Among the modified hydrolysates, two novel efficient ACE-inhibitory peptides, which are namely PNVA and PNLG, were purified and identified by a sequential approach combining a sephadex G-15 gel column, reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), before we conducted confirmatory studies with synthetic peptides. The ACE-inhibitory activity assay showed that PNVA and PNLG exhibited lower IC50 values of 8.18 ± 0.24 and 13.16 ± 0.39 μM than their corresponding truncated analogs (NVA and NLG), respectively. Molecular docking showed that PNVA and PNLG formed a larger number of hydrogen bonds with ACE than NVA and NLG, while the proline at the N-terminal of peptides can affect the orientation of the binding site of ACE. The method developed in this study may potentially be applied to prepare efficient ACE-inhibitory peptides, which may play a key role in hypertension management.Natural angiotensin converting enzyme (ACE)-inhibitory peptides, which are derived from marine products, are useful as antihypertensive drugs. Nevertheless, the activities of these natural peptides are relatively low, which limits their applications. The aim of this study was to prepare efficient ACE-inhibitory peptides from sea cucumber-modified hydrolysates by adding exogenous proline according to a facile plastein reaction. When 40% proline ( Hypertension is one of the leading causes of global disease burden . In 2015Acaudina molpadioidea). The sea cucumber is a particularly good source of bioactive peptides and is rich in proteins that result in high yields of peptides [Acaudina molpadioidea protein hydrolysates by a facile plastein reaction [Currently, chemically synthesized ACE inhibitor drugs, such as captopril, enalapril and lisinopril, are being widely used in clinical applications ,6,7. Howpeptides ,14. Recereaction ,16,17. Preaction , includireaction  or ACE-ireaction .A number of structure–activity relationship studies have showed that peptides, such as APP , KPLL 2 and VYPFStructural bioinformatics show that the activity of ACE-inhibitory peptides is related to both their molecular masses and amino acid sequences . Some stAcaudina molpadioidea protein hydrolysates to enhance the activity of natural ACE-inhibitory peptides has not been previously discussed. The aim of this study was to prepare efficient ACE-inhibitory peptides from sea cucumber-modified hydrolysates by adding exogenous amino acids according to a facile plastein reaction. Furthermore, we aimed to identify the ACE-inhibitory peptides with high activities by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). In addition, molecular docking simulations were conducted to investigate the nature of interactions between the peptides and ACE. This study can provide previously unknown information on the effect of exogenous amino acids on the activity of natural ACE-inhibitory peptides and provide an insight into the peptide structure−ACE inhibition relationship.To the best of our knowledge, the addition of exogenous amino acids to p < 0.05). These results demonstrate that the plastein reaction modified by the addition of exogenous Pro was capable of improving ACE-inhibitory activity. The obtained results are consistent with Sun et al. [As shown in n et al. , who repn et al.  reportedn et al.  reportedw/w, proline/free amino groups) was added, the ACE-inhibitory activity obtained was higher compared to other proportions after 6 h of reaction (IC50 = 0.93 ± 0.05 mg/mL) (p < 0.05) (50 = 0.59 ± 0.03 mg/mL) after 1 h of reaction (The plastein reaction is mainly affected by substrate concentration and reaction time . Therefo < 0.05) B. Meanwhreaction C. After p < 0.05). The fractions with the same molecular mass peaked at the same time in the Sephadex G-15 gel column [Chromatographic fractionation is a method that is often used for peptide elution. A Sephadex G-15 gel column was used to fractionate the ACE-inhibitory peptides, before the fractions were pooled to obtain fractions A−H at 220 nm A. After l column . Therefo50 = 27 ± 2 µg/mL) (p < 0.05). Fraction E3 was purified by 21.85-fold using a two-step purification process  was significantly higher than the activity of PNLG (IC50 = 13.16 ± 0.39 μM). Meanwhile, NVA (IC50 = 12.69 ± 1.50 μM) exhibited a significantly higher activity than NLG (IC50 = 17.45 ± 0.89 μM). Our observations are consistent with those of Jang et al. [Subsequently, four peptides, which were namely PNVA, PNLG, NVA and NLG, were synthesized by a chemical method and their ACE-inhibitory activities were determined. As shown in g et al. , who repg et al.  reportedg et al. , suggestg et al. ,31,33. HTo understand the molecular interaction mechanism of the four peptides  against ACE, molecular docking analysis was performed using Molecular Operating Environment (MOE) software. The docking scores of PNVA, PNLG, NVA and NLG were −7.13, −6.77, −5.14 and −5.12 kcal/mol, respectively , which sThe modes of action of ACE-inhibitory peptides include competitive, noncompetitive, uncompetitive and mixed modes. Most ACE-inhibitory peptides act as competitive inhibitors . The bin2+. These results indicate that the Pro at the N-terminal of the peptides could reform the rigid structure of the peptides, further altering the binding sites between peptides and ACE. These are similar to the observations made by Liu et al. [Intriguingly, the Pro of PNVA forms two hydrogen bonds with Asp354 and Thr358, while the Pro of PNLG forms two hydrogen bonds with Asp354 and Glu262. These results indicate that the Pro at the N-terminal of both PNVA and PNLG plays an essential role in determining their binding affinity. Similarly, Min et al.  reported50 = 8.18 ± 0.24 µM) and PNLG (IC50 = 13.16 ± 0.39 μM) were much lower than synthetic ACE inhibitor captopril (IC50 = 23 nM) [A. molpadioidea were characterized as novel peptides, which are derived from a food source that is eaten daily. Therefore, the ACE-inhibitory peptides (PNVA and PNLG) will be very useful in the preparation of antihypertensive drugs. Meanwhile, their activities were much higher than that of other biopeptides, AGPPGSDGQPGAK (IC50 = 420 ± 20 µM) [50 = 956.30 µM) [50 > 1000 µM), suggesting the peptides that we prepared have higher ACE-inhibitory activities. The ACE-inhibitory peptides derived from sea cucumber-modified hydrolysates by adding exogenous Pro will play a key role in hypertension management.Numerous studies on hypertensive human volunteers have demonstrated that ACE-inhibitory peptides significantly reduce blood pressure . In addi± 20 µM) , AV (IC5Acaudina molpadioidea) was purchased from a local market  and its water content was determined to be 12.65% per 20 g on average. Hippuryl histidyl leucine (HHL), rabbit-lung ACE and Sephadex G-15 were purchased from Sigma Chemical Ltd . Papain (from papaya) and trypsin (from bovine sources) were purchased from Nanning Pangbo Biological Engineering Ltd . O-Pthaldialdehyde (OPA) was purchased from Beijing Soularbio Technology Ltd. . Proline (PubChem CID 145742), valine (PubChem CID 6287), tyrosine (PubChem CID 6057), tryptophan (PubChem CID 6305), leucine (PubChem CID 6106) and phenylalanine (PubChem CID 6140) were purchased from Sinopharm Chemical Reagent Ltd. . All other chemicals were obtained from local commercial sources and were of the highest purity available.Sea cucumber  to cut into pieces. During stirring, the protein was extracted at 45 °C over a period of 8 h and centrifuged  to obtain water-soluble protein solutions. After this, these solutions were freeze-dried using a lyophilizer .The protein present in the body wall of h et al. . BrieflyAcaudina molpadioidea body wall protein was initially dissolved in water and its pH was adjusted to 7.0. After this, trypsin (2.5 kU/g protein) and papain (2.5 kU/g protein) were added to this solution and digested in a 50 °C water bath for 4 h. Subsequently, the enzymes were inactivated at 95 °C for 15 min. The suspension was then centrifuged at 8000× g and 4 °C for 20 min, after which the supernatant was removed and ultrafiltration precipitation was carried out with a 5 K membrane . The obtained precipitate was freeze-dried  and stored until further use.The freeze-dried Acaudina molpadioidea protein hydrolysates for the plastein reaction. The reaction conditions were as follows: substrate concentration of 40% (w/v), temperature of 45 °C and papain dosage of 2.5 kU/g protein. Furthermore, the effect of different proportions and reaction time on the ACE-inhibitory activity was determined.Different concentrations of exogenous Pro, Phe, Trp, Tyr and Leu (at pre-determined weight ratios of exogenous amino acid to free amino groups) were added to the Acaudina molpadioidea hydrolysates to the same volume of OPA reagent (40 mg/mL). After 5 min, the absorbance of the resultant solution was measured at 340 nm using a UV spectrophotometer . L-leucine was used as the standard. The equation of the standard curve is Y = 0.3037X + 0.0078, where X is the concentration of free amino acids and Y is the absorption value of the sample.The OPA method , with so50 value was defined as the concentration of inhibitor that was needed to inhibit 50% of the ACE activity.The ACE-inhibitory activity of the hydrolysates was measured according to a previously reported protocol  with sliACE-inhibitory peptides were purified using a Sephadex G-15 column (2.6 cm × 65 cm) and eluted with double distilled water at a flow rate of 1.2 mL/min. The absorbance of the eluent was monitored at 220 nm.The ACE-inhibitory activity of peptides in the hydrolysates was analyzed by RP-HPLC on a Zorbax SB-C18 column  equipped with an Agilent 1260 infinity HPLC system  at a flow rate of 1 mL/min. An acetonitrile gradient from 5% to 30% was used for 40 min to separate different groups of peptides. Chromatographic separation was carried out at 35 °C. The components were collected at the absorbance of 220 nm and their ACE-inhibitory activity was measured.The molecular weights of peptides in the filtered hydrolysates were analyzed by MALDI-TOF . The amino acid sequences were determined in the positive ion mode by de novo sequencing. Subsequently, the peptides were synthesized using the solid phase method and purified by HPLC . The synthesized peptides are listed in http://www.rcsb.org) for the docking studies. The induced-fit docking approach was applied to analyze the side-chain flexibility of residues at the binding sites.The initial peptide structures of PNVA, PNLG, NVA and NLG were produced using the xleap module of AMBER16  and were subjected to minimization using the Molecular Operating Environment (MOE) software (version 2016) . Moleculp < 0.05).All the data (average of three replicates) are reported in the form of mean ± standard deviation and the results were validated by one-way analysis of variance (ANOVA). Significant differences between the means of the parameters were determined by Duncan’s multiple range tests  possessed higher ACE-inhibitory activity than the original hydrolysates. Furthermore, the free proline content in the reaction system decreased significantly (p < 0.05). In addition, two novel ACE-inhibitory peptides, PNVA and PNLG with IC50 values = 8.18 ± 0.24 and 13.16 ± 0.39 μM, were identified for the first time in Acaudina molpadioidea protein hydrolysates. Molecular docking showed that PNVA and PNLG form more hydrogen bonds with ACE than NVA and NLG, while the presence of proline at the N-terminals of the peptides can affect the orientation of the binding sites of ACE, leading to a reduction in ACE activity. The exogenous proline is assembled into ACE-inhibitory peptides, with this phenomenon partially contributing to the increase in the ACE-inhibitory activity of natural peptides. In this study, we demonstrated that the addition of exogenous proline to Acaudina molpadioidea protein hydrolysates through the plastein reaction is a promising method to enhance the activity of natural ACE-inhibitory peptides. Further studies are necessary to investigate the detailed assembly of exogenous proline using isotopic tracer methods and to prove the in vivo efficacy of ACE-inhibitory peptides in lowering blood pressure. The results of the present study also highlight the potential applications of plastein reaction in preparing antihypertensive drugs.Upon the addition of exogenous amino acids, it was possible to enhance the ACE-inhibitory activity of


