Streptococcus thermophilus. We further showed that rsfAFP interacts with the membrane and ice crystals to cover the outer layer of cells, forming a dense protective layer that maintains the physiological functions of S. thermophilus under freezing stress. These findings provide the scientific basis for using rsfAFP as an effective antifreeze agent for lactic acid bacteria cryopreservation or other frozen food.Cryogenic machining is one of the most commonly used techniques for processing and preserving in food industry, and traditional antifreeze agents cannot regulate the mechanical stress damage caused by ice crystals formed during recrystallization or thawing. In this study, we successfully developed an express system of a novel recombinant snow flea antifreeze peptide (rsfAFP), which has significant ice recrystallization inhibition ability, thermal hysteresis activity and alters ice nucleation, thus regulating extracellular ice crystal morphology and recrystallization. We showed that rsfAFP improved the survival rate, acid-producing ability, freezing stability, and cellular metabolism activity of  Cryopreservation is one of the most effective techniques for food storage2. However, frozen food continuously suffers from the damage caused by ice crystals growth or recrystallization, which damages the cell and tissue structure, so that weight loss, discoloration, protein denaturation, and nutrient loss of the products during freezing or freezing storage3. Moreover, cell-based frozen foods, such as freeze-dried probiotics are susceptible to mechanical damage caused by ice crystals, which eventually leads to reduced cell viability4. Therefore, an antifreeze agent is inevitably for frozen food to reduce mechanical damage caused by ice crystals during cryogenic machining or cryopreservation. At present, skim milk, sugars and phosphates are the most widely used antifreeze agents for the cryopreservation of food, while glycerol and dimethyl sulfoxide (DMSO) are typically used for cells5. Although very successful and widely used, these antifreeze agents are not the perfect solutions since they cannot regulate the formation of ice crystals during recrystallization or thawing. The mechanical stress damage of the ice crystals formed during recrystallization or thawing is even greater than during the freezing process6. In addition, traditional commercial antifreeze agents like sugar and phosphates will bring sweet taste, high salt content, and high calorie, while organic solvents like glycerol and DMSO, can potentially have cytotoxic effects and cause irreversible damage to cells7. Therefore, studies are needed to investigate innovative cryopreservation methods.Due to the high-water activity in fresh foods, chemical reactions and microbial growth are prone to occur, leading to food deterioration8. Thermal hysteresis activity (THA), ice recrystallization inhibition (IRI) ability, and ice nucleation isomerization ability are key properties of AFPs12, making them promising novel cryoprotectants for application in frozen foods, cryobiology, or biological materials16. However, the large-scale applications of AFPs purified from natural organisms are difficult due to low yield and high purification costs3. Recently a glycine-rich AFP has been isolated from Canadian snow fleas17, and its molecular structure was reported by Pentelute et al.18 and Treviño et al.19. The snow flea AFP belongs to the family of hyperactive insect AFPs20, and its THA is 1–2 orders of magnitude higher than most AFPs found in plants and fish21, and could potentially be an excellent antifreeze agent for the cryopreservation of lactic acid bacteria (LAB)22. In the past, the research on snow flea AFP mainly focused on the interaction mechanism with ice crystals24. However, the protective mechanism of AFP on microorganisms under freezing stress is rarely studied.Antifreeze proteins (AFPs) are a unique class of proteins that enable organisms to survive in freezing and subfreezing environmentsB. subtilis is a non-pathogenic microorganism with high safety and can be used in the food and medicine industry25. However, to our knowledge, there have been no reports about the expression and production of hyperactive snow flea AFP in Bacillus subtilis. Moreover, the cryoprotective mechanism of snow flea AFP on microorganisms is still unclear. Therefore, the present study attempts to express and produce a recombinant snow flea antifreeze peptide (rsfAFP) using B. subtilis (WB800N). The effects of rsfAFP on ice crystals morphology, TH, and IRI were investigated. Furthermore, the cryoprotective effect of rsfAFP on the physiological functions of Streptococcus thermophilus under freezing stress, and the mechanism by which rsfAFP interacts with these cells and ice were also studied. Findings from this work will provide new insights into the mechanism by which rsfAFP regulates some cell physiological functions and the formation of ice crystals under freezing stress. In addition, rsfAFP as an antifreeze agent has great potential value in food processing and storage.Currently, the expression of heterologous proteins in bacteria is one of the main methods to overcome the low yield and high purification costs for the production of natural proteins. B. subtilis strains by electroporation. Recombinant B. subtilis clones were identified by PCR and the results are presented in Fig. DNA sequence and the corresponding translated peptide sequence is shown in Supplementary Fig. β-d-1-thiogalactopyranoside (IPTG). The target protein was further isolated and purified by affinity Ni-Charged resin chromatography with a final purity of over 95%. SDS-PAGE displayed a clear single target protein band ) in the control groups, rsfAFP showed a significant TH exothermic peak , 1.0 mg/mL skim milk (65.39%), and 15% glycerol (79.80%) were used as cryoprotectants. During the freezing process, the extracellular water was first freezing into ice. The pressure difference between the inside and outside caused dehydration of the cell, increasing the intracellular electrolyte concentration. This elevated intracellular solute led to the denaturation or inactivation of sensitive enzymes as well as certain proteins, affecting the metabolic activity of cells. The metabolic vitality of S. thermophilus with different cryoprotectants is shown in Fig. S. thermophilus in the rsfAFP group was greatly improved when compared with the commercial cryoprotectant groups.The survival rate of cryopreserved S. thermophilus cells, so the cryoprotective effect of different cryoprotectants on bacterial cells can be reflected by measuring the acid production. The acid production and the growing stability of S. thermophilus with different cryoprotectants after freezing stress are shown in Fig. S. thermophilus significantly improved the freezing stability. After 14 days under freezing conditions, the survival rate in the PBS group decreased by 97.88%. The survival rate in the glycerol group, the most commonly used commercial cryoprotectant (glycerol group), still decreased by 61.57%. However, the survival rate of the rsfAFP group decreased only by 38.50% under the same storage conditions.The acid production capacity is related to the metabolic activity of S. thermophilus cells without cryoprotectants were significantly damaged during frozen storage, and most of the cells had no intact cellular structure after freezing stress treatment. In addition, due to the rupture of the cell structure, the contents of S. thermophilus cells leaked out and deposited on the surface of the cells, making the cells adhere to each other without clear boundaries , AFPs, with the advantages of less toxicity and non-cell membrane penetration, are becoming the most promising cryoprotectants for the cryopreservation of cells38. The regulation effect of insect AFPs on ice crystals conforms to the “Janus” effect, which includes non-ice-binding faces (NIBF) and ice-binding faces (IBF)39. The IBF is attributed to the orderly arrangement of methyl and carboxyl groups on AFPs to form an ice-like hydration layer. The NIBF is a disorderly arrangement of a large number of charged side chains and hydrophobic side chains, and the interface water on the IBF is disordered. During the freezing process, the IBF of AFPs will be adsorbed on the growth interface of ice crystals, and the NIBF is exposed to water, disrupting the orderly arrangement of water at the interface and inhibiting the formation of ice nucleus. The IBF and the NIBF work together to inhibit the growth of ice crystals39. Snow flea antifreeze protein has a Polyproline type II Helix conformation19, which also includes IBF and NIBF . The restricted endonuclease BamHI/XbaI and DNA purification kit were purchased from SanGon Biotech Co., Ltd . S. thermophilus used to investigate the damage caused by freezing stress was obtained from Shanghai Jiao Tong University . Other analytical reagents used in this work were purchased from Sigma-Aldrich, St, Louis, MO, USA.18, which includes 175 amino acids. The nucleotide sequence was optimized according to B. subtilis WB800N codon preference, and the cDNA sequence encoding for rsfAFP was synthesized by Kingsley Biotech. Co., LTD. . This rsfAFP gene was double-digested with BamHI (ggatcc) and XbaI (tctaga) and cloned into pHT43 vector, which was renamed as pHT43-SF-P, and transformed into WB800N by electroporation. The pHT43-SF-P plasmid was extracted from WB800N and confirmed by PCR.rsfAFP is a peptide consisting of two repeats sequences of 6.5 kDa snow flea AFPB. subtilis medium (5 mL) for 12 h at 30 °C, and then transferred to 600 mL of B. subtilis medium supplemented with erythromycin resistance  for 3 h at 37 °C. When the OD600 reached 0.8–1.0, the medium was induced with 1 mM IPTG at 37 °C for 4 h. Cells were collected by centrifugation , lysed in native buffer, centrifuged (C1) and the native protein extract recovered in the supernatant. The pellet harvested from C1 was resuspended in denaturing buffer (Urea 8 M) and centrifuged . The supernatant contained the denatured protein extract, semi-purified from the culture medium. The proteins extracted from the native buffer, denaturing buffer and culture medium were analyzed by Tricine-SDS-PAGE and western blot using methods previously established by Zhang et al.22.WB800N competent cells (previously stored in a glycerin tube) were sub-inoculating in 2+ resin. Briefly, a slurry of affinity versus His-tag was packed in a Ni2+ resin column (10 × 2.5 cm) and equilibrated with 5-column volume of equilibrating buffer and TBS buffer . After harvesting the culture medium, 200 mL was loaded into the column. The column was washed 3 times with TBS buffer with 0, 30, and 50 mM imidazole, and the fractions were identified as W1, W2, and W3, respectively. The bound proteins were eluted using gradient elution with the same buffer containing 200 mM and 400 mM imidazole at a flow