Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol , suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a “hinge” located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in  DSF was purchased from Sigma-Aldrich . In these experiments urease reactions were started by the addition of urea to a final concentration of 2 mM. The residual activity in percentage of the initial was plotted against the time of incubation and fitted to an exponential decay equation with a plateau (Equation (2)).To evaluate the time and concentration-dependence of the inhibition of urease activity, the initial rates of urea hydrolysis were measured in the standard reaction mixture in the presence of DSF (tetraethylthiuram disulphide), : Enzyme inactivation,To evaluate the saturation of CVU with inhibitors, enzyme was incubated for one hour at 37 °C with different concentrations of inhibitors in the standard reaction mixture, the reactions were started by the addition of urea to a final concentration of 2 mM, followed by the measurement of the residual activity. Percentages of residual activity were plotted against inhibitor concentration. Since two types of behavior were observed, two different equations were used to fit the data. In one case, we observed a trend that produced a full loss of activity (Equation (3)), and in the other case a partial inhibition is observed, meaning that at enzyme saturation activity value is non-zero (Equation (4)).h, is the Hill coefficient; IC50 is the apparent inhibition constant; [I], is the inhibitor concentration. Residual activity and A0 is the same as in Equation (2).Equation (3): Full-Inhibition Saturation Kinetics,plateau is included.Equation (4): Partially-Inhibition Saturation Kinetics,For the inhibition pattern urea saturation kinetics experiments were performed at fixed/variable concentrations of DSF and data were globally fitted to Equation (5).Ki, is the Non-Competitive Inhibition Constant, and all other parameters and variables are previously defined.Equation (5): Non-Competitive Inhibition Mechanism,S)-1-(3-Mercapto-2-methyl-1-oxopropyl)-l-proline), bithionol ) and quercetin were performed in a similar manner as for inhibition experiments with DSF. These inhibitors were purchased from Sigma-Aldrich . Inhibition kinetics data were fitted to the indicated equation. The protection of the activity by a given inhibitor , at a concentration equal to the IC50 value, over the inhibition produced by DPS was monitored measuring changes in initial velocity and plotting the percentage of inhibition for each treatment and expressed as mean ± SEM. Student t test was used to estimate statistical significance. Measurements of variance were performed using a one-way ANOVA. Differences were considered significant when p was less than 0.05. All experiments were performed at least three times, and in all cases the SEM justifies the accuracy of the values, except in the case of inhibition kinetics of bithionol and quercetin in which SEM is higher than 10% of the estimated value.Inhibition of CVU by Aldrithiol , captopril . CVU was excited at 290 nm, and fluorescence intensity was recorded at 340 nm after an hour of incubation at 37 °C in the absence or presence of different concentrations of DSF. In order to discard solvent fluorescent changes, a control experiment was performed in which the different volumes of solvent (same as in the titration volumes used in ligand additions) were added to urease. Fluorescence intensity changes were plotted against the DSF concentrations and fitted to Equation (5).ΔIF, is the change in fluorescence intensity at 340 nm; Bmax, is the maximum ΔIF and corresponds to the maximum binding; [DSF], is the disulfiram concentration and Kd, is the dissociation constant.Equation (6): Fluorescence binding saturation,N,N-diethyldithiocarbamate (DTC). JBU three-dimensional structure was downloaded from PDB, and DSF was downloaded from Zinc Database (http://zinc.docking.org/substance/1529266). Three-dimensional structures where dock prepared using USCF-Chimera (https://www.cgl.ucsf.edu/chimera/), followed by the function Autodock Vina which was used to perform docking using a grid that corresponds to the whole tetrameric urease structure. The most abundant auto-validated complex model was analyzed, and the best model is presented.In order to test the possibility of DSF interacting with Cys592 of urease, docking experiments in JBU (PDB code: 3LA4) were performed by using AutoDock Vina and analyzing the 9 best scored solutions  with DSFOur kinetic experiments, docking models and comparative study of available urease sequences support the potential use of disulfiram to control urease activity. On the other hand, disulfiram and other compounds that contain thiol reactive groups can be used to redesign better and specific urease inhibitors. Here the docking approach seems to be adequate to predict whether compounds of this kind can interact with relevant Cys residues.


Both Gαq and the downstream hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) inhibit TRPM8. Here, I demonstrate that direct Gαq gating is essential for both the basal cold sensitivity of TRPM8 and TRPM8 inhibition elicited by bradykinin in sensory neurons. The action of Gαq depends on binding to three arginine residues in the N terminus of TRPM8. Neutralization of these residues markedly increased sensitivity of the channel to agonist and membrane voltage and completely abolished TRPM8 inhibition by both Gαq and bradykinin while sparing the channel sensitivity to PIP2. Interestingly, the bradykinin receptor B2R also binds to TRPM8, rendering TRPM8 insensitive to PIP2 depletion. Furthermore, TRPM8-Gαq binding impaired Gαq coupling and signaling to PLCβ-PIP2. The crosstalk in the TRPM8-Gαq-B2R complex thus determines Gαq gating rather than PIP2 as a sole means of TRPM8 inhibition by bradykinin.Activation of Gα  •q governs the cold sensitivity of TRPM8 and TRPM8 inhibition by bradykininGα•q gates TRPM8 through engaging three basic arginine sites on TRPM8Gα•q gating sites but not PIP2 signalingTRPM8 inhibition by bradykinin depends on Gα•2 sensitivity of TRPM8 channelsBradykinin receptor prevents the PIP q, contributing to pain and inflammation. Zhang reveals Gαq gating sites on TRPM8 and shows that bradykinin receptor solely uses Gαq gating sites for TRPM8 inhibition upon activation, while depriving the channel of sensitivity to PIP2.TRPM8 channels are inhibited by receptors coupled to Gα TRPM8 channels detect a wide range of cold temperatures, spanning from innocuous cooling to noxious cold . It is aHowever, TRPM8 was inhibited during inflammation due to the actions of released inflammatory mediators such as bradykinin (BK) and histamine . Inhibitq-coupled receptors. Initially, TRPM8 inhibition induced by BK was attributed to dephosphorylation of TRPM8 by activated protein kinase C (PKC) , which is essential for TRPM8 activation (2 hydrolysis-mediated TRPM8 inactivation has been implicated in TRPM8 desensitization and cold adaptation (BK and histamine act on Gα C (PKC) . Anothertivation , leadingaptation .q directly inhibits TRPM8 in vitro  1–3 in TRPM8 as Gαq gating sites. Mutation of these sites entirely abolished TRPM8 inhibition by activated Gαq and BK and also significantly reduced TRPM8 inhibition by histamine. I further elucidated the mechanism for the lack of a role of PIP2 in TRPM8 inhibition by BK, and I found that it is determined by a bidirectional crosstalk in the TRPM8-Gαq-B2R complex. Our data also suggest independent and cooperative modulation of TRPM8 by Gαq and PIP2.In this research, I have found that direct Gαq directly inhibits TRPM8 channels in vitro  mice, in which Gα11 remains