Understanding feedbacks between the Greenland Ice Sheet (GrIS) and the Atlantic Meridional Overturning Circulation (AMOC) is crucial for reducing uncertainties over future sea level and ocean circulation change. Reconstructing past GrIS dynamics can extend the observational record and elucidate mechanisms that operate on multi-decadal timescales. We report a highly-constrained last glacial vertical profile of cosmogenic isotope exposure ages from Sermilik Fjord, a marine-terminating ice stream in the southeast sector of the GrIS. Our reconstruction reveals substantial ice-mass loss throughout the Younger Dryas (12.9-11.7 ka), a period of marked atmospheric and sea-surface cooling. Earth-system modelling reveals that southern GrIS marginal melt was likely driven by strengthening of the Irminger Current at depth due to a weakening of the AMOC during the Younger Dryas. This change in North Atlantic circulation appears to have drawn warm subsurface waters to southeast Greenland despite markedly cooler sea surface temperatures, enhancing thermal erosion at the grounding lines of palaeo ice-streams, supporting interpretation of regional marine-sediment cores. Given current rates of GrIS meltwater input into the North Atlantic and the vulnerability of major ice streams to water temperature changes at the grounding line, this mechanism has important implications for future AMOC changes and northern hemisphere heat transport. Modelling studies suggest that increased freshwater input from Greenland has the potential to slow down AMOC in the future3, thus substantially weakening heat transport to the North Atlantic and potentially driving positive ice sheet-ocean feedbacks4.In today’s warming climate, the persistent North Atlantic cooling anomaly off the southeast coast of Greenland is thought to be caused by the accelerating input of meltwater from the Greenland Ice Sheet (GrIS) and considered an indicator of a weakening of the Atlantic Meridional Overturning Circulation (AMOC)5) was a period of sustained Northern Hemisphere atmospheric cooling of up to 10 °C 7 that is considered to have been initiated by freshwater input into the Arctic Ocean and/or North Atlantic from the expanded Laurentide Ice Sheet8. In contrast to widespread terrestrial glacier expansion in Europe during the YD9, ice streams and local glaciers in southern Greenland may have reacted in antiphase to atmospheric temperature changes9; local terrestrial glaciers near Scorsby Sund, east Greenland, show evidence of Younger Dryas standstill10, whilst reconstructions of the Jakobshavn Isbrae and Kangerlussuaq ice streams (west and east Greenland respectively) suggest possible retreat14. However, this intriguing potential non-linear response is poorly constrained by current field data, and is not presently captured in ice sheet models, the latter perhaps owing to an underrepresentation of ocean processes15. These discrepancies are, however, consistent with suggestions of divergent ocean circulation behaviour across the North Atlantic, with steady or even decreasing marine 14C reservoir ages southeast of Greenland17 at a time of substantial increase across the wider North Atlantic18. Intriguingly, marine sediment cores from the southeast Greenland continental shelf appear to record sustained freshwater spikes from 12.8-12.0 ka that have been interpreted as a consequence of significant meltwater input into the North Atlantic possibly sourced from the GrIS19, but due to its fragmentary nature, terrestrial evidence from ice-free areas of the GrIs is at present inconclusive and contradictory. Thus, the YD may provide an extreme scenario that could allow atmospheric temperature effects to be disentangled from ice sheet-ocean interactions, and given current uncertainties, developing new and detailed terrestrial records of deglaciation is crucial.Unfortunately, contemporary observations of the GrIS and North Atlantic are too short (<100 years) to inform on our understanding of long-term trajectories and feedbacks over multi-decadal to centennial timescales. Periods of large-scale rapid climate reorganisation in the recent geological past offer considerable potential for understanding ice-sheet-ocean dynamics, thereby extending the observational window. The Younger Dryas  outlet glaciers of the GrIS terminated off-shore on the continental shelf in deep cross-shelf troughs at depths of ~600 m23.Over 80 km long and up to 12 km wide, Sermilik  is the largest fjord in southeast Greenland, with the classic steep walls and geomorphology of a former ice sheet outlet. The lower part of the fjord trends roughly north-south, whilst the upper 30 km coincides with a narrowing and shallowing of the fjord Fig. . Bathyme21 coincided with the abrupt Northern Hemisphere atmospheric warming following the YD24. However, in order to fully understand the dynamical response of the ice stream to environmental change, it is important to determine the timing and magnitude of thinning as well as the rate of terminus or lateral retreat. Here we present the results of new in situ cosmogenic isotope analysis of vertical transects which we combine with published lateral transects up Sermilik Fjord21 and examine possible drivers of the dynamic changes we discover using transient experiments performed with an Earth system model.The rapid retreat of the GrIS outlet in Sermilik during the early Holocenein situ cosmogenic nuclide 10Be, and exposure ages calculated using the Northeast North American production rate , between ~40–430 m above present day sea level in Sermilik Fjord, as well as two additional discrete sites at lower elevations Fig. . Samples14C ages from elevated lakes on the eastern side of Sermilik26, using OxCal 4.127 (see Methods). This analysis reveals that ice stream drawdown was underway by 13.0 ± 0.4 ka (1 s.d.), and continued into the Holocene until 10.3 ± 0.2 ka (1 s.d.), prior to its retreat upstream, indicating the ice ungrounded and underwent surface lowering of ~376 m. This continued throughout the Younger Dryas, with ~280 m of the of the thinning occurring during this period  caused by the 11 ka peak in obliquity31 may also have contributed, but the YD expansion of a local terrestrial ice cap in the far north of Greenland32 implies that this effect was not strong enough to drive major ice stream retreat.Our observations raise the question: what could have driven substantial retreat of a major outlet glacier of the GrIS despite a marked drop in atmospheric temperature at the Younger Dryas? Rising sea levels can cause ice-stream instability and retreat; however, although global sea level was rising prior to and during the YD34. Reconstructions of Arctic sea ice at the time are limited and contradictory35, but modelling suggests that winter sea ice at 12 ka was expansive36  between 13 and 12.2 ka B.P (see Methods). Despite a marked simulated North Atlantic sea surface temperature decline and sea ice expansion , it is vital that this feedback is included in ice-sheet modelling studies to constrain projections of future sea-level rise and northern hemisphere heat transport.Such a mechanism could have significant implications for future ice-sheet dynamics. A number of major outlets of the present-day southern GrIS have grounding lines at depths of 500–800 m b.s.l., including Helheim (which feeds into Sermilik Fjord), Kangerlussuaq (east Greenland) and Jakobshavn Isbrae19, and suggest that ice sheet mass loss was driven by sub-surface warming driven by altered deep ocean convection in the Norwegian Sea and a subsequently weakened AMOC. This positive feedback mechanism could have significant implications for present-day GrIS/AMOC interactions1 and highlights complex and non-linear ice-sheet–atmosphere–ocean dynamics that must be incorporated into future projections.Our study reveals substantial Greenland ice stream collapse in the Younger Dryas during a period of considerable atmospheric and sea-surface cooling in the North Atlantic. Our finding supports the interpretation of marine sediment cores off the SE GrISin situ cosmogenic isotope analysis from ice-free areas along Sermilik Fjord, southeast Greenland during fieldwork in June/July 2009. Sample sites were chosen to span the greatest possible range within the time available for fieldwork, both geographically and attitudinally. Geological maps were consulted prior to sampling to ascertain ideal sampling areas for the quartz-rich rock types needed for 10Be measurement. Accessibility was also a key control when selecting sample areas; sites were accessed by boat and foot, so terrain and local conditions had to be considered. Where possible, samples were taken in bedrock-erratic pairs or triplets (i.e. bedrock-erratic or bedrock-erratic-erratic), to allow for any inconsistencies in glacial history to be detected. Samples were selected based on vegetation cover, potential for snow cover  and their stability . Erratic samples were taken either as whole cobbles (up to ~20 cm) or as subsample of larger erratic boulders (up to 1 m), in which case the top surface was taken. Bedrock samples were taken from glacially streamlined surfaces. Samples were taken up to 12.5 cm thick, although thinner samples were preferred where possible. Samples from lower elevations were above the local marine limit (~40 m a.s.l. (ref.47)). Samples were taken along two vertical transects, from an island in the northern sector of the fjord  and from the east wall of the fjord , as well as from two additional sites ; see Fig. Rock samples were collected for dating by Samples were taken by hand using a four-pound lump hammer and chisel; all samples were labelled and photographed before being securely wrapped and relabelled, and detailed field notes on lithology, vegetation/snow cover and local geomorphology were made. Inclination measurements of the surrounding topography were measured to ascertain topographic shielding, using a handheld clinometer, every thirty degrees on the azimuth, with additional measurements taken if this missed significant features. Latitude, longitude and elevation readings were taken using a handheld GPS device (accurate to ±10 m in elevation).48 and Ditchburn and Whitehead49, modified by C. Fogwill and T. Barrows/J. Stone, respectively. Samples were then ignited to Be oxide, mixed with Nb, loaded into copper cathodes, and 10Be/9Be measured by accelerator mass spectrometry (AMS) at Scottish Universities Environmental Research Centre (SUERC). AMS measurements were normalized to the NIST 27900 standard . Procedural blank ratios were 5.16 × 10−15, 5.48 × 10−15, 3.65 × 10−15 and 1.83 × 10−15. Blank ratios were 0.5–10.7% of sample ratios (mean: 3.4%). The isotopic ratio of the procedural blanks for each batch of samples was subtracted from the sample ratio, with errors in sample and background propagated in quadrature. See Table Beryllium purification chemistry was undertaken at the University of Exeter Cosmogenic Isotope Laboratories. Chemistry followed protocols adapted from Ivy-Ochs50 (http://hess.ess.washington.edu), version 2.2, constants version 2.2.1, using the recently revised 10Be half-life (1.387 Ma refs52) and Be isotope ratio standardization of Nishiizumi et al.53. Age calculation assumed standard pressure, zero erosion and sample density of 2.62 g.cm2. Samples were scaled using the time-dependent Lal/Stone scaling scheme55, following similar studies and the recent analysis by CRONUS-Earth which shows the Lal scheme to provide a better fit to calibration date than the other, neutron-monitor based, schemes56; using other scaling schemes produces exposure ages which differ by 1–4% (~120–480 yrs). For our analysis, we use the production rate from the North American (NENA) calibration set 25, in line with other studies in the area, but also because of the robustness of this calibration set for studies in Greenland57. NENA exposure ages for all samples are shown in Figure 58 (mean = 2.6%/318 yr older than NENA); Western Norway59 (mean = 4.9%/560 yr younger than NENA); and Baffin Island/Arctic60 (mean = 1.5%/173 yr younger than NENA); see Table −3, and we made no correction for