase might activate LRRK1 kinase activity through phosphorylation because LRRK1(Y944F) overexpression can suppress the ULK1 and ULK2 double-depletion phenotype C,D. TherRRK1(KM) , indicatBecause ATG101 was not required for Rab7 Ser-72 phosphorylation on damaged mitochondria A,B, we iin vitro  rescues the defect in CCCP-induced phosphorylation of Rab7 at Ser-72 caused by ULK1 and ULK2 double-depletion. Furthermore, we demonstrated that ULK1 phosphorylates LRRK1, suggesting that ULK is involved in LRRK1 activation through phosphorylation. In contrast, the ectopic recruitment of ATG13 alone to the mitochondria cannot induce Rab7 Ser-72 phosphorylation. However, the co-expression of LRRK1(Y944F), but not wild-type LRRK1, in cells expressing mitochondria-targeted ATG13 can induce the pS72-Rab7 signal on mitochondria. These results provide insights into the mechanism through which the ULK complex plays a role in LRRK1 activation in Parkin-mediated mitophagy. Therefore, mitophagy activation induces the recruitment of the ULK complex and LRRK1 to the damaged mitochondria, where ULK kinase is activated by local clustering, thereby phosphorylating and activating LRRK1. Thus, the localization of LRRK1 on the mitochondria by the ULK complex indicates the LRRK1 activation is linked to spatial regulation. This model simultaneously resolves the timing and location of Parkin-mediated mitophagy induction.In this study, we found that in Parkin-mediated mitophagy, ATG101 is required for the clearance of depolarized mitochondria but not for Rab7 Ser-72 phosphorylation on depolarized mitochondria. These results suggest that the ULK complex mediates mitophagy through both LRRK1-dependent and LRRK1-independent pathways, whereas ATG101 is involved in only the latter pathway . AccordiRecently, What is the role of Rab7 Ser-72 phosphorylation in Parkin-mediated mitophagy? When Rab7 is recruited to damaged mitochondria in response to mitochondrial depolarization, it plays a critical role in correctly targeting ATG9-bearing vesicles to induce mitophagosome formation around damaged mitochondria . MoreoveLRRK2 engages in the regulation of mitochondrial homeostasis in a manner dependent on its kinase and pathogenic mutations . LRRK2 fMycoplasma contamination. The antibodies and their suppliers were as follows: anti-TOM20 , anti-GFP , anti-Flag , anti-Myc , anti-HA , anti-ATG9 (Abcam), anti-LC3 , anti-C-III core 1 (Invitrogen), anti-ATG13 , anti-ULK1 , anti-TBK1 , anti-ATG101 (Abcam) and anti-PDH E2/E3bp (Abcam). Affinity-purified rabbit antibodies against pS72-Rab7 were produced according to a previously described method . These cell lines were obtained from either the American Type Culture Collection or the Japanese Collection of Research Bioresources and were regularly evaluated for d method . CCCP an22875 (deposited by Noboru Mizushima). siRNA-resistant LRRK1 was generated as previously described , GFP–LRRK1(Y944F) and pHAGE-mt-mKeima-P2A-FRB-Fis1 were generated as previously described . GFP–LRRescribed . siRNA f2, 0.5 mM DTT, 5 µCi [γ-32P]ATP and 100 µM ATP. Samples were incubated for 20 min at 30°C, and the reactions were terminated by the addition of Laemmli sample buffer, followed by boiling. Samples were resolved by SDS-PAGE and analyzed using autoradiography.The recombinant protein GST–ULK1 was purchased from SignalChem. GFP–LRRK1 proteins were expressed in HEK293 cells and immunopurified using an anti-GFP antibody . Kinase g  for 12 min. The supernatant was added to 50 µl (1.5 mg) of Dynabeads Protein G (Invitrogen) with the indicated antibodies (10 µg) and rotated for 2 h at 4°C. The beads were then washed three times with ice-cold phosphate-buffered saline and subjected to immunoblotting. Raw data for western blots are provided in Cells were lysed in RIPA buffer , followed by centrifugation at 15,000For immunofluorescence staining, the cells were grown on coverslips, treated as indicated, fixed in 4% paraformaldehyde for 15 min at 37°C or methanol for 2 min at −20°C, and permeabilized in 0.5% Triton X-100 for 5 min. Subsequently, the cells were incubated with primary and secondary antibodies. The primary antibodies used were as follows: mouse anti-Flag at 1:500, anti-Myc at 1:200, rabbit anti-TOM20 at 1:200, anti-pS72-Rab7 at 1:250, anti-ATG9 at 1:100, anti-LC3 at 1:500, anti-C-III core 1 at 1:100, and anti-PDH E2/E3bp at 1:500. The secondary antibodies used were as follows: Alexa-Fluor 488-, 555-, or 647-goat anti-mouse IgG or anti-rabbit IgG antibodies (Invitrogen). Confocal microscopy was performed using a Zeiss LSM800 microscope. In quantifying the percentage of cells with pS72-Rab7 or PDH E2/E3bp signals in Flag–Parkin-decorated structures, GFP–LRRK1(Y944F)-decorated