intact and will take over the activation of downstream PLCβ-PIP2 signaling.We have previously shown that activated Gαin vitro , but it +. TRPM8-independent firing responses were excluded from further analysis.Cell-attached patch clamping was used to record TRPM8-mediated firing responses in small-to-medium sensory dorsal root ganglia (DRG) neurons, because the cell-attached mode preserves intracellular modulatory factors, minimizing artificial disruption of intracellular signaling ; furtherq-deficient mice. I first validated the ablation of Gαq, but not Gα11, in Gαq-lacking DRG neurons (q-deficient DRG neurons responded to two consecutive cold ramps similarly to wild-type (WT) neurons , consistent with the finding of the basal inhibition of TRPM8 channels by endogenous Gαq  -coupled N or C terminus of TRPM8 was used to pull down different chimeric Gαq proteins in which different functional regions in the distal C terminus of Gαq are replaced by Gαi .In principle, the mutation of Gαt effect B and 4C. ablated D  on N8 both significantly enhanced TRPM8 currents   J. Howeve68Q) , and three positively charged arginine residues  within these regions function as Gαq effector sites. These experiments also support the notion that gating of TRPM8 by menthol and voltage is separable through independent mechanisms, though they are allosteric during TRPM8 gating.Altogether, Gαqi on TRPM8 currents elicited by menthol  , supporting the idea that increased sensitivity of TRPM8-TM is due to relief of basal inhibition from endogenous Gαq/11 and not caused by indirect alterations to the intrinsic biophysical properties of the channel. These data are also consistent with the basal inhibition of TRPM8 by endogenous Gαq in DRG neurons  . As expected, TRPM8-TM exhibited much larger currents than the WT channel and completely abolished the inhibitory effects of 3Gα menthol A and 5B. 5.3 μM) C. Howeve neurons C–1G. Notage ramp D. A furt 5.9 mV) E–5G. It channels G  in the N terminus of TRPM8 are Gαq effector sites responsible for the Gαq gating of TRPM8.Altogether, TRPM8-TM completely abolished the inhibitory effects of 3Gαq binding TRP domain and the bottom S6 from the pore domain form the dome of the cytoplasmic cavity. The height of the cavity measured from the bottom of S6 to R368 is 83 Å. The distance of R470 from two opposite subunits is 68 Å, and the distance of two opposite R368 residues is 82 Å . Both activated Gαq and concomitant PIP2 hydrolysis cause TRPM8 inhibition. The identified TRPM8-TM selectively deficient for Gαq gating but without impairment in PLCβ signaling and PIP2 sensitivity (discussed later) will be an excellent tool for determination of the relative role of direct Gαq gating and PIP2 signaling in TRPM8 inhibition during inflammatory signaling.The inflammatory mediators BK and histamine inhibit TRPM8 through binding, respectively, to B2R and H1R, two Gα2+-free conditions , allowing for intact completion of intracellular signaling before entering the whole-cell configuration for monitoring TRPM8 currents. In HEK293 cells co-expressing TRPM8 and B2R, a short pretreatment with BK elicited a robust inhibition of the inward and outward currents of TRPM8 , and the involvement of other mechanisms is unlikely.Similar findings were also observed when TRPM8 was activated by membrane voltage. q gating of TRPM8 channels is the sole mechanism for BK-induced inhibition of TRPM8, without a significant role for PIP2 hydrolysis. However, both Gαq gating and PIP2 signaling contribute to histamine-triggered TRPM8 inhibition.Taken together, direct Gαq-coupled GCPRs. How can PIP2 signaling play a role in the modulation of TRPM8 initiated by H1R, but not by B2R, though both receptors use Gαq gating for modulation of TRPM8? To elucidate this question, I used the inducible PIP2 depletion system in which 5-phosphatase (5-ptase) coupled to FK506-binding protein 12 (FKBP12) is rapidly recruited to the membrane through dimerization with a membrane-targeted fragment of mammalian target of rapamycin (mTOR) by rapamycin, resulting in the depletion of membrane PIP2    A and 5I. (<10 Å) . However (<10 Å) I. Thus, 10 Å)   .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xuming Zhang . The male and female mice and their littermates were maintained on a C57BL/6 background housed in a 12h light/dark cycle with food and water ad libitum. The experiments on mice were approved by the ethical review committee of Aston University and UK home office and carried out in accordance with the Animal Scientific Procedures Act 1986 in the UK. The knockout mice are viable but exhibit impaired motor coordination and defective platelet activation  as described previously  as described previously . Brieflyq (GNA0Q00000) were from the Missouri cDNA resource center. PLCβ-His (6x) cNDA is a kind gift from Dr. Elliott Ross . The membrane lipid PIP2 depletion was induced using mRFB-FKBP-5-ptase catalytic domain together with membrane targeted PM-FRB-CFP , as described ) at 4°C overnight. Bound Gαq proteins were eluted by boiling in sample buffer followed by separation in 10% SDS-PAGE gel and detection with primary anti-Gαq  and secondary HRP-linked rabbit antibody . The blots were stripped and redetected with HRP-conjugated anti-GST .GST pull down assay was performed as described previously . Brieflyq binding to hexahistidine-tagged PLCβ . All the blots shown are representative of at least three independent experiments.Nickel beads pull down assay was conducted as described previously with slight modifications . Brieflyged PLCβ E. In thiq and Gα11 in Gαq−/− mice was detected using western blot with anti-TRPM8 , anti-Gαq  and anti-Gα11 , respectively. The blots were also stripped and redetected with anti-β-tubulin  for verifying equal loading across samples.The expression level of TRPM8, Gα2+-free extracellular solution was used and contained (in mM): 140 NaCl, 4 KCl, 10 HEPES, 1 MgCl2, 5 EGTA, 5 Glucose, pH7.4 with NaOH. Ca2+-containing Hanks solution was largely similar to Ca2+-free solution except that 5mM EGTA in Ca2+-free solution was replaced with 2mM CaCl2. For studying the effect of GPCR and PIP2 signaling on TRPM8 in All electrophysiology recordings were obtained using an Axopatch 200B patch amplifier (Molecular Devices) as described previously . Briefly1/2) was calculated using the Boltzmann equation as described previously (The current-voltage (I-V) relationship of TRPM8 was determined using a ramp depolarization from −120mV to +120mV in 650 msec. Voltage-dependent activation of TRPM8 was further investigated from currents evoked by depolarization voltage steps from −120mV up to +200mV in 20mV increments with every step lasting for 100msec.The half-maximal activation voltage (Veviously .2+-containing Hanks solution (see above). Cell-attached patch clamping maintains the intracellular contents of cells allowing for reliable and long lasting recording of action potentials. As TRPM8 is mainly expressed in small-to-medium sized DRG neurons .Imaging of Tubby-R332H-YFP in live cells were performed using a Leica confocal microscope (Leica) as described previously . Fluoresq (PDB: 3OHM) was analyzed and modeled using Discovery studio 4.5 (BIOVIA).The structure of TRPM8 (PDB: 6BPQ) and GαFiring events of DRG neurons and TRPM8 currents were quantified and analyzed using the Clampfit 10.2 software (Molecular Devices). The number of cells was denoted in parentheses above the bars of each figure and derived from at least three different days of preparations. All the data passed the normality test and are shown as mean ± SEM. Significance between groups was determined using Student’s t test or one-way ANOVA, and considered significant at p < 0.05.