inheritance, vegetation or seasonal snow cover, all of which are assumed to be zero or minimal owing to our sampling methods and geomorphic assessment. We assume an erosion rate of zero; using an erosion rate of 2 × 10−4cm yr−1 increases ages by ~2%61.Exposure ages were calculated using the CRONUS-Earth online age calculatorC_Combine function in the chronological software OxCal 4.1 (ref.62); this performs a chi-square test on bedrock-erratic pairs/triplets, highlighting and discarding the outlier in those pairs/triplets that failed. The function then combines the remaining individual samples within each pair/triplet into one event using a cumulative probability density function. This process led to the exclusion of five samples (Table 26)) using the Bayesian modelling of the T_Scaled analysis function within OxCal 4.1 (ref.27). Using Bayes theorem with a Poisson process deposition model, the algorithms employed sample possible solutions with a probability that is the product of the prior and posterior likelihood probabilities. Taking into account the deposition model  and the actual 10Be measurements, the posterior probability densities quantify the most likely age distributions; the outlier option was used to detect ages that fall outside the calibration model for each group and, where necessary, down-weight their contribution to the final age estimates. In this analysis, none of the remaining samples were considered outliers. This analysis generates the thinning profile of the ice stream shown in Fig. 10Be exposure ages, particularly at the lower end of the profile. Of the seventeen 10Be exposure ages included in the model, only five were from unpaired events; of these five, only two were bedrock, one of which (SF0925) was younger than its discarded erratic pair, and therefore not experiencing inheritance. Although the remaining unpaired bedrock sample  is used to help constrain the upper end of the profile, numerous other samples at this elevation and below (e.g. SF0936/37 (bedrock-erratic), SF0943 (erratic), SF0920/21 (bedrock-erratic)) have 10Be exposure age envelopes well within or prior to the onset of the YD. As such, we can be confident that our thinning trajectory is not significantly skewed towards older ages by any potential 10Be inheritance within bedrock samples. The ages resulting from the Bayesian modelling in OxCal are shown in Table −1, or ~0.23 m yr−1 during the Younger Dryas alone (i.e. that due only to non-atmospheric drivers). This is on a comparable order of magnitude to the thinning seen in the fastest-flowing outlet glaciers of Greenland today (~0.84 m yr−1 in glaciers flowing faster than 100 m yr−1 (ref.63)); it is however important to note the difference in observational timescales when making these comparisons.Samples were taken in two vertical transects along the fjord  as well as two additional discrete sampling sites from lower elevations ; comparing the box and whisker plots of the dates from both transects Fig.  shows thes Table : four be43). LOVECLIM comprises an ocean general circulation model and a thermodynamic-dynamic sea ice model, with an horizontal resolution of 3° × 3° and 20 vertical levels, coupled to a spectral T21quasi-geostrophic atmospheric model64. Starting from the background conditions of the Last Glacial Maximum, the model is forced with the time-varying evolution of solar insolation31, ice-sheet topography65, high latitude albedo and atmospheric CO2 (ref.66) for the period 21 ka to 10 ka B.P. The millennial-scale variability of the last deglaciation is simulated by the addition of meltwater in the North Atlantic, Arctic and/or the Southern Ocean. A weakening of the AMOC during the YD is simulated by a 0.25 Sv freshwater flux applied in the Arctic Ocean  between 13 and 12.2 ka B.P.A transient experiment of the last deglaciation was performed with the Earth System model LOVECLIM (ref.The data supporting this manuscript is available in the Supplementary information.Supplementary Information


The build-up of ice on aircraft, bridges, oil rigs, wind turbines, electrical lines, and other surfaces exposed to cold environments diminishes their safe and effective operation. To engineer robust surfaces that reduce ice adhesion, it is necessary to understand the physics of what makes a surface icephobic (“ice-hating”) as well as the relationship between icephobicity and ice adhesion. Here we elucidate the molecular origin of icephobicity based on ice-surface interactions and characterize the correlation between material icephobicity and liquid wettability. This fundamental understanding of icephobic characteristics enables us to propose a robust design for topologically textured, icephobic surfaces. The design identifies the critical confinement length scale to sustain liquid water (as opposed to ice) in between roughness features and can reduce the strength of ice adhesion by over a factor of twenty-seven compared to traditional hydrophobic surfaces. The reduction in ice adhesion is due to the metastability of liquid water; as ambient ice cleaves from the textured surface, liquid water leaves confinement and freezes – a process which takes the system from a local energy minimum to a global energy minimum. This phase transition generates a detachment force that actively propels ambient ice from the surface. Hence, there is a need for engineered surfaces that suppress ice formation and reduce ice adhesion.Ice formation presents a persistent problem in processes where structures are subject to cold conditions. Ice buildup on aircraft wings can cause a 25% increase in drag and 90% reduction in lift3. However, surface defects and homogeneous nucleation can eventually trigger the formation of ice on the substrate4. Water can also condense and freeze or desublimate in between surface texture, leading to ice occupation of the roughness and significantly increasing ice adhesion to the material5. Lastly, de-icing cycles erode surface roughness and degrade the ice-repellency of the substrate6. A robust, icephobic design that minimizes ice adhesion to the surface must therefore ensure that ambient ice does not fill the space between surface texture by impalement or phase change, despite surface defects or damage.Topologically textured superhydrophobic surfaces have been shown to reduce both ice nucleation and adhesion by enhancing liquid droplet roll-off and sustaining ambient ice on top of the texture7) and slippery liquid-infused porous surfaces (SLIPS8) can decrease ice adhesion by introducing a lubricant that more favorably wets a textured surface than water. In these methods, the lubricant must be constantly replenished.De-icing a substrate has also been facilitated by introducing other species onto the surface. Liquid impregnated textured surfaces  and a solid substrate to uncover the molecular origin of icephobicity. The relationship between the various intrinsic contact angles of a substrate shows that both hydrophobic and hydrophilic substrates exhibit icephobic properties and can be textured appropriately to reduce ice adhesion.11, this study examines periodic arrays of pores and pillars at the interface between ambient ice and the material substrate. The confinement length scales for two-phase equilibrium predicted from classical nucleation theory are shown to agree closely with simulation results for both types of texture at the interface, suggesting the underlying theoretical predictions may be generally applicable. We further elucidate how liquid water confined in equilibrium within surface roughness impact the physics of ice adhesion through an analysis of free energy and non-equilibrium steered molecular dynamics. These procedures inform the rational design of textured, icephobic surfaces that reduce ice adhesion to a minimum.Although pores enclosed entirely inside the substrate have been simulated with molecular dynamicsThe mechanism for the reduction in ice adhesion is found to be due to the metastability of liquid water. We demonstrate that for icephobic surfaces sustaining confined liquid water, initiating cleavage of ice adhering to the textured surface activates a persistent force propelling the adhered ice away from the surface. This is due to the change in free energy associated with the phase transition of the liquid water to ice as it leaves confinement. As a result, the strength of ice adhesion to the textured surface is decreased by over a factor of twenty-seven from that to a perfectly smooth hydrophobic surface. A robust, de-icing surface exhibiting strong resistance to defects and damage can be achieved by texturing an icephobic substrate with deep pores or tall, periodic features with spacing below the prescribed critical confinement length scale.The critical confinement length scale below which liquid water is sustained in between surface texture can be determined from heterogeneous nucleation theory. The theory describes the change in free energy associated with the formation of an ice particle from ambient, liquid water near a solid surface.hF is the enthalpy of fusion12, TF the bulk freezing temperature of water associated with the saturation pressure, and T the temperature of the liquid water in thermal equilibrium with the ice particle. The subscripts I, L, S denote the ice, liquid, and substrate, respectively, such that ρI is the density of ice13 and σIL the ice-liquid surface energy. The term g(θIL)  adjacent to bulk ice. The subsequent analysis assumes that the height of the pore is much larger than its radius. Although the cylindrical pore is not a typical anti-icing surface roughness explored in current experiments, it will be shown later that the critical pore radius derived from considering a cylindrical geometry provides a good estimate of the critical confinement length scale for general surface texture. In addition, the cylindrical geometry of the pore is a meaningful approximation for porous substrates; the present study will show that porous surfaces can demonstrate robust de-icing performance, motivating their use in future anti-icing experiments.For simplicity, the surface texture considered will be a cylindrical pore on an otherwise flat, icephobic surface  and the subscript V denotes the vapor. vC the specific volume, pV the vapor pressure, and pS the saturation vapor pressure at temperature T over the condensed phase. Mechanical equilibrium between the condensed phase and water vapor across a curved interface is described by the Young-Laplace equation:To explore other possible phase transition pathways that could result in ice filling the space between surface texture, we examine the coexistence condition between water vapor and condensed water (ice or liquid), which describes the condition for desublimation or condensation in confinement. The critical radius Analogous to equation , the cripV (vapor pressure) or pC is known depending on whether the ambient phase outside of the pore is vapor or condensed water, respectively.Therefore, the equilibrium confined phase is vapor if the pore radius pC = 1 atm, and the confined phase is vapor; for θLV = θIV = θIL = 120°. This does hold not true in general for a specific substrate; however, a relationship between these contact angles has not been established experimentally or computationally in the literature. The assumption is not made for subsequent analyses. Instead, this study will examine the relationship between the three intrinsic contact angles using molecular dynamics.Figure The critical confinement length scale for the reverse case, in which ambient vapor outside the pore exists in equilibrium with the confined condensed phase, is shown in Supplementary Fig. The critical confinement length scale for freezing θIL (icephobic or icephilic) with the liquid-vapor contact angle θLV (hydrophobic or hydrophilic) of a substrate. Since both θIL and θLV are functions of temperature and pressure, the choice of ambient conditions may be somewhat arbitrary. We have chosen to compute θIL at T = 255 K and pC = 1 atm, corresponding to the lower range of freezing depression induced by salts; and θLV at T = 300 K and pC = 1 atm, corresponding to standard laboratory conditions. The contact angles were calculated from equation  shows that this icephobicity occurs due to the