structures, GFP–ATG13-decorated structures or TOM20-positive mitochondria, the colocalization of these signals in immunofluorescence images was manually counted. For each series of experiments, the microscopic settings were optimized for the brightest unsaturated images and maintained during the analysis.Mitochondrial clearance was quantified using ImageJ . First, the region of the cell to be analyzed was outlined with a closed polygon, and the immunofluorescence intensity of C-III core 1 within this region was determined. Then, the net fluorescence intensity of each was calculated by subtracting the background immunofluorescence. The relative C-III core 1 fluorescence intensity for each cell was obtained by dividing the value of C-III core 1 immunofluorescence by the region. Cells with a signal of 0 were defined as cells with mitochondrial clearance and summarized in a bar graph.t-test. Data are combined from three independent experiments. Error bars represent standard deviation (s.d.). Detailed n values for each figure panel are stated in the corresponding legends. No statistical method was employed to predetermine the sample size. GraphPad Prism was used for the analysis of statistical significance.Statistical analysis was conducted using either Dunnett's multiple-comparison test or Welch's Click here for additional data file.10.1242/joces.260395_sup1Supplementary informationClick here for additional data file.


The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. In silico docking modeling was performed to confirm the main binding site of ergotamine. (3) Results: The ergotamine-mediated inhibitory effect on the NR1a/NR2A subunits has concentration-dependent, reversible, and voltage-independent properties. The major binding sites were V169 of the NR1a subunit and N466 of the NR2A subunit. (4) Conclusion: Ergotamine effectively inhibited NR1a/NR2A subunit among the subtypes of NMDAR. This inhibition effect can prevent excessive Ca2+ influx, which prevents neuronal death.(1) Background: The N-methyl-D-aspartate receptors (NMDARs) mediate fast excitatory currents leading to depolarization. Postsynaptic NMDARs are ionotropic glutamate receptors that mediate excitatory glutamate or glycine signaling in the CNS and play a primary role in long-term potentiation, which is a major form of use-dependent synaptic plasticity. The overstimulation of NMDARs mediates excessive Ca All the values are presented as the standard error of the mean. The differences between the means of the control and application values were determined using one-way ANOVA with a Tukey test. p <0.01 was considered statistically significant.To investigate the concentration–response curves of the role of ergotamine on glutamate-stimulated inward current (I2O-injected oocytes were not affected by the application of glutamate (100 µM). The effects of ergotamine (30 and 10 µM) on glutamate (100 µM)-evoked current are shown in Glu is caused by the application of (30 µM) ergotamine in the presence of glutamate (100 µM) on each type of NMDAR subunit  was recorded at 65.2 ± 6.5%, 45.5 ± 8.5%, 10.2 ± 4.3%, and 18.6 ± 5.3%, respectively. The coapplication of ergotamine with glutamate (100 µM) had nearly no impact on IGlu on NR1a/2C and NR1a/2D subunits. On the NR1a/2B subunits, the coapplication of ergotamine with glutamate could only slightly reduce the intensity of the currents elicited by glutamate compared to treatment with glutamate only; however, it markedly inhibited IGlu on the NR1a/2A subunit receptor in the same condition. These results implied that the coapplication of ergotamine on inward currents of NR1a/2A subunits has an inhibitory manner, and the inhibition was reversible.To assess the impact of ergotamine on various NMDAR subunits, 100 µM glutamate was supplemented into a bath solution to stimulate a large inward current in oocytes that were injected with NMDAR subunit  mRNAs. Glu were analyzed. The results demonstrated that ergotamine-mediated inhibition of glutamate-induced inward currents for NR1a/2A and NR1a/2B subunits was concentration-dependent. As shown in 50 values for each recombinant NR1a/NR2A–D were 15.3 ± 6.1, 15.7 ± 4.8, 6.5 ± 3.6, and 13.8 ± 6.4 µM, respectively; the values of the Hill coefficient for recombinant NR1a/NR2A–D were 2.1 ± 1.5, 2.1 ± 1.0, 0.8 ± 0.5, and 1.2 ± 0.6, respectively, corroborating previous analysis results. In summary, the potent inhibitory effect of ergotamine on glutamate-evoked currents on the NR1a/NR2A subunits was highest compared to that on other NMDAR subunits. A further study on the NR1a/NR2A subunits focused on clarifying the role of the inhibition of ergotamine in NMDARs is required.Previous data showed the inhibitory effect of ergotamine on the application of