Pedicle screw loosening is a common complication after spine surgeries. Traditionally, it was assessed by radiological approaches, both X-ray and CT (computed tomography) scan, while reports using mechanical method to study screw loosening after spine surgery are rare. The primary objective was to study the prevalent of pedicle screw loosening according to extraction torque during screw removal surgery and access the sensitivity and specificity of both X-ray and CT scan for diagnosing screw loosening. The second objective was to identify the risk factors for low extraction torque of pedicle screw that might lead to loosening.Thirty-three patients who underwent pedicle screw removal surgery after at least 2 years from primary surgery were evaluated preoperatively for fixation stability by X-ray and CT scan. In total, 236 screws were taken out, and the extraction torque data was recorded and analyzed to identify the sensitivity and specificity of both imaging studies for screw loosening. Furthermore, risk factors that might contribute to low extraction torque were also studied.p = 0.009); meanwhile, screws of non-fusion surgery had lower extraction torque when compared to those in fusion surgery (p = 0.001). BMD  and age had low but significant linear relationship with screw extraction torque .The mean extraction torque of removed screws was 1.55 ± 1.00 Nm; a torque force of less than 1.02 Nm was used to define a screw as loosened. According to such criterion, the loosening rate was found to be 33%. X-ray had a sensitivity of 24% and a specificity of 98%, while CT scan had a sensitivity of 22% and a specificity of 96%. Extraction torque of pedicle screws inserted in fractured vertebrae was significantly lower than those in non-fractured vertebrae (Our findings showed that both X-ray and CT scan had high specificity for screw loosening detection, but their sensitivities were relatively low. Surgeons needed to be more cautious when assessing screw loosening merely according to radiological examination, and aware of that screws in fractured vertebrae or non-fusion surgery were vulnerable to loosening. Pedicle screw fixation is widely used in spine surgery for numbers of indications, such as degenerative disease, trauma, tumor, infection, and deformity. It reduces the range of motion of the stabilized spine, increases the fusion rate, and is generally considered to be safe with relatively low complication rate associated with the device , 2.One of the typical complications widely reported in literatures is screw loosening, which may lead to fixation failure and require revision surgery . The keyThe limitation of radiological approaches led to a great variety of screw loosening rate in literatures. Some papers showed relatively low loosening rate, less than 1% in non-osteoporotic patients evaluated by X-ray , 5, 13, Extraction torque, as an objective mechanical indicator, has been used to evaluate the mechanical fixation of pedicle screws in animal model . HoweverThis was a prospective designed single-center study. Patients who underwent pedicle screw removal surgery were screened for eligibility. The indications for screw removal included the following: (1) pedicle screw fixation for thoracolumbar fracture without fusion and imaging confirmed solid fracture union, (2) patients required screw removal that presented persistent axial para-midline back pain to palpation or abnormal foreign body sensation due to pedicle fixation with imaging confirmed solid fusion, and no other cause found, e.g., infection. Those with significant pedicle malplacement or destructive spine disorders, such as bone metabolic disease, were excluded from our study. Informed consent was obtained from all individual participants included in the study.The titanium alloy pedicle screws  with diameter of 4.0 to 6.5 mm and length of 30 to 55 mm were used in the primary surgeries. Conventional lateral and anteroposterior radiographs were taken before and at 3 to 6 months after primary surgery. The same X-ray and CT scan were scheduled before screw removal surgery to evaluate fracture union and spine fusion, as well as stability of instrumentation. Bone mineral density (BMD) was tested using dual energy X-ray absorptiometry. The maximum extraction torque was recorded while unscrewing the screw using torque gauge with a range of 0.06 to 6.00 Nm . The same surgeon (J.S. senior resident) with sufficient practice made all the extraction torque recordings. The radiographs were evaluated by a senior spinal surgeon (X.W. senior attending doctor), who was blinded to the extraction torque and patient information. X-ray criteria for screw loosening were a radiolucent zone surrounding the screw thicker than 1 mm and/or the “double halo” sign. CT scan criterion of loosening was a no signal zone surrounding the whole body of screw on the CT image. Because of metal artifact, no signal zone was usually seen around screw tail, which could not be interpreted as screw loosening . The Chi-square test was used for categorical variables. Mann-Whitney Thirty-three patients, 10 females, and 23 males, aged from 17 to 66 years (average age 38 years) at the time of implant removal, were included; patient characteristics are shown in Table p = 0.863) or specificity (p = 1.00) between X-ray and CT scan. The mean torque of screws, which were diagnosed as loosening by X-ray, was 0.53 ± 0.65 Nm, which was significantly lower (p < 0.0001) than others (1.65 ± 0.98). While the mean torque of loosened screws diagnosed by CT was 1.03 ± 1.00 Nm, which was also significantly lower (p = 0.008) than others (1.71 ± 1.00 Nm).Using X-ray radiographs taken the day before removal operations, radiolucent area (wider than 1 mm) around the screw and/or the double halo were detected in 20 screws. Using CT scan, the no density zones were found surrounding 17 screws. As shown in Tables p = 0.673) as shown in Fig. p < 0.001) as shown in Fig. n = 54) was 2.14 ± 1.12 Nm, which was significantly higher than those placed in other segments. There were 26 screws placed in 13 fractured vertebrae. The mean torque of screws placed in fractured vertebrae was 1.03 ± 0.63 Nm, which was significantly lower (p = 0.009) than those in non-fractured vertebrae, as shown in Fig. p = 0.437) between screws placed in the end segments (n = 88) and those in the mid segments (n = 103), as shown in Fig. n = 142) were significantly higher than those in non-fusion surgery (n = 84) (p = 0.001), as shown in Fig. The risk factors of screw loosening were analyzed. Regarding extraction torque, there was no significant difference between polyaxial and monoaxial screws p = 0.67 as shownp = 0.010, R2 = 0.304, F = 8.296) as well as age , indicating pedicle screw in aged patients or those with low bone density may be less stable according to mechanical measurement, as shown in Figs. p = 0.848), weight (p = 0.196), BMI (p = 0.125), and time interval between the primary surgery and screw removal surgery (p = 0.965).Linear regression analysis was used to estimate the relevance between the extraction torque of pedicle screw and other clinical findings. Our results showed a low but significant linear correlation between extraction torque and BMD  and specificity (p = 1.00). It was observed in one case that CT scan failed to show a clear gap around screw, even though an obvious double halo was found on X-ray and the extraction torque was 0.06 Nm  had significant higher extraction torque than those in other segments. A possible explanation could be that surgeries performed in lumbosacral spine were mainly for degenerative disc diseases, which often required spinal fusion, while in the thoracolumbar spine, where operations were more likely for vertebral fracture, fusion were not always necessary.As expected, linear regression analysis showed that the stability of pedicle screw correlated positively with BMD and negatively with patients’ age. The relatively low related coefficients might be due to the existence of numerous confounding factors, and these results indicated that the failure risk of instrumentation increased with age and osteopenia/osteoporosis, which was widely credited but rarely proven in vivo with mechanical measurement.Some reports showed that increasing length and diameter could increase the stability of pedicle screw , 25, whiThere are several limitations in our study. First, although this was a prospective and blinded designed study, the relatively small number of patients’ enrollment and the heterogeneity of screw size and position might render our findings susceptible to confounding factors. Hence, further studies with larger sample size and stratified data according to different factors will be needed to better understand the prevalent of pedicle screw loosening. Second, the lack of pedicle torque during primary surgeries made it unable to observe the longitudinal change of torque and the influence of inserting torque on screw loosening.In general, we found a 33% pedicle screw showed an extraction torque less than 1.02 Nm, which might be considered to be loosening according to our data distribution. Both X-ray and CT had high specificity to detect screw loosening, but their sensitivities could be overestimated. Surgeons need to be more cautious when assessing screw loosening based on radiological examination, since a considerable fraction of low extraction torque screws might have been underestimated. Pedicle screws of non-fusion surgery placed in fractured vertebrae had significantly lower extraction torque and, therefore, could be vulnerable to loosening. Pedicle screws in aged patients or patients with lower BMD might be less stable due to lower extraction torque.


OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.


Their chemical structures were elucidated through extensive spectroscopic analyses, including 1D and 2D NMR experiments and HR-ESI-MS analysis and comparison with previously reported data. All the isolated compounds were assessed for soluble epoxide hydrolase (sEH) inhibitory activity. Among them, erigeside II (5) showed inhibitory activity with an IC50 value of 58.5 µM.Two new phenolic glucosides, pipercroside A and B ( Piper belongs to the Piperaceae family and comprises around 700 species distributed across both hemispheres + Pipercroside B (2): Colorless gum; C17H24O9; c 0.05, MeOH); 1H-NMR  and 13C-NMR data , see m/z 395.1318 [M + Na]+ .The soluble epoxide hydrolases (sEH) inhibitory assay, Bis-Tris (B9754), and albumin (A8806) were purchased from Sigma Aldrich . The human recombinant soluble epoxide hydrolases , PHOME (10009134) and 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) (10007972) were purchased from Caymanchem . The 96-well white plate was purchased from Costar . The fluorescence intensity measurement was conducted on using a Tecan infinite F200 microplate reader .40 − S0/C40 − C0] × 100. Where C40 and S40 are the fluorescence of control and inhibitor after 40 min, and S0 and C0 are the fluorescence of inhibitor and control at 0 min. In this testing, AUDA was used as a positive control.The enzymatic assays were performed according to the methods modified from previous research . The 130Piper crocatum Ruiz & Pav, including two new phenolic glucosides, pipercroside A and B. Their structures were elucidated through NMR and mass spectrometry, and their sEH inhibitory activity was evaluated to find that erigeside II (5) exhibited inhibitory activity, with an IC50 value of 58.5 µM.In conclusion, for the first time 12 phenolic compounds were isolated from the leaves of


The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation. The kinase domains (KD) of all the AGC kinases share the same tertiary structure, characterized by an amino-terminal small lobe (N-lobe) and a carboxy-terminal large lobe (C-lobe), as originally described for PKA3. The two lobes form a pocket that binds one molecule of ATP as phosphate donor during substrate phosphorylation.The 61 human AGC kinases form a monophyletic group of serine/threonine kinases that preferably phosphorylates residues in close proximity of basic amino acids such as Arg (R) and Lys (K)4. The DFG sequence is part of the ATP binding site whose orientation defines the active (DFG-in)3 and the inactive (DFG-out) states of AGC kinases5. The AL contains, in 43 out of 61 AGC kinases  spanning from Asp-Phe-Gly (DFG) to Ala-Pro-Glu (APE) sequences, and including the activation loop (AL) and the P + 1 loopses Fig. , a key p14, a sequence (consensus F-x-x-F/Y-S/T-F/Y) localized on the C-terminal tail and present in 53 out of 61 AGC kinases  the phosphorylated HM serves as docking site for the PIF binding pocket of PDK1 which in turn phosphorylates the AL; 2) several AGC kinases  harbor a phosphate binding pocket, next to the hydrophobic pocket, that interacts with its own phosphorylated HM. This interaction contributes, in cooperation with phosphorylated AL, to reorient the αC-helix in the active conformation15. Besides the AL and HM, some of the AGC kinases have another phosphorylatable site involved in the regulation of their activation, the turn motif, which is localized in the C-terminal tail, preceding the HM. Once phosphorylated, this site helps the C-tail to wrap the N-lobe and addresses the HM to the hydrophobic pocket site16. In summary, the phosphorylation events on the above-mentioned sites are among the major events concurring to AGC kinase activation.Moreover, the αC-helix forms, together with the N-lobe, a regulatory hydrophobic pocket. This site mediates the interactions between the N-lobe and the hydrophobic motif (HM)ses Fig. . The HM 17. At the intracellular pH, the phosphate group is only partially deprotonated and −1 and −2 charged species coexist. Thanks to these negative charges, phosphate groups can act as donors for salt bridges with positively charged amino acids, such as arginine (Arg) and lysine (Lys). Moreover, both protonated and deprotonated phosphate oxygens can form hydrogen bonds with different amino acids18. Phosphorylation can affect the activity and the function of proteins in different ways: (1) by favoring the disordered-ordered transitions; (2) by allosteric regulation at the level of tertiary and quaternary structures; (3) by changing the recognition properties of protein binding sites; (4) by regulating post-translational modifications19.In general, the addition of a phosphate group confers novel chemical properties to different amino acids, above all serine (Ser), threonine (Thr) and tyrosine (Tyr)20. Therefore, for more than 30 years, protein phosphorylation has been artificially mimicked in the lab by phosphomimetic substitutions of phosphorylatable sites with Asp and Glu21. Remarkably, such substitution is frequently found throughout the evolution of eukaryotes. Moreover, the opposite process has also been shown to occur: phosphorylatable residues can emerge by mutation of preexisting phosphomimetic amino acids20.The carboxyl group of aspartate (Asp) and glutamate (Glu) is also deprotonated at intracellular pH and can mimic the phosphate group, especially for the −1 charged speciesin silico studies on the AGC kinase RSK2. Whereas the phosphorylated AL interacts with three key residues, Arg114, Arg192 and Lys216 in RSK2, the phosphomimetic amino acid substituted to the phosphorylatable residue in the AL binds only to Arg192 and Lys216. The inability of the phosphomimetic substitution to correctly interact with all three residues compromises the molecular conformational transitions required for the activation of the enzymatic activity and explains the failure of the phosphomimetic substitution. Combining this result with a phylogenetic analysis, we highlight the unfeasibility of the phosphomimetic substitution on the AL of AGC kinases.Here, we focus our attention on the unusual low frequency of phosphomimetic substitution on the AL, compared to the HM, in the evolution of AGC kinases. To investigate the reasons of this AL distinctive feature we performed biochemical, mutational and Phosphorylation on Ser or Thr in the activation loop (AL) and in the hydrophobic motif (HM) of AGC kinases represents a critical event leading to their activation. To investigate whether these key serine/threonine residues have been replaced by phosphomimetic amino acids (Asp or Glu) during protein kinase evolution, we aligned the protein sequences of all human AGC kinases. 60 out of 61 AGC kinases show a conserved AL sequence and 53 out of 61 AGC kinases show a HM sequence, although in some kinases, including PKA, there is only a truncated form of it Fig. . The comNaegleria gruberi, a unicellular eukaryote, has evolved a phosphomimetic substitution in the HM of the orthologue of human p70S6K  and three isoforms of PKN  the phosphorylatable residue is replaced by Glu and Asp, respectively Fig. . The numS6K Fig. . Likewis AL Fig. .23, Dictyostelium discoideum AKT24, MSK225, PDK126, PKA27, PKCt28, p70S6K29, SGK130 and RSK131. The only exception is PKCd where a phosphomimetic substitution in the AL resulted in a modest increase of the kinase activity28. This is in agreement with a previous report showing that phosphorylation of PKCd AL was not required for its kinase activity32.The lack of phosphomimetic substitutions in the AL raises the question whether the negative charges of Asp and Glu are actually able to mimic the negative charges of phosphorylated amino acids in the AL. Since phosphomimetic substitution is widely used as a tool for biochemical studies, we carefully analyzed published data reporting the effects of such substitutions on the kinase activity Fig. . In the 23, AKT233, Dictyostelium discoideum AKT24, MSK225, PKCt28, p70S6K and SGK134. This analysis suggests that phosphomimetic residue in the AL are unable to correctly simulate the phosphorylation of Ser or Thr.Contrariwise, phosphomimetic substitutions in the HM do not hamper or promote the kinase activity of AKT137. The CTKD is phosphorylated and activated by ERK that binds RSK on a specific docking site localized in the C-terminal tail38. The activation of NTKD is instead a classic two-steps process: first the active CTKD phosphorylates the HM in Ser380 for RSK1 and Ser386 for RSK2, then 3-phosphoinositide-dependent kinase 1 (PDK1) binds to the phosphorylated HM and phosphorylates the AL on Ser221 for RSK1 and Ser227 for RSK240.To elucidate why the phosphomimetic substitution in the AL is functionally inactive, we experimentally studied this amino acid substitution in RSK protein kinases. RSKs are composed by two kinase domains: a N-terminal kinase domain (NTKD), belonging to AGC kinase family, and a C-terminal kinase domain (CTKD), belonging to the CaMK  family42  and in the presence of PDK1 inhibitor GSK2334470 (3 μM), MEK inhibitor