lower density of water in the contact layer (within 5 Å from the substrate surface) compared to bulk water density; water molecules favor positions in the bulk rather than at the interface with the solid substrate. This lower contact layer density penalizes the ice phase by deforming the lattice structure and introducing strain energy. On the other hand, the liquid phase exhibits significantly greater mobility in the contact layer compared to ice as indicated by the higher mean squared displacement <u2> of liquid water molecules , the temperature of both phases remain constant and equal , and the ice-liquid contact angle with the vertical pore wall is constant .These trajectories along with Supplementary Fig. θLV and θIL, Supplementary Table θIL and the operating conditions.For two substrate materials with different Due to the latent heat of fusion released  with phase change from liquid water to ice, the presence of the liquid water in between the surface texture should make this equilibrium state metastable. We show subsequently that sustaining liquid water in between surface texture corresponds to a local energy minimum. It can be further shown that this metastability is useful in reducing the strength of ice adhesion to the surface.20. The method uses a history dependent potential to bias the system away from local minima associated with particular values of the collective variable.We now wish to sample the energy landscape of the system in search of a possible global minimum away from the local minimum corresponding to confinement of liquid water in the pore. Metadynamics was used to reconstruct the free energy landscape in the space of a collective variable (CV) representing the vertical distance between the centers of mass of the ambient ice and the substrateNote that the choice of the CV does not fully capture the complexity of de-icing pathways, which for instance can be expected to exhibit energy barriers in orthogonal shear directions or may involve some degree of interfacial premelting. Establishing a free energy landscape in the directions orthogonal to the ice/substrate interface necessitates the use of orthogonal CVs. Interfacial premelting occurs at low undercoolings and must be examined near the freezing point. Both analyses are outside the scope of this study.Although the collective variable selected does not provide the most general description of possible ice/substrate dynamics, it does effectively describe the change in free energy associated with the removal of the ambient ice from the substrate. The free energy landscape in the vertical direction is used to probe the existence of local vs global energy minimia for porous surfaces, in contrast to the single global energy minimum expected for a flat substrate. Thus, the vertical distance between the centers of mass of the ambient ice and the underlying material is an appropriate choice for the collective variable in this context. The kinetics of ice detachment arising through shearing between the ambient ice and the substrate will be explored subsequently in this study using nonequilibrium simulations.Figure The 5 nm cylindrical pore satisfies Thus, the change in free energy elucidates the mechanism by which liquid water confined between surface texture reduces ice adhesion. Along with passively lubricating the interface, the liquid water confined in between surface texture actively does work in pushing ambient ice off the substrate as the metastable phase undergoes a freezing transition during the detachment process.As the pore radius increases past the critical confinement length scale (σSV < σSL) from a surface energy perspective. One may thus be tempted to attribute the decrease in free energy associated with the detachment of the ambient ice from a porous substrate sustaining confined liquid water to this decrease in surface energy (σSV − σSL < 0). However, note that for an icephobic substrate, the liquid phase is preferred inside the pore compared to the ice phase (σSL < σSI). Based on this surface energy analysis, the detachment of the ambient ice from the substrate with the 10 nm cylindrical pore should therefore demonstrate the largest decrease in free energy, since the initial confined phase inside the pore is ice (σSV − σSI < σSV − σSL). However, this is not the case; the largest decrease in free energy occurs for porous substrates in which the confined phase is liquid. Thus, it is the phase transition of confined liquid water to ice that is responsible for producing the smallest energy barrier between the local (attached) and global (detached) energy minima for porous surfaces.For a hydrophobic substrate, the vapor phase is preferred inside the pore compared to the liquid phase  with roughness satisfying Supplementary Fig. ion Fig.  occurs wθIL < 90°), the texture may not necessarily dewet during ice detachment. Supplementary Fig. For hydrophilic substrates  and θIL = 134.0° (computed at T = 255 K) at T = 255 K and pC = 1 atm is 96.75 kPa, which is on the same order of magnitude as experimental values21. The strength of ice adhesion for the pillared surface is less than for the flat surface when the pillar spacing ≲4.5 nm, which matches well with theory  and surface wettability. The confinement of metastable liquid in between surface texture corresponds to a local energy minimum; as the ambient ice is sheared from the substrate, the liquid water escapes confinement and freezes, generating a detachment force driving the system to a global energy minimum where the ambient ice is mostly detached from the substrate. Icephobic surfaces textured according to the critical confinement length scale therefore undergo a permanent decrease in ice adhesion, either by the above mechanism or by ambient ice remaining suspended atop the surface roughness in the unimpaled state.Surfaces that sustain liquid water in confinement are robust against local defects, which negligibly affects the global de-icing performance. As long as the confinement length scale is satisfied on average by the surface texture, the surface will continue to reduce ice adhesion until the texture is completely depleted. Porous or periodically textured surfaces are prime candidates for de-icing applications.23; silylated MCM-41 has a liquid vapor contact angle of 133°24. Similarly, metallic organic frameworks such as MOF 5 have been polymerized to achieve liquid vapor contact angles of 135°25, and carbonized to achieve pore diameters around 6 nm26. MOF pores are interconnected, but as the periodic surface textures show, a continuous film of liquid water can be sustained between surface texture such that the ice/water interface area exceeds the ice/substrate interface area. Such appropriately textured, icephobic materials may be applied as robust surface coatings in a wide range of anti-icing and de-icing applications.Materials with such properties include mesoporous zeolites and MOFs. A zeolite such as MCM-41 has pore diameters between 1 and 10 nmIn this work, molecular dynamics is used to provide a quantitative characterization of material icephobicity and to probe the utility of liquid water sustained between surface roughness in reducing ice adhesion.17 water model was used for computational efficiency and for precision capture of the hexagonal, cubic, and amorphous phases of ice. mW, proposed by Moore and Molerino, is a single particle model that accurately reproduces the structure, energetics, and phase transitions of water. The model comprises a re-parameterization of the Stillinger Webber potential, which incorporates a three-body term that penalizes non-tetrahedral configurations17:The course-grained mW27.Ice nucleation and liquid diffusion are several times faster in mW than in water due to the absence of hydrogen atoms and long-range electrostatic calculations allows crystallization studies using mW to access longer time and length scales28 software. A periodic domain of water was initially crystallized at T < 273.15 K and pC = 1 atm. A solid substrate material was constructed from mW molecules in the ice phase, similar to previous studies27. Molecules defining the substrate were tethered to their equilibrium positions. The interaction between the substrate and water was governed by a 6–12 Lennard Jones pair potential11; water-water as well as surface-surface interactions were governed by the three-body mW potential.Molecular dynamics simulations were implemented using LAMMPSet al. differentiates cubic, hexagonal, and interfacial ice as well as liquid water and low density glass (amorphous ice). The local structures of the four closest neighbors of a mW molecule are projected onto a basis of spherical harmonics. The alignment of the local structure centered on each neighbor characterizes the central mW molecule as belonging to a specific ice or liquid phase27.The CHILL algorithm developed by Moore σ between the various phase interfaces were calculated using the stress tensor method17 in molecular dynamics simulations:Lz is the length of the simulation domain normal to the interface, PN = Pzz is the normal component of the stress tensor with respect to the interface, and θLV, θIL, θIV) interaction with the substrate:The surface energies mentclasspt{minima11. It would be of interest, but not in the scope of the current study, to examine how surface energy changes with surface polarity.The surface energy between water and the substrate is changed to modulate surface hydrophobicity/icephobicity by altering the Lennard Jones energy parameterSupplementary Information


Skin reaction may develop at the site of vaccine administration. A 54-year-old woman who developed a cellular blue nevus at the site of the combined tetanus, diphtheria, and acellular pertussis (Tdap) vaccine injection four years prior to presentation is described. In addition to blue nevus, other reactions at combined tetanus, diphtheria, and pertussis vaccine injection sites include abscess, deep reactive nodular infiltrates of mixed inflammation, and necrotizing granuloma. In conclusion, blue nevus can be added to the list of cutaneous events that can occur at Tdap vaccination sites. Adverse cutaneous events can occur in vaccine injection sites . These iA 54-year-old woman presented with a new lesion on her left arm. Four years prior to presentation, an acquaintance of the patient developed pertussis, which prompted the patient to seek immunization. The patient had not been vaccinated against pertussis in childhood. Based on the Center for Disease Control age recommendations, Tdap was administered on her left deltoid; the patient subsequently developed a new lesion over the previous site of the vaccine administration.Cutaneous examination revealed a 5 x 5 mm papule on her prior Tdap vaccination site Figure . A punchCorrelation of the clinical history, lesion morphology, and pathology established the diagnosis of a cellular blue nevus developing at the site of the Tdap vaccination. The residual lesion was excised. There has been no recurrence.Bordetella pertussis bacilli and licensed in 1914 . T-7. TMycoOur patient developed a cellular blue nevus on the site of the injection after she received a Tdap vaccination . To the A postulated pathogenesis for the formation of a new cutaneous disease at a site of prior cutaneous insult has been attributed to the creation of an “immunocompromised district”-a localized site of immune destabilization in the skin leading to increased risk of developing dermatoses . ThroughThe administration of vaccinations is extremely common. The development of cutaneous adverse reactions at the site of vaccination is a rare occurrence. Although the rate for cutaneous adverse reactions at the vaccination sites remains to be established, the estimated rate would be very low based on the published literature of these events.Adverse events may occur in local vaccination sites. Our patient developed a cellular blue nevus at the site of the Tdap vaccine administration. The development of a melanocytic nevus at the site of Tdap vaccination has not been previously described as a post-vaccination sequela. We suggest that this vaccination site-related tumor may have resulted from the creation of a local immunocompromised district in the skin following Tdap vaccination.


