Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC These two highly hydrophilic compounds are not only potent DXR inhibitors, but show strong antibacterial effects as well3 is somewhat effective in treating malaria caused by Plasmodium falciparumet al. have found that the leaf extracts of Cercis siliquastrum, a Mediterranean plant, exhibit strong inhibitory activity against DXRUp to date, 2-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of terpenoids has been found and establishedE. coli DXR. The aims of the study are to disclose the possible antibacterial mechanism of the theaflavins and to seek new DXR inhibitors.As the most popular functional drink worldwide, tea has aroused widespread interest due to its potential benefits to human health691012The theaflavins are unstable compounds, especially under a basic condition50 of 0.27 μM. These data are in good agreement with the previously published results5We evaluated the reliability of the HPLC method in the measurement of DXR inhibitory activity of the theaflavins. The results show that use of VC and the theaflavins would not interfere with the quantification of DXP  derivatization and HPLC profiles, see The other factor that could influence the determination of inhibitory activity of the theaflavins against DXR would be the AP because we must use it to dephosphorylate DXP before DNPH derivatization. We thus measured whether VC and the theaflavins might display any inhibition against this enzyme. We find that VC has no effect on the activity of AP at 2 mM and the four theaflavins also have no inhibition against AP at all , even at50 values could thereby be calculated and listed in 50 of 162.1 μM and TF3,3′G exhibits the highest (IC50 14.9 μM).With the validated HPLC method, we also evaluated whether VC could affect DXR activity. The result shows that 2 mM VC had no effect on the activity of DXR. Then, we evaluated the DXR inhibitory activity of all four theaflavins at a final concentration of approximately 150 μM, employing 2% DMSO as a co-solvent and VC as a protective agent. As expected, the positive control fosmidomycin showed complete inhibition of DXR activity at 1.0 μM. The results reveal as well that all of the compounds were active in suppressing DXR activity at the selected concentration: TF exhibited approximately 40% inhibition against DXR, whereas the other three completely inhibited the activity of the enzyme. Encouraged by these experiments, we further determined the DXR inhibitory effects of the theaflavins at different concentrations. From the results depicted in 50 value and the other was about twice of it. We also selected baicalein as a positive control since it has been demonstrated to be a non-specific inhibitor of DXRIt has been reported recently that some flavonoids could produce promiscuous inhibition against DXR when using DMSO as a co-solvent171850 value in tris-HCl buffer , in the absence or presence of DXR (4 μg/mL). Baicalein was referenced as a positive control owing to the same reason as mentioned above. The particle size evaluation was performed by a DLS analyzer and the results display that except for baicalein, the theaflavins produce no detectable particles. The data are shown in DLS is a widely utilized technique in materials science to measure particle sizes in solutions20Ki values against NADPH and DXP were calculated accordingly and are listed in To determine the modes of the inhibitory action of the theaflavins against DXR, the initial enzyme kinetics were investigated over fixed inhibitor concentrations and at different DXP/NADPH concentrations employing the HPLC method described above. Lineweaver-Burk (LB) graphical charts were obtained by plotting the reciprocal of the reaction velocity against the reciprocal of the concentration of DXP/NADPH. The results, as shown in The molecular docking results evidenced that both compounds locate at the gateway of the hydrophobic pocket of DXR, next to the binding position of NADPH and quite far away from the DXP location , and metabolic liability, which have precluded its  h, and m50 larger than 100 μM, whereas the other three with at least one gallate side chain show stronger inhibition against the target than TF, with IC50 values in the range of 14.9 to 29.2 μM. Thus, the DXR-inhibitory activities of the theaflavins apparently correspond to the gallate side chain in the structure. The same phenomenon has been observed on the suppressive capacity of these compounds against Bacillus cereusHaving overcome the stability issue of the theaflavins under the DXR assay conditions and validated the HPLC method, we measured the inhibition of the tea polyphenols against DXR, and the data indicate that compound TF, lacking a gallate side chain, exhibits the lowest DXR inhibitory activity among the four theaflavins, with an ICAlthough there have been many publications on the bioactivities of flavonoids617202123241618From view of the chemical structures, the theaflavins are polyphenolic compounds containing a flavanol moiety. Accordingly, they could possibly exert promiscuous inhibition on DXR. We thus carried out extra assays to judge whether the inhibitory manner of the theaflavins is specific, under the guidance of Shoichet’s criteriaHaving elucidated that the theaflavins exerted specific suppression against DXR, we determined their inhibition kinetics using Lineweaver-Burk double-reciprocal plots. The results show that all four theaflavins are non-competitive inhibitors of DXR with respect to DXP and un-competitive inhibitors with respect to NADPH. The non-competitive inhibition mode of the compounds against DXP is predictable because they share no structural similarity with DXP, and their un-competitive inhibition mode versus NADPH is reasonable because DXR has been determined to mediate the conversion of DXP to MEP via an ordered Ping Pong mechanism, with NADPH binding firstTo obtain more information about the interaction between the theaflavins and DXR, we performed some docking experiments to simulate the binding modes of TF3,3′G and TF to the enzyme. The docking results support the conclusions we obtained from the other experiments. Furthermore, from the docking results we can conclude the interaction between TF3,3′G and DXR is much stronger than that between TF and the enzyme because there are more H-bonds and more close contact between TF3,3′G and DXR. This could not only explain why compound TF3,3′G is a much more potent inhibitor of DXR than TF but also accounts for the importance of the gallate side chain for the DXR inhibitory activity of the theaflavins.+, and thus suppressing the activity of the enzymeCombining all the above observations, we could deduce that after NADPH binds to DXR, TF3,3′G (or another theaflavin compound) could specifically bind to the enzyme in the vicinity of the binding sites of NADPH, forming a complex that could retard the departure of the oxidized product NADPIn summary, the data obtained in this study show that the polyphenolic compounds theaflavins are a novel DXR inhibitory chemotype which can specifically inhibit the activity of DXR with weak to medium effect. Based on these data, we might only partially disclose the antibacterial mechanism of theaflavins. The modest DXR inhibition of these polyphenolic compounds may suggest that the theaflavins would exert their antibacterial effect also via suppressing the functions of other targets which need further elucidation.E. coli DXR was carried out in accordance with a published methodTheaflavins were purchased from Chengdu Biopurify Phytochemicals, Ltd. , and stock solutions of them (10 mg/mL) were prepared in 40% aqueous DMSO or 0.1% Triton X-100 just before use. NADPH was purchased from GEN-VIEW SCIENTIFIC INC. . Fosmidomycin was from Toronto Research Chemicals, Inc. . Calf intestine AP was purchased from Takara . Triton X-100 and DNPH were purchased from Sinopharm Chemical Reagent Co., Ltd. . DXP was synthesized according to a procedure previously reported2 and 2% (W/V) DMSO to a final concentration of 100 μM in the absence and presence of 2 mM VC. The mixtures were subsequently incubated at 37 °C for 35 min before they were centrifuged at 6000 rpm for 3 min and analyzed. HPLC conditions: Column, Shim-pack VP-ODS column . Detection wavelength: 280 nm. Injection volume: 20 μL. The mobile phase consists of 60% solvent A [2% acetic acid in water (v/v)] and 40% solvent B (acetonitrile). Flow rate: 0.7 mL/min. Column temperature: 25 °C.Stability of the theaflavins in Tris-HCl buffer was evaluated using an Agilent 1200 HPLC equipped with a DAD detector. The theaflavins were separately diluted into 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgClE. coli DXR was determined using the pre-column derivatization HPLC method published by this group with minor modifications2, 2 mM VC, 0.5 mM NADPH, 2% (W/V) DMSO or 0.01% Triton X-100, and 4 μg/mL DXR in a final volume of 100 μL. The theaflavin compounds or baicalein were added to the mixtures separately before the addition of DXP  to start the reaction. In a control assay, fosmidomycin (3) was used instead of the theaflavins. The reaction mixture was incubated at 37 °C for 30 min before the DXR was inactivated by boiling for 3 minutes. Subsequently, 10 μL AP buffer  and 2 U AP were added, and the mixture was incubated at 37 °C for 120 min. 10 μL DNPH  and 280 μL methanol were supplemented. After incubated at 37 °C for another 45 min, the mixture was centrifuged at 6000 rpm for 3 min and the supernatant was used for HPLC analysis on the Agilent 1200 HPLC system. HPLC conditions: Column, Shim-pack VP-ODS column . Injection volume: 60 μL. Gradient: 0 min, 40% acetonitrile; 17 min, 80% acetonitrile; 18–20 min, 40% acetonitrile. Detection wavelength: 360 nm. Flow rate: 0.7 mL/min. Column temperature: 30 °C.The inhibitory activity of the theaflavins and baicalein against 50 value, and the other 500 μM) were added to the mixtures separately before the addition of DXP  to start the reaction. The reaction mixtures were incubated at 37 °C for 120 min, and the AP was inactivated in boiling water for 3 min. Then, the reaction mixture was derivatized with DNPH and analyzed with the HPLC method.The HPLC method described above was also used to determine the inhibitory activity of the theaflavins against AP. The reaction mixture comprises 2 mM VC, 10 μL AP buffer and 2 U AP in a final volume of 100 μL. The theaflavin compounds  containing 2 mM VC and 2.0% DMSO (V/V) was measured in the absence and presence of DXR  on a DLS analyzer . The detector time was set to 10 min. The concentration of each of the theaflavins was 3–5 times of its IC2+-fosmidomycin quaternary complex were obtained from Protein Data Bank (PDB accession code 2EGH). Fosmidomycin was removed, and subsequently DXP was docked into the binding sites of fosmidomycin because it had been suggested that DXP could be superposed exactly onto fosmidomycin2+-DXP quaternary complex as a receptor.Autodock 4.2.6 software was used for docking experiments, and the results were shown by Chimera 1.10.1 software. The profiles of the crystal of the DXR-NADPH-MgHow to cite this article: Hui, X. et al. Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway. Sci. Rep.6, 38945; doi: 10.1038/srep38945 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