PD98059 (50 µM) or RSK inhibitor BI-D1870 (3 µM) Fig. . The pho42 Fig. .Figure 245, RSK2 is characterized by high basal levels of phosphorylation on all its internal residues , as well as at the level of phosphorylation of its substrate YB-1 on Ser102 . The inhibition of PDK1 determined a complete inactivation of RSK2 in vitro enzymatic activity  substituted with an Asp (D) in position 227 (S227D). As a negative control, we used a kinase-dead mutant of the NTKD, i.e. K100A, where the mutation is located in the ATP-binding site in the NTKD, which leads to inactivation of the kinase activity of the protein48. The lack of activity of S227D is comparable to that of the kinase dead K100A mutant  determines failure of kinase activation. This is due by the inability of such phosphomimetic substitution to recapitulate the bonds formed by the phosphorylated Thr51.Since phosphomimetic mutants have been widely exploited for scientific research purposes, the compromised kinase activity of RSK2 S227D mutant prompted us to investigate the underlying molecular reasons of this failure. Indeed, while the low kinase activity of phosphomimetic mutant in the AL has already been described in other kinases of the AGC kinase family, the underlying mechanism has never been further explored. However, a study on PAK1 (p21-activated kinase 1), which does not belong to AGC kinase family but harbors an AL phosphorylated by PDK1RMSD) value (0.954 Å) (PDB ID 4NW5) in comparison with the active Akt2 (PDB ID 1O6K). The starting protein showed that pSer227 is part of a hydrogen bond network which involves Arg114, Arg192 and Lys216 in RSK2  and the Root-Mean-Square Fluctuations (RMSF).In order to investigate the effect of the phosphomimetic substitution on the dynamics of the AL and of the entire protein, we performed molecular dynamics (MD) simulations of the active wild type RSK2 (with the phosphorylated Ser227) and of RSK2 harboring the mutation S227D. As a readout of MD simulation, we used the Root-Mean-Square Deviation  and 212–218 (corresponding to the loop carrying the DFG motif) are more flexible for the Asp227 mutant rather than the pSer227 one  and the activation loop (AL). The HM is a prototypical example of the interchangeability between acidic amino acids and phosphorylatable residues, both found in different AGC paralogues and orthologues Fig. . This siCompared to the HM phosphorylation site, the AL one is very different: (1) there is no trace of interchangeability between acidic amino acids and phosphorylatable residues, neither in AGC kinase orthologues nor in paralogues; (2) experiments of phosphomimetic substitutions determine the loss of kinase activation; (3) MD simulations showed that the phosphate on the AL forms a complex interaction with three spatially distant positively charged amino acid that cannot be mimicked by a negatively charged amino acid; (4) the interactions of the phosphate on the AL with these three amino acids are necessary to allow the conformational rearrangements required for the activation of the protein; (5) from a phylogenetic point of view the existence of basic amino acids in the αC-helix and in the β9 strand is interconnected with the existence of a phosphorylatable AL.54. It is conceivable therefore that AGC kinases during their history evolved a strict dependence on the presence of a phosphate on the AL, as neither a phosphomimetic amino acid nor other residues are able to recapitulate the complex set of interactions orchestrated by the phosphate group on the AL.Such irreversibility of phosphorylatable to phosphomimetic conversion in the AL might be explained by the long evolutionary path of this regulatory site in AGC kinases. It is well known, in fact, that the reversibility evolution path decreases with the time passed after an amino acid substitution due to the accumulation of other additional substitutions in the same protein. After a long evolutionary period, the reversion of the initial substitution would indeed find a structural situation that is completely different from the original one, and therefore the restoration of the original function of that amino acid would be impossible55. Similarly, GPRKs lack a phosphorylatable residue in the AL and do not depend on the AL for their activation. The function of this site has instead been substituted by the binding with G-Protein Coupled Receptors (GPCRs), their upstream activators, capable of determining the transition in the active conformation13.However, evolutionary paths have followed an alternative way to elude the dependence on AL phosphorylation by simply eliminating the AL role in kinase activation. This is the case of the seven members of the Rho-activated protein kinases that lack a regulation mediated by the phosphorylation of AL: in ROCK1 and ROCK2 the phosphorylatable site is present but its phosphorylation does not play any role in kinase activation; in the other five proteins this site is absent. In this family, activation is achieved after protein dimerization, which determines kinase rearrangement in the active conformation, including the stabilization of the αC-helixAnother possible explanation of the different propensity for an effective phosphomimetic substitution in the AL and HM could be found on the different function of the phosphorylation on such sites. The phosphorylation on the AL is mainly involved in disordered/ordered transition and allosteric regulation in the tertiary structure. On the contrary the phosphorylation on the HM modifies the protein binding properties of such sites and is involved in the stabilization of the αC-helix or oligomerization in Rho-associated kinases. It is conceivable that the functions of disordered/ordered transition and allosteric regulation associated with the AL phosphorylation require a more complex set of interactions compared to the HM and explains the lower propensity to phosphomimetic substitution of the first site.The irreversibility of phosphomimetic substitution in the AL has also a strong implication in biochemical experimentation. Since AL constitutive phosphorylation cannot be mimicked by a phosphomimetic amino acid, it is not possible to separate the activation of AGC kinases from the phosphorylation of their AL by the upstream kinase PDK1. Therefore, research on PDK1-mediated activation of downstream kinases by phosphorylation on the AL has to rely exclusively on loss of function approaches, such as the substitution of the AL phosphorylatable residue in Ala or PDK1 inhibition, silencing or knock-out.In summary, we have investigated the effects of amino acid substitutions in the AL in RSK, highlighting the structural and evolutionary reasons of AGC kinases’ addiction on the AL phosphorylation.Cell lines were handled using standard cell culture conditions. HeLa were obtained from the ATCC resource center and 293T cell lines were cultured in DMEM medium supplemented with 10% FBS (Gibco), 200 U/ml of penicillin, and 200 μg/ml streptomycin (Sigma-Aldrich). All experiments were performed on cell lines that had been passed for <3 months after thawing. Transient gene expression in HeLa was obtained by using Lipofectamin (Thermo-Fisher scientific).PDK1 inhibitor GSK2334470, MEK inhibitor PD98059 and RSK inhibitor BI-D1870 were purchased from Sigma-Aldrich and dissolved in DMSO to a final concentration of 10 mM. The obtained solutions were aliquoted and stored at −80 °C.To mutate RSK1 and RSK2, the following primers were designed:RSK1S221D FW 5′-CCACGAGAAGAAGGCCTATGATTTCTGCGGGACAGTGG-3′;RSK1 S221D RE 5′-CCACTGTCCCGCAGAAATCATAGGCCTTCTTCTCGTGG-3′;RSK1 K210A FW 5′-CTGACTTTGGCCTGAGCGCAGAGGCCATTGACCACGA-3′;RSK1 K210A RE 5′-TCGTGGTCAATGGCCTCTGCGCTCAGGCCAAAGTCAG-3′;RSK1 R108A FW 5′-AAGTACGTGACCGCGTCGCGACCAAGATGGAGAGAGA-3′;RSK1 R108A RE 5’-TCTCTCTCCATCTTGGTCGCGACGCGGTCACGTACTT-3’;RSK2 S227D FW 5′-CCATGAAAAGAAGGCATATGATTTTTGTGGAACTGTGG-3′;RSK2 S227D RE 5′-CCACAGTTCCACAAAAATCATATGCCTTCTTTTCATGG-3′;RSK2 K216A FW 5′-CAGATTTCGGCCTAAGTGCGGAGTCTATTGACCATGA-3′;RSK2 K216A RE 5′-TCATGGTCAATAGACTCCGCACTTAGGCCGAAATCTG-3′;RSK2 R114A FW 5′-AAGTTCGAGACCGAGTTGCGACAAAAATGGAACGTGA-3′;RSK2 R114A RE 5′-TCACGTTCCATTTTTGTCGCAACTCGGTCTCGAACTT-3′.56.RSK2K100A and RSK2Y707A was instead provided by Professor Ramos JW57 to obtain their overexpression in HeLa cells.RSK2WT, RSK2S227D, RSK2K100A, RSK2Y707A and RSK2S227D/Y707A mutants were cloned into pCCL.sin.cPPT.polyA.CTE.eGFP.minhCMV.hPGK.Wpre lentiviral plasmidAll mutants  were cloned in pDONR/zeo using Gateway BP Clonase (Thermo Fisher Scientific). These constructs were then transferred through Gateway LR Clonase in the pDEST27  to obtain fusion proteins.1. Sequence alignment was performed by AlignX (VectorNTI 11) using BLOSUM62 similarity matrix. The guide tree was built using the Neighbor Joining method (NJ) developed by Saitou and Nei56. The phylogenetic tree was generated with FigTree v1.4.2 software.The phylogenetic tree was obtained from sequence alignment of the kinase domains of human AGC kinases from kinome.com project2O2 and luminol. The peroxidase linked to secondary antibody oxidizes, in the presence of H2O2, the luminol, which decays from an excited state, generating light. Generated light is used to impress a photographic film. Loading control was performed using β-actin immunodetection.In order to verify RSK2 regulation and activation, HeLa cells overexpressing RSK2WT were treated