rate of 0.5 mL/min, the recovered fractions labeled E1 and E2, respectively. The eluate obtained from the culture medium was analyzed by SDS-PAGE. Briefly, 4 μL of the eluate was mixed with an equal volume of 2× sample buffer solution , and then incubated at 95 °C for 5 min. The samples were then quantitatively by SDS-PAGE grayscale analysis.The recombinant target protein was purified by affinity versus His-tag on Ni42. A Mascot MS/MS Ion Search was conducted with MS/MS data from Swiss-Prot and UniProt-Avena-1 databases.Nano LC-MS/MS was applied to identify the rsfAFP. The rsfAFP band in the SDS-PAGE was excised and then submitted to in-gel digestion with trypsin. The peptide sequencing was carried out and its molecular weight was measured using Nano LC-MS/MS. The detailed protocol for Nano LC-MS/MS analysis was as described by Liu et al.43.The THA of rsfAFP was evaluated with a 8500 DSC with Proteus Thermal Analysis software , the detailed procedures having been previously described46. Briefly, a small drop (3 μL) of pH 7.4 TBS buffer  with or without rsfAFP (0.1 mg/mL) was placed between two circular glass coverslips. The coverslips were placed in the chamber of the cooling stage, and then rapidly cooled from room temperature to −50 °С at a rate of −10 °C/min and held for 5 min. The microscopic images of a sample at −50 °С were recorded through CCD camera, and then gradually the temperature was increased from −50 °С to −14 °С at a rate of 5 °C/min. The sample was then held at −14 °C for 1 min and the temperature increased to −12 °C at a rate of 1 °С/min. Afterward, a cycle of temperatures between −14 °C and −12 °С at a rate of 1 °C/min was performed 5 times, and the microscopic images were recorded for each sample.IRI activities of rsfAFP were determined with a polarizing microscope equipped with an optical CCD camera  using a cold stage  with minor modifications from the previously described methods47. Briefly, 0.5 nL of rsfAFP (0.1 mg/mL) was injected into a temperature-controlled sample holder full of silicone oil using a microsyringe and quickly frozen to −20 °C, and then slowly warmed to the melting temperature (Tm). Once a single ice crystal appeared, the temperature was slowly decreased to the freezing temperature (Tf) at which ice started to grow. The temperature was controlled within a margin of error of 0.01 °C, and throughout, the manipulations were protected by nitrogen  to prevent air liquefaction to form a condensate. The evolution of an ice crystal was recorded by an optical CCD camera.The morphology and growth rate of a single ice crystal were observed using a nanoliter osmometer with a stereomicroscope. The detailed protocol for the Nanoliter osmometer experiments was provided in the method described by Geng et al.S. thermophilus was previously sub-cultured in 20 mL of M17 broth  at 200 rpm and 37 °C for 18 h, followed by inoculation in 20 mL of M17 broth  and culture at 200 rpm 37 °C for 4 h until the cell density OD600 reached 1.0. Cells were harvested during the mid-log growth phase by centrifugation (6000 rpm for 10 min at 4 °C) and washed twice with 20 mM PBS (pH 7.4). The population of S. thermophilus in the suspension was approximately 108 CFU/mL. To determine whether antifreeze agents effectively played a cryoprotective role in S. thermophilus after cold stress, the bacterial culture was diluted in PBS (pH 7.4) with 10 folds gradient dilution. 30 μL of diluted cells were added to 270 μL rsfAFP (0.1 mg/mL) in a 1.5 mL cryovials. Commercial cryoprotectants, including 1.0 mg/mL sucrose, 1.0 mg/mL skim milk and 15% glycerol (v/v), were used as positive groups. The phosphatic buffer solution  was used as a negative group. Aliquots (0.3 mL) of each bacterial suspension were frozen at -20 °C for 24 h and samples were subjected to 2 cycles of freeze-thaw in a 2-hour interval.Briefly, S. thermophilus cells in each sample after cold stress were analyzed using the OD600 rapid detection according to the methods of Chen et al.5. The survival rate was expressed as ODA/ODB × 100, where ODA was the number of viable cells after freezing stress, and ODB was the number of viable cells before freezing.The viability of the S. thermophilus in each sample before and after cold stress was evaluated according to the method described by Meneghel et al.33.The acidifying activity of S. thermophilus was investigated according to Chen et al.5 using INT Analysis Kits.The metabolic activity of S. thermophilus were freeze-dried using a lyophilizer  using a freezing/thawing protocol with or without cryoprotectant. The microscopy of S. thermophilus was performed by SEM  after a small amount of gold was plated onto the samples.The cells of 5 Briefly, the samples  were mixed with potassium bromide 1 to 100. The spectra of each sample were analyzed using the OMNIC 8.2 software .The interactions between rsfAFP and the cell membrane were analyzed using egg yolk lecithin as a model according to the methods reported by Chen et al.S. thermophilus after freezing stress were dropped into grids . Then vitrification was performed on the samples with a Vitrobot (FEI). These samples were observed under a Talos F200C G2 TEM (FEI). The images were captured with a CCD camera (Ceta 4 K*4K) under low dose conditions. A concentration of 0.1 mg/mL rsfAFP was used for the Cryo-TEM analysis and 20 mM PBS was used as a negative control.Cell solutions (5 μL) of P < 0.05 was considered significant.Data were analyzed by SPSS 17.0  and reported as mean ± standard deviation from at least three to four independent experiments. Duncan’s multiple range test was used for statistical analysis and Supplementary Material


Histone H3 lysine 9 di-methylation H3K9me2) is an epigenetic repressive histone modification that was found at aberrant states in Alzheimer’s disease (AD) patient samples . The add is an epWe first tested whether G9a/GLP inhibition could rescue long-term potentiation (LTP) deficits in CA1 area of hippocampal slices of APP/PS1 mice  induced by weak stimulation (WTET) is stabilized into a long-lasting LTP (late-LTP) by a strong stimulation (STET) in another independent pathway within a specific time frame –5. In APThen we perfused APP/PS1 hippocampal slices with G9a/GLP inhibitors  in the AD-like condition. This contradicts prior studies reporting upregulation of synaptic genes and synaptic transmission in Aß-impaired cortical neurons and familial AD (FAD) mice in vivo . This diSince G9a/GLP inhibition supported late-LTP maintenance through facilitating protein synthesis in response to plasticity induction, we tested whether G9a/GLP inhibition could also facilitate chemically induced LTP by using the chemical forskolin, an activator of adenylyl cyclase, which is involved in late-phase LTP and AD pathophysiology .Bath application of forskolin  is typically sufficient to induce potentiation in wild-type hippocampal slices . In contFurthermore, we measured de novo protein synthesis after forskolin application using the SUnSET (surface sensing of translation) assay. Co-application of BIX or UNC with forskolin significantly increased de novo protein synthesis in hippocampal area CA1 (Fig. Recent studies suggest that high-frequency stimulation-induced LTP and forskolin-induced LTP recruit distinct molecular pathways . Here, GOpen questions remain. Can G9a/GLP inhibition lead to the downregulation of repressive histone mark H3K9me2, thereby upregulating transcription? Are other functions of G9a/GLP, such as methylation of non-histone proteins, altered in AD? More studies are needed to clarify the precise mechanism underlying the G9a/GLP inhibition-induced upregulation of activity-dependent expression of PRPs in the APP/PS1 hippocampus.Additional file 1. Materials and Methods.


The most rapid global sea-level rise event of the last deglaciation, Meltwater Pulse 1A (MWP-1A), occurred ∼14,650 years ago. Considerable uncertainty regarding the sources of meltwater limits understanding of the relationship between MWP-1A and the concurrent fast-changing climate. Here we present a data-driven inversion approach, using a glacio-isostatic adjustment model to invert for the sources of MWP-1A via sea-level constraints from six geographically distributed sites. The results suggest contributions from Antarctica, 1.3 m , Scandinavia, 4.6 m (3.2–6.4 m) and North America, 12.0 m (5.6–15.4 m), giving a global mean sea-level rise of 17.9 m (15.7–20.2 m) in 500 years. Only a North American dominant scenario successfully predicts the observed sea-level change across our six sites and an Antarctic dominant scenario is firmly refuted by Scottish isolation basin records. Our sea-level based results therefore reconcile with field-based ice-sheet reconstructions. Meltwater Pulse 1A was the most rapid global sea-level rise event during the last deglaciation, but the source of the freshwater causing this rise is debated. Here, the authors use a data-driven inversion approach to show that the North American and Eurasian Ice Sheets were the dominant contributors. It was driven by the collapse of vulnerable ice sheet sectors, and was concurrent with rapid Northern Hemispheric warming and disruptions in oceanic and atmospheric circulation4. The ice-ocean-climate feedbacks operating during this period are not well understood largely due to a lack of consensus on the sources of MWP-1A6, which, in turn, were likely to be a key driver in stimulating rapid deglacial climate change9.Meltwater Pulse 1A (MWP-1A) was the largest and most rapid global sea-level rise event of the last deglaciation, characterised by ∼20 m global mean sea-level (GMSL) rise within 500 years12. This approach is restricted by large uncertainties regarding palaeo ice-sheet boundary conditions, climatic conditions, and ice-sheet model parameters. Ice histories derived using this approach do not always match sea-level observations11. Conversely, the second method seeks to reconcile ice-sheet change with spatially variable records of sea-level change using a glacio-isostatic adjustment (GIA) model15, an approach often termed sea-level fingerprinting17. The primary limitation of sea-level fingerprinting is the spatial and temporal scarcity of sea-level records across MWP-1A. Commonly, only three sites are used , resulting in an under-constrained problem and strongly non-unique solutions18. Other techniques, e.g., analysis of the oceanographic20 or isotopic21 effects of specific meltwater sources, add further constraints, but the primary source of MWP-1A remains controversial with three ice sheets proposed as the major contributor, namely, the North American Ice Sheet, including Greenland (NAIS)22, the Antarctic Ice Sheet (AIS)13 and the Scandinavian and the Barents Sea Ice Sheet (henceforth denoted together as SIS)23.Two major techniques have been used to constrain the sources of MWP-1A. The first uses physics-based models, constrained by field-based