During year-to-year El Niño events in recent decades, major sea surface warming has occurred frequently in the central Pacific. This is distinct from the eastern Pacific warming pattern during canonical El Niño events. Accordingly, the central-Pacific El Niño exerts distinct impacts on ecosystems, climate and hurricanes worldwide. The increased frequency of the new type of El Niño presents a challenge not only for the understanding of El Niño dynamics and its change but also for the prediction of El Niño and its global impacts at present and future climate. Previous studies have proposed different indices to represent the two types of El Niño for better understanding, prediction and impact assessment. Here, we find that all popularly used indices for the central-Pacific El Niño show a dominant spectral peak at a decadal period with comparatively weak variance at interannual timescales. Our results suggest that decadal anomalies have an important contribution to the occurrence of the central-Pacific El Niño over past decades. Removing the decadal component leads to a significant reduction in the frequency of the central-Pacific El Niño in observations and in Coupled Model Intercomparison Project Phase 5 simulations of preindustrial, historical and future climate. However, recent observations show that major warm SST anomalies of El Niño events in recent decades have frequently been confined to the central Pacific (CP) with less warm or even slight cold anomalies in the east1267891053513451314151617181920El Niño, the most important driver for year-to-year global climate variations, is classically referred to as substantial sea surface temperature (SST) warm anomalies in the equatorial central-eastern Pacific  that approximately match the locations of the maximum SST warm anomalies of the CP and EP El Niño, respectively. However, the Niño4 and Niño3 indices themselves are highly correlated  and thusnew = N3normalized − 0.5*N4normalized and CPnew = N4normalized − 0.5*N3normalized. The EPnew and CPnew index has a high correlation (about 0.9) with the EPtN3N4 and CPtN3N4 index, respectively. Their correlations with the other indices are also high  with the N3 and N4 index, respectively  El Niño indices actually represent two phenomena at distinct timescales, rather than at the same interannual timescale . While this method may misrepresent the influence of a mixed type of El Niño, it appears to be effective and has been widely adopted to identify the two types of El Niño22325Previous studies have suggested that climate change in response to anthropogenic forcing might affect the occurrence and characteristics of El Niño events27Model results confirm the importance of the decadal and longer timescale signals on the CP El Niño . WithoutIn summary, our results find that all of the popularly used CP El Niño indices display a major spectral peak at a decadal period rather than at interannual timescales, probably owing to the orthogonality or independence constraint that acts to mathematically minimize the correlation between the CP and EP SST anomalies. Note that many of the CP indices are able to capture weak peak variances at about two and four years; this suggests that these indices may still be useful for examining interannual variability if their decadal components are removed20253031It is worth noting that mechanisms of the Pacific decadal/multi-decadal anomalies are complicated and remain to be poorly understood. Different hypotheses have been proposed to explain the decadal/multi-decadal anomalies in the tropical Pacific20232425263435363738394041422535363823244023402634Disentangling the complex mechanisms for the tropical Pacific decadal/multi-decadal anomalies remains to be a long-standing challenge. This is beyond the scope of this study and warrants further investigations. Improved understanding of the decadal and longer timescale anomalies, induced by either nonlinear processes and rectification of ENSO, Pacific internal mechanisms, decadal background-ENSO interactions, stochastic processes, inter-basin interactions, external radiative forcing, or the combination of these factors202324252630343536373839404142To identify EP and CP El Niños over 1950–2013, we use the Hadley Centre Sea Ice and Sea Surface Temperature (HadISST) data setFor the observations and model simulations, when both the five-month running mean Niño3  and Niño4  SST anomalies exceed 0.5 °C, the EP El Niño is defined if the Niño3 SST anomaly is greater than the Niño4 anomaly. When the Niño4 SST anomaly is larger, it is defined as a CP El Niño2https://www.ncl.ucar.edu, where details for each method are available). Power spectral analysis is performed based on the 5-month running mean Niño3, Niño4, CP and EP El Niño indices. The statistical confidence interval of the power spectra is estimated with a theoretical Markov red-noise spectrum using the lag-1 autocorrelation. The power spectra of both the Niño4 and all CP El Niño indices display a decadal peak that is marginally significant at 10% significance level .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Hoof shape is linked to an increased risk of lameness in the horse and has been shown to adapt to different loading patterns associated with the workload and shoeing interval length. This study investigated how different measurements of the hoof wall and the hoof pastern axis angle changed with work in riding school horses, across a four to six week shoeing/trimming interval. The dorsal hoof wall, and weight bearing and coronary band lengths reduced in size post-shoeing/trimming. This, combined with the increase to the inner and outside hoof wall heights on the digital images despite trimming, suggests that shoeing/trimming increased the vertical orientation of the hoof during the shoeing interval investigated. At the same time, increases in the dorsal hoof wall angle, heel angle, and heel height occurred, promoting a more correct dorsopalmar balance. The changes observed are consistent with the workload of the horses studied. The results suggest that a regular farriery interval of no more than six weeks could prevent excess loading of the structures within the hoof, reducing long term injury risks through cumulative, excessive loading in riding school horses.Variation in equine hoof conformation between farriery interventions lacks research, despite associations with distal limb injuries. This study aimed to determine linear and angular hoof variations pre- and post-farriery within a four to six week shoeing/trimming interval. Seventeen hoof and distal limb measurements were drawn from lateral and anterior digital photographs from 26 horses pre- and post-farriery. Most lateral view variables changed significantly. Reductions of the dorsal wall, and weight bearing and coronary band lengths resulted in an increased vertical orientation of the hoof. The increased dorsal hoof wall angle, heel angle, and heel height illustrated this further, improving dorsopalmar alignment. Mediolateral measurements of coronary band and weight bearing lengths reduced, whilst medial and lateral wall lengths from the 2D images increased, indicating an increased vertical hoof alignment. Additionally, dorsopalmar balance improved. However, the results demonstrated that a four to six week interval is sufficient for a palmer shift in the centre of pressure, increasing the loading on acutely inclined heels, altering DIP angulation, and increasing the load on susceptible structures . Mediolateral variable asymmetries suit the lateral hoof landing and unrollment pattern of the foot during landing. The results support regular (four to six week) farriery intervals for the optimal prevention of excess loading of palmar limb structures, reducing long-term injury risks through cumulative, excessive loading. Equine distal limb lameness is commonly associated with poor foot conformation and hoof imbalance ,2,3,4, wResearch has identified that the biomechanical function of the distal limb can alter as a result of changes in the hoof shape. Consequently, this influences the forces acting on the hoof’s structural components ,10, as wAnecdotally, within the equine industry, shoeing/trimming interval lengths are commonly between four to eight weeks. However, intervals can vary beyond eight weeks, especially within the leisure horse population. Within research, the length of recommended shoeing/trimming intervals vary between four to six weeks, or six to eight weeks ,7,18,19.Twenty-six horses of mixed breed, gender, age (12 ± 9.9 years), and height (157 cm ± 2.3 cm), which did not display any stereotypic behaviours and were resident at Hartpury College, Gloucestershire, UK, were selected for inclusion in the study. All subjects were on loan for use within the College’s riding school and were subjected to the same exercise and management regime: two 45-minute flat, jump, or lunge lessons per day , with one day off per week, and stabled (rubber matting and shavings) with restricted grass turnout. The horses included in the study were deemed in good health and functionally fit for work by experienced equestrian professionals who had daily access to an equine veterinary surgeon, to examine horses presenting with lameness. The horses declared unfit for work by the veterinary surgeon were removed from the study. Each individual received regular farrier treatment  by one main farriery team (WCF (Worshipful Company of Farriers) qualified), at shoeing intervals between four and six weeks. Farriery was performed by one of four farriers under the direction and supervision of a lead farrier, to promote a consistent approach. All horses had been previously exposed to farrier treatment and were not undergoing any corrective farriery. Ethical approval for the study was granted by the University of the West of England (Hartpury) Ethics Committee (Project Identification Code: ETHICS2016-03).Data were collected between September and December, when horses presented for their next farrier treatment. Digital images were taken of each horse’s left forelimb and hoof, to enable a comparison of morphometric measurements of key anatomical features and angles  pre- andPrior to data collection, horses had their rugs removed and limbs cleaned. Key anatomical landmarks were used to orientate the placement of circular markers on the left forelimb and hoof, to ensure that the same anatomical regions were measured within all of the horses  and to fThe photographic images were imported into Dartfish™ software version 7 , to facilitate the angular and linear measurements outlined in new is the length of the dorsal hoof wall post-farriery, αnew is the hoof angle post-farriery, aold is the length of the dorsal hoof wall pre-farriery (after four to six weeks), and αold is the hoof angle pre-farriery. d is the predicted