with following conditions: 1) SF medium overnight, followed by a 15 min stimulation with EGF (10 ng/ml); 2) SF medium supplemented with the PDK1 inhibitor GSK2334470 (3 μM) overnight, followed by a 15 min stimulation with EGF (10 ng/ml); 3) SF medium supplemented with the MEK inhibitor PD98059 (50 μM) overnight, followed by a 15 min stimulation with EGF (10 ng/ml); and 4) SF medium supplemented with the RSK inhibitor BI-D1870 (3 μM) overnight, followed by a 15 min stimulation with EGF (10 ng/ml). Moreover, to test RSK2 activation, HeLa cells overexpressing or not RSK2WT, RSK2S227D, RSK2Y707A, RSK2S227D/Y707A or RSK2K100A were cultured in serum free medium overnight, and then treated or not with phorbol myristate acetate  or EGF (10 ng/ml) for 5 minutes. Finally, to test and compare RSK1 and RSK2 mutants’ activation, HeLa cells overexpressing GST, GST-RSK1WT, GST-RSK1S221D, GST-RSK1K210A, GST-RSK1R108A, GST-RSK2WT, GST-RSK2S227D, GST-RSK2K216A, GST-RSK2R114A, GST-RSK2K100A and GST-RSK2Y707A were cultured in normal culture medium overnight and, then, starved for 3 h and treated with EGF (10 ng/ml for 15 min). After treatment, cells were washed twice with PBS 1X and lysed with 300 µl of “Lysis Buffer” . Lysates were then sonicated for 10 sec, in order to break DNA strand and to reduce viscosity. After that, samples were heated at 95 °C for 5 min. 10 µl each sample were used for BCA quantification at 562 nm wavelength and variable volumes (corresponding to 5 or 10 µg of proteins), were loaded in 7.5% or 4–15% SDS-PAGE wells. Proteins were run in polyacrylamide gel at constant voltage (200 V) for 35 min. Then, proteins were transferred on a PVDF membrane, using Trans-Blot Turbo BIORAD. The PVDF membranes were let dry, and then hydrated with TBS  0.1% Tween for 30 min at room temperature (RT). Afterwards, membranes were incubated with primary antibody (Ab) under gentle agitation overnight at 4 °C. Monoclonal Ab rabbit anti-pT359RSK, monoclonal Ab rabbit anti-pS380RSK, monoclonal Ab rabbit anti-pS102YB1 and monoclonal Ab rabbit anti-YB1 were purchased from Cell Signaling Technology. Polyclonal Ab rabbit anti-pS221/227RSK1/2 was purchase from Thermo Fisher Scientific. Polyclonal Ab mouse anti-RSK2, polyclonal Ab rabbit anti-RSK1 and polyclonal Ab goat anti-β-actin were purchase from Santa Cruz Biotechnology. Each antibody was diluted 1:1000 in TBS, 0.1% Tween, 5% BSA. After primary Ab incubation, the membrane was washed 3 times with TBS 0.1% Tween, then incubated at RT under gentle agitation with secondary Ab , which are conjugated to Horseradish Peroxidase (HRP) enzyme. The secondary antibody was used at 1:10000 dilution in TBS 0.1% Tween. After secondary Ab incubation, the membrane was washed 3 times with TBS 0.1% Tween, and incubated for 1 min in the Western Lightning TM Chemiluminescent Reagent Plus ECL (PerkinElmer), containing H2, 1 mM PMSF, 1.5 mM MgCl2, 0.5% Triton X-100, 20 mM Na-β-glycerophosphate, 1 mM Na3VO4, 0.2 mM EDTA, and 1:1000 protease inhibitor cocktail; Sigma-Aldrich). GST-tagged proteins were isolated through Glutathione Sepharose 4B beads . The GST-tagged proteins isolated from 293T bound to glutathione beads were used to pull-down proteins from 293T extracts and they were washed with the same buffer used before but containing 1 M NaCl2. The pulled-down proteins were dissociated using a reducing buffer  and analyzed by immunoblot. RSK kinase assay was performed by using Abcam p70 S6K Activity Kit (ab139438), which is based on a solid phase enzyme-linked immuno-absorbent assay (ELISA) that utilizes a specific synthetic peptide as a substrate for p70-S6K, but also for p90-S6K (RSK), and a polyclonal antibody that recognizes the phosphorylated form of the substrate. Purified GST-RSK2WT and GST-RSK2S227D were diluted 1:30 in reducing buffer . Then, Kinase Assay Dilution Buffer was added to samples, as reported in the assay procedure, and transferred in appropriate wells of the kit plate. To initiate the kinase reaction 0.5 mM ATP was added in each well and the plate was incubated at 30 °C for 1 h. Next steps were performed as described in the assay procedure. Finally, the reaction was stopped and absorbance of each sample was measured at the wavelength of 450 nm.pDEST27-RSK2WT and pDEST27-RSK2S227D plasmids were transfected in 293T cells with calcium phosphate (Promega). Then, cells were treated or not with the PDK1 inhibitor GSK2334470 (3 µM). After 16 h, cell lysates were extracted in ice using cold lysis buffer  compared to active Akt/PKB (PDB ID 1O6K). Moreover, the loop carrying Ser227 was phosphorylated and visible in this structure. The Ser227 was mutated into Asp (Asp227) to obtain the phosphomimetic, using UCSF Chimera software. The most favorable rotamer for Asp227 was considered. The Mg-ATP bound state was generated by using the coordinates of Mn2AMP-PNP from the structure of Akt/PKB complexed with a GSK3 peptide (PDBID 1O6K).The crystal structure of N-terminal RSK2 kinase in complex with 2-amino-7-substituted benzoxazole (PDB ID 4NW5) was taken into account since it shows the lowest 58 and AMBER14 force field at 1 atm, pH 7.4 and 298 K of temperature. The simulations were set up with 1.25 fs time step under periodic boundary conditions. The timescale of the molecular dynamics simulations (20 ns) was chosen to see the local structural effects of the substitution of pSer to Asp, especially for what concerns the salt bridges network. Such timescale however prevented us to simulate large movements of the kinase lobes, that happen in the order of microseconds and that would require a much larger computing power, not available with the current technology60.A 20 ns MD simulation was run on the protein containing pSer227 and on the molecule carrying S227D mutation using the Yasara packageAll the data are represented as the mean value of at least three experimental replicates and error bars represent standard deviations. Statistical analysis was performed using GraphPad Prism software (GraphPad Software). Statistical significance was determined by a Student’s t test with P < 0.05 considered significant.Supplementary material.


The ice recrystallization rates (kd) of AFGP1–5 and type III AFP samples prepared at a fixed overall ionic strength of 100 mM progressively decreased following the Hofmeister series for anions. Our results demonstrate that the performance of AF(G)Ps is significantly influenced by additives present in common cryopreservation media. It is thus important to conduct excipient compatibility experiments to identify potential incompatibilities between additives and AF(G)Ps in cryopreservation formulations.Antifreeze (glyco)proteins (AF(G)Ps) have received increasing attention as potential cryopreservation agents since their discovery in the 1970s. While cryopreservation strategies for specific cells (such as red blood cells) are successful and widely implemented, preservation of other cell types, tissues and whole organs remains challenging. This is due to the multifactorial nature of the freeze-thaw damage, the complexity of preserving biological matter and the (country-to-country) variability of the employed procedures and regulations. AF(G)Ps are well-known for their ability to modulate ice crystal growth morphology and ice recrystallization inhibition (IRI), both of which are considered key contributors to freeze-thaw damage. To date, however, the impact of AF(G)Ps on cell survival remains at best partially understood as conflicting results on the benefits or disadvantages of including AF(G)P in cryopreservation strategies remain unelucidated. We hypothesize that variability in the additives in the cryopreservation media contributes to the observed discrepancies. To critically examine this idea, we monitored the inhibition of ice recrystallization by AF(G)P in the presence of various salts using a quantitative analysis of optical microscopy images via the Lifshitz-Slyozov-Wagner (LSW) theory for Oswald ripening. We found that the addition of salts, which are used in culture and cryopreservation media, enhances the IRI activity of AF(G)Ps, and that the magnitude of the enhancement was in line with the Hofmeister series. The size of ice crystals grown in AFGP While cryopreservation strategies for specific cells (such as red blood cells) are successful and widely implemented, preservation of other cell types, tissues and whole organs remains challenging. This is due to the multifactorial nature of freeze-thaw damage, the complexity of preserving biological matter and the (country-to-country) variability of the employed procedures and regulations. In many cases, cellular damage to cryopreserved cells, tissues and organs is induced by ice recrystallization during thawing ,2. Smallkd) [At the end of the 19th century, Franz Hofmeister observed and quantified egg white protein precipitation triggered by the addition of different salts containing the same cation. Hofmeister’s original work pointed towards ion specificity in the precipitation of proteins, where anions had a more pronounced effect than cations . Direct kd) ,21,22. W3) > 99%, sodium phosphate (NaHPO4) > 89%, sodium citrate (Na3C6H7·2H2O), disodium tetraborate (Na2B4O7·10H2O) > 99.5% and sodium chloride (NaCl) > 99% (Acros Organics) were purchased from Fischer Scientific. AFGP1–5 and recombinant type III AFP QAE HPLC12 samples were prepared by mixing the