glacio-geological evidence, to simulate regional ice sheet change during the last deglaciation13, our results show good agreement with most recent regional ice-sheet reconstructions26, and may lead to a reconciled solution of MWP-1A sources.In this work, we combine a data-driven inversion approach with sea-level fingerprinting to simultaneously determine probability distributions for the magnitude and sources of MWP-1A based on six sea-level sites spanning the far, intermediate and near field. The results indicate a 17.9 m  global mean sea-level rise during MWP-1A, which consists of a dominant NAIS contribution , a substantial SIS contribution (20-35%) and a minor AIS contribution . Unlike previous sea-level fingerprinting studies18, namely, coral reef records from Tahiti27 and Barbados30 and sedimentary indicators from Sunda Shelf31. The geographical distributions of these sites do not permit the separation of meltwater sources from the AIS and SIS 18. This is challenged by a recent chronological reinterpretation of the SIS ice history that proposes the SIS was a major MWP-1A contributor23. Such a large SIS contribution has not yet been tested using sea-level fingerprinting.To robustly fingerprint the sources of MWP-1A, three main challenges need to be overcome. First, the above-mentioned non-uniqueness problem. Previously, only three sea-level sites showed sufficient temporal resolution for fingerprinting studies across MWP-1A37, and isolation basin stratigraphies from Northwest Scotland40  at Hydrographer’s Passage (HYD) and Noggin Pass (NOG)d40 Fig. . Where n17, also known as the elastic component of RSL change18. Assuming the NAIS, AIS and SIS were the only contributors to MWP-1A, the global pattern of RSL change caused by melt from these ice sheets can be identified as a linear combination of three spatially variable sea-level fingerprints . The first only affects our Northwest Scotland data. RSL change here contains a large local GIA signal associated with changes to the British-Irish Ice Sheet (BIIS). We determine a local GIA correction for all these SLIPs to isolate the GIA signal associated with non-local ice sheet change41 . We calculate chronological probability distributions following the methodology of Hibbert et al.43, accounting for multimodal, asymmetric 14C age distributions and age reliability screening (see Methods).The standardised SLIPs constrain local MWP-1A magnitude at each site. We use a conservative time window of 14.65-14.00 ka BP to select SLIPs at each site that clearly mark the initiation and termination of MWP-1A, enabling us to capture the full magnitude of MWP-1A sea-level rise. Ideally, only records with mean ages within this window will be selected, but for sites with insufficient temporal coverage we include records whose 2σ age error bars extend into it. We estimate local MWP-1A magnitude from the SLIPs using a MC linear regression method, which captures any asymmetric depth and age uncertainties of different types of sea-level indicators by randomly sampling their uncertainty distributions. We use two approaches to represent indicative depth distributions of coral sea-level indicators; an empirically-derived distribution from modern coral analogues  and a uniform distribution, using palaeo-water depths from original publications (the uniform scenario)MC linear regression, using randomly selected data points and a weighted least square method, determines the local MWP–1A RSL rise rate at each site (see Methods). We convert this to local MWP-1A magnitude by multiplying by the assumed duration of MWP-1A (500 years in this study). We exclude, as implausible, regressions producing a reverse slope . Repeating this process 20,000 times (excluding the implausible iterations) produces distributions of local MWP-1A magnitude for each site Figs. . Because44; Supplementary Fig. 15. We recommend both viscous signals be considered in future meltwater source inversion studies.The viscous component of RSL change has a significant effect on local sea-level change during MWP-1A. Far-field localities  will have experienced local sea-level fall associated with the redistribution of water to regions experiencing peripheral bulge subsidence and due to the ocean load-induced continental levering effect (we refer to the combined effect as ocean siphoning46). Most of the other Tahiti coral samples were identified as Porites sp.1. Modern analogues  suggest a bimodal depth-habitat distribution concentrated at 0–15 m and 40–50 m43. This bimodal empirical distribution was generated from a global compilation, and given insufficient modern observations at Tahiti, we consider it a maximum vertical depth range for this species. Comparatively, the palaeo-water depths derived from coral-algal assemblages (our uniform scenario) suggest depths of 0–10 m or even 0–20 m47. Therefore our empirical scenario yields a larger uncertainty range for the MWP-1A magnitude  than the uniform scenario 52, with only minor uncertainty associated with Earth parameters  within a 95% confidence range of 3.9–17.3 m Fig. . The maj53 to optimise the contribution of the NAIS, AIS and SIS to MWP-1A based on sea-level fingerprints generated using realistic deglaciation geometries55 , which consists of a dominant NAIS contribution of 5.6–15.4 m , a substantial SIS contribution of 3.2–6.4 m (20–35%) and a minor AIS contribution of 0–5.9 m (0–35%) with median values of 12.0, 4.6 and 1.3 m, respectively , similar to our estimate. We suggest a substantial SIS contribution is essential to reconcile the gap between regional ice-sheet reconstructions and global sea-level fingerprinting  of GMSL rise during this period. Rapid disintegration of the NAIS and SIS has been proposed to be consistent with the operation of ice-sheet saddle collapse62) and generated two sea-level fingerprints using the ICE6G_C model22. We solve for the contribution of these two NAIS sectors to MWP-1A separately along with the AIS and SIS. The results yield 4.4 m  and 8.7 m (0–16.8 m) contributions from the Western and Eastern NAIS, respectively, with little change to the AIS and SIS contributions . The impact on the inferred MWP-1A contribution from each ice sheet is <0.3 m . RSL change takes place due to changes in the shape of the solid Earth and the sea surface, with the latter being defined by the shape of Earth’s gravity field. Because the solid Earth behaves viscoelastically over the timescale of interest, RSL change at location φ can be divided into a component that reflects the instantaneous response of the solid Earth and sea surface to an influx of meltwater (∆RSLElastic) and a component that reflects ongoing perturbations to these surfaces due to past surface load change (∆RSLViscous):F(φ) terms denote ice-sheet-specific, site-specific sea-level fingerprint values. The term sea-level fingerprint describes the normalised elastic component of RSL change triggered by a given pattern of ice loss17. Fi(φ) is insensitive to the value of ESLi and the choice of Earth rheology69. Therefore, it can be calculated using existing ice sheet reconstructions and treated as a known parameter (see Sea-level fingerprint). The three ESL terms are the unknown parameters in our inversion. We assume that the NAIS, AIS, and SIS are the only contributors to MWP-1A, where any Greenland contribution is included in the NAIS.We solve for the meltwater contributions from the NAIS, AIS, and SIS that bestfit observations of RSL change across MWP-1A (∆RSLPreViscous(φ) and ∆RSLDurViscous(φ) are the changes associated with the viscous effects of ice melt prior to and during MWP-1A respectively (see Viscous component of sea-level change).The viscous component of RSL change can also be decomposed into two terms:Elastic at our six field sites we create a set of equations that can be inverted to yield the ESL contribution to MWP-1A from each of the three ice sheets considered here (see Inversion for MWP-1A sources).Substituting Eq. Elastic at each site by computing the fit to probability distributions of all viscous-corrected sea-level index points (SLIPs) at that site that lie within MWP-1A (see Monte Carlo linear regression). Prior to carrying out the linear regression, the SLIPs are also corrected for any local GIA effects  and any spatial gradient of RSL that exists between sites that are combined to estimate sea-level change at a single location . We assume the thermosteric contribution to RSL change during MWP-1A is negligible.Monte Carlo linear regression is used to estimate probability distribution of the ∆RSLPreViscous(φ) is controlled by ice melt prior to MWP-1A it can be calculated using an existing global ice model (see below). In contrast, ∆RSLDurViscous(φ) depends on the unknown ESL parameters, which makes Eq. Elastic(φ) at six sea-level sites (see main text) using a Monte Carlo linear regression method that assumes ∆RSLDurViscous(φ) is zero. (ii) Invert for the three ESL values using the ∆RSLElastic(φ) values calculated in step i (for the first iteration) or step v . (iii) Correct the bias within the inversion results using jackknife resampling (see details below). (iv) Calculate ∆RSLDurViscous(φ) using the bias-corrected ESL inversion from step iii (see details below). (v) Computing ∆RSLElastic(φ) using the ∆RSLDurViscous(φ) values obtained in step iv. (vi) Repeat step ii-v until convergence of ESL values has been achieved.Because ∆RSL71, which uses a gravitationally self-consistent theory that accounts for migrating shorelines and Earth rotational feedback75. The Earth is represented by a spherically symmetric, radially stratified , self-gravitating Maxwell body comprising an elastic lithosphere, and an upper and lower mantle extending to 670 km and from 670 km to the core-mantle boundary, respectively. The elastic and density structure of the Earth is derived from the preliminary reference Earth model76. GIA modelling is used to calculate sea-level fingerprints, the local GIA signal in Northwest Scotland, spatial sea-level gradients, and the viscous component of sea-level change.Sea-level change and the solid Earth response to changes in surface loading are computed using the CALSEA software package69, we use realistic melt geometries across MWP-1A from two recent regional ice-sheet reconstructions of Lambeck et al.24 and the BRITICE-CHRONO project  for the NAIS and SIS, respectively  they provide conservative estimates on ice history uncertainties. These ice models were combined with Earth parameters that reflect the rheological properties beneath the British Isles (denoted as near-field rheology). Specifically, we use elastic lithospheric thicknesses of 65, 72 and 80 km, upper mantle viscosities of 4, 4.5, 5, 5.5 and 6 × 1020 Pa s and lower mantle viscosities of 1, 1.5, 2, 3, 4, 5, 7 and 10 × 1022 Pa s. These ranges are constrained by previous GPS analysis and are largely independent of the assumed ice history80. We