location of CoP at midstance, relative to the point of rotation at the toe.Van Heel et al.’s  predictip ≤ 0.05.Using the Van Heel et al.  equationSignificant differences were reported after shoeing for the majority of the lateral view, anterior view, and HPA measurements .p = 0.01) than the mean MDHWL (3.57 ± 0.69 cm), prior to farriery. Medio-lateral variation in DHWL demonstrated an increased significant difference after shoeing (p = 0.0001), with the mean LDHWL (4.09 cm ± 0.80 cm) being 7% longer than the mean MDHWL (3.84 ± 0.74 cm). Similar results occurred for the hoof angle; significant differences were found between the mean MHA and mean LHA prior to farriery (p = 0.0001), with the MHA (78.96° ± 5.81°) being 7% greater than the LHA (73.17° ± 4.20°). Again, a more significant difference was recorded after shoeing (p = 0.0001), with the direction of difference remaining consistent, with MHA (80.80° ± 5.41°) being 10% greater than LHA (72.79° ± 4.07°).Significant medio-lateral variation was reported between the linear and angular measurements in the hoof . The meaThe calculated shift in the CoP distance found that the predicted CoP was located, on average, 0.5 cm back from the point of rotation of the toe across the cohort after shoeing.Significant correlations were found between twenty-eight pairs of variables measured prior to farriery treatment, and these ranged in strength and direction . After sCorrect  hoof conformation is essential to prevent lameness . For exan = 40) during an eight to ten week shoeing interval. Extrapolation from these results would suggest that the four to six week interval applied here would have been expected to reduce DHWL by 0.5 to 0.75 mm, which is much greater than was found (0.25 ± 0.97 cm). The discrepancies between these results are likely reflective of the differing study durations  reported a DHWA ranging from 48° to 57° and a mean angle of 51.8°; and Dyson et al. [n = 19) reported a mean DHWA of 52.4°, ranging 43.4°–64.7°.DHWA is defined as the angle formed at the junction of the DHW and the weight bearing surface of the foot ,26 and an et al.  (n = 19)The decrease in DHWA associated with a four to six week interval  is analogous to the 3.3° decrease reported by Moleman et al. . Force dChanges reported in the lateral view suggest that angular modifications of the distal limb are occurring at the level of both the MCP and DIP joints. The reduced DHWL, increased DHW and heel angle, the decrease in the weight bearing length, and increase in the palmar coronary band height, suggest that although the toe shortened, the length of the heel was not altered during the shoeing/trimming procedure. Following trimming, the weight bearing length decreased in the anterior view, whilst the medial and lateral wall lengths, as measured from the digital images, increased. The changes which occurred in the MCP and DIP joint angulations across the lateral view could also explain the increase in medial and lateral wall lengths from the anterior view images. We believe that the decrease in the coronary band lengths observed from both the lateral and anterior view 2D images occurred as a result of the change in positioning of the hoof capsule post-shoeing/trimming. The dorsal wall shortens by a relatively greater amount than the heels, which results in the hoof assuming a more vertical orientation. This is supported by the differences found between the lateral and medial hoof wall length; 73% of participants had a longer lateral wall pre-shoeing, and this increased to 88% of the population post-shoeing. Furthermore, the mean difference between the two sides increased following routine shoeing, from 7% to 10%. Guidance in achieving mediolateral balance of the equine foot refers to the trimming of the medial and lateral walls, to ensure that the live sole of the foot is level with the ground  and the Pre-shoeing, only 31% of horses presented with a greater LHA, though this decreased to 12% post-shoeing. The more slanted lateral wall presented a more acute angle with the sole compared to the angle on the more upright medial wall, which was more obtuse, by between 1° and 7°. Approximately 25% of participants presented with a longer medial than lateral wall pre-shoeing, suggesting that the loading pattern that they exhibited differed from normal lateral landing and unrollment. These differences potentially infer a more medial landing posture, indicating that some degree of low level lameness was inherent in the study population; a potential inherent career risk of riding school horses . FurtherInterestingly, although MHAs and LHAs changed post-shoeing, these changes were not significant. A similar number of horses presented with an increase and decrease in LHA; however, a greater proportion of the sample (58%) demonstrated an increase in MHA post-shoeing. The lack of significant results is attributed to the large individual variations seen between the horses examined.The hoof pastern axis (HPA) is defined by plotting a line down from the middle of the metacarpophalangeal joint, through the centre of the proximal and distal interphalangeal joints, and through the axis of the phalanges ,26. A stThe forelimb CoP is known to deviate in a palmar direction across an eight week shoeing interval, whilst breakover remains relatively consistent ,17. The Moleman et al.  identifiThe current study applied a pragmatic and observational research approach to investigate the impact of shoeing practices on hoof morphology in riding school horses. Whilst the study has strong external validity, it should be noted that the real-world design possesses a number of limitations, and therefore, the results should be interpreted with caution. Many factors can influence hoof growth. These include, but are not limited to, horse breed, height, diet, disease, time of year, the environmental effects of pasture quality, and lameness. The horses integrated here were subject to consistent management practices and were considered fit for riding school work by experienced equestrian professionals and the supporting veterinary team. The presence of low grade lameness has been previously identified in riding school horses to affect loading patterns and, subsequently, hoof growth, which could explain some of the variation observed within our results . SubjectAfter shoeing, hoof-surface interface interactions result in dynamic responses in the hoof promoting adaptation over time within the hoof’s structural components, affecting linear and angular measurements. In the shod horse, the presence of the shoe prevents toe wear. Therefore, the adaptation which occurs over time will be influenced by the shoe. Even with shoeing intervals of four to six weeks, changes are observed in key parameters associated with foot balance. Our results suggest that, for the majority of horses, the weight bearing length of the foot increases (as the toe grows), causing a decrease in the heel angle and an increase in hoof angle displacement as the hoof pastern axis becomes more broken backwards, negatively influencing dorsopalmar balance. Changes also occur in the dorsal wall associated with loading during locomotion; in the shod horse, foot placement demonstrates a lateral-medial unrollment pattern. Therefore, increased loading may occur in the lateral hoof wall, which would result in lateral hoof wall length increasing and the lateral angle decreasing over time. In contrast, and in agreement with previous research , a four Caution is advised in the interpretation of these results, as a high degree of inter-subject variation was found across the measurements undertaken and as a number of variables could not be controlled due to the horses’ working schedule. This variation could represent individual conformation differences in the hoof or distal limb, could be a cumulative result of previous farrier treatments , or may


Arabidopsis thaliana Regulator of G Signaling protein 1 (AtRGS1) modulates G protein activation and is phosphorylated by several RLKs and by WITH-NO-LYSINE kinases (WNKs). Here, a combination of in vitro kinase assays, mass spectrometry, and computational bioinformatics identified and functionally prioritized phosphorylation sites in AtRGS1. Phosphosites for two more RLKs (BRL3 and PEPR1) were identified and added to the AtRGS1 phosphorylation profile. Bioinformatics analyses revealed that RLKs and WNK kinases phosphorylate plant RGS proteins within regions that are conserved across eukaryotes and at a high frequency. Four phospho-sites among 14 identified are proximal to equivalent mammalian phosphosites that impact RGS function, including: pS437 and pT267 in GmRGS2, and pS339 and pS436 in AtRGS1. Based on these analyses, we propose that pS437 and pS436 regulate GmRGS2 and AtRGS1 protein interactions and/or localization, whereas pT267 is important for modulation of GmRGS2 GAP activity and localization. Moreover, pS339 most likely affects AtRGS1 activation.Heterotrimeric G proteins function in development, biotic, and abiotic stress responses, hormone signaling as well as sugar sensing. We previously proposed that discrimination of these various external signals in the G protein pathway is accomplished in plants by membrane-localized receptor-like kinases (RLKs) rather than G-protein-coupled receptors.  Arabidopsis thaliana, the heterotrimeric G protein complex contains only one canonical Gα subunit (AtGPA1), one Gβ, and three Gγ subunits (In subunits . Paradoxsubunits . Heterotsubunits . In animsubunits  showing subunits  and the subunits . Phosphosubunits .Glycine max RGS2 (GmRGS2) by Nod factor receptor 1 (NFR1), a LysM receptor kinase  in plants  revealedr kinase  was showin vitro. BRL3 and AtRGS1 function together to fine tune growth inhibition and ROS activation  encodes a member of the RLK/Pelle kinase family that physically interacts with the heterotrimeric G protein Gβ to regulate the division of zygote and the cell fate of its daughter cells  informatics method – SAPH-ire – to enable quantitative analysis of PTM hotspots in protein families. Unlike traditional methods of PTM analysis, SAPH-ire comprehensively integrates all modification data within a protein family, a method that has been validated to improve functional prioritization of PTMs . AnalysiEscherichia coli BL21 (DE3) pLysS cells , and then incubated at room temperature for 8.5 h to achieve saturation. Approximately, 1 μg of kinase domain and 2.7 μg of His6-RGS-J5 were added into each reaction. The reaction was stopped by adding 10 μl of 5x Laemmli sample buffer. The kinase and His6-RGS-J5 proteins were separated on a SDS-PAGE gel, and the radio-labeled phosphate transferred on proteins was visualized with a phospho image analyzer.Complementary DNAs encoding the complete cytoplasmic domain  for BRL3 (amino acids 773–1164) and PEPR1 (amino acids 770–1123) were cloned into a modified pET Gateway vector for expression of His-tagged recombinant protein in sS cells . BRL3 ansS cells . PurifieAmborella was chosen because of its position at the base of the angiosperms. Pinus, Selaginella, and