protein with the different salted buffers in the right amounts to reach fixed protein concentrations of 5 nM (AFGP1–5) and 500 nM (rQAE), which are the IRI activity inflection points in the absence of added salts according to literature [Salt stock solutions of 1 M were prepared by dissolving the desired amount of salt in ultrapure water. Sodium nitrate (NaNO1–5) and 500 nM (rQAE). We prepared these by sandwiching 2 µL of sample between two cover slides, which was rapidly frozen (20 °C/min) to −40 °C in a Linkam LTS420 stage attached to a Nikon ECLIPSE Ci-Pol Optical Microscope (To test the effect of specific ion interactions on the IRI activity of AF(G)Ps that bind the primary prism plane, we selected the sucrose sandwich assay. Classical quantitative methods for studying IRI activity, such as the splat assay and the capillary method, are not sensitive enough to detect small differences in IRI. The liquid volume fraction is too low and the ice volume fraction too high to reliably avoid false positives  molecules without ice affinity as inhibitors of ice recrystallization). In a sucrose sandwich assay, the liquid volume fraction is increased by addition of the innocuous solute sucrose, allowing molecules without ice affinity to freely defuse. IRI experiments are performed at a fixed concentration of 5 nM . Normal distributions of ice crystal radii were assessed by Shapiro–Wilk tests to properly analyze the statistical differences between data sets. The non-parametric Mann–Whitney–Wilcoxon test was used to identify statistical differences between the median values for the ice crystal radii at the end point of the experiment; p-values lower than 0.05 indicated statistical differences between compared sets.1–5 from Antarctic tooth fish. We analyzed the impact of sodium nitrate, sodium chloride, sodium borate, sodium phosphate and sodium citrate on their IRI activity by optical microscopy using the so-called sucrose sandwich assay, which allowed us to determine and monitor ice crystal grain size and ice growth rates over time.Aiming to elucidate the effect of various salts commonly used in cryopreservation media, we compared the ice recrystallization inhibition (IRI) activity of antifreeze (glyco)proteins in the presence of sodium salts from the Hofmeister series for anions at a fixed overall ionic strength of 100 mM. We selected two widely studied fish antifreeze proteins, which varied three orders of magnitude in IRI activity in 30 wt% sucrose solutions. These are the globular fish type III antifreeze protein QAE HPLC12 (rQAE) from the ocean pout, and the glycosylated polypeptide AFGPR> was smallest for sodium phosphate (<R> = 8.0 µm) and largest for sodium nitrate (<R> = 8.4 µm). Similarly, ice recrystallization rates (kd = ∂<R>3/∂t) were smallest for sodium phosphate (kd = 7.1 µm3 min−1) and largest for sodium nitrate (kd = 7.9 µm3 min−1). All five anions increased the ice recrystallization rate by roughly 30%, from ~5.5 µm3 min−1 for unsalted to 7.5 ± 0.3 µm3 min−1 for salted 30 wt% sucrose solutions .First, a series of control experiments were performed to examine the impact of the added salts on the dimensions and growth rates of ice crystals in 30 wt% sucrose solutions without AF(G)Ps. At the endpoint of the experiments, after an annealing time of 90 min, Shapiro–Wilk tests identified log-normal distributions of ice crystal radii ranging in size between 0.8 and 14.4 µm for all salted solutions without proteins a. The nuolutions b. As themparison c. As exp1–5 was examined  in the salted sucrose solutions with AFGP1–5 (R> ~6.9 µm). In accordance with LSW theory, we observed a linear increase in <R>3 as a function of time for all 5 nM AFGP1–5 samples . In fact, AFGP1–5 showed the same ice growth rate in the presence of sodium borate as without any salt in the solution. The complete loss of TH activity of AFGPs upon addition of borate ions is well-documented. It is caused by the cross-linking of AFGPs through esterification of the glycosylated threonine amino acids, which are crucial for both TH and IRI activity [kd values for sodium borate containing AFGP1–5 samples should have been similar to those of the borate control solution (~7.2 µm3 min−1). We attribute this differential impact of borate on TH and IRI activity to a borate concentration-dependent decrease in the adsorption rate of AFGPs to ice. Indeed, an earlier study demonstrated that AFGPs exposed to 0.3 M of borate still adsorbed to ice seeds, albeit with three times less affinity, while TH activity was completely lost [Next, the impact of the salts on the IRI activity of AFGPexamined . The iceg-normal . Most di AFGP1–5 a for all samples a. The soC6H6O73− c. This tactivity ,24. As bely lost . As ice-kd. The ice crystal radii distributions of most salted rQAE samples (with the exception of sodium nitrate) skewed towards smaller ice crystal sizes than the reference rQAE sample without added salt (R> 11.4 µm) were comparable to those of the AFGP1–5 IRI assay at a 100-fold lower concentration (0.7 <R> 10.7 µm). Ranking IRI activity for rQAE from weak to high based on kd at φ0, we found NO3− < Cl− < B4O72− < HPO42− < C6H6O73−  of cryopreservation media can thus significantly impact the inhibition of ice recrystallization by AF(G)Ps, and may  explain the apparently conflicting results in the scientific literature on the moderate success of AF(G)Ps as cryoprotectants. We attribute these salt effects to the same mechanisms put forward to rationalize the ‘salting-out’ of proteins, namely a salt-induced reduction in solubility due to specific interactions between salt ions and surface-accessible residues. For the antifreeze (glyco)proteins studied here, this effect may be augmented due to a salt gradient in the water–ice interface, which would further favor AF(G)P adsorption onto the ice crystals. Alternatively, specific ion–protein interactions could stabilize structural elements critical to the binding of AF(G)Ps to their respective ice crystal planes.Interestingly, our quantitative analysis of the ice crystal growth rates when rescaled to vanishingly small ice crystal volume fractions demonstrated that the IRI activity of both rQAE and AFGPay <R>20 . The sal1–5 in the presence of sodium borate, sodium salts boosted both IRI and TH in accordance with the Hofmeister series for anions. By contrast, sodium borate raised the IRI activity of AFGP1–5, while it simultaneously diminished its thermal hysteresis (TH) activity [Importantly, our findings also reveal a new strategy to boost the performance of AFGPs as cryoprotectants. In all cases except for AFGPactivity . This is1–5 in accordance with the Hofmeister series for anions, with one exception, as previously reported by others for the TH activity of the insect antifreeze protein RiAFP and AFGP1–5 [1–5 despite its reaction with the hydroxyl functionalities of AFGP1–5, which is known to diminish TH activity. The presence of salts clearly affected the performance of AF(G)Ps, which may find use as additives in cryopreservation media to inhibit ice recrystallization and thereby alleviate cryoinjury. Our findings contribute to an improved understanding of the physical mechanisms that underpin the differential impact of excipients, specifically salts, on the performance of cryopreservation formulations.In conclusion, we have quantified the impact of sodium salts at a fixed ionic strength of 100 mM on the ice recrystallization inhibition activity of two types of antifreeze (glyco)proteins. We found that all salts enhanced the IRI activity of rQAE and AFGP AFGP1–5 ,26. Sodi


The present study was aimed to determine the prevalence of infectious bronchitis virus (IBV) as well as virus isolation, identification, and molecular characterization of various strains circulating in Bangladesh.A total of 371 swabs and organ samples were collected from four types of chicken including layer, Sonali , broiler, and broiler breeder under eight districts  during 2014-2016 in Bangladesh.Out of 371 samples, 65 samples were positive in reverse transcriptase polymerase chain reaction (RT-PCR) for molecular identification of IBV. The overall prevalence was 17.52% recorded and among the selected types of chicken, the highest prevalence of IBV was found in layer that was 42.22% followed by 17.24% in Sonali, 14.93% in broiler breeder, and lowest prevalence was 11.94% in broiler chicken, respectively. Moreover, the prevalence of IBV was recorded highest in aged chicken at 41-60 weeks, which was 54.55% in layer, 27.27% in Sonali, and, afterward, 14.68% was found in broiler breeder, respectively. Frequency of IBV more frequently in winter (22.67%) followed by rainy (15.87%) and summer season (11.58%). The highest prevalence of IBV was found Tangail district (41.67%) followed by Mymensingh (24.42%), Gazipur (19.32%), Dhaka (15.38%), Jamalpur (16.67%), Bogura (13.68%), Cumilla (5.88%), and Rangpur (9.26%), respectively. Samples that were found high positive in IBV RT-PCR  were subjected to inoculation into chicken egg embryo to observe characteristic changes in chicken embryo. Swabs and organ samples were processed and passaged in 9-day-old embryonated chicken eggs through allantoic cavity route. IBV virus suspected samples inoculated into chicken egg embryos after 3-5 passages showed dwarfing and curling of the embryos which are characteristic lesions of IBV. Allantoic fluid was