NMDA subunit (NR1a/2A and NR1a/2B) receptors. Different concentrations of ergotamine, ranging from 1–100 μM, and different potencies for IGlu, may allow ergotamine to interact with specific amino acids of NR1a/NR2A subunits via hydrogen bond formation.The homology models of wild-type NR1a/NR2A subunits and mutant channels were built to predict the possible ligand-binding site of ergotamine on the NR1a/NR2A subunits. The in silico docking of ergotamine to wild-type and mutant models of the subunit was analyzed. Using the docking tool AutoDock, we predicted the binding affinity of ergotamine to NR1a/NR2A subunits based on the binding free energy of each site. The values of all the binding free energies are shown in Glu current on oocytes expressing these mutants was further analyzed to determine whether ergotamine interacted with the channel on these residues as the binding site or not. The values of the inhibitory response of ergotamine on IGlu for each mutant type are shown in To validate the best near-native pose for the selected residues from the NR1a/NR2A subunits with ergotamine, each residue was mutated into alanine to elucidate its functional role. Thus, the inhibitory effect of ergotamine on the Imax values for the NMDAR subunits NR1a/NR2A, NR1a/NR2B, NR1a/NR2D, and NR1a/NR2C were recorded at 75.2% ± 9.7%, 53.7% ± 5.8%, 24.0% ± 5.1%, and 13.9% ± 3.0%, respectively. To compare these results with mutant-type, the inhibition percentage of ergotamine applied to each mutation type was expressed as a sigmoid curve using the Hill equation, and the results are shown in max value of 13.9% ± 0.7% and IC50 of 7.8 ± 1.1 µM. The Hill coefficient (nH) of 1.3 ± 0.2 are shown in Moreover, the mutation in mutant 1 A NR1a WF NR1a VA, the Im4, (4) haloperidol, (5) H2O2, and (6) ischemic damage. Several factors are involved in neuronal loss; however, neuronal loss can also be caused by ROS [2+ accumulation results in the overproduction of ROS owing to a partial reduction of oxygen by mitochondrial electron transport chains, cytoplasmic lipoxygenase, and peroxisome flavoprotein oxidases. The balance of detoxification and generation of ROS induces them to play their original role. The transient generation of localized ROS plays an important role in receptor-mediated cellular signaling [2+ regulation is affected, leading to oxidative stress caused by excess free radicals, which is associated with various diseases [2+ at an optimal concentration is essential.Various neuropathological diseases begin with the neuronal loss . Neuronad by ROS ,26. ROS d by ROS . Excessiignaling ,28. Howediseases . Therefo2+ regulation, and (3) interacts with various neurotransmitter receptors [Neuropathological disorders caused by ROS overproduction include Huntington’s disease, Parkinson’s disease, and AD. The pathogenesis for AD was hypothesized as follows: brain cell death due to cytotoxicity is caused by the accumulation of beta-amyloid. Aggregates of beta-amyloid are found in AD patients in the form of peptides ranging from 39 to 43 amino acids in length . These aeceptors .2+ accumulation in the cell increases ROS generation [2+. As a result, beta-amyloid aggregates interact with nerve cell membranes and induce the accumulation of ROS in the cells [Recently, it has been shown that neuronal cell dysfunction and oxidative cell death are related to AD-associated beta-amyloid protein accumulation. Therefore, the importance of intracellular redox regulation is emphasized. Neuronal depolarization is induced when the activity of the respiratory enzyme deteriorates owing to aging, the ROS scavenging ability decreases, and the energy metabolism process is impaired. Neuronal depolarization induces activation of NMDARs in the brain, and excessive Caneration . These hhe cells . Therefo2+ overload are associated and lead to neuronal death and synaptic dysfunction. Moreover, these reviews reported the role of NMDARs in aggravating chronic diseases, especially AD and Parkinson’s disease [NMDARs are involved in many diseases, including neurodegenerative and psychiatric disorders. NMDAR subunits exhibit different functions depending on their combination. The subunit NR1 can assemble in pairwise combinations with one of four possible NR2 subunits (di-heteromeric NMDARs) or can assemble as a complex of NR2 and NR3 (tri-heteromeric NMDARs) ,32,33,34 disease ,39. It wThe expression of NR2A and NR2B subunits, found mostly in the cerebral cortex and hippocampus, was reported to be involved in the impairment of several functions of the brain, including cognitive performance and synaptic function ,41,42,432+ concentration by inhibiting NR1a/NR2A subunits in vitro, and this result suggests that ergotamine may regulate redox