calculate the age-specific local GIA signal for each SLIP and subtract this from the original RSL reconstruction to give the non-BIIS GIA signal. The uncertainty for this procedure is considered within the inversion strategy by sampling the 360 local GIA correction values  and adding the standard deviation of the corrections to the original depth uncertainty in quadrature. After removing the local GIA signal, there is a distinct RSL jump recorded between 14.5 and 14.2 ka BP, which is consistent with the MWP-1A signal observed in far-field sea-level records . We apply a correction for this spatial gradient that enables us to determine the RSL change across MWP-1A at a single locality for each region. We quantify this gradient by testing 240 parameter combinations to incorporate uncertainties associated with ice history and Earth rheology. Specifically, we combine the two global ice models ICE6G_C22 and ANU2 with 120 different Earth models. The Earth models each have an elastic lithospheric thickness of 60, 72 or 90 km, an upper mantle viscosity of 1, 3, 5, 6 or 7 × 1020 Pa s, and a lower mantle viscosity of 0.1, 0.2, 0.3, 0.5, 0.6, 0.7, 0.9 or 1 × 1022 Pa s for ICE6G_C, or 0.7, 0.9, 1, 1.5, 3, 4, 5 or 7 × 1022 Pa s for the ANU model (we made different choices for lower mantle viscosity because the two ice models have different preference ranges). Because the local GIA signal in Northwest Scotland is removed separately we do not include the BIIS component of ICE6G_C and ANU when calculating the spatial gradient for Northwest Scotland to avoid a double correction. The ensemble mean of the 240 parameter combinations is used to determine the time-dependent data-specific spatial sea-level gradient  will be approximately linear over MWP-1A18, we quantify this linear rate by considering the viscous response to ice sheet change between the end of the last interglacial and the start of MWP-1A (14.65 ka BP). We assume no melting after 14.65 ka BP and calculate the linear rate of RSL change during the following 1,000 years  signal that is specific to the age and location of each SLIP, assuming the viscous contribution is 0 at the initiation of MWP-1A (14.65 ka BP). For all sites except Northwest Scotland we use the mean value derived from a 240-member GIA model ensemble, as described in the previous section. For Northwest Scotland, since the dominant viscous signal here relates to SIS-induced peripheral bulge subsidence , which primarily depends on the local rheology of the British Isles, we use the near-field Earth parameters described in the section on Northwest Scotland. Again, to avoid a double correction, we did not include the BIIS in the global ice model when calculating ∆RSLPreViscous(φ) for Northwest Scotland.To correct for the viscous signal across MWP-1A, we estimate the viscous contribution of RSL change to each SLIP, accounting for their specific age and location. Given that the viscous response to ice melt prior to MWP-1A (∆RSLDurViscous(φ) terms depend on the ESL values in Eq. Elastic(φ) (less than 10%), neglecting them will not significantly alter the inversion result during the first iteration. Beginning from the second iteration, we scale the ice melt geometries that are used to generate the sea-level fingerprints  is then calculated assuming a linear rate of ice melt throughout MWP-1A. A range of Earth models are used, as for ∆RSLPreViscous(φ) above, and the ensemble mean of each set is used to determine the ∆RSLDurViscous(φ) terms, accounting for the age and location of each SLIP  in Eq. 43.To quantify the elastic-induced local MWP-1A magnitude at each site . Where possible, we use a spatially variable local coral depth-habitat distribution instead of a global compilation for each coral species. This method is denoted as the empirical scenario. Alternatively, we use the coral palaeo-water depth determinations  from the original publications for different coral species. For this method, we assume a uniform distribution, in that the indicator may occur equally anywhere within the given range43. We denote this method as the uniform scenario. For non-coral SLIPs , we use the indicative range or facies formation range as determined by the original authors, which is also assumed to be uniformly distributed. Furthermore, when sampling the depth distributions of all SLIPs, we considered the error distribution associated with each of the GIA corrections described above and elevation measurement uncertainties due to coring, levelling and tectonic correction if necessary. We exclude any data explicitly stated as not in situ by the original authors.For the coral sea-level indicators, we test two methods for representing their indicative depth distributions. First, we use an empirically-derived taxon-specific depth-habitat distribution for each coral-based sea-level indicator81 to obtain the calibrated age probability distribution by recalibrating the conventional radiocarbon age and uncertainty using the latest calibration curves: IntCal2082 for Northern Hemisphere terrestrial samples; SHCal2083 for Southern Hemisphere terrestrial samples and Marine2084 for all marine samples. For marine samples, we apply appropriate, updated  local marine reservoir corrections . For all other samples, U-series ages have been recalculated where necessary, assuming a closed system with the latest decay constants85. Only U-series ages passing the following age reliability screening criteria  are considered. A normal distribution is adopted for U-series ages, whereas our radiocarbon ages use the full age probability distribution43. For any replicated ages, we use the inverse weighted mean value/distribution of each replicate group.The chronological probability distributions depend on the method used to date each SLIP. For samples that are radiocarbon dated, we use OxCal version 4.4w for each SLIP is calculated byy and σx are standard deviations estimated from the depth and age distributions, respectively, and dy/dx is the gradient of global sea-level change at the sampled age obtained from Lambeck et al.2. The last term is used to convert the effective contribution of age uncertainty into depth uncertainty. Regressions that produce a reverse slope  are excluded as implausible. The process is repeated 20,000 times (excluding the implausible iterations) to produce a distribution of local MWP-1A sea-level rise rates for each site. Lastly, the local MWP-1A magnitude is obtained by scaling this linear rate to the MWP-1A duration. We use a 500-year MWP-1A duration in this study since it leads to more comparable results with the MWP-1A magnitude from regional ice-sheet reconstructions, but our inversion results can be linearly scaled to any assumed MWP-1A duration for comparison.For each of our six sites, we use MC simulation to randomly sample the age and depth distributions of each selected SLIP, and for each MC sampling, we use a weighted least square method to compute an optimum straight line to fit the randomly sampled points. The slope of this line is the averaged RSL rate across MWP-1A (units m/ka), which is assumed to be temporally linear throughout MWP-1A. Note that, since the lack of temporal resolution and uneven temporal distribution of sea-level data prohibit our ability to capture the maximum rates of sea-level rise at each site, our results should be interpreted as the averaged rate of RSL change at each site across our MWP-1A time window (14.65–14.0 ka BP). Within each weighted least square calculation, the weighting factor 36; see their Fig. 4a). In order to constrain RSL at the end of MWP–1A, we adopt a data extrapolation approach that uses the large number of SLIPs between ∼13.0 and 12.0 ka BP at the two sites to extrapolate RSL backwards in time. To ensure the accuracy of the data extrapolation we only use SLIPs that pertain to a shallow, high-energy/exposed reef edge environment , i.e. SLIPs which have a relatively small depth uncertainty. The data extrapolation was implemented using the same MC linear regression method as above, in combination with the uniform scenario ) estimated above, the inversion for MWP-1A sources can be made by identifying the optimum ESL parameters in Eq. 53 since we assume that ice sheet growth during MWP-1A would be implausible. The weighting factor for each site is given by w(φ) = 1/σ(φ)2 where σ(φ) is the standard deviation estimated from the local MWP-1A magnitude distributions.Based on the site-specific elastic-induced local MWP-1A magnitude distributions  was created by leaving the ice history prior to MWP-1A unchanged, but assuming that ice loss during MWP-1A followed the magnitude and spatial pattern of ice loss represented by the mean of our inversion result: 13.1 m NAIS, 3.3 m SIS, and 1.5 m AIS. The rate of ice melt during MWP-1A was assumed to be linear. In the original ANU model, there is not enough ice for the SIS to melt 3.3 m during MWP-1A. We therefore decrease the rate of ice melt between 16.5 and 14.5 ka BP (the latter is the time of MWP-1A initiation in the ANU model) to ensure there is enough ice to provide 3.3 m ESL melt during MWP-1A (Fig. We modified the deglaciation history during MWP-1A in the ANU ice model-1A Fig. . The synSupplementary InformationPeer Review File


The successful synthesis of the BCD-modified silica was confirmed by FT-IR and TGA. Using contact angle measurements, BET analysis and SEM characterization, a possible formation mechanism for the generation of silica particles bearing BCD derivatives on their surface was highlighted. The obtained modified silica displayed the capacity to remove bisphenol A (BPA) from wastewater due to the presence of the BCD moieties on the surface of the silica. The kinetic analysis showed that the adsorption reached equilibrium after 180 min for both materials with qe values of 107 mg BPA/g for 2-BCD-OHSiO and 112 mg BPA/g for 2-BCD-NH2SiO. The process followed Ho’s pseudo-second-order adsorption model sustaining the presence of adsorption sites with different activities. The fitting of the Freundlich isotherm model on the experimental results was also evaluated, confirming the BCD influence on the materials’ adsorption properties.This study presents the synthesis of silica particles bearing two beta-cyclodextrin (BCD) (beta-cyclodextrin-BCD-OH and diamino butane monosubstituted beta-cyclodextrin-BCD-NH Cyclodextrins (CDs) are a class of three-dimensional 3D) cyclic oligosaccharides composed of 6, 7, or 8 D-glucopyranosyl units linked by α-D-1→4) bonds with amphiphilic characteristics given by the hydrophilic surface and the hydrophobic internal hollow [→4 bonds D cyclic In terms of air, soil, or water decontamination, CDs’ properties were exploited by creating host–guest inclusion complexes based on the hydrophobicity of the inner cavity that facilitates the encapsulation of targeted molecules . DiffereMany CDs-based polymers have been used to remove organic pollutants