Protein phosphorylation, mediated by protein kinases, plays a crucial role in cellular regulation. One of the most important protein kinases is protein kinase A (PKA). N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) is often used as a “PKA specific inhibitor” to study the involvement of PKA in signaling pathways. However, evidence from cell-free experiments has suggested that H89 can also inhibit other protein kinases. In this study, previously generated PKA-null and PKA-intact mouse cell lines derived from mpkCCD cells were treated with H89 over a range of concentrations commonly used in the literature, followed by mass spectrometry-based phosphoproteomics to globally assess changes in phosphorylation. From a total of 14,139 phosphorylation sites quantified, we found that 571 and 263 phosphorylation sites with significant changes in abundance in PKA-intact and PKA-null cells, respectively. Analyses of sequence logos generated from significantly decreased phosphorylation sites in PKA-intact and PKA-null cells both revealed a preference for basic amino acids at position −3 and −2. Thus, H89 appears to inhibit basophilic kinases even in the absence of PKA. Likely H89 targets include basophilic protein kinases such as AKT, RSK, AMPK and ROCK. We conclude that, in intact cells, H89 can affect activities of protein kinases other than PKA, and therefore responses to H89 should not be regarded as sufficient evidence for PKA involvement in a signaling process. Regulation of protein phosphorylation is mainly controlled by various kinases, a class of enzymes that catalyze the transfer of the phosphoryl group from adenosine triphosphate (ATP) to the hydroxyl group of proteins. Among over 500 known kinases2, protein kinase A (PKA) is one of the most studied. PKA catalytic subunits are coded by two separate genes, Prkaca and Prkacb. Functions of PKA span a wide-range of biological processes, including cell proliferation, differentiation, cell cycle control, metabolism, and transport3. Knowledge of PKA kinase-substrate relationship is thus essential to fully understand cellular signaling in both health and disease.Protein phosphorylation is one of the most important post-translational modifications. This reversible modification plays a crucial role in cellular signal transductionin vitro suggests that H89 may not be specific for PKA. Based on such in vitro kinase assays, H89 was shown to inhibit at least 8 other protein kinases  by more than 80% at 10 µM4. In fact, H89 caused even greater inhibition of MSK1, RSK1, and ROCK2 than of PKA. A remaining question is to what extent H89 inhibits other kinases in intact cells. The recent development of mouse collecting duct cell lines in which CRISPR-Cas9 was used to delete both PKA catalytic subunit genes (PKA-null cells) allows this question to be addressed5. Here, we used large-scale quantitative phosphoproteomics analysis to test the effects of H89 in PKA-null and PKA-intact cells. Our results show that H89 causes broad effects on the phosphoproteome of PKA-null cells establishing that H89 has actions that are not specific to PKA.To determine if PKA is involved in a given cellular process, many investigators have relied on the use of small molecule kinase inhibitors. One such inhibitor is N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89). It is widely claimed to be a potent and specific PKA inhibitor and is thus commonly used. However, evidence using purified recombinant protein kinases 6 in which Prkaca and Prkacb gene expression was previously deleted (“PKA-null” cells) by introducing mutations using CRISPR-Cas95. “PKA-intact” cell lines did not have deletion of either gene. We used three different PKA-null lines and three different PKA-intact lines in separate biological replicates. For each biological replicate, we also included up to three technical replicates. Cells were initially maintained in complete medium, DMEM/F-12 containing 2% serum and other supplements . Cells were seeded on permeable membrane supports (Transwell) and grown in complete medium containing 0.1 nM 1-desamino-8-d-arginine-vasopressin  for 4 d. Then, the medium was changed to simple medium  with 0.1 nM dDAVP and maintained for 3 d. Transepithelial resistance (>2 kΩ-cm2) was measured by EVOM (WPI) prior to cell harvest to assure confluence and polarization. Cells were treated with 0.1, 1 or 10 µM H89 in DMSO  for 30 min added to both apical and basal media. Controls were in 0.1% DMSO for 30 min.The study utilized immortalized mpkCCD cellsCells were lysed with Laemmli buffer . Samples were homogenized using a QIAshredder (Qiagen). Protein concentration was measured using the BCA assay method. Samples were added with 5× loading buffer  and incubated at 65 °C for 10 min. The denatured samples were subjected to SDS/PAGE. The proteins were transferred to nitrocellulose membranes and probed with anti-PKA antibodies (CST #4782). Blocking buffer and infrared fluorescence-conjugated secondary antibodies were obtained from LI-COR. Fluorescence images were visualized by a LI-COR Odyssey System.Cells were washed three times with ice-cold PBS and then lysed with 8 M urea buffer  and scraped into Eppendorf tubes, followed by sonication to solubilize proteins. Protein concentrations were measured using Pierce™ BCA Protein Assay Kit. Protein lysates were reduced with 20 mM dithiothreitol for 1 hour at 25 °C, and then alkylated with 40 mM iodoacetamide for 1 hour at 25 °C in the dark. The samples were diluted eight-fold with 20 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5) to reduce the urea concentration prior to digestion with Trypsin/LysC (Promega) (1:20 wt/wt.) overnight at 37 °C. The peptides were desalted using hydrophilic-lipophilic-balanced (HLB) extraction cartridges (Oasis) and quantified using Pierce™ Quantitative Colorimetric Peptide Assay. For each replicate, equal amounts of each sample (160–400 μg of peptide) were labeled using TMT10Plex Mass Tag Labeling Kit (Thermo Scientific) following the manufacturer’s instructions. Samples were combined and, after taking an aliquot for total proteomics, phosphopeptides were sequentially enriched following Sequential Enrichment from Metal Oxide Affinity Chromatography protocol . We then fractionated the samples (12 fractions) using high pH reverse phase chromatography (Agilent 1200 HPLC System). Samples were then vacuum-dried and stored at −80 °C until analysis.The dried peptides were re-suspended with 0.1% formic acid in LC-MS grade water (J.T. Baker) before mass spectrometry analysis. Total and phosphopeptides were analyzed using a Dionex UltiMate 3000 nano LC system connected to an Orbitrap Fusion Lumos ETD mass spectrometer equipped with an EASY-Spray ion source (Thermo Fisher Scientific). Peptides were introduced into a peptide nanotrap at a flow rate of 5 μL/min. The trapped peptides were fractionated with a reversed-phase EASY-Spray PepMap column  using a linear gradient of 4 to 32% acetonitrile in 0.1% formic acid (120 min at 0.3 μL/min).Mus musculus UniProtKB7 reference proteome  using MaxQuant8 1.6.2.3. Reporter ion MS3 with TMT10plex was specified as labeling type and carbamidomethyl (C) was configured as fixed modifications. Lot-specific TMT isotopic impurities correction factors were used as recommended in TMT product data sheets. Variable modifications included phospho (STY), oxidation (M), and acetyl (Protein-N-term). Site localization scores generally exceeded 0.95  is undefined, we arbitrarily set the value of XH89 in control group (no H89) to zero. P values for βH89 were derived from moderated t-test against zero, implemented in Linear Models for Microarray Data (LIMMA) R package10. A significant change in phosphorylation for a phosphosite was defined as |βH89| > 0.15 and P < 0.05.For each replicate i, after logarithmic transformation of the data, we fit the equationH89. Centralized peptide sequences (±10 amino acids) in each category were used to generate sequence logos using an updated version of PhosphoLogo11 (filtering χ2 < 0.01). Phosphosites with site localization probability (MaxQuant) less than 0.75 were excluded. The list of all quantified phosphosites, whether changed or not, were used to create a position-specific background matrix of amino-acid frequencies.The phosphosites that were affected by H89 in each cell type were then categorized as increased or decreased based on the sign of β12 was then performed on βH89 of PKA-null and PKA-intact cells. Statistical test for enrichment of each term is based on MANOVA test on rank of each βH89. P-values were adjusted with Benjamini-Hochberg method to control false discovery rate at 0.01.To test whether effects of H89 treatment on protein phosphorylation are enriched for any protein annotation terms, each protein was annotated with HPRD PhosphoMotif, gene ontology, KEGG, Pfam, GSEA, Corum, InterPro, PRINTS, PROSITE, Reactome, and SMART terms using Perseus software. 2D annotation enrichment analysisin vitro inhibition constant (Ki) value between 0.048 and 0.135 μM13. Consequently, in this study, we tested H89 concentrations ranging from 0 to 10 μM.We reviewed the literature to determine the concentrations that have been used to investigate the role of PKA in various cellular processes  using the SMOAC phospho-enrichment protocol followed by LC MS/MS combined with high pH fractionation allowed quantification of 14,507 phosphosites including phosphosites in mono-phosphopeptides and combinations of sites in multi-phosphopeptides. This corresponds to 4,730 phosphoproteins. More than 90% of peptides identified were phosphopeptides after SMOAC. Pre-SMOAC enrichment samples were also analyzed, identifying 9,291 proteins.H89) representing the slope of the log2(peptide intensity) versus log10(H89 concentration) lines, and P values were obtained using a moderated t-test for βH89. Figure Myl12b), that are physiologically important in the regulation of non-muscle myosin II-A and II-B14. The phosphorylation is seen to be inhibited by H89 in both PKA-intact and PKA-null cells. This finding is consistent with the view that H89 inhibits one or more kinases among myosin light chain kinase, rho kinase and Pak kinases that have been documented to phosphorylate these sites17. The second example is a phosphorylation site, S677 in SCY1-like protein 2  which was inhibited by H89 only in the PKA-intact cells. Phosphorylation at this site was previously shown to increase in response to vasopressin18 and was decreased in PKA-null cells versus PKA-intact