Chara were included as divergent sequences outside the angiosperm, represented by a gymnosperm, a lycophyte, and a green alga, respectively. There are few monocot genomes available outside the cereals but the available five non-cereal monocots were included. The remaining were eudicots covering six classes. The plant sub-alignment profile consisting of AtRGS1 and GmRGS2 was then extracted from this alignment and included in a profile-to-profile alignment with the PTM sub-alignment from SAPH-ire, which is restricted to family members with at least one PTM and/or a high-resolution 3-D structure. MSAs were generated with MUSCLE using default parameters  as the most appropriate homology target for AtRGS1 (PMID 16301204). Next, we used the 2GTP structure as a template for alignment of the RGS domain model and AtGPA1 (PDB: 2XTZ). Interface residues were identified within four angstroms using PyMol . Interface data (binary classified as 0 or 1) were tabulated with PTM feature data from SAPH-ire and an Integrating Score calculated as follows to enable comprehensive relation of all features for each RGS domain MAP follows:∗ (PC) ∗ (PRC) ∗ W1(NKC) ∗ W2(PPI)IS = (CPC) where,      IS = Integrative score      CPC = Cluster PTM count      PC = PTM count      PRC = PTM residue conservation      NKC = Neighbor known function count      PPI = Protein–protein interface residence      W1 = Conditional weight factor 1      W2 = Conditional weight factor 2.Each feature was chosen based on extensive modeling of the relationship between MAP features and biological functionality for PTMs . The resin vitro including BAK1, BRL3, and PEPR1  . The peptide mass tolerance was set as 100 ppm and 0.02 Da for MS/MS tolerance. Sequence coverage identified by mass spectrometry and in vitro kinase reactions are demonstrated in Supplementary Figure S1. As summarized in Figure 1, the identified AtRGS1 phosphorylation sites  are listed along with previously reported WNK8-mediated phosphorylation of AtRGS1  and NFR1-mediated phosphorylation sites on GmRGS2 .A screen of 70 active, recombinant arginine–aspartate type LRR RLKs revealed that more than 10 RLKs phosphorylate AtRGS1 ctively) . AlthougLong-standing evidence from animal systems revealed that RGS proteins are prominent targets of PTM. In many cases, site-specific phosphorylation of animal RGS proteins has also been shown to serve a regulatory function . In contSeveral common features  were shown to be predictive for phosphorylation sites that are regulatory for specific protein families. These include but are not limited to several features derived from MSA of modified proteins, including phospho-acceptor residue conservation, phosphorylation observation frequency (PTM count), phosphorylation site clustering, among others as reviewed in Supplementary Figure S2A). Second, we analyzed the conservation of phospho-acceptor residues in the RGS sub-family comprised of structurally resolved proteins that harbor experimentally verified PTMs (PTM sub-family) . Third, we used SAPH-ire to compare the plant phosphorylation sites identified here with the experimental PTM sites that were contained within the PTM sub-family.While the volume of experimental PTM data from animal RGS proteins is sufficient to enable feature-to-function modeling by SAPH-ire, the relative sparsity of data observed in 7TM-RGS proteins, which are also found outside the canonical RGS family, makes this type of analysis difficult. As a result, the RGS protein family was not included in previous iterations of SAPH-ire reported in the literature . To overFigure 2A). The alignment also conforms well with previous studies that include several additional plant sequences not yet curated by Uniprot as can be seen by direct comparison  , with interspersed well-conserved sequence islands.Multiple sequence alignment of plant RGS proteins revealed high overall sequence conservation (% identity) across 7TM-RGS family members (ure S2B) . Thus, PFigure 2B). Within this region, only 5 of 14 sites were located within the RGS domain itself, three of which  were located in alignment positions with 100% phospho-acceptor residue (S/T/Y) conservation. Highly conserved phosphosites were not necessarily restricted to the RGS domain, but were also found in the region between the 7TM and RGS domain as well as the C-terminal sequence islands that were well-conserved for the entire family . Alignment positions harboring a phosphosite were also more conserved on average compared to all other alignment positions in which phosphorylation is possible (63% vs. 49%), and 8 of 14 sites were found in positions with greater than 65% phospho-acceptor residue conservation  . Thus, RLK and WNK-mediated phosphorylation is enriched at positions in which most 7TM-RGS proteins can also be phosphorylated.We focused further analyses on the cytoplasmic region (outside the 7TM domain) which harbored all 14 plant phosphorylation sites . To enable this comparison and preserve AtRGS1 and GmRGS2 sub-alignment, we aligned the sequence profile of the plant proteins  to the MSA for the PTM sub-family. As a result, the relative relationship between AtRGS1 and GmRGS2 phosphosites was retained.We next determined whether plant phosphosites were also well-conserved in the context of the PTM sub-family comprised of structurally resolved RGS proteins that harbor at least one PTM . Not surprisingly, the highest degree of residue conservation was found within the RGS domain . In addition, four short conserved sequence regions (SCR) were also observed flanking the RGS domain including: SCR-A , SCR-B , SCR-C , and SCR-D  . Plant-specific phosphosites were located specifically within the most well-conserved regions in the PTM sub-alignment – including SCR-B, SCR-C, SCR-D, and the RGS domain located between alignment positions 900–1800, and were not found within regions between these positions . In comparison, PTMs observed in non-plant proteins were broadly dispersed throughout the entire sequence length of the family . Thus, we conclude that RLK and WNK-mediated phosphorylation occurs in cytoplasmic regions of 7TM-RGS proteins that are more, rather than less, conserved between plants and mammals with the caveat of the ascertained bias.Sequence conservation in the PTM sub-family (% identity) is lower overall compared to the plant sub-family, with five distinct conserved regions that are dispersed along the sequence length of the alignment . Five of the eight phosphosites were localized within or immediately adjacent to the RGS domain  which harbors 13 possible sites of phosphorylation (in AtRGS1) resulting in a 1.6-fold enrichment over random expectation. One site, AtRGS1-pS436, was located precisely within SCR-D, but was not enriched beyond random expectation. Finally, the two remaining sites (AtRGS1-pS428 and AtRGS1-pS453) were close to, but outside, SCR-D and therefore not enriched within a conserved region of the sub-alignment. Unlike AtRGS1, phosphorylation of GmRGS2 was enriched in SCR-B (fourfold), SCR-C (threefold), and SCR-D (twofold), but was not enriched over random expectation in the RGS domain. Thus, phosphorylation of AtRGS1 and GmRGS2 occurs within regions that are conserved across eukaryotes and at a frequency that is higher than expected by chance. Whereas the RGS domain is the predominant target of RLK-mediated phosphorylation in AtRGS1, regions flanking the RGS domain are the predominant targets of phosphorylation in GmRGS2.To estimate the degree to which RLK- and WNK-mediated phosphorylation sites were targeted randomly, we quantified the enrichment of plant phosphosites over random expectation . We restricted the enrichment analysis to serine and threonine (S/T) residues found within the cytoplasmic portion of the plant RGS proteins, because all mapped plant phosphosites were found on only these two residue types. In AtRGS1, 29 S/T sites were located throughout the cytoplasmic region of which eight were detectably phosphorylated a priori as positive correlates of biologically functional PTMs  – a computational informatics tool that enables functional prioritization of PTMs by compiling multiple feature-to-function relationships for modified alignment positions  . PTM datnal PTMs .Supplementary Table S1). The likelihood that a MAP is biologically functional  increases proportionally with an increase in the number of PTMs found within ±2 residues of the site in question – which we defined previously as the cluster PTM count . We observed several distinct regions that exhibit a peak cluster PTM count of four or more, most of which corresponded with SCRs B, C, D, and the RGS domain. Notably, 4 of the 14 plant phosphosites corresponded precisely with non-plant PTMs  and were also at or near the peak of PTM clusters, including: GmRGS2-pT267, AtRGS1-pS405, GmRGS2-pS437, and AtRGS1-pS436 . In some cases, plant phosphosites were also co-incident with or in proximity to non-plant PTMs that are known to be biologically functional .A total of 242 RGS family MAPs were identified by SAPH-ire, of which 76 were located in clusters within ±2 alignment positions of the 14 plant MAPs . We found that 8 of the 14 phosphosites were localized in PTM clusters comprised almost entirely of phosphorylation – suggesting that these sites are utilized by several different organisms. Three of these  are particularly interesting because they are part of dense phosphorylation clusters  that harbor functional PTMs . GmRGS2-pT267 neighbors a site in human RGS2  that is phosphorylated by cGMP-dependent protein kinase  and necessary for activation of the RGS protein and resultant attenuation of receptor-mediated vascular contraction . The structural homology model and orientation of this model with AtGPA1 was produced by 3-D alignment with the structure of human RGS1 bound to activated Gαi1 (PDB: 2GTP) (methods). We then determined the interface residues observed between the two proteins in the model and combined this information with the MAP feature data from SAPH-ire. As we showed previously, integrating multiple features to provide a single scoring factor enables the direct and quantitative comparison of each MAP to one another in a protein family .The RGS domain of RGS proteins defines the primary function of the protein family and the structure of the domain is well-characterized. Structural projection of PTM hotspots – a feature embedded within SAPH-ire – enables visualization of hotspots in the context of high-resolution X-ray crystal structures and provides meaningful context about the local three-dimensional environment of PTMs . Using tn family . TherefoFigure 5A). For contrast, we projected the five plant phosphosites along with 46 RGS domain PTMs from the PTM sub-alignment , which revealed widespread coverage across most of the domain . Visualizing the PTM sites after surface rendering illustrates that only GmRGS2-pS405 and AtRGS1-pS365 are readily accessible . AtRGS1-pS365 is not found in a dense PTM cluster and is not well-conserved in non-plant RGS proteins, indicating that this region