from contaminated waters for higher absorption efficiency, but recycling procedures were proven to be limited sometimes since the structural integrity of the polymer chains gave rise to problems after repeated recovery cycles . On the Ever since bisphenol A (BPA) was discovered as a synthetic estrogen in 1890, its application as monomer in the plastic industry was intensively developed in food packaging, epoxy and polyacrylate resins, unsaturated polyesters, polysulfones, or polycarbonate plastics . AlthougDifferent methods have been applied for the removal of BPA from contaminated environments involved in membrane separation processes, advanced oxidation procedures, or biological treatments ,34. The 2/g) or porous structures depending on the synthesis method [2) comes from the possibility to modify its surface with functional groups that enhance the adsorption process of targeted molecules inside the CDs’ cavities. For instance, byproducts from the incomplete fuel combustion, namely, polycyclic aromatic hydrocarbons (PAHs) were removed from water using two types of CDs (hydroxypropyl-β-cyclodextrin (HPBCD) and β-cyclodextrin (BCD)) and tetraethyl orthosilicate (TEOS) as SiO2 precursor [2 nanoparticles [2 composite by the surface functionalization of SiO2 with BCD that was used to remove methyltestosterone hormone from contaminated waters. Methacryloyl-BCD was used also to functionalize SiO2 NPS to remove p-nitrophenol from contaminated waters, with kinetic studies confirming that fast adsorption processes occur in the case of BCD-SiO2-based composites and that the adsorption mechanism is determined by the concentration of the organic pollutants and the structure of the hybrid CDs-based material [Silica-based materials have been used in numerous applications including pharmacy, medicine, catalysis, food, paints, and coatings ,42,43,44s method . Anotherrecursor . The silarticles . Anotherarticles , involvematerial .2 particles for enhanced BPA removal from contaminated water. The BCD-SiO2 derivatives are aimed to improve the interaction between the pollutant and the adsorbent. Therefore, our goal was to provide a facile synthesis route to access SiO2-BCD materials and to investigate the maximum complexation capacity of the BCD-SiO2 hybrid composites towards BPA. Thus, adsorption kinetics, complexation mechanism, and isotherms were taken into consideration for our study.Thus, the novelty of our work involves the use of BCD-modified SiOPyridine (Aldrich) was dried on molecular sieves of 3Å. The β-cyclodextrin (BCD-OH)  was vacuum dried before use for 24 h. Glucose (Aldrich anhydrous), phenol (Merck), sulfuric acid , bisphenol A (Merck), ethanol (Chimopar), 3-(triethoxysilyl)propyl isocyanate, tetraethyl orthosilicate (TEOS) (Aldrich), cetyltrimethylammonium bromide (CTAB), and sodium hydroxide (Fluka) were used as received.2BCD-NH were presented in our previous study [2BCD-NH was as follows: 5.5 g mono tosylated-BCD (4.266 mmol) were dissolved in 166 mL of 1,4-diaminobutane (DAB), slowly warmed up to 70 °C, and kept at this temperature for 24 h. At the end of the reaction time, the solvent was vacuum distilled and the solid that resulted was dissolved in a minimum amount of water. This syrup was added dropwise into 150–200 mL of acetone, which precipitated a white solid. At least two acetone precipitations are needed to obtain a white powder solid; otherwise, the product seems oily. The final product was obtained by drying the sample in a vacuum oven at 40 °C for 2 days with a 54% yield. NMR and MALDI characterization were presented in our previous study [The synthesis procedure and characterization of us study . Brieflyus study .2) was dissolved in 20 mL of dry pyridine by stirring at 70 °C. After 2.5 h of stirring, 1.1 mmol (0.27 mL) 3-(triethoxysilyl) propyl isocyanate were added to the BCD solutions. After 24 h, the solvent was removed by vacuum evaporation and the residue was washed three times with n-hexane. The obtained yield was over 95% for both intermediaries. The product was used directly without any other purification for the synthesis of SiO2-BCD-OH or SiO2-BCD-NH2 (please see One mmol of BCD derivative (1.14 g BCD-OH or 1.2 g BCD-NH2) (1 mmol) was introduced, and the reaction was stirred for an additional 4 h at 90 °C. After, the SiO2-BCD-OH and SiO2-BCD-NH2 were isolated by centrifugation and washed thoroughly with diluted HCl solution (0.01 M), water, ethanol, and acetone. The final products were dried at 70 °C for 24 h, affording around 4.2 g for each material (yield 85%).One-half g of cetyltrimethylammonium bromide (CTAB) was dissolved in 160 mL of water. The solution was heated at 90 °C and 2 mL of NaOH 2 mol/L were added and stirred for 15 min. Then, 60 mmol (13.4 mL) TEOS were added while continuously stirring the reaction mixture. After 10 min from the introduction of TEOS , the silane-BCD precursor  Nova NanoSEM 630 (FEI) .2I2 were used with a drop volume of 20 µL. The measurement of each contact angle was made within 10 s after each drop to ensure that the droplet did not soak into the film. The contact angles reported were the mean of 10 determinations. To prepare the SiO2 and BCD-modified SiO2 films, solutions (5% weight) in isopropanol were prepared by sonication and then deposited by drop-casting on microscope glass plates (five successive deposition/drying cycles) to ensure a complete coverage of the glass plates.A KSV CAM 200 apparatus was used for static contact angle measurements performed on dried films. Water droplets or CH−1 in 4000–600 cm−1 region. The samples were analyzed using the attenuated total reflection (ATR) technique.FT-IR spectra were recorded on a Bruker VERTEX 70  spectrometer using 32 scans with a resolution of 4 cm2 particles were determined with a gas (N2) porosimeter-type Gemini V based on the BET method.The values for the specific surface areas of the modified SiO−1. The BCD content was quantified by determining the reducing sugars of the silica using concentrated H2SO4 acidolysis and phenol colorimetric analysis [2-BCD-OHSiO or 2-BCD-NH2SiO) in 2 mL H2O. Then, 7 mL of concentrated sulfuric acid and 1 mL of 4% phenol standard solution were added and the solution was incubated at 80 °C to ensure hydrolysis and phenol coupling reaction. The glucose level was assessed using the calibration curve, and the BCD content was calculated as follows (Equation (1)):The UV-Vis spectra were recorded using a V-550 Able Jasco spectrophotometer, using a bandwidth of 1 nm and a scanning speed of 1000 nm minanalysis . The met2 (2-BCD-OHSiO and 2-BCD-NH2SiO) was conducted using batch equilibrium technology in a mixed solution of ethanol and doubly distilled water (v:v 3:7) at the desired concentrations and a pH value of 5. In general, a 0.01-g sample of modified silica was dispersed thoroughly in 50 mL of solution of bisphenol A at various concentrations (5–30 mg/L) and shaken in a thermostatic bath (shaker operated at a speed of 180 rpm at 25 °C). After equilibrium was reached, the solid sample was separated by centrifugation. The concentration of bisphenol A in the residual solution was determined using fluorescence spectrophotometry (bisphenol A: λex/λem 274/307). The amount adsorbed onto the BCD-modified polymer spheres was determined using Equation (2):0 (mg L −1) and ce (mg L −1) are the initial and final solution concentrations of bisphenol A, and m is the mass of BCD-modified polymer particles (mg).The fluorescence spectra were registered using a FP-6500 Able Jasco spectrofluorometer. The adsorption of bisphenol A onto BCD-modified SiO−1; samples mass, ~3 mg; temperature range, room temperature −700 °C; and heating rate, 10 °C min−1 in an alumina crucible.The thermogravimetric analyses (TGA) were performed using Netzsch TG 209 F3 Tarsus equipment considering the next parameters: nitrogen atmosphere flow rate, 20 mL minBCD-OH, respectively, 2BCD-NH. The synthesis approach involved the synthesis of the BCD silane derivative by reacting the BCD-OH and 2BCD-NH with 3-(triethoxysilyl) propyl isocyanate (TESPIC) (step A) followed by the hydrolysis–condensation together with TEOS (step B)  and 23.49 (weight %) for The morphological investigation of the materials was performed by scanning electron microscopy .2 particles was visible, but their dimension varied between the samples. In the case of 2-BCD-OHSiO, the particle diameter was about 300–350 nm, while for 2-BCD-NH2SiO and 2SiO it was around 150–200 nm. The silica generation process included several steps involving the formation of the initial “clouds” followed by densification, resulting in the primary particles that underwent collapse or association, leading to assemblies that undertook the growth process [Si-BCD-OH, the growth of the assemblies was favored. In contrast, for 2Si-BCD-NH, the process was much more similar to 2SiO. In the case of Si-BCD-OH, there was the chance of di-silyl derivatives being formed as the result of similar reactivity between the -OH groups. In contrast, in the case of 2Si-BCD-NH, due to the higher reactivity of -NH2, the monosubstituted silyl formation was favored. Thus, the presence of di-silyl Si-BCD-OH may be the explanation for the alteration of the silica formation process by promoting the growth of the assemblies.From the SEM images, the spherical shape of the SiO process . In our 2 particles, BET analysis was performed. The BET analysis afforded a surface area of 81, 53, and 90 m2/g for SiO2 unmodified, 2-BCD-OH,SiO and 2-BCD-NH2SiO, respectively. Thus, the analysis of the results indicated that the surface area in the case of 2-BCD-NH2SiO was almost double compared to 2-BCD-OHSiO and only slightly higher than the unmodified SiO2. The results sustain the mechanism of particle generation and are in accordance with the SEM analysis.For further information about the characteristics of the modified SiO2SiO), 16° (2-BCD-NH2SiO), and 14°(2-BCD-OHSiO). Due to the small difference between the samples, we also performed contact angle measurements using CH2I2. The values for CH2I2 were 11° (2SiO), 15° (2-BCD-NH2SiO), and 27° (2-BCD-OHSiO). Thus, it was noticed that the hydrophilic characteristic of SiO2 increased [To ascertain this assumption, contact angle measurements towards water were performed on the unmodified silica and on the two BCD-modified samples. The obtained contact angles values were 19°  and qt (mg g −1) are the amounts of bisphenol A adsorbed per unit mass of BCD-modified SiO2 (2-BCD-OHSiO and 2-BCD-NH2SiO) at equilibrium and t (minutes), respectively, k1 is the pseudo-first-order adsorption rate constant (min −1), k2 is the pseudo-second-order adsorption rate constant (g (mg min)−1, and Kp is the intraparticle diffusion constant (mg g−1 min−0.5).After 180 min of interaction at 25 °C using a bisphenol A solution of 100 mg/L , an equi2, the Lagergren pseudo-first-order adsorption model failed to explain the materials’ response during the adsorption process, since the qe, calc value was very different than the experimental. The Lagergren pseudo-first-order model assumes that the rate of change of solute uptake with time is directly proportional to the difference in saturation concentration and the amount of solid uptake with time, which is generally applicable over the initial stage of an adsorption process. Therefore, we opted to investigate the suitability of Ho’s pseudo-second-order adsorption model  indicates the amount of adsorbate at equilibrium, maxq (mg g−1) indicates the maximum amount of adsorbate at equilibrium, ec (mg L−1) is the equilibrium concentration of the adsorbate in the solution, LK (L mg−1) and FK (L mg−1) are the Langmuir and Freundlich constants, respectively, and n is the heterogeneity factor.The study of the adsorption isotherms offers information on the interaction between the adsorbate and the adsorbent when the adsorption process reaches equilibrium and allows the determination of the adsorption capacity of the material, which is an important parameter for system evaluation. The most intensively used isotherm adsorption models are Langmuir (Equation (6)) and Freundlich (Equation (7)).2 0.949 and 0.994) of the Langmuir isotherms (Equation (6)), the value for the intercept is negative, resulting in negative values for KL and qmax for 2-BCD-OHSiO and 2-BCD-NH2,SiO confirming that the Langmuir isotherm is not well suited to explain the adsorption at equilibrium for both materials. Since the Langmuir isotherm does not apply to the materials’ adsorption at equilibrium, it means that the monolayer adsorption of BPA did not take place. The Langmuir model can used to describe most of the adsorption processes that involve predominantly chemical interactions between solute and sorbent and, with some restrictions, for physical sorption processes. The primary condition for this model validity is the existence of only one type of active site on the surface of the sorbent [FK is the Freundlich constant, which represents adsorption capacity as well as the n term, which is an empirical constant indicating the adsorption intensity of the system was larger for 2-BCD-NH2,SiO indicating both a stronger adsorption and higher capacity of the material, probably due to the presence of amino functionality, which led to a different access to the BCD inner cavity.The linearization of the two selected isotherms in Equations (4) and (5) and the parameters calculated are presented in  sorbent . Thus, t sorbent . The res sorbent . In this2-BCD-NH2SiO displayed a stronger interaction, as suggested by the slightly shorter period until the equilibrium state was attained and from FK and n values of the Freundlich isotherm.Comparing the data presented in 2). The obtained modified silica displayed the capacity to adsorb organic molecules such as bisphenol A due to the presence of the BCD moieties on the surface of the silica. The chemical attachment of the BCD derivatives (BCD-OH and BCD-NH2) to the surface of the silica particles was qualitatively confirmed by FT-IR spectroscopy and quantitatively by acidolysis and phenol colorimetric analysis. The degrees of silica functionalization using the BCD-OH and BCD-NH2 derivatives were 23.97 and 23.49 weight %. The morphology of the obtained silica particles was investigated by SEM and BET analyses. Using contact angle measurements, a possible formation mechanism for the generation of silica particles bearing hydrophile groups on their surface was highlighted.This study presents the synthesis of silica modified with two BCD derivatives (BCD-OH and BCD-NHe values of 107 mg BPA/ g for 2-BCD-OHSiO and 112 mg BPA/ g for 2-BCD-NH2.SiO The kinetics followed Ho’s pseudo-second order adsorption model, sustaining the critical influence of the BCD moieties on the materials’ properties during the adsorption process and its participation in the process through the introduction of the BPA in the BCD inner cavity. This aspect was also demonstrated by the fitting of the Freundlich isotherm model on the experimental results. The materials’ adsorption capacity and the process kinetics make them suitable candidates for wastewater treatment processes.The kinetic analysis of the adsorption revealed that the process reached equilibrium after 180 min for both materials, with q


Protein kinases lacking Arg in the catalytic loop HxD motif  are associated with innate immune signaling across kingdoms. Phosphorylation activates plant immune receptor kinases (RKs), but the mechanistic details of activation are limited. Using the non-RD immune RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we investigated the role of the receptor cytoplasmic domain in immune signaling and found that the catalytic activity of EFR is dispensable for antibacterial immunity. Nevertheless, ligand-induced EFR-mediated signaling is initiated by activation loop phosphorylation, but not via the catalytic activity of the receptor protein kinase domain. We propose that leucine-rich repeat-receptor kinase complexes containing a non-RD kinase are activated through phosphorylation-dependent conformational changes of the receptor cytoplasmic domain. Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex. The translation of extracellular stimuli into intracellular signaling activities is carried out by myriad receptors, localized primarily at the plasma membrane. In metazoans, this role is fulfilled by proteins with diverse molecular architectures, which includes ligand-perceiving G protein-coupled receptors, receptor tyrosine kinases (RTKs), Toll-like receptors, integrins, and ligand-gated ion channels. In plants, plasma membrane-localized receptor kinase (RK) complexes are the primary receivers of extracellular molecular signals, and their importance in environmental adaptation and plant development is underscored by the evolutionary expansion of RK gene families in plant genomes –5. RKs a6111317Early work on the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1), an LRR-RK, established that phosphorylation of both the ligand-binding receptor and coreceptor was critical for activating BR responses –23. In v20Targeted analysis of phosphorylation by tandem mass spectrometry (MS/MS) of recombinant or affinity-purified proteins identified a large number of phosphorylation sites throughout LRR-RK cytoplasmic domains –47. Thes48Arabidopsis thaliana  LRR-RK family, and a variant of FLAGELLIN SENSING 2 (FLS2) carrying a Thr-to-Val mutation at this position (T867V) does not undergo ligand-induced endocytosis  influx (and activation of Ca2+-dependent protein kinases), and BIK1-independent initiation of mitogen-activated protein kinase (MAPK) cascades that collectively drive transcriptional reprogramming to ultimately halt pathogen ingress  and elongation factor thermo-unstable , respectively  and perceive pathogen-associated molecular patterns (PAMPs) or host-derived damage-associated molecular patterns (DAMPs) , 63. PAM ingress –68. In Aectively , 70. Botectively –74. Phosectively , 62, 74.ectively , 43, 75,ectively , 74.Because of the exogenous nature of their cognate ligands, PRRs serve as a useful model to understand the biochemical mechanisms regulating receptor activity since it is possible to study acute responses to ligand perception. We previously reported on the unidirectional phosphorylation of EFR by BAK1 in vitro and on the critical role of EFR Tyr phosphorylation in receptor complex activation , 62. BuiCD) is capable of autophosphorylation in vitro following purification from Escherichia coli, and similar to RD-type protein kinases, mutation of either the proton acceptor to Asn (D849N) or the catalytic loop Lys that participates in substrate coordination (K851E) (CD (D849N were immunopurified from transgenic Arabidopsis seedlings expressing green fluorescent protein (GFP)-tagged EFR variants treated with mock or 100 nM elf18 for 10 min, and the partially purified receptor complexes were then incubated with γ[32P]ATP to assess their protein kinase activity. As in previous studies , but we cannot exclude that other protein kinases in the immunoprecipitate could be responsible.The cytoplasmic domain of EFR contains a non-RD–type protein kinase domain with Cys (C848) in place of Arg in the catalytic HRD motif, suggesting that the EFR protein kinase domain does not require activation loop phosphorylation for function . Neverth (K851E)  compromi51E)  . Like the WT receptor, both EFRD849N and a second catalytically deficient mutant, EFRK851E, could activate an elf18-induced oxidative burst in N. benthamiana leaves but with reduced intensity and delayed maxima compared to WT EFR .Because EFR protein kinase activity was not required for its phosphorylation in the isolated active receptor complex, we tested whether different catalytic site mutants of EFR could trigger the elf18-induced oxidative burst following transient expression in ve burst  , indicating that S612 is an autophosphorylation site in the active receptor complex, consistent with our observation that EFR catalytic activity is not required for BAK1-S612 phosphorylation. We next measured the oxidative burst in response to elf18 treatment in the same transgenic lines. As was observed in N. benthamiana, both catalytic site mutants could activate an elf18-induced oxidative burst similar to the WT receptor, but with reduced intensity or with delayed maxima (K851E compared to either WT or EFRD849N (Inset); however, this difference might be attributed to reduced accumulation of the receptor in the EFRK851E transgenic line  signaling through accumulation of the SA reporter protein PATHOGENESIS-RELATED GENE 1 (PR1) , 79 by iic lines D, consisic lines , 80. TheAgrobacterium-mediated transient transformation of Arabidopsis leaves and elf18-induced resistance to Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) infection  transgene (Agrobacterium/pBIN19g:GUS). As a proxy for transformation efficiency, we measured GUS activity in leaf extracts using a quantitative fluorometric assay  or 1 μM elf18, and then 24 h later pressure-infiltrated Pto DC3000. After 2 d, we measured pathogen levels by colony counting  and long-term  immune responses in transgenic plants expressing EFRSI Appendix, Table S1) , suggesting that they may be involved in the regulation of EFR-mediated immune signaling.Given that EFR is phosphorylated in the active elf18-EFR-BAK1 receptor complex, we aimed to identify the sites of phosphorylation and to test whether phosphorylation regulates elf18 responses in a site-specific manner. To identify phosphorylation sites on EFR, we carried out in vitro protein kinase assays on EFR-GFP immunopurified from transgenic seedlings treated with mock or 100 nM elf18 and subsequently performed phosphorylation site discovery by liquid chromatography-MS/MS. In total, we identified 12 high-confidence Ser and Thr phosphorylation sites