cells5, suggesting that it is a PKA target site. The reader can examine any particular phosphorylation site interactively at https://esbl.nhlbi.nih.gov/H89/phosphosite.To determine the effect of H89 on protein phosphorylation in each cell line, multiple linear regressions for phosphopeptide abundance versus H89 concentration and replicate number (to account for batch effect) were performed for each phosphosite (see Method). This effect was quantified as the coefficient  phosphosites that were significantly increased and 402 (2.8%) that were significantly decreased in response to H89 in PKA-intact cells Fig. . In PKA-% phospho11. Sequence logos based on information content for each subset of significant phosphosites were created as shown in Fig. 19. Additional 2D annotation enrichment analyses  is inhibited by H89, thereby resulting in activation of downstream kinases Fig. . Additio20 and transcriptomics studies21, prior knowledge about kinase specificities compared to the sequence logos of inhibited sites shown in Fig. 23. Table Melk) and Aurora-related kinase 2 (Aurkb) kinases are the basophilic kinases that are increased in abundance in the PKA knockout, providing a potential explanation for sites inhibited by H89 in PKA-null but not PKA-intact cells.What non-PKA protein kinases are inhibited by H89 to account for the extensive decreases in phosphorylation observed in PKA-null cells? Several types of information are pertinent to this question, namely the identification of kinases that are expressed in mpkCCD cells from prior proteomicsKnowing protein kinase/substrate networks is crucial to the understanding of cellular signal transduction. Over the years, H89 has been used to infer a role for PKA in many processes. In this study, we utilized unbiased phosphoproteomics and showed that many protein phosphorylation sites were altered in abundance by H89 treatment, both in PKA-intact and PKA-null cells. Because the peptide hormone vasopressin is the main stimulus to PKA activation in mpkCCD cells, the studies were done in the presence of the vasopressin analog dDAVP.in vitro approaches are useful to study direct effects of small molecule inhibitors like H89 on each specific kinase, they may not predict the effects of H89 in living cells for a variety of reasons. The mechanism of H89-mediated kinase inhibition is believed to be through competition with ATP4. Therefore, differences in ATP concentration locally within the cell would modify H89 effects. In addition, the complex environment of the cell with multiple interacting proteins may modify responses to H89. To analyze the specificity of H89 toward PKA within cells, we took advantage of PKA-null mpkCCD cells previously generated in our lab using CRISPR-Cas95 and their parent PKA-intact cells to investigate the effects of H89 on the phosphoproteome. Indeed, H89 in a concentration range of 0.1 to 10 μM had clear cut effects even in the PKA-null cells. Thus, care should be taken in formulating conclusions about the role of PKA in cellular processes based on H89 experiments.Although other investigations have already shown possible non-PKA inhibiting effects of H89, they were performed in cell-free systems. While these in vitro data points to MSK1, PKBα, SGK, RSK1, RSK2, ROCK2, AMPK, and CHK124. The sequence logos generated from the current studies suggest as well that H89 may inhibit one or more basophilic kinases, which include AKT, RSK, AMPK ROCK, CHK, MARK, MELK, and AURB. As expected in in vivo studies, some of the effects of H89 on phosphorylation were likely indirect , also known as RSK1, was decreased at an activating site, viz. S380 [AGAHQLFRGFpSFVATGLMEDD], which likely accounts for secondary decreases in phosphorylation of other proteins. This may include a Eukaryotic translation initiation factor 4B (Eif4b) double phosphopetide (S422 and S425), a known target of RSK125, whose abundance was found to decrease.What protein kinases are inhibited beyond PKA? The ect Fig. . For exaet al. previously reported that less than 50% of all possible protein kinases were detected in mpkCCD cells using deep proteomic profiling20. Thus, in other cell types, H89 may inhibit a different set of kinases and consequently have distinct effects on the phosphoproteome.One limitation of our approach is that kinase inhibition activity of H89 can only be detected if that kinase is both present and active in the cells. For that reason, it is likely that some phosphorylation sites that would be targeted by H89-sensitive kinases in other cell types, but which are unexpressed in mpkCCD cells, were unchanged. Yang In conclusion, we showed that H89 not only inhibits PKA, but also affects many protein phosphorylation events independent of PKA. Hence, a change in phosphorylation in response to H89 cannot alone be considered as sufficient evidence for a role for PKA.Supplementary figures and tablesSupplementary dataset S1Supplementary dataset S2Supplementary dataset S3


Prinsepia utilis Royle fruits using molecular docking analysis to delineate their inhibition toward digestive enzymes. A total of 20 phenolics was identified and quantified. Rutin, quercetin-3-O-glucoside, and isorhamnetin-3-O-rutinoside were the major phenolic compounds in the total phenolic fraction and flavonoid-rich fraction. The anthocyanin-rich fraction mainly contained cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. All of the fractions exhibited strong radical scavenging activities and good inhibition on cellular reactive oxygen species (ROS) generation in H2O2-induced HepG2 cells, as evaluated by DPPH and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. Moreover, the powerful inhibitory effects of those fractions against pancreatic lipase and α-glucosidase were observed. The major phenolic compounds that were found in the three fractions also showed good digestive enzyme inhibitory activities in a dose-dependent manner. Molecular docking analysis revealed the underlying inhibition mechanisms of those phenolic standards against digestive enzymes, and the theoretical analysis data were consistent with the experimental results.The present study investigated the phenolic profiles and antioxidant properties of different fractions from  In recent years, incidences of diseases related to metabolic syndrome have risen rapidly around the world, such as obesity, cardiovascular disease, and diabetes . A high-Plant phenolics, a group of plant secondary metabolites, are characterized by the presence of an aromatic ring containing one or more hydroxyl substituents that are widely distributed in plants . AccordiPrinsepia utilis Royle, which belongs to the Rosaceae family, is a deciduous perennial shrub. This plant mainly grows in high-altitude areas (1000–2500 m) in northern India and southwest China in the Himalayan mountain district −m/z = 609.1476) was positively characterized as rutin by comparing it with the corresponding authentic standard. The characteristic ion fragment (m/z = 300.0282) of compound 9 resulted from the loss of rutinose moiety. Compound 12 ([M − H]−m/z = 623.1636) was positively identified as isorhamnetin-3-O-rutinoside by a commercial phenolic standard. The loss of rutinose moiety of isorhamnetin-3-O-rutinoside produced a characteristic ion at m/z = 315.0519. However, compound 10 was detected with the highest peak area in FF, which was tentatively identified as quercetin-3-O-glucoside ([M − H]−m/z = 463.0896), and the fragment ion (m/z = 300.0282) was produced from the loss of glucose moiety from compound 10. Compound 15 ([M]+m/z = 449.1074), which was positively characterized as cyanidin-3-O-glucoside, was the main phenolic compound detected in the AF: the loss of glucose moiety from compound 15 resulted in the production of a fragment ion with m/z = 287.0548. Most of the phenolic compounds that were detected in the present work, especially anthocyanins, have not been reported in this plant to date. However, protocatechuic acid, rutin, quercetin-3-O-glucoside, and isorhamnetin-3-O-rutinoside, all of which were detected with higher peak areas in the present research, were also reported by a previous work in the seeds of P. utilis  × 100. Each test was measured in triplicate.The antioxidant activities of all of the fractions from  earlier . The absP. utilis fruits were measured by ABTS assay as described earlier [control − ODsample)/ODcontrol] × 100. Each test was measured three times.The antioxidant activities of all of the fractions from  earlier . The abs (DMEM) medium containing 10% fetal bovine serum (FBS), 1% antibiotic mixture of streptomycin (100 mg/mL) and penicillin (100 U/mL) at 37 °C with a humidified 5% CO2 atmosphere. The methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was applied to evaluate the cytotoxicity of the sample to HepG2 cells [5 cells per well, and incubated for 24 h. Thereafter, DMEM with or without tested samples was added into each well and removed at the end of the incubation period (24 h), followed by being attacked with one mM of H2O2 for four h (except for the control group), and each group had three repetitions. Then, cells were harvested and washed twice with phosphate buffer solution (PBS). The cells of all of the groups were incubated with 10 μM of DCFH-DA for 20 min, and washed with serum-free medium to remove excess DCFH-DA by centrifuging at 156× g for five minutes. Subsequently, the intracellular ROS of each group was measured by flow cytometry with guava® easyCyte 6-2L  within 45 min. All of the experiments were measured in triplicate.HepG2 cells were supplied by Kunming cell bank, Chinese Academy of Sciences , which were cultured in normal Dulbecco’s Modified Eagle MediumG2 cells . All of G2 cells  with minP. utilis fruits, rutin, isorhamnetin-3-O-glucoside, and cyanidin-3-O-glucoside against pancreatic lipase activity were measured according to the method reported earlier [The inhibitory effects of different fractions from  earlier . The absP. utilis fruits, rutin, and isorhamnetin-3-O-glucoside were evaluated as previously described [The α-glucosidase inhibition activities of different fractions from escribed  with somO-glucoside, and cyanidin-3-O-glucoside) and orlistat (a pancreatic lipase inhibitor) to pancreatic lipase, and two phenolic standards (rutin and isorhamnetin-3-O-glucoside) and acarbose (α-glucosidase inhibitor) to α-glucosidase, were performed by using SYBYL-X 2.1.1 , according to previous studies [https://www.ncbi. nlm.nih.gov/pccompound), and the PDB format of the porcine pancreatic lipase (PDB