is not commonly utilized as a regulatory phosphosite in non-plant RGS proteins . The site is also poorly conserved in plants . However, despite this, AtRGS1-pS365 (which is conserved in GmRGS2) is located precisely at the interface modeled between AtRGS1 and AtGPA1 . Thus, AtRGS1-pS365 may provide a more restricted but direct effect on Gα subunit interactions for AtRGS1 and GmRGS2, specifically. Other AtRGS1 phosphosites found in the RGS domain  are somewhat buried in comparison, including pS339 that is proximal to RGS4-103, which when phosphorylated, inhibits its interaction with Gαq  and one from GmRGS2 (pS405) showed that they exist on opposite ends of the domain . Indeed, the MAP harboring pS405 ranks within the top 7 of all RGS domain MAPs, three of which are known to be biologically functional . Moreover, pS405 is one of several MAPs that contribute to a continuous domain surface comprised of six MAPs, including one that harbors two functional PTMs (hRGS5-pS166 and hRGS7-pS434) . Both human phosphosites regulate RGS protein interactions. Phosphorylation of hRGS5-S166 by PKC abolishes its binding capacity for Gα subunits . Lending further support to this hypothesis, we found that GmRGS2-pS405 and AtRGS1-pS406  are indeed within five residues of lysine residues GmRGS2-K410 and AtRGS1-K411, both of which are 88% conserved  in the plant 7TM-RGS sub-family. As the role of these proximal phosphorylation and ubiquitination sites has yet to be determined, further work will be required to establish the validity of this intriguing hypothesis.GmRGS2-pS405 may also be indirectly involved in ubiquitin-mediated regulation of the 7TM-RGS protein. Functional crosstalk between phosphorylation and ubiquitination events that control protein degradation, trafficking, among other aspects of protein regulation, are often near each other such that the probability of a functionality increases dramatically when the phosphosite is within five residues of the ubiquitination site. We found that the MAP harboring the plant phosphosite is in a PTM cluster comprised exclusively of phosphorylation and ubiquitination sites observed in human RGS10, RGS13, RGS14, and RGS19 . By using mass spectrometry and bioinformatics approaches, we found 14 phospho-acceptor sites, three of which, GmRGS2 S267, S437 and AtRGS1 339, are suggested to be important for regulation of GAP activity, stability and localization due to their close proximity to functional mammalian RGS sites. The phosphorylation island containing the highly conserved GmRGS2 S437, T428, and AtRGS1 S428, S435/436 is a key site for regulation of RGS protein interactions or its localization. Here, we prioritized them for rigorous testing.This study provides the most comprehensive profiling and analysis of plant RGS PTMs to date. Furthermore, the data are analyzed in the context of all eukaryotic RGS modification data, and thereby reveal several structural and functional PTM relationships in RGS proteins from both plants and animals. A summary of the cumulative dataset, including kinase/G protein-linked pathways and physiological responses are provided for context  involved in systemic inflammation, reduces serine S434 phosphorylation of RGS7 and the interaction of RGS7 with 14-3-3 (S405D) did not change GAP activity in vitro , RGS7 (S434), and RGS16 (Y168). Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Gα subunits than did the unphosphorylated RGS5 . The phoh 14-3-3 . Phosphoh 14-3-3 . Phosphoin vitro  but thisin vitro phosphorylation experiments and MT analyzed the results via SAPH-ire; and MT-O, AJ, and MT wrote and approved the manuscript.MT-O, DU, AJ, and MT designed the experiments; MT-O, BL, and DJ performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Køge Bugt, in southeast Greenland, hosts three of the largest glaciers of the Greenland Ice Sheet; these have been major contributors to ice loss in the last two decades. Despite its importance, the Holocene history of this area has not been investigated. We present a 9100 year sediment core record of glaciological and oceanographic changes from analysis of foraminiferal assemblages, the abundance of ice-rafted debris, and sortable silt grain size data. Results show that ice-rafted debris accumulated constantly throughout the core; this demonstrates that glaciers in Køge Bugt remained in tidewater settings throughout the last 9100 years. This observation constrains maximum Holocene glacier retreat here to less than 6 km from present-day positions. Retreat was minimal despite oceanic and climatic conditions during the early-Holocene that were at least as warm as the present-day. The limited Holocene retreat of glaciers in Køge Bugt was controlled by the subglacial topography of the area; the steeply sloping bed allowed glaciers here to stabilise during retreat. These findings underscore the need to account for individual glacier geometry when predicting future behaviour. We anticipate that glaciers in Køge Bugt will remain in stable configurations in the near-future, despite the predicted continuation of atmospheric and oceanic warming. It is thought that this increase in ice loss was triggered by submarine melting at tidewater glacier termini, in turn caused by the warming of the subpolar North Atlantic6. However, the precise mechanisms controlling marine-terminating glacier retreat are not well-understood, largely because of the difficulties in monitoring these extremely inhospitable environments. This knowledge gap makes it difficult to accurately predict future glacier behaviour and to assess the contribution of glaciers in southeast Greenland to global sea-level rise. Recent work has also highlighted that this region of Greenland is important in modulating global thermohaline circulation; this occurs through variation in the mixing of ocean currents on the continental shelf 7, and through the enhanced influx of glacial meltwater9. Despite its obvious importance, relatively little is known about the glaciological and oceanographic history of southeast Greenland. Direct observations of glacier behaviour and ocean conditions only extend back to the early 20th Century11; these provide brief snapshots of ice-ocean interactions. We present results from the first sediment core obtained from Køge Bugt, the drainage portal for one of the largest glacial systems in Greenland12. We analyse ice-rafted debris abundance, foraminiferal assemblages, and sortable silt grain size to constrain glacier behaviour and oceanographic conditions through the last 9100 years. We use these results to assess the stability of glaciers in Køge Bugt in response to changing ocean and climatic conditions during the Holocene. These new data provide a baseline against which to assess the significance of current glaciological changes. They are also useful for improving predictions of future behaviour.Southeast Greenland has experienced dramatic glaciological changes over the last two decades. Glaciers across the region simultaneously retreated, accelerated, and thinned2 of the Greenland Ice Sheet. These glaciers are amongst the fastest in Greenland3; velocities near calving termini can exceed 12 km yr−1. Glaciers here experienced significant, dynamically-induced thinning in the early-2000s1. This was followed by a period of glacier stabilisation and thickening during the latter half of the 2000s13. Nevertheless, between 2000 and 2012 glaciers in Køge Bugt were the third largest contributor to sea-level rise from Greenland, with a collective discharge anomaly of 67 Gt12.Køge Bugt (Ikeq) is a large glacial embayment in the centre of southeast Greenland Fig. . Three m14 and subglacial topography data16 show that this glacial trough extends through the bay, but rises above sea-level only a few kilometres inland of the present-day ice margin  trough in the centre of Køge Bugt Fig. . Aerogra20  to a depth of around 300 m, these are underlain by warmer (~4 °C) water masses , approximately 50 km from the present-day ice margin Fig. . Sedimen23 of sediments in core ER1116  was calculated to provide a measure of the variability in iceberg production through time  in core ER1116 was deposited exclusively by icebergs calved from the local tidewater glaciers. In some high-latitude environments coarse material can also be transported by sea-ice24 and kelp fronds25. However, both mechanisms are negligible in Køge Bugt. The absence of sediments in the littoral zone here inhibits the transport of coarse material by sea-ice. Similarly, kelp-plucking does not occur here as shallow marine areas are perpetually abraded by sea-ice and icebergs, and this prevents kelp colonisation. Icebergs from glaciers further north in the region could potentially deliver coarse material to the core site. Non-local icebergs are prevented from entering Køge Bugt by an area of islands and shallow bathymetry to the north of the bay, this obstructs iceberg transit along the inner-shelf26. Icebergs further offshore are advected past the bay as they are entrained in fast, southerly-flowing ocean currents  can provide a record of the relative changes in hydrodynamic energy at the seabed35. This technique has been widely used in deep-sea and continental slope environments, in these settings the variability reflects changes in the selective deposition of silt grains36. A reduction in current speed allows finer particles to settle out of suspension during long-distance transport; this results in a smaller mean sortable silt grain size at distal sites35. In contrast, at the core location in Køge Bugt, sediment is primarily deposited in situ by iceberg-rafting; this deposits sediment across the grain size spectrum (from clay to boulders). We argue that silt sorting in these settings primarily occurs after sediment deposition through the preferential erosion of fine- grained sediment (winnowing).Measuring the mean grain size of the sortable silt fraction ; in areas with lower sedimentation rates well-sorted sediments can be produced under just a few days of fast-flow conditions (i.e. 10 cm s−1) a year35. Our results suggest that increases in the mean grain size of the sortable silt fraction are primarily caused by increased current flow at the seabed; the degree of sorting of the silt fraction (σ) increases concurrently with the mean grain size  and high abundances of Cibicides lobatulus . The Irminger Current is the only source of warm, saline water masses in southeast Greenland42; we postulate that subsurface waters in Køge Bugt were well-ventilated by the Irminger Current during the early-Holocene. This interpretation is supported by high sortable silt grain sizes and the abundance of Cibicides lobatulus during this interval , glaciers here lost an estimated 127 Gt (141 km3) relative to the present-day configuration  for these glaciers12. Maximum Holocene glacier retreat of 6 km (relative to the present-day) is broadly comparable to changes during the 20th and 21st Centuries; between 1931 and 2012 the central outlet, Køge Bugt Glacier, retreated 5 km53.The ice-rafted debris record displays variability