distributed throughout the EFR cytoplasmic domain (able S1) . Severalable S1) , and sevanalysis . InteresArabidopsis plants expressing nonphosphorylatable (Ser/Thr-to-Ala) or phospho-mimic (Ser/Thr-to-Asp) mutants of EFR in the efr-1 background and tested whether the mutants could trigger activation of MAPK cascades in response to elf18 treatment . Based on this screen, we identified two phosphosite mutants that completely lacked elf18-induced MAPK phosphorylation, namely EFRS753D and EFRS887A/S888A. Transgenic plants expressing either the EFRS887A or EFRS888A single-site mutant had reduced but not completely abolished MAPK activation, suggesting that phosphorylation of either residue could fulfill a putative regulatory function. In separate experiments, we tested the capacity of EFR phosphorylation site mutants to trigger BAK1-S612 phosphorylation and confirmed loss of MAPK activation for both the EFRS753D and EFRS887A/S888A receptor variants . In these experiments, we occasionally observed reduced sensitivity of plants expressing EFRS753A to 0.2 nM elf18; however, this effect was small, and was neither consistent between independent transgenic lines nor across independent experiments. Collectively, the loss of elf18 responses in EFRS753D and EFRS887A/S888A mutants indicates that the novel S753 and S887/S888 phosphorylation sites of EFR are negative and positive regulators of receptor complex activation, respectively.Finally, we hypothesized that specific EFR phosphorylation sites might regulate distinct downstream pathways in a manner reminiscent of animal RTKs . We therve burst A and C ave burst B and D, hibition C and D, The activation of transmembrane receptors in response to exogenous and endogenous signals is a critical biochemical process in all aspects of organismal development and stress response. The plant plasma membrane is decorated with a diverse suite of RKs that perceive a wide range of ligands. The largest family of RKs in plants, the LRR-RKs, fulfill critical roles in plant development and environmental response. Members of the LRR-RK family function coordinately with coreceptors from the SERK family to activate intracellular signaling following ligand perception. While the details of ligand perception have been quantitatively described –15, 18, bak1-4 background has a dominant negative effect on the elf18-induced oxidative burst . By comparison, a catalytic mutant of XA21—a non-RD PRR from rice—confers partial immunity to Xanthomonas oryzae pv. oryzae  signaling reported in previous studies may thus be consequence of poor receptor accumulation under transient expression or in stable transgenic lines. Collectively, this suggests that the dispensibility of catalytic function might be a common feature of non-RD protein kinases that function in immunity.Our observation that the catalytic activity of EFR is dispensable for all elf18-induced immune responses  and 3 ar. oryzae . FLS2 is. oryzae –90; howe lacking . In the In the absence of a direct catalytic role, we foresee two possible functions for EFR in the receptor complex. First, EFR could serve as a protein–protein interaction scaffold to define specificity in activating downstream responses. In support of this, studies of chimeric receptor kinases indicate that the cytoplasmic domain of the ligand binding receptor defines signaling specificity , 16. ThiS753D or EFRS887A/S888A we observed a loss of both BIK1-dependent (oxidative burst) and BIK1-independent (MAPK) signaling events, suggesting that the defect for both mutants occurs at the level of receptor complex activation. Interestingly, both the S753 and S887/S888 phosphorylation sites localize to subdomains that are important for regulatory conformational dynamics of protein kinases . These residues are well conserved in Arabidopsis subfamily XIIa LRR-RKs and in PLANT ELICITOR PEPTIDE 1 RECEPTOR 1 (PEPR1), all of which are known or hypothetical PRRs , suggesting that these sites might be important in regulating immune signaling. Activation loop phosphorylation serves as a key regulatory switch of RD protein kinases (93Even though EFR kinase activity is not required, EFR phosphorylation is critical for immune signaling . Here, w kinases , 91, 92.cal PRRs , 31, 32, kinases , 93–95, inases 93–98, the S887A/S888A is largely devoid of any phosphorylation (In a typical RD protein kinase, activation loop phosphorylation triggers conformational changes that establish a catalytically competent active state of the protein kinase domain . Based orylation , includiS887A/S888A nonphosphorylatable mutant, an EFRS753D phospho-mimic mutant also abolished elf18 responses, but not complex formation with BAK1 (S887A/S888A, elf18-induced phosphorylation of EFRS753D was similar to the WT (S753A mutants did not display constitutive activation of any PTI responses. Although a possible mechanism to explain the impact is less clear compared to S887/S888, S753 phosphorylation could disrupt order–disorder transitions of the EFR ɑC-helix, explaining impaired activation of the EFRS753D-containing receptor complex. Indeed, intrinsic ɑC-helix disorder can promote an inactive state of some protein kinases, including plant RKs (Like the EFRith BAK1 –6. Howevo the WT , indicato the WT . Consistlant RKs , 102, leCollectively, identification and characterization of EFR phosphorylation sites in the present work and in previous work from our laboratory suggests that phosphorylation-dependent conformational changes of the EFR cytoplasmic domain regulate receptor complex activation. We propose a model  where inSI Appendix, Experimental Procedures. Oligonucleotide sequences used for cloning are available in SI Appendix, Table S3 and details of antibodies and immunoblotting conditions are available in SI Appendix, Table S4. All materials and detailed protocols are available on request from the corresponding author.Detailed experimental procedures used in this study including plant growth conditions, PAMP treatments, cloning and plant transformation, recombinant protein expression and purification, protein extraction, in vitro protein kinase assays, SDS/PAGE and immunoblotting, pathogen infection assays, coimmunopurification, and homology modeling are available as Supplementary File


Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech  The clas3-Cys5) .Coagulation factor XI (FXI) is a zymogen of FXIa , which cThe leech is a kind of arthropod with important medicinal value in traditional and modern medicine. There are a large number of antithrombotic components in the salivary glands of leech, and they are divided into two categories. One is anticoagulant proteins, including thrombin inhibitors and FXa inhibitors; the other is anti-platelet aggregation proteins. The thrombin inhibitors include Hirudin , HaemadiWhitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115 and their inhibitory activity against FXIa were tested. Among them, WPK5 showed strong inhibitory activity to FXIa while showing negligible inhibition to other proteases of the coagulation cascade. Then, loop replacement was used to improve the potency of WPK5 to FXIa. The selectivity of WPK5-Mut to other coagulation factors, in vitro anticoagulant and in vivo antithrombotic activities were further evaluated.In this study, five Kunitz-type peptides were obtained from the salivary gland transcriptome of the leech 3 was from Sin-opharm Chemical Reagent Co, Ltd. ; All other chemicals used in this study were of analytical grade.Factor Xa, Thrombin, Trypsin, Factor XIIa, Pefachrome FXIIa/TH 5253 (H-D-CHA-Gly-Arg-pNA·2AcOH), CS-11(22) (Bz-Ile-Glu(γOCH3)-Gly-Arg-pNa and Bz-Ile-Glu(γOH)-Gly-Arg-pNa), CS-31(02) , CS-21(66) (p-Glu-Pro-Arg-pNa·HCl) and CS-01(38)  were from Hyphen-Biomed, ; HFXIa was from Enzyme Research Laboratories, ; Kallikrein was from AssayPro LLC, ; APTT and PT reagent were from STEELLEX ; FeClC57BL/6J mice  were purchased from Comparative Medicine Centre of Yangzhou University . All animals were housed under controlled temperature (23–25 °C), humidity (30–40%) and light , with free access to food and water. All animals were tested after one week of adaptive feeding. Human venous blood was obtained from healthy adult volunteers in accordance with the Declaration of Helsinki. All protocols used were approved by the Ethics Committee of the China Pharmaceutical University.Whitmania pigra was built previously in our lab [The transcriptome of the salivary gland of the leech  our lab . Kunitz-Pichia pastoris GS115 cells using an Electroporator . The positive clones were cultured in BMMY medium, and the expression was induced by adding 1% (v/v) methanol for 72 h. The fermentation broth was purified by column previously loaded with UniNTA-80Ni , and target peptide was eluted by 20 mmol/L PBS including 200 mmol/L imidazole.The gene sequences of WPK1-WPK5 and WPK5-Mut were synthesized and then enriched by PCR. pPIC9K was cleaved with EcoRI and NotI to obtain a linearized vector, and the target gene fragment was recombined into pPIC9K by homologous recombination to obtain a full-length plasmid. Then, the target gene in pPIC9K was confirmed by DNA sequencing. The plasmid contains an open reading frame, α factor signal peptide sequence, a 57 amino acid Kunitz-type peptide sequence and a C-terminal 6 *(His) tag. The plasmids were linearized with the SacI restriction enzyme (TaKaRa). The linearized plasmids were then transformed into 18 column . Eluent A consisted of 0.1% TFA in distilled water (v/v), and eluent B consisted of 0.1% TFA in 100% acetonitrile (v/v). Elution was carried out according to the following process: 0–20 min, B 10–70%; 20–22 min, B 70–90%; and 22–25 min, B 90% at a flow rate of 1 mL/min and the absorbance of the elution fractions was monitored at 214 nm. WPK5 and WPK5-Mut were qualitatively analyzed by using LC-Q-TOF-MS. The analysis was performed on an Agilent Zorbax SB-C18 (4.6 mm × 250 mm) and the mobile phase consisted of 0.1% formic acid in MilliQ water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B) at a flow rate of 0.5 mL/min. The mobile-phase gradient (10–70% B) was applied. Source nitrogen gas temperature was 325 °C, sheath gas flow was 12 L/min, and nebulizer pressure was 40 psig. Voltages were set at 4000 (capillary) and 175 V (fragmentor). Positive ions were acquired in the range of 100–3200 m/z for MS scans. Internal mass correction was enabled by using two reference masses at 121.0509 and 922.0098 m/z. Data acquisition and instrument control were performed using Agilent MassHunter Workstation software (B.06.01 SPI).WPK5 or WPK5-Mut was loaded to RP-HPLC on a C0 represents the slope of the control and Vi represents the slope of different concentrations of Kunitz-type peptide. IC50 was defined as the concentration of inhibitor required to inhibit the activity of FXIa by 50%.The inhibitory activity of purified Kunitz-type peptides to FXIa were tested as previously