ID:1ETH) and isomaltase (PDB ID: 3A4A) was downloaded from RCSB PDB (http://www.rcsb.org/pdb/home/home.do). The homology modeling of α-glucosidase (S. cerevisiae) was performed by SYBYL-X 2.1.1 due to the structural information of α-glucosidase (S. cerevisiae) not being available. The homology modeling result showed that isomaltase from the same organism shares high similarity with α-glucosidase. Therefore, isomaltase was used as the template in the current α-glucosidase docking analysis. The Surflex-Dock Geom (SFXC) was used as the docking mode. All of the water substructures were deleted in the “remove substructures” option. Hydrogens (Random) and charges (Gasteiger–Huckel) were added. Subsequently, a lipase–ligand file was generated and saved as a molecular SD format. C-Score calculations were applied during the binding procedure in order to select the better results for analysis.The molecular docking assays of three predominant phenolic standards  ± standard deviation (SD) and analyzed by using one-way ANOVA. Tukey’s procedure was applied to determine the significance of differences (p < 0.05). All of the analyses were performed by using Origin 8.5 software . The Pearson’s correlation coefficient was examined by using SPSS 20.0 .Data are presented as mean values (P. utilis fruits was analyzed by UHPLC-ESI-HRMS/MS. A total of 20 phenolics were identified and quantified. Rutin, quercetin-3-O-glucoside, and isorhamnetin-3-O-rutinoside were the major phenolic compounds in the TPF and FF. The AF mainly contained cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. All of the fractions showed good radical scavenging activities and the strong inhibition of cellular ROS generation of in H2O2-induced HepG2 cells. Meanwhile, those fractions exhibited powerful inhibitory effects against pancreatic lipase and α-glucosidase. The major phenolic compounds that were detected in those fractions also exerted good digestive enzymes inhibitory activities in a concentration-dependent manner. Molecular docking analysis revealed the underlying inhibition mechanisms of those phenolic standards against digestive enzymes, which were consistent with the experimental results.In the present study, the phenolic composition in the TPF, FF, and AF from


Canis familiaris allergen 1 to 7 (Can f 1–Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated  In particular, the domestic dog, Canis familiaris, is a significant source of indoor allergens, found in its saliva, dander, sweat, and urine, that cause allergy symptoms such as asthma and allergic rhinitis. Due to an increase in indoor breeding leading to frequent and intimate contact between dogs and humans, dog allergies have become increasingly prominent worldwide, especially in advanced nations4.Exposure to pet-derived allergens is a major risk factor for allergy development7. On the other hand, Can f 3, Can f 5, and Can f 7 have been classified as serum albumin, urinary kallikrein, and epididymal secretory protein E1/Niemann pick type C2 protein, respectively10. In allergology, lipocalins represent a significant protein group as major mammalian respiratory allergens from dogs, horses, cats, rats, and mice belong to this family12. Lipocalins are characterised by a common tertiary structure exhibiting an eight-stranded, antiparallel β-sheet closed back on itself forming a continuous hydrogen-bonded β-barrel12. The tertiary structures of Can f 2 and Can f 4 have been previously determined14, while those of Can f 1 and Can f 6 have not. Of the lipocalin allergens, Can f 1 was reported as a major dog allergen, where no less than 50–75% of dog-allergic subjects were sensitised12. Can f 6, a recently identified lipocalin allergen produced from the submaxillary gland and detected in dander7, shows a high amino acid sequence identity with Fel d 4 (67%) and Equ c 1 (55%), major cat and horse lipocalin allergens, respectively15. Consistent with this finding, sera from dog-sensitized patients with immunoglobulin E (IgE) to Can f 6 show cross-reactivity with Equ c 1 and Fel d 416. It has been reported that 38% of dog- and cat-sensitised patients possess Can f 6-reactive IgE15. These reports suggested that Can f 6 and its homologous allergens possibly contribute to multi-sensitization, leading to allergic symptoms commonly caused by contact with different mammals.Seven proteins designated serially as Can f 1 to Can f 7 have been identified as dog allergens. Can f 1, Can f 2, Can f 4, and Can f 6 are members of the lipocalin family, which is composed of various secretory lipid-transport proteinsin silico based on its structural information and the presence of charged residues, a frequent feature of epitopes identified previously. Moreover, we demonstrated the validity of the prediction using mutated rCan f 6 proteins generated by site-directed mutagenesis.In this study, we produced a recombinant protein of the dog lipocalin allergen Can f 6 (rCan f 6) and determined its three-dimensional structure at 2.35 Å resolution. We then predicted the IgE epitopes of Can f 6 Purified rCan f 6 yield was calculated to be 21 mg/L using the absorbance at 280 nm. Gel filtration chromatogram of rCan f 6 exhibited a single peak Fig. , indicatTo investigate the assembly state of rCan f 6 in solution, we performed analytical ultracentrifugation-sedimentation velocity (AUC-SV), which provides the molecular mass of a protein or association state for reversibly-interacting proteins in solution. The majority (more than 90%) of rCan f 6 species had a sedimentation value  of 2.0 S Fig. . The est17. It was also demonstrated by western blotting that rCan f 6 reacts with IgE in the serum from patient 16, but not sera from the 6 non-dog-allergic donors  of 0.63 Å for main-chain atoms. Supplementary Fig. 18, an eight-stranded antiparallel β-barrel  interrupted by β-hairpin-forming loops and an accessory α-helix  were substituted with triple alanine  Fig. . When se22, it is conceivable that structured regions of allergens are necessary for accurate epitope determination.To the best of our knowledge, this is the first report describing the crystal structure of the dog lipocalin allergen Can f 6. Using structural information, we determined the Can f 6 epitopes possibly involved in cross-reactivity with Fel d 4 and Equ c 1, cat and horse lipocalin allergens, respectively. Since accumulating evidence suggests that conformational epitopes are important in IgE cross-reactivity15. Although the rate of Can f 6-reactive sera was slightly higher in the present study (47%), this difference can be explained by the cross-reactivity between Can f 6 and Fel d 4 that was demonstrated previously15. The possibility that Fel d 4-sensitised cat-allergic patients were counted as dog-allergic patients, or vice versa, complicates the accurate diagnosis of pet allergies. This is supported by a previous study showing that 61% of sera from a mixture of cat- and dog-exposed allergic patients are reactive to Can f 67.A previous study demonstrated that sera from 38% of dog-allergic patients possessed Can f 6-reactive IgE23. Hilger et al.7 modelled the tertiary structure of Can f 6 and Fel d 4 in silico based on the crystal structure of Equ c 1 and showed that these allergens share highly conserved structural characteristics as well as amino acid sequence identity. Consistent with these findings, the X-ray crystal structure characteristics of Can f 6 remarkably resembled those of Equ c 1  encompasses the mutated sites of both Can f 6-mu-2 (KEK) and mu-3(EEN). The fact that IgE reactivity of denatured rCan f 6-mu-2 was diminished compared with rCan f 6 regardless of serum , 597-nucleotide (nt) open reading frame (ORF) was found in the cDNA and the first 69-nt sequence of the ORF was predicted to encode a 23-amino acid (aa) signal peptide. Thus, the region encoding a putative and mature Can f 6 protein was amplified by PCR using the primer sets listed in Table BamHI and SalI restriction enzymes and inserted into the expression plasmid pGEX4T-2, which produces rCan f 6 harbouring an additional 2 amino acid residues (Gly-Ser) at its N-terminus. After sequence validation of the cloned PCR product, BL21 (DE3) Escherichia coli cells were then transformed with the plasmid construct pGEX4T-2-rCan f 6.The nucleotide sequence of Can f 6 and Fel d 4 cDNAs was obtained from GenBank (accession numbers HE653774 and AY497902). Using Signal IP4.1 software  and disrupted by ultrasonication followed by centrifugation. The supernatant was filtered using 0.45-µm disk filters and loaded onto a Glutathione Sepharose 4B column . The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin  was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg  with PBS. Protein concentration was determined by measuring the absorbance at 280 nm. Mutated rCan f 6 proteins were expressed and purified as described for rCan f 6.To express the recombinant Glutathione S-transferase (GST)-fused rCan f 6 and rFel d 4, 3CN (1 µL) mixed with 2 µL of a matrix solution (10 mg/mL) and then air-dried on the sample plate.The molecular mass of rCan f 6 proteins was determined by the AXIMA confidence spectrometer  in the positive ion mode. rCan f 6 matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was carried out in linear mode using sinapinic acid  as matrices. The lyophilised sample (0.1–0.5 nmol) was dissolved in 0.05% trifluoroacetic acid (TFA)/50% CHC(s) distribution model using the SEDFIT 14.4 software26. We used a resolution of 500 increments between 0 and 15S as well as maximum entropy regularization (p = 0.68).AUC-SV experiments were performed using the ProteomeLab XL-I Analytical Ultracentrifuge . Sample concentration was adjusted to 50 µM. Runs were carried out at 60,000 rpm with a temperature of 20 °C using aluminium double-sector centrepieces and a four-hole An-60 Ti analytical rotor equilibrated to 20 °C. Sedimentation behaviour was monitored with UV detection optics at 280 nm. All SV raw data were analysed by the continuous 27. Processing statistics are summarised in Table All crystallisation experiments were performed at 20 °C using the sitting-drop method of vapour diffusion. Under optimised conditions, a crystallisation drop containing 2 µL rCan f 6 protein solution  and 2 µL precipitant solution (1.0 M ammonium dihydrogen phosphate and 20% [w/v] PEG3350) was equilibrated against 200 µL precipitant solution. Crystals suitable for X-ray diffraction appeared within one week in crystallisation drops  as the search model; this template shares 61% sequence