through the Holocene that reflects changes in both iceberg production and oceanographic conditions. However, when examining glacial behaviour, the most significant observation is that ice-rafted debris is present continuously throughout core ER1116 Fig. . This deons Fig. . We calc54. Small land-terminating glacier systems in the region also retreated substantially (3–5 km) during the peak of the Holocene Thermal Maximum55. These new results constrain the frontal position of glaciers in Køge Bugt, but do not provide a direct record of the calving flux, which likely varied through the Holocene. Nevertheless, our results demonstrate that the glaciers here are relatively stable, even when subjected to large external forcings. We attribute this behaviour to their unique bathymetry and subglacial topography; the bed slopes upwards rapidly inland from the present-day margin. This configuration allows the glaciers to adjust to new stable positions with only a minor retreat56. The steep surface profile of the ice sheet in Køge Bugt, which reflects the subglacial topography  X-ray images were obtained at ~20 cm intervals, these were spliced into a single composite image using functions in Adobe Photoshop CS6.The core was split lengthways prior to analysis. Half of the core was subjected to non-destructive analysis using X-ray imaging  and line scanning , this was then archived. X-ray imaging was used to examine sedimentary structures in ER1116. Individual high-resolution (0.09 mm px210Pb and five 14C age determinations. Bulk sediment samples (~10 cm3) for 210Pb dating were taken at ten centimetre intervals from the uppermost 50 cm of ER1116  and the Beta Analytic laboratory, USA (n = 4). Radiocarbon dates were calibrated with CALIB 7.0  using the Marine13 calibration curve58. A marine reservoir correction of 400 years (ΔR = 0) was applied to all 14C dates59, as the benthic foraminiferal assemblages used for dating indicate an Atlantic water origin.The chronology for ER1116 was built from five 116 Fig. . Radioca3 of sediment, representing a 1 cm core slice, at 2 cm intervals, from the full-length of ER1116. Water content was determined prior to sieving by freeze-drying and weighing. Sediment was wet-sieved through a stack of Retsche sieves . The >2 mm fraction was excluded from the percentage calculations to remove large, anomalous peaks caused by the presence of large individual clasts.The grain size distribution of sediment in ER1116 was determined by wet-sieving and laser diffraction analysis. Material was sampled from 10 cmμm34). Analysis was undertaken with a Malvern Mastersizer 3000. Samples were pretreated with tetrasodium pyrophosphate (0.01 M Na4P2O7 · 10 H2O) and submerged in an ultrasound bath for two minutes to disaggregate particles prior to measurement37. Analysis of a sample typically takes ~10 seconds and produces measurements with errors of less than ±1%.Laser diffraction analysis was used to determine the size distribution of fine-grained sediments (0.3–63 3). Subsamples were wet-sieved and the 100 μm to 1 mm size fraction was selected for analysis60. The 63–100 μm size fraction was also checked for foraminifera content. Foraminifera tests were concentrated by flotation using heavy liquid  and then dry picked from a graticuled tray using a stereo microscope. Both planktonic and benthic foraminifera tests were picked. Where possible at least 300 individual benthic tests were picked, this enables statistically robust analysis61. In many intervals foraminifera tests were sparse. Samples with fewer foraminifera provide less-robust data. Nonetheless, these yield valuable information about environmental conditions. The percentage abundance of individual species was calculated against the total of agglutinated or calcareous tests in each sample.Foraminiferal assemblage analysis was undertaken every 8 cm in ER1116. Additional subsamples were collected at 4 cm intervals in areas of the core where rapid shifts in species assemblage occur. Foraminifera tests were obtained from 1 cm slices of core material (~9 cmh2003–201253) and retreat (dr2003–2012). This was then extrapolated inland to the 50 m bed contour (dr2012–HolMin), the minimum possible extent of glaciers here during the Holocene . The 50 m contour was used to account for isostatic depression of the crust in the early-Holocene. Field observations62 and a compilation of relative sea-level limits63 demonstrate that the maximum isostatic depression in the Køge Bugt area was between 20 and 40 m. We conservatively use the 50 m contour for all calculations. Modern glacier retreat was calculated from Landsat 7 imagery with a modified centre-line method64 using the mean of three flow lines orientated perpendicular to the glacier front. The relationship between glacier retreat and thinning was established from the central outlet glacier (Køge Bugt Glacier) as this has experienced the largest rates of modern retreat and thinning. This glacier also has a simple margin geometry. The volume of ice loss during minimum Holocene glacier extent was calculated for each grid cell (1 km × 1 km) in the Køge Bugt glacial basin (13_a)53 using:Mass loss from glaciers in Køge Bugt was calculated by establishing a linear relationship between modern glacier thinning  and multiplied by 0.9 to obtain an estimate of ice mass loss (Gt). This approach assumes that the surrounding outlet glaciers in Køge Bugt behaved synchronously. It also relies on the assumption that glacier thinning between 2003 and 2012 had equilibrated to changes in margin position, and that the relationship between calving front position and thinning has remained constant throughout the Holocene. This method provides a first-order approximation of maximum ice loss from glaciers in Køge Bugt over the last 9100 years.Individual grid values were then summed to obtain an estimate of volume loss from the glaciological basin (kmSupplementary material


The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. Our cells contain a network of filaments made up of a protein called actin. Just like the skeleton that supports our body, the actin ‘cytoskeleton’ gives a cell its shape and strength. Actin filaments are also critical for many other processes including enabling cells to move and divide. The assembly of actin filaments must be properly controlled so that they are formed at the right time and place within the cell.A complex of proteins known as the WAVE Regulatory Complex (WRC) promotes the assembly of actin filaments. The complex contains a region called the VCA, which is able to bind to and activate another protein to make the new actin filaments. The WRC regulates filament assembly by controlling the availability of the VCA in a way that is similar to opening and closing a safe box. When new actin filaments are not needed, the safe box is closed and the VCA is not available. However, when cells need to make new actin filaments, the WRC is opened to release the VCA region so that it is able to bind to the filament-producing protein.Previous studies have shown that a protein called Rac1 acts as a key to open the WRC and trigger actin filament assembly. But it remains unclear how this works. A major obstacle to studying this process is that Rac1 and the WRC only weakly interact with each other, which makes it difficult to capture the interaction under experimental conditions.To overcome this obstacle, Chen et al. tethered a Rac1 molecule to the WRC in order to make the interaction more stable. A technique called cryo-electron microscopy was used to study the three-dimensional shape of this Rac1-WRC complex. Unexpectedly, Rac1 was attached to a different part of the WRC than the site predicted by previous studies. Further experiments showed that Rac1 needs to bind to both of these sites at the same time in order to open the WRC and release VCA, similar to using two keys to open one safe box for increased security.Some cancers, neurological disorders and other diseases can be caused by defects in WRC and Rac1 activity. Therefore, these findings could lead to new ways to treat these conditions in human patients. Dynamic rearrangements of the actin cytoskeleton play central roles in cellular processes, ranging from cell migration and adhesion to endocytosis and intracellular vesicle trafficking . In manyThe WASP family members WAVE1, WAVE2 and WAVE3 are essential to actin dynamics needed for normal development and function of most eukaryotic organisms, and are also implicated in many cancers . In cell3, phosphorylation on both Ser and Tyr residues, and membrane receptors containing WIRS motifs. Among these, the GTPase Rac1 functions as a canonical activator, whose direct interaction with the WRC is necessary, and in some reports, sufficient to drive activation , together with complementary mutagenesis and biochemical analyses of the complex. Surprisingly, Rac1 is not found on the binding site we predicted previously. Rather, it binds to a conserved site on the opposite end of Sra1. Our biochemical data, including quantitative pull-down, membrane recruitment, and analytical ultracentrifugation (AUC) assays with WRC mutants, confirm that both the original site and the new site can bind Rac1, the latter with approximately 40-fold higher affinity. Actin assembly experiments further reveal that both sites contribute to activation of the WRC toward the Arp2/3 complex. These data lead to a model in which maximal WRC activation requires simultaneous engagement of two Rac1 molecules on the membrane. This bivalent activation model suggests a mechanism by which cells may precisely control actin dynamics at membranes by sensing the density of active Rac1 produced by upstream signaling pathways.6. The previous crystal structures (and the cryo-EM structure reported here) showed that WAVE1 is only ordered to residue L184 within the WRC, affording the system here an effective linker of 65 amino acids. This linker,~230 Å if fully extended, would provide ample length to connect Rac1 to the previously demonstrated binding site, which is approximately 70 Å from residue L184 of WAVE1. Based on experiments measuring binding to immobilized GST-Rac1, the tethered Rac1 protected the WRC from binding to GST-Rac1 much more efficiently than the un-tethered Rac1 protected the wild type WRC, suggesting the tethered Rac1 binds the WRC in a physiologically relevant manner  and Switch II (a.a. 57–75), to bind and activate downstream signaling proteins in a nucleotide-dependent manner . In our Adjacent Site), and the site observed in the EM structure the ‘D Site’ (for Distant Site).In our structure Rac1 is not located at the site predicted by our previous biochemical/mutagenesis analyses. That site is a conserved surface patch on Sra1 near the α4-α6 helices of the WAVE1 meander region, adjacent to the VCA binding site  Chen et. InsteadHow can the previous biochemical analyses be reconciled with the location of Rac1 found in the present structure? In our previous work, we found that the A Site was one of the few highly conserved surface patches on