described . In brieWPK5 or WPK5-Mut and serine protease (trypsin (0.5 nM), FXIIa (1 nM), kallikrein (0.5 nM), FXa (2.5 nM), or thrombin (1.42 nM)) were mixed with different molar ratios in TBS-BSA, and then incubated for 60 min at 37 °C. The chromogenic substrate (CS-11(22) (0.44 mM), TH 5253 (0.25 mM), CS-31(02) (0.25 mM), CS-11(22) (0.44 mM), CS-01(38) (0.10 mM)) was added into the corresponding protease well, and absorbance at 405 nm was then continuously tested for 1 h using a microplate reader (FEL-1).2 solution was added to initiate clotting and the fibrin formation time was recorded in duplicate using Semi-automatic coagulation analyzer (SC40 (LG-PABER-I), STEELLEX, Beijing, China). For PT assay, 40 μL PPP and 10 μL different concentrations of WPK5 or WPK5-Mut were added to the test cup and incubated at 37 °C for 3 min, followed by the addition of 100 μL PT reagent to initiate clotting and the fibrin formation time was recorded in duplicate using Semi-automatic coagulation analyzer (SC40 (LG-PABER-I), STEELLEX, Beijing, China).For APTT assay, 40 μL PPP and 10 μL different concentrations of WPK5 or WPK5-Mut were added to the test cup and incubated at 37 °C for 3 min, followed by the addition of 50 μL APTT reagent, and then incubated for more 3 min. Finally, 50 μL of pre-warmed CaCl3-induced carotid artery thrombosis model in mice [3 solution was placed on the carotid artery for 3 min and then removed, the blood flow was observed for 30 min. The occlusion time was defined as the time from the removal of the FeCl3 saturated filter paper to the blockage of the vessel for at least 3 consecutive min [The in vivo antithrombotic activity of WPK5-Mut was evaluated by using a FeCl in mice . Mice . Results were expressed as mean ± SD values. The statistical differences among multiple-samples were evaluated by using one-way ANOVA analysis followed by Dunnett’ s multiple comparison test. Whitmania pigra salivary gland has been built in our lab [The transcriptome of the leech  our lab . Five KuPichia pastoris GS115, and purification by UniNTA-80Ni column. Tricine-SDS-PAGE analysis showed that there is one band. The purity of WPK5 or WPK5-Mut was more than 95% by HPLC analysis . WPK5 did not prolong PT even at 160 μM  and WPK5-Mut (0.75 mg/kg) treated group . At a dose of 1.5 mg/kg, WPK5-Mut significantly prolonged occlusion time . The occlusion did not happen within 30 min in three mice, while in other three mice, unstable thrombus formation was observed, as demonstrated that the blood vessel was gradually blocked at the beginning, and then flushed away by the blood flow and blocked again. At a dose of 3.0 mg/kg, occlusion did not happen within 30 min, which was similar to that of heparin sodium (2.7 mg/kg).To evaluate anti-thrombotic efficacy of WPK5-Mut in vivo, a FeCln = 6).To evaluate the bleeding risk of WPK5-Mut, a mice tail cutting assay was used. Doses were designed as 3 and 6 mg/kg, which represent 2 and 4 times the effective antithrombotic dosage, and heparin sodium (2.7 mg/kg) was used as a positive control. As shown in Whitmania pigra. Their inhibitory activity to FXIa were tested by the chromogenic substrate method. Among them, WPK5 showed strong inhibitory activity to FXIa. The potency of WPK5 was improved through loop-replacement strategy, and the mutant, WPK5-Mut, was further evaluated for the extensive bioactivity including in vitro anticoagulant activity and in vivo antithrombotic activity. WPK5-Mut prolonged APTT in both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid thrombosis in mice. Interestingly, WPK5-Mut did not show significant bleeding risk even at 4 times the effective antithrombotic dosage in a mice tail cutting assay, indicating that WPK5-Mut is a promising candidate for the development of antithrombotic drug.We disclosed five Kunitz-type peptides from the transcriptome of the salivary gland of the leech Theromyzon tessulatum [Haementeria officinalis [Placobdella ornata [Whitmania pigra [50 of 978.20 ± 52.15 nM, and showed negligible inhibition to other proteases that exist in the coagulation cascade. FXIa is an intrinsic coagulation factor, and the inhibition of it prolong the APTT without affecting PT. The anticoagulant effect of WPK5 was evaluated by APTT and PT assay. At a dose of 80 μM, WPK5 prolonged 1.4-fold APTT, and did not prolong PT even at a dose of 160 μM. Based on our knowledge, this is the first FXIa inhibitory peptide from leeches. Some FXIa inhibitory peptides derived from other animals have been reported, such as rFasxiator [Bungarus fasciatusm with a IC50 of 1.5 μM, which is weaker than WPK5, while Desmolaris, a Kunitz-type protein from the salivary gland of a vampire bat, has poor selectivity .The salivary glands of leeches contain many anticoagulant and antiplatelet components, such as Theromin, a thrombin inhibitor from leech ssulatum ; Antistaicinalis ; Ornatina ornata . FXIa inia pigra . In the asxiator  and Desmasxiator . rFasxia50 of 8.34 ± 0.20 nM, which is about 100-fold higher potency than WPK5 in inhibitory activity to FXIa. The protease specificity data showed that WPK5-Mut effectively inhibited the activity of FXIa at a molar ratio of 50:1, with 86.21% inhibition. When the molar ratio of WPK5-Mut to kallikrein or FXIIa or FXa was 775:1, WPK5-Mut produced weak inhibitory activity with 25.25%, 21.50%, and 28.25%, respectively, and when the molar ratio of WPK5-Mut to thrombin was 1550:1, it showed no inhibitory activity, indicating that WPK5-Mut is selective for these enzymes. However, when the molar ratio of WPK5-Mut to trypsin was 39:1, WPK5-Mut could effectively inhibit the activity of trypsin, with an inhibition rate of 85.20%.Domain swapping, loop replacement, and site-directed mutations are effective strategies to enhance the potency of proteins . To imprAlthough, the loop 1 and loop 2 of WPK5-Mut come from PN2KPI, and the inhibitory activity to FXIa of WPK5-Mut and PN2KPI is similar (8.34 ± 0.20 nM vs. 8.01 ± 3.92 nM), WPK5-Mut showed a stronger effect on murine plasma APTT than that of PN2KPI. WPK5-Mut prolonged APTT by 2 times at a concentration of 3.47 μM compared with saline treated group, while PN2KPI prolonged APTT by 1.5 times at the concentration of 19 μM . The ant3-induced carotid artery thrombosis model in mice was used to evaluate the antithrombotic effect of WPK5-Mut and PN2KPI [A FeCld PN2KPI  in vivo.Whitmania pigra. By loop replacement strategy, WPK5-Mut was obtained, and its ability to inhibit FXIa was increased by 100-fold compared to WPK5. WPK5-Mut prolonged plasma APTT in vitro, and significantly inhibited FeCl3-induced carotid artery thrombosis without bleeding risk. Taken together, WPK5-Mut is the first Kunitz-type FXIa inhibitor identified from leech Whitmania pigra with potent antithrombotic activity and could be a candidate for the development of clinical antithrombotic agent.We identified a novel Kunitz-type FXIa inhibitor, WPK5, from leech


NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC  and active (neddylated) forms of cullins . High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na Sout mice ,53, and NK4 mice . In addiade WNK4 ,56,57.+ intake alter the activity and stability of WNK kinases [+ modulates the abundance or activity of cullins, and whether inhibition of cullin activity alters K+-mediated effects on NCC. In an attempt to address this knowledge gap, here we asked three questions: (1) Do long-term alterations in dietary K+ intake change cullin abundance or their neddylation status? (2) Are short- or long-term K+-dependent changes in NCC abundance or phosphorylation independent of systemic hormones mediated by alterations in cullin abundance or activity? (3) Are the effects of a long-term high dietary K+ intake on NCC altered in a mouse model of FHHt. Our results show that in mice fed various K+ diets, the neddylation levels of renal Cul1, 3, 4, and 5 are negatively correlated with pNCC and NCC expression. Furthermore, the K+ inhibitory effects on NCC phosphorylation in the long term are attenuated in CUL3-Het/Δ9 mice or in the presence of MLN4924 (neddylation inhibitor) in isolated renal tubules. Together our study indicates that Cul3 and potentially other cullin family members are important for modulating the overall response of the DCT to altered extracellular K+ and ultimately NCC activity  or components of the Cullin E3–ligase complex (E2 ligase and substrate receptors) could alter cullin activity. Interestingly, the NEDD8 E3 ligase DCN-like protein 4 (DCNL4) was recently identified as a K+-regulated protein modulating SPAK activity in kidney intercalated cells [In kidney samples from mice receiving 0%, 1%, or 5% Kncreased . These cncreased . In contC levels . Togetheed cells , but whe+ media , NCC phosphorylation was still reduced despite the presence of MLN4924, suggesting that acute reductions in NCC phosphorylation are independent of cullin and potentially WNK activity. This could suggest that in the short term, the “on switch” for NCC and SPAK phosphorylation  does not need to be “turned off” for the high K+-mediated effects to occur. However, acute effects of potassium on NCC phosphorylation are absent in Cl−-insensitive WNK4 mice and in SPAK knockout mice [+, systemic factors, and NCC that is absent in the ex vivo tubules. Such an idea is supported by acute K+ loading only decreasing NCC phosphorylation in mice that were prior K+-restricted for 1 day, but not in mice that were chronically K+-restricted for 5 days [After 30 min’ incubation of tubules in higher extracellular Kout mice ,64, sugg+ on NCC phosphorylation were attenuated in the presence of MLN4924, fitting well with studies in the FHHt CUL3-Het/Δ9 mice, where the ability of a high dietary K+ intake to reduce NCC phosphorylation was also limited (+ on NCC phosphorylation are reduced, but not prevented, suggesting that NCC dephosphorylation is also important. This is supported by studies in Cl−-insensitive WNK4 mice [+ loading, with early effects being driven by reductions in the activity of the WNK–SPAK pathway, whereas later effects may be driven by alterations in the WNK–SPAK activity but also by changes in NCC dephosphorylation. Further studies are required to examine this possibility, but unfortunately, the phosphatase responsible for NCC dephosphorylation subsequent to a high K+ load is still unclear with inconsistent results using PP1, PP2, or PP4 phosphatase inhibitors [In contrast, in tubules the long-term effects of high K limited . Thus, wNK4 mice , where thibitors ,64,65,66+ loading effects on NCC partially depend on the actions of one or more cullins. Deciphering the importance of individual members and how their actions converge and combine with other regulatory mechanisms to exert overall effects on NCC will require the generation and phenotyping of mice where individual cullins are deleted only in the DCT.In conclusion, our results indicate that chronic K