identity with Can f 6. Although four molecules of Can f 6 were found in the asymmetric unit, non-crystallographic symmetry restraints were not applied during the model refinement. Iterative rounds of model building and refinement were carried out using COOT30 and Refmac531 in the CCP4i package, respectively. Graphical representations were prepared using PyMOL software (http://www.pymol.org). Refinement statistics are summarised in Table The structure was solved by molecular replacement using MOLREP® High Sensitivity Streptavidin-HRP  diluted 1:10,000 times with PBS was added (100 µL/well). For detection of allergen-IgE complexes, TMB Substrate Reagent  was added (100 µL/well) and incubated for 15 min at room temperature. To stop the reaction, 1 N H2SO4 was used (100 µL/well). Absorbance at 450 nm was measured using the BioTekTM EonTM Microplate Spectrophotometer .rCan f 6 and mutants were immobilised onto ELISA plates  overnight at 4 °C. After washing with PBS, the plate wells were blocked with 3% skim milk in PBS for 1 h at room temperature. Sera from 38 dog-allergic patients and 6 non-dog-allergic donors were diluted 1:250 with PBS, added to the wells (100 µL/well), and incubated for 1 h at 37 °C. Subsequently, biotin-labelled goat anti-human IgE antibody  diluted 1:5,000 with PBS was added to the wells (100 µL/well) and incubated for 1 h at 37 °C. Next, Pierce32. rCan f 6 proteins were separated by 12% native polyacrylamide gel electrophoresis (PAGE) and 15% SDS-PAGE, transferred to 0.2-µm polyvinylidene fluoride (PVDF) membranes , and then stained with Ponceau S . After blocking for 1 h at room temperature in 3% skim milk, the membrane was incubated with diluted (1: 100) sera from dog-allergic patients for 16 h at 4 °C. After washing with Tris-buffered saline/Tween 20 (TBST), the blots were incubated with a biotin-labelled antibody against human IgE (SeraCare) and HRP-conjugated streptavidin (Thermo Fisher Scientific). Immunoreactivity was visualised using the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and a LAS4000 imaging device .Western blotting was performed as described previouslySupplementary figures and tables


This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI).Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG.N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition (p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone .Significant inhibition with DI was possible with average inhibition of 23% (Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients. Three serological criteria for APS are included: IgG and IgM anti-cardiolipin (aCL) and anti-beta-2-glycoprotein I (aβ2GPI) ELISAs, and the lupus anticoagulant test. Whilst IgG and IgM antiphospholipid antibodies (aPL) are well recognized, the importance of IgA aPL is still controversial.24 but who have typical clinical features of APS (seronegative APS). Part of the controversy regarding IgA aCL and aβ2GPI is due to the variable prevalence reported in cohorts.5 Some groups found significant clinical associations with thrombosis and APS diagnosis,6 whilst others did not.7Diagnostically IgA aPL has been evaluated in several studies and found even in patients negative for the classical diagnostic antibodies8 implying potential pathogenicity in patients in the absence of IgG and IgM aPL antibodies. Other studies have suggested this pathogenicity has already been seen in patients.10In vitro studies of IgA aPL have shown pathogenic potential in a mouse model of APS11 though these have been shown to have their own risks.12 This has led to a spate of research into novel therapeutics for APS.1315The standard of treatment for APS patients is lifelong anticoagulation either by warfarin, heparin or, more recently, using direct oral anticoagulants such as rivaroxaban,These novel therapeutics target β2GPI rather than the clotting cascade, and the majority of research has been carried out on the IgG subclass. This study used one of these novel therapeutics (Domain I) and its modified forms (PEGylated DI) to test whether it was possible to disrupt the binding of patient IgA antibodies to β2GPI immobilized in an ELISA.2 Data on positivity for aCL and lupus anticoagulant (LA) were taken from clinic records. Patients with known IgA positivity were selected for this study.Thirteen patients were selected from the University College London Hospital (UCLH) and University of Istanbul cohorts of patients with APS, with or without systemic lupus erythematosus (SLE). Patient sera were tested previously for IgG, IgM and IgA aβ2GPI and Anti-Domain I positivity.13 Briefly: protein was produced bacterially in Escherichia Coli and folded manually through dilution. Purification was by Immobilised Metal Affinity Chromatography (IMAC), Cation Exchange Chromatography (CEX) and gel filtration. PEGylation was carried out using TheraPEGR targeting disulphide bonds. Two conjugates were produced using 20 kDa and 40 kDa poly(ethylene glycol) (PEG). Expression tag was removed by FXa cleavage and protein was dialysed against phosphate-buffered saline before quantification and storage. Concentrations of PEGylated conjugates were expressed in terms of mass of DI.DI was prepared as previously and PEGylated as described.g, 10 min, 4℃) and supernatant stored for analysis. Samples were washed with 500 µl of binding buffer, centrifuged and bound IgG was eluted with glycine  rotating for 1 h before centrifugation and neutralization with 10 µl of Tris  per 100 µl of glycine.Serum samples of IgG/IgA aβ2GPI positive patients were depleted using protein G beads. Serum samples were diluted 1:1 with binding buffer  and applied to 100 µl of protein G beads (Sigma) for 1 h rotating at room temperature. Samples were centrifuged . Purification was as for serum IgG depletion with the following exception: serum was diluted 2:1 with 10 mM sodium phosphate pH 7.2 and loaded onto a 1 ml column.2 The IgA aβ2GPI inhibition ELISA was carried out as in McDonnell et al.13 with the following alterations: secondary antibody was an anti-human IgA (Abcam) at a dilution of 1:10,000 and substrate was applied for 20 min before being stopped. DI was used at concentrations ranging from 25 µg/ml to 100 µg/ml, 20 kDa and 40 kDa PEGylated proteins were used at 50 µg/ml. Purified IgA was used between 12.5 µg/ml and 25 µg/ml of IgA, depleted serum was used at dilutions between 1:12.5 and 1:25 to achieve an optical density (OD) of between 0.4 and 0.8.Direct ELISAs for IgG and IgA aβ2GPI were as previously described.t-tests.PRISM and Stata programmes were used to carry out one-way analysis of variance and non-parametric Mann–Whitney N = 13) were screened for inhibition with increasing doses of DI (0–100 µg/ml). As shown in n = 4), low inhibition  and high inhibition . Dose-dependent inhibition for clusters 2 and 3 can be seen in Initially serum samples from patients (p < 0.05) enhancement of inhibition for both PEGylated forms in comparison with non-PEGylated DI.Inhibition with 20 kDa PEG-DI and 40 kDa PEG-DI was carried out at 50 µg/ml. Inhibition can be seen in Another interesting result was the difference in inhibition for patients with high and low IgG and IgA. Patients in cluster 1 with low inhibition also had the lowest levels of both IgG and IgA aβ2GPI (5.08 GBIU and 44.48 ABIU) and aDI . Those patients with moderate inhibition in cluster 2 had high IgA  but lower IgG  whereas those in cluster 3 with the highest inhibition had both high IgG and IgA aβ2GPI  and aDI (53.65 ADU and 52.08 GDU).To analyse further the high-level inhibition of IgA aβ2GPI binding in serum from patients in cluster 3 we carried out further tests on serum depleted of IgG and on purified IgA samples.Samples from three of the four cluster 3 patients . Two of these (1 and 4) showed dose-dependent inhibition between 25 and 100 µg/ml of DI. This can be seen in In this study we have shown that it is possible to inhibit the binding of both purified and serum IgA from APS patients from binding to β2GPI.17 These results are in contrast to our previous research using IgG aβ2GPI antibodies where significant inhibitory potential of DI was retained but not enhanced after PEGylation.13Interestingly, the inhibitory effect seen with DI was enhanced by PEGylation. This is an unusual phenomenon; the majority of PEGylated proteins lose their natural affinity for ligands. One possible explanation is that this apparent increase may also be due to stabilizing the epitope on DI or due to an increased purity of DI. An alternative theory may be that DI has aggregated and as such is presenting multiple epitopes. However, we studied this possibility via a gel-filtration method and also with native polyacrylamide gel electrophoresis and saw no aggregation. The potential for aggregation is also higher in the non-PEGylated protein as PEGylation has been shown to reduce aggregation.The strongest inhibition was seen in those patients with both high IgA aDI/aβ2GPI and high IgG aDI/aβ2GPI. To distinguish the effects of IgG and IgA inhibition we carried out experiments using IgG-depleted serum and purified IgA. The results obtained with total serum, IgG-depleted serum and purified IgA cannot be compared directly in a quantitative way due to the differences in sample preparation. However, a qualitative comparison shows that inhibition of IgA aβ2GPI was seen in IgG depleted samples and in purified IgA for all but one sample. This sample (from patient 3) was notable because it had particularly high levels of IgG and IgA aβ2GPI binding and because it was not possible to deplete the IgG aβ2GPI. It is possible that the poor inhibition in this sample was because of a particularly high level of antibody binding (both isotypes).This study utilized patients from two different centres with different ethnic backgrounds; however, the numbers are small. Further studies with larger cohorts would aid in interpreting these results.This study shows it is possible to inhibit the binding of IgA from patients to β2GPI in an ELISA using DI and PEG-DI and suggests that the most efficient inhibition is seen in patients with dual positivity for IgG and IgA antibodies. It also raises the interesting result that PEGylation may increase the ability to inhibit IgA from patients; however, larger studies are required for this to be confirmed.Domain I of beta-2-glycoprotein I is capable of inhibiting IgA antibody binding.Biochemical modification of Domain I can increase antibody binding.Patients’ serum most inhibited by Domain I contained both high IgA and IgG.