the WRC. We showed that mutating any of three surface residues , or one pair of residues (E434K/F626A), at the A Site disrupted Rac1 binding in qualitative GST pull-down experiments, and that the two mutations analyzed in a quantitative equilibrium dialysis experiment (R190D and E434K/F626A) both decreased affinity . In compWe first examined the ability of immobilized GST-Rac1 to retain the WRC mutants. GST-Rac1 loaded with GMPPNP  could efficiently retain wild type (WT) WRC as well as one of the six D Site mutants, D1122A/E1123A . All othWe obtained similar results under different reaction conditions, employing the high-affinity mutant Rac1 (Q61L/P29S) at a higher salt concentration  . Under t8 tag to Rac1  and the A Site mutations disrupted binding through allosteric effects on the D site. To examine this possibility, we used Multi-Signal Sedimentation Velocity Analytical Ultracentrifugation (MSSV AUC) to directly measure the stoichiometry of the interaction between Rac1 (Q61L/P29S) and ΔWRC230  Balbo e. To sepaD of 0.27 μM as determined below), the binding stoichiometry only reached 1:1 (D ~11.5 μM) of the A Site as determined below (The MSSV AUC data for the mutants were quite different. For the A Site mutant (C179R), at a nearly saturating concentration of EGFP-Rac1 . Fitting to a simplified sequential two-site binding model or to a single-site model resulted in a similar dissociation constant for the D Site , and yielded a dissociation constant for the A Site for the D Site-bound WRC ) of 0.262 ± 0.044 μM, suggesting positive cooperativity between the A and D Sites compared to the dissociation constant for the A Site alone . Such cooperativity likely results from avidity, as two nearby Rac1 molecules, either partners in a GST fusion or adjacent on the solid matrix, bind the same WRC.The above data all suggest that the A Site has a weaker affinity than the D Site. To quantify this difference, we used an equilibrium pull-down (EPD) assay to determine the binding affinity of GST-Rac1 (Q61L/P29S) for each site  Lee et . In thisD ~7 pM, ) to retaD,D of 0.265 ± 0.004 μM for the D Site , fit well to a single-site binding model, yielding a KD,A for the A Site 2-WRC assembly. These data would be consistent with strong positive cooperativity between WRC binding events when Rac1 only binds to the A Site. The better fit afforded by this model does not authenticate the molecular mechanism it describes. We note, however, that the potential for cooperative interactions between closely-positioned WRCs has been suggested by the crystal structure of miniWRC. There, the N-terminal helix of Sra1, which is not observed in the EM density of the dilute WRC studied here, bound between adjacent WRC assemblies in the crystal lattice (For the D Site mutant (Y967A), the strong shift of the data to higher Rac1 concentrations clearly shows that the interaction that recruits the WRC to the beads is appreciably weaker than that for the wild type or A site mutant WRCs. But the isotherm did not fit well to either a single- or a two-site binding model, although both yielded a similar K lattice . Future bona fide binding sites for Rac1, the latter with ~40 fold higher affinity than the former. The two sites can act together to recruit the WRC to membranes containing active Rac1. In retrospect, in our previous equilibrium dialysis experiments, the A Site mutants did not reach saturation at the highest concentrations of Rac1 used (8 μM), behavior we interpreted as indicating reduced affinity at a single site  could produce actin-assembly rates equivalent to that of the constitutively active WAVE1 VCA peptide (dashed cyan curve in 1 complexes (A or D site engaged) and WRC-(Rac1)2. The lack of response to WRC-Rac1 concentrations up to 80 nM suggests suggests that engagement of a single site  is not sufficient to drive WRC activation. Further, plotting actin assembly rate versus the concentration of WRC-(Rac1)2 yields a linear relationship  caused phenotypes in mice including decreased response to cocaine and a reduced number of dendritic spines in neurons of the nucleus accumbens . We foun3, its basic surface will lie against the membrane and its acidic surface will face the cytoplasm. In this orientation, both Rac1 binding sites can be positioned to enable a bound GTPase to bury its prenylated tail in the bilayer, consistent with an activation model involving simultaneous engagement of both sites. This orientation is also compatible with other potential interactions. For example, VCA can be readily released from above the membrane to interact with the Arp2/3 complex further in the cytoplasm . This behavior could be harnessed by cells to determine precisely where and when to turn on actin assembly. Analogous ideas have been suggested previously, based on observations that Rac1 and the Arf GTPase can cooperatively recruit and activate the WRC at membranes . Given t6-Rac1(1-177)(Q61L/P29S) in ΔWRC230-Rac1, WAVE1(1–217) in ΔWRC217, WAVE1(1–230) in ΔWRC230, WAVE1(1–217)-(GGS)6-(485-559) in WRC217VCA, and WAVE1(1–230)-(GGS)6-(485-559) in WRC230VCA. The WRCs were expressed and purified essentially as previously described for the mini-WRC (TM (DE3)RIL cells (Stratagene) at 10°C , and Abi2 (1-158), in addition to different WAVE1 constructs, including WAVE1(1–230)-(GGS)mini-WRC , except  at 10°C .Based on the following arguments the mutant WRCs all appear to be properly assembled and stable. (1) All mutations were made to surface residues, except the S967F mutation, which is buried immediately underneath the D Site (and nevertheless did not negatively affect Rac1 binding/activation). Such mutations likely only disrupt the local binding surface, rather than the overall folding of the WRC, especially since the WRC is ~400 kDa in size and is held together through multiple, extensive inter-subunit interactions. (2) Reconstitution of the recombinant WRC is a multi-step process, involving purification of individual proteins from different host cells and assembly/purification of subcomplexes and ultimately the WRC by a variety of affinity, ion exchange and gel filtration chromatography steps. All the mutant WRCs behaved identically to the wild type WRC during each step of the reconstitution (see example in 6-Rac1(1-188)-His8 (mCherry-Rac1-His8 for short) and MBP-tagged mEGFP-(GGS)6-Rac1(1-188) (EGFP-Rac1 for short) were expressed in BL21 (DE3) T1R cells at 18°C overnight and purified using amylose beads (New England Biolabs). After the MBP tag was removed by TEV-protease cleavage, the protein was further purified by Ni-NTA agarose beads , followed by cation-exchange chromatography through a Source SP15 column . All constructs were verified by DNA sequencing. Other proteins, including Arp2/3 complex, actin, WAVE1 VCA, GST-Rac1, Rac1 and TEV protease, were purified as previously described (MBP-tagged mCherry-(GGS)escribed .2 and 2 mM TCEP. The protein sample (3.5 μl at 0.1 mg/mL) was applied to glow-discharged Quantifoil holey carbon grids , which were then flash frozen in liquid ethane using a Gatan CryoPlunge 3. Grids were imaged with an FEI Tecnai F20 operated at an acceleration voltage of 200 kV and a calibrated magnification of 40,410x , yielding a pixel size of 0.62 Å on the specimen level. A Gatan K2 Summit direct detector device (DDD) camera was used to collect dose-fractionated image stacks in super-resolution counting mode using the UCSF –/Å2/s. Of a total of 2173 image stacks, 30 frames were recorded with 200 ms per frame  for 1366 stacks, 34 frames were recorded with 300 ms per frame  for 407 stacks, and 51 frames were recorded with 200 ms per frame  for 400 stacks. The frames were binned over 2 × 2 pixels (yielding a pixel size of 1.24 Å), aligned to each other using motioncorr  to remove glycerol from previous purification steps and exchange buffer to 10 mM HEPES pH 7.0, 100 mM NaCl, 1 mM MgCltioncorr , and sumThe defocus parameters of each micrograph were determined with CTFFIND3 . ParticlAll images were then automatically picked with Relion1.3 , yieldinFor particle polishing in Relion , image s2 and 5 mM β-mercaptoethanol) at 4°C for 30 min, followed by three washes using 1 mL of the binding buffer. Bound proteins were eluted with GST elution buffer  glycerol, 2 mM MgCl2, 5 mM β-mercaptoethanol and 30 mM reduced glutathione) and examined by SDS-PAGE. Non-equilibrium GST pull-down experiments in 2, and 5 mM β-mercaptoethanol) and stored as a 50% (v/v) slurry. All protein samples were dialyzed against EPD buffer overnight at 4°C to maximize buffer match. After dialysis, GST-Rac1(1-188) (Q61L/P29S) was concentrated to ~500 μM using an Amicon Ultra centrifuge concentrator . All proteins were centrifuged at ~21,000 g at 4°C for 10 min to remove denatured proteins before use. Each reaction was assembled in 100 μL total volume of EPD buffer in a 200 μL PCR tube (Axygen), which contained 0.1 μM ΔWRC230  and MBP-WAVE1(1–230)) varying concentrations of GST-Rac1(1-188) (Q61L/P29S), 30 μL of the Glutathione Sepharose beads  slurry using a wide-bore pipette tip), and 0.05% Triton X100 to facilitate mixing. The reactions were gently mixed at 4°C on a rotary mixer for 30 min. After a brief centrifugation  to pellet the beads, 40 µL of the supernatant was immediately transferred to 8 µL of 6X loading buffer  SDS, 60% (w/v) glycerol, 0.00012% (w/v) bromophenol blue, and 140 mM freshly added 2-mercaptoethanol), and analyzed by Coomassie blue-stained SDS-PAGE gels. The gels were imaged by a ChemiDocTMXRS + system (BioRAD) using its standard protocols. Total intensity of the Sra1 and Nap1 bands was quantified by ImageJ (FIJI) to determine the unbound WRC. The derived fractional occupancy from 2 to 5 independent experiments was directly merged to obtain the binding isotherms. The program DynaFit  experiments were performed essentially as previously described . Glutath (BioKin ) was use2+-NTA DGS (Avanti Polar lipids) were prepared as previously described  were added to cleaned wells equilibrated with 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP, and incubated at 40°C for 1 hr.Supported lipid bilayers (SLBs) containing 98% POPC and 2% Niescribed , with th2, 1 mM TCEP and 1 mg/mL BSA. mCherry-Rac1-His8 protein  mounted on a Leica DMI6000 microscope with a 100 × 1.49 NA objective . Images were acquired at 488 nm for EGFP-WRC and 561 nm for mCherry-Rac1. Images were processed using ImageJ (FIJI). Signal from the 488 nm channel from samples with only mCherry Rac1 and binding buffer was used as background for EGFP-WRC measurements. Signal from the 561 nm channel from samples with only SLBs and EGFP-WRC was used as background for mCherry-Rac1 measurements. EGFP/mCherry signals were calculated from 10 images from two independent experiments. Data are reported as mean ± SEM. The autofocus module on our microscope was used to find the focal plane for all measurements.2 for EGFP-Rac1) to remove potential aggregates. The proteins were aliquoted, flash frozen in liquid nitrogen, and stored at −80°C. Before use, proteins were thawed in a water bath at room temperature