status and may be a potential treatment for several neurological diseases.Consequently, the development of NMDAR-selective antagonists proves its immense therapeutic potential. Although ergotamine is an ergot alkaloid known to exhibit high efficacy in cardiovascular disease and migraine treatment , its neuGlu in cells expressing NR1a/NR2A subunits was clarified. The results of double mutations of V169 of the NR1a subunit and N466 of the NR2A subunit showed that both residues are important binding sites for ergotamine. The potential mechanisms by which ergotamine blocks the channel were visualized by performing homology modeling and ligand docking to the transmembrane domain of the NR1a/NR2A subunits. Excessive activity of NMDARs induces excessive Ca2+ influx into cells and may lead to neuronal death. These results suggest that the inhibition of NMDAR by ergotamine may have a neuroprotective function. Through in vitro molecular study and in silico docking modeling, ergotamine effectively inhibited NR1a/NR2A, a subtype of NMDAR, and it was found that it could be a drug that can potentially modulate redox status by regulating Ca2+ influx into the neurons.The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. The ergotamine-mediated inhibitory effect on the current evoked by glutamate on oocytes expressing NR1a/NR2A subunits was concentration-dependent and reversible and worked as voltage-independent. The pharmacological mechanism of ergotamine-mediated suppression of I


Inhibitory impairments may persist after abstinence in individuals with alcohol use disorder (AUD). Using traditional statistical parametric mapping (SPM) fMRI analysis, which requires data to satisfy parametric assumptions often difficult to satisfy in biophysical system as brain, studies have reported equivocal findings on brain areas responsible for response inhibition, and activation abnormalities during inhibition found in AUD persist after abstinence. Research is warranted using newer analysis approaches. fMRI scans were acquired during a Go/NoGo task from 30 abstinent male AUD and 30 healthy control participants with the objectives being (1) to characterize neuronal substrates associated with response inhibition using a rigorous nonparametric permutation-based fMRI analysis and (2) to determine whether these regions were differentially activated between abstinent AUD and control participants. A blood oxygen level dependent contrast analysis showed significant activation in several right cortical regions and deactivation in some left cortical regions during successful inhibition. The largest source of variance in activation level was due to group differences. The findings provide evidence of cortical substrates employed during response inhibition. The largest variance was explained by lower activation in inhibition as well as ventral attentional cortical networks in abstinent individuals with AUD, which were not found to be associated with length of abstinence, age, or impulsiveness. Inhibition, working memory, and cognitive flexibility are three core dissociable executive function (EF) components ,2,3. HigAlcohol use disorder (AUD) is a clinical/mental health condition resulting from heavy, chronic, and maladaptive use of alcohol and is characterized by cognitive, affective, and social impairments shown to be related to its adverse effects on brain structure and function ,13,14,15Among the cognitive processes adversely affected by alcohol, impairments in inhibitory processing are thought to be responsible for impulsiveness and disinhibitory behaviors, considered to be the cardinal characteristics of externalizing psychiatric disorders such as AUD . TherefoUntil recently, a dominant view in the functional magnetic resonance imaging (fMRI) literature was the “right frontal” hypothesis, which proposes that response inhibition activates a network of regions within the right frontal brain areas, in which right inferior frontal cortex (rIFC) is a key component . Most studies that have reported persistent inhibitory impairments despite long periods of abstinence from alcohol have used neuropsychological tests ,22,23,24Most fMRI studies have used Statistical Parametric Mapping (SPM) for Blood-Oxygen-Level-Dependent (BOLD) analysis. SPM requires data to fulfil parametric assumptions, which often is difficult to achieve in biophysical systems such as the brain  and may It is, therefore, clear that literature involving brain regions responsible for different types of inhibition, the adverse effects of heavy and maladaptive alcohol use on inhibitory processes, and associated brain regions are challenged by myriad complexities, ranging from the effects of alcohol, its quantity, clinical conditions, course