and subjected to extensive dialysis (for 3 continuous days with multiple buffer exchanges at 4°C) in the same beaker containing AUC buffer  to remove glycerol  and to maximize buffer match. After dialysis, the samples were centrifuged at ~21,000 g for 10 min at 4°C to remove denatured proteins. Approximately 400 µL of the samples were introduced into the sample sectors of dual-sector Epon centerpieces that had been placed between sapphire windows in a standard AUC cell. The reference sectors were filled with the same volumes of AUC buffer. The cells were inserted into an An50Ti rotor and put under vacuum for temperature equilibration for a minimum of 2 hr. Subsequently, centrifugation was commenced at 50,000 rpm. Data were acquired using both absorbance at 490 nm and interference optics. All experiments were performed overnight at 20°C. Interferometric molar signal increments for the two protein species were calculated based on their respective amino-acid compositions  distributions as described below.The Multi-Signal Sedimentation Velocity (MSSV) Analytical Ultracentrifugation (AUC) assay was performed as previously described , to measositions , resultifficient  at 490 n M−1cm−1 , likely  M−1cm−1 . A data kc(s) distributions that reported on the concentrations of WRC and EGFP-Rac1 as a function of sedimentation coefficient. By integrating these distributions over sedimentation coefficient ranges where co-sedimentation was evident, the concentrations of the individual proteins in the complex could be calculated, and thus the molar ratios of the complexed WRC and EGFP-Rac1 were derived. Sedimentation coefficient values for the individual proteins and complexes were determined by a weighted integration scheme ) or WRC230VCA (6-(485-559)) or VCA, and various amounts of Rac1 Q61L loaded with GMPPNP.Actin polymerization assays were performed at 22°C using a PTI Fluorometer  as previously described . Reactio In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tony Hunter as the Senior Editor.Thank you for submitting your article "Rac1 GTPase Activates the WAVE Regulatory Complex Through Two Distinct Binding Sites" for consideration by The Reviewing Editor has drafted this decision to help you prepare a revised submission.Our view is that this is a well-executed study that provides novel information about the regulation of actin assembly and membrane dynamics. In a previous round of review, there was a question around mutagenesis: did the authors make any measurements to verify that the mutant proteins are stable and well folded? This control would verify that the mutations are interfering with binding rather than protein structure.The authors responded with good arguments that the mutated proteins are folded. Doing a simple urea-denaturation experiment while monitoring the intrinsic fluorescence of the complex might have revealed some loss of stability, which is different from being folded. Examples exist of apparently innocuous surface mutations that change the stability a protein.We ask the authors to present their arguments why the mutants are properly folded in the manuscript and show gel filtration profiles for the most important mutants. Moreover, input lanes for the most important mutants along with wild type must be shown in the pull-down gels. We ask the authors to present their arguments why the mutants are properly folded in the manuscript and show gel filtration profiles for the most important mutants.We have now included in the Materials and methods section a paragraph containing our arguments that the mutant WRCs are most likely properly folded and assembled, with a call-out in the main text (second paragraph in subsection “Rac1 Binds to Two Distinct Sites on the WRC with Different Affinities”). We have also added gel filtration chromatography traces for three additional mutants in a new Moreover, input lanes for the most important mutants along with wild type must be shown in the pull-down gels.We have moved the input gel for the pull down experiments into the main We have also gone through the manuscript carefully to make sure that all Rac and WRC constructs used in each experiment are clearly indicated in the text, methods and figure legends.


IgE antibodies serve as the gatekeeper for the release of mediators from sensitized (IgE positive) mast cells and basophils following a relevant allergen exposure which can lead to an immediate-type hypersensitivity  reaction. Purified recombinant and native allergens were combined in the 1990s with state of the art chip technology to establish the first microarray-based IgE antibody assay. Triplicate spots to over 100 allergenic molecules are immobilized on an amine-activated glass slide to form a single panel multi-allergosorbent assay. Human antibodies, typically of the IgE and IgG isotypes, specific for one or many allergens bind to their respective allergen(s) on the chip. Following removal of unbound serum proteins, bound IgE antibody is detected with a fluorophore-labeled anti-human IgE reagent. The fluorescent profile from the completed slide provides a sensitization profile of an allergic patient which can identify IgE antibodies that bind to structurally similar (cross-reactive) allergen families versus molecules that are unique to a single allergen specificity. Despite its ability to rapidly analyze many IgE antibody specificities in a single simple assay format, the chip-based microarray remains less analytically sensitive and quantitative than its singleplex assay counterpart . Microgram per mL quantities of allergen-specific IgG antibody can also complete with nanogram per mL quantities of specific IgE for limited allergen binding sites on the chip. Microarray assays, while not used in clinical immunology laboratories for routine patient IgE antibody testing, will remain an excellent research tool for defining sensitization profiles of populations in epidemiological studies. IgE antibody was identified in 1967 as the molecular gatekeeper which controls the elicitation of allergic symptoms in humans . AntibodThe detection and quantitation of the levels of allergen-specific IgE antibody in human serum was made possible in 1967 with the discovery of IgE as a unique immunoglobulin isotype . PurifieOver the years, significant technological developments have allowed the use of (a) non-isotopic poly- and monoclonal anti-human IgE Fc conjugates to detect bound IgE antibody; (b) the World Health Organization IgE reference preparation  to allowMicroarray technology has been only recently applied to the field of diagnostic allergy serology. The first report of chip-based microarray technology being applied to the diagnosis of human allergic disease occurred in 2002. Hiller et al. immobilized 94 purified recombinant and natural allergen molecules at 0.3 mg/mL in 150 mM sodium phosphate buffer in triplicate onto a pre-activated amine reactive coated glass slide . The sliThere are reports of the use of microarray technology in the detection of IgE antibody for a number of special patient care related services. Reported applications include the assessment of poly-sensitization , clarification of the allergen triggers for patients with atopic dermatitis and anaphylaxis and the selection of appropriate allergens for use in immunotherapy ,13,14,15Hevea brasiliensis natural rubber latex). Second, there is an increase in analytical specificity when selected molecules are used to detect IgE antibodies. For instance, Ara h 2 for peanut; Cor a 9 and 14 for hazelnut detect IgE antibody profiles that aid in determining a clinical risk for a systemic reaction and clarifying the need for an oral food challenge. Third, molecular allergens allow the identification of cross-reactivity  in Timothy grass, lipid transfer proteins in plant food and pollens [The advantage of the IgE antibody chip-based microarray rests in its conservation of serum volume, increased speed of analysis, reduced technician intervention and the use as an optimal configuration for point-of-care tests. The availability of recombinant allergenic molecules has enhanced IgE antibody assay performance in a number of ways. First, it has permitted an increased analytical sensitivity by supplementing extracts that are deficient in a particular allergen specificity . And fo2. Agreement studies with the ISAC, singleplex ImmunoCAP and skin prick testing involving the most prevalent allergen specificities from cat, dog, mite, Timothy grass, Birch tree and peanut produced correlation coefficients between 0.73 and 0.95.While the ISAC was the first and it has been the most visible microassay for the detection of IgE antibody, other groups have reported different assay formats. Unlike the ISAC that is a manual assay, Williams et al. reported an automated microarray system called the Microtest that involves the use of a self-contained biochip and reagent cartridge . It measn = 7 to 16) of immobilized purified indoor areoallergens [The multiplex Luminex xMAP technology-based microarray reported by King involves a magnetic xMAP bead set with a discrete number . The use of molecular allergens can enhance the limit of quantitation of an IgE antibody assay by supplementing allergens that are missing or in low abundance in extracts. They can enhance analytical specificity by detecting IgE antibodies to particular stable allergens that signal a high risk for a systemic allergic reaction. They can identify IgE antibody responses to cross-reactive allergens present in foods and pollens. Finally molecular allergens can identify genuine primary sensitization which is not possible with the use of allergen extracts. IgE antibody microarrays have the advantage of increased speed of analysis with reduced turn-around time and conservation of sample volume which is attractive for pediatric testing. The microarray assays tend to be simpler in design, with fewer reagents that can reduce cost and technician time. All these advantages come with the constraint of a lower analytical sensitivity, more complex quality control, and less quantitative results than are generated by singleplex IgE antibody assays. The panel format of the chip forces IgE antibody analyses to all 112 allergens on the microarray which can encourage abusive testing that results in the measurement of unneeded or unwanted IgE specificities. Finally, due to the limited amount of allergenic protein that is deposited on the chip per dot, high levels of allergen-specific IgG antibody may interfere by competing with the binding of IgE antibody of the same specificity, and thus reduce the accuracy of IgE antibody measurements. The overall conclusion is that the current diagnostic microarray assays for IgE antibody will remain excellent research tools for epidemiological studies. They will, however, not compete in the clinical laboratory arena with singleplex assays that have superior analytical sensitivity, quantitation and the ability to use allergen extracts that are viewed as comprehensive in terms of containing the principal allergens in any given allergen specificity.