of illness, and recovery, on one hand, and methodological differences and analytical limitations on the other, e.g., . While iage = 41.42 years, SD = 7.31) and 30 healthy controls . Recent studies on the same sample can be referred to for further details  and negatively associated with the Go performance accuracy  but not significantly correlated with the NoGo performance accuracy  when the entire sample was assessed. However, none of the correlations with age were found to be significant for either group separately, except for average reaction time of the controls . BIS-11 impulsiveness scores and length of abstinence were not found to be significantly correlated with any of the BOLD activation outcome variables and, therefore, are not reported.Data presented in this study is included as a 3; a total of p < 0.05, p < 0.01, and p < 0.001. The cluster signs and sizes, MNI coordinates of peaks/troughs, and corresponding brain regions are given in Thresholding the group-level BOLD response map at levels U test showed that the first PC scores , whereas examining the next nine PC scores r(58) = 0.20, p < 0.132], suggesting age did not contribute to the variance explained by the first PC score (The direction of the first eigenimage response  contribuarse PCA . In thisThe main findings of the present study are as follows: (1) the nonparametric fMRI analysis of BOLD responses across both abstinent AUD and control groups on successful NoGo trials showed cortical activation in the bilateral anterior insula, right inferior, middle, and medial frontal gyri, which include the pre-SMA and paracingulate cortical regions, the right superior parietal lobule, and the middle temporal gyri a; (2) th regions a, inferi regions b, superi regions c. Age waBased on the results of fMRI studies examining neural correlates of response inhibition conducted in the last two decades, several models of inhibitory control have been proposed . AmoHampshire and colleagues, on the other hand, proposed that there are no inhibitory modules in the frontal lobe. They presented response inhibition as an example of a broader class of control processes ,31,32,33In the context of these hypotheses of inhibitory functions and their brain substrates, the NoGo activation findings across groups in the present study tend to conform more to the common network mechanism model proposed by Hampshire and colleagues. While findings of predominantly right frontal brain area activation  drawn from the sample used in the present study. The results were largely consistent with the main analysis, and the trends and directions of mean differences and associations were replicated. However, given the crucial importance of age-related brain changes in AUD, it is important that future studies account for these confounding factors and confirm the current findings with age-matched designs. Similarly, the effect of the level of educational attainment and appropriate measures of intelligence need to be controlled for in future studies to avoid possible model-fitting errors and to confirm the current findings, as there are equivocal findings on the effect of level of educational attainment on brain structure and function after controlling for IQ at age 11, e.g., [Sex differences in functional brain activation patterns as well as in alcoholism are well documented in the literature. The present study involved males and, therefore, the findings of the present study may be generalized accordingly. It is, however, important to conduct similar studies in females to confirm findings. Age was not found to be associated with the first PC score 1, e.g., . While wIn summary, the findings of right fronto-parieto-temporal and ventral attentional network activation during response inhibition obtained in the present study seem to support the common network mechanism hypothesis. Abstinent individuals with AUD have significantly lower positive BOLD responses during response inhibition when compared to controls, mainly in the right fronto-parieto-temporal cortical regions of the brain, except for in the bilateral anterior insula. Our findings provide evidence of neural substrates of inhibitory processing deficits observed in abstinent individuals with AUD. The negative correlation of age with overall behavioral performance on the task suggests that the association may not be attributed to the effect of AUD. On the other hand, no significant correlation of age with the first PC score that captures the variance due to different levels of activation between abstinent individuals with AUD and control groups is indicative of low BOLD related activation in the fronto-parieto-temporal cortical regions that may be attributed to the persistent adverse effect of AUD.