collected from all inoculated eggs and performed partial sequencing of S1 gene for three isolates. After sequencing, the phylogenetic tree was constructed from the nucleotide sequences of IBV isolates. Two of the isolates are 4/91 IBV and another one matched with QX-like IBV.st time as which will help for IBV control strategy.The results revealed that the three isolates from different places in Bangladesh were identified for the 1 The poultry industry in Bangladesh has grown leaps and bound since the past two decades. Poultry farming not only assists in upgrading the financial condition but also makes a substantial contribution to human nutrition . The totGamma coronavirus [One of the most economical important diseases of poultry is infectious bronchitis virus (IBV) . It is honavirus . The spionavirus  and alsoonavirus .IBV can affect chicken of all ages . HoweverThe present study was aimed to determine the prevalence of IBV as well as virus isolation, identification, and molecular characterization of various strains circulating in Bangladesh.The Animal Ethics Committee of Bangladesh Livestock Research Institute approved the study.Oropharyngeal, tracheal, and cloacal swab samples were collected in virus transport media containing falcon tube and organ samples  which were collected in sterilized zipper-lock bag from the breeder, broiler, layer, and Sonali  chicken that was showing clinical respiratory sign from each sampling area. The chickens were vaccinated with IBV vaccine against MA5, H120, and IB 4/91 strain according to standard vaccine schedule from a commercial source. A total number of 371 swab and organ samples were collected from the respective farm. Among them, broiler breeder (n=134), layer (n=45), Sonali (n=58), and broiler (n=134) were considered for sampling. After collection, the sample transported in ice box and stored at −20°C refrigerator in the laboratory. The organ samples were taken into sterile pestle and homogenized with the help of sterile scissors and mortar and added 20% phosphate-buffered saline to make a suspension and then centrifuged at 1000 rpm for 10 min. Supernatant fluid was taken by syringe for RNA extraction. Swab samples were homogenized by vortexing at 2-3 s and selected for RNA extraction.RNA extraction was done with Purelink RNA Mini Kit, P/N: 12183018A by Invitrogen, USA, as per manufacturer’s instructions.et al. [The one-step RT-PCR was performed using S1 gene-specific primer and probe with thermal condition described by Callison et al.   were subjected to inoculate at the 9dwarfing  in chickdwarfing . After 4Allantoic fluid in FTA card was sent to the CEVA Phylexia Laboratory, Hungary, for Sanger sequencing of IBV. Sequences of the S1 gene of the Bangladeshi IBV isolates were compared with published IBV sequences deposited in the GenBank database using a BLAST. Sequence identities by BLAST analysis were included in alignment and phylogenetic construction. A phylogenetic tree of the nucleotide sequences was constructed using R version 3.4.0 and under package phangorn version 2.4.0, and package ape version 5.1   Figure-.A total of 371 samples were collected and tested for IBV by the real-time RT-PCR test. The overall prevalence of IBV was found to be 17.52% (65/371). Among the selected types of chicken, the highest prevalence of IBV was found in commercial layer chicken that was 42.22% (19/45) and the lowest prevalence was showed broiler chicken as 11.94% (16/134). The prevalence of 17.24% (10/58) and 14.93% (20/134) was showed in Sonali and broiler breeder chicken, respectively .According to the age of the chicken in both commercial layer and Sonali chicken, the highest prevalence was found in age between 41 and 60 weeks, which was 54.55% (6/11) and 27.27% (3/11), respectively. However, in case of broiler breeder chicken, it was highest at 21-40 weeks of age than 41-60 weeks of age, which was 16% (4/25) and 14.68% (16/109), respectively. On the other hand, in broiler chicken, age divided into two groups due to short life cycle and found prevalence 12.5% (1/8) at 0-16 days of age and prevalence 11.9% (15/126) at 17-32 days of age .Three years (2014-2016) were observed during the study period, among three seasons, highest 22.67 (34/150) and lowest 11.58% (11/95) prevalence was found in winter and summer season correspondingly. In rainy season, 15.87% (20/126) prevalence was found .The identified IBV samples were inoculated into 9-day-old SPF ECE through chorioallantoic membrane route. The chicken embryos were shown characteristic lesions of growth such as curled and dwarfed embryos with covered by a thickened amnion after the third passage propagation. Then, IBV was isolated and reconfirmed by RT-PCR.The partial nucleotide sequence analysis of S1 glycoprotein gene was performed in the isolate CK/BD/IBV/NPHL1, CK/BD/IBV/NPHL2, and CK/BD/IBV/NPHL3 and found further defined genetic relationship with two IBV subtypes; one isolate was QX-like IBV and another two isolates were 4/91 IBV circulating in the selected regions, shown in phylogenetic tree . The seqThe phylogenetic analysis demonstrated that the isolates of QX-like IBV are 99% related with isolated of China (accession no. KX107695.1) and two isolates of IBV 4/91 were related with Indian isolates (KF809773.1) and Poland isolates (KT886452.1). The results indicate that both QX-like IBV and 4/91 strains are circulating in chickens in Bangladesh.et al. [et al. [et al. [In this study, the prevalence and characterization of IBV in between 2014 and 2016 were identified. The prevalence of IBV was widespread in the selected areas of Bangladesh and overall 17.52% prevalence was found by identification of S1 gene in quantitative PCR (qPCR). Overall, 58% seroprevalence of IBV were demonstrated by Barua  [et al.  finds th [et al. . They reet al. [Of three types of chicken, commercial layer chickens were the highest (42.22%) prevalence of IBV and lowest prevalence was in broiler chickens. For other two types, it was 17.24% in Sonali and 14.93% in broiler breeder chicken. Except broiler, the other three types of chickens reared for more than 60 weeks of age that increased the chances of harbor of IBV infection. Jackwood  and SegeThe prevalence of IBV had shown a relation in age of chicken. The highest prevalence of IBV was found in the age between 41 and 60 weeks of age of commercial layer and Sonali chicken. On the other hand, it was highest at 21-40 weeks of age in broiler breeder and at 0-16 days of age in broiler.et al. [Javed et al.  demonstret al.  and it met al. ,26.In the present study, the prevalence of IBV becomes highest during winter season (November-February) and it was 22.67%. Researchers shown that the incidence of IBV increased during winter season due to cold weather is more favorable to IBV for survival and spread through droplets and dust ,23,27.et al. [The samples for this study were collected from eight districts of Bangladesh, in which Tangail district revealed the highest prevalence of IBV and it was 33.30% and the second highest prevalence found in Mymensingh district as 24.42%. It has been mentioned by Sylvester et al. , that thet al. .Genetically, the S1 gene sequenced data identified that the two serotypes of IBV were circulating during the period of 2014-2016 that is QX-like IBV and 4/91 strains. The comparative analysis S1 gene hypervariable region of IBV in nucleotide sequence is a strong tool for the identification of circulating field variants of IBV ,31.The study focused on the overview of the prevalence and molecular characterization of IBV circulating in Bangladesh. The overall 17.52% prevalence with highest in commercial layer chickens comparatively Sonali, broiler, and broiler breeder chickens. The age of chicken over 41 weeks shows more prevalent. The prevalence of IBV ubiquitous round the year but increase prevalence during winter season. According to location, it was highly prevalent in Tangail district. Finally, the sequencing and phylogeny analysis of S1 gene of IBV demonstrated the QX-like IBV and 4/91 strain of IBV were circulating in the environment.ZAB, MZA, MG, and ZUMK: Conceived and designed the study. ZAB, MZA, and MMM: Collected samples and performed the experiments and laboratory analyses. MZA: Performed the data analyses. ZAB and MZA: Drafted the manuscript. MMM, MG, and ZUMK: Revised the manuscript critically. All authors read and approved the final manuscript.


Recent thinning and loss of Antarctic ice shelves has been followed by near synchronous acceleration of ice flow that may eventually lead to sustained deflation and significant contraction in the extent of grounded and floating ice. Here, we present radiocarbon dates from foraminifera that constrain the time elapsed between a previously described paleo-ice-shelf collapse and the subsequent major grounding-line retreat in the Whales Deep Basin (WDB) of eastern Ross Sea. The dates indicate that West Antarctic Ice Sheet (WAIS) grounding-line retreat from the continental shelf edge was underway prior to 14.7 ± 0.4 cal kyr BP. A paleo-ice-shelf collapse occurred at 12.3 ± 0.2 cal kyr BP. The grounding position was maintained on the outer-continental shelf until at least 11.5 ± 0.3 cal kyr BP before experiencing a 200-km retreat. Given the age uncertainties, the major grounding-line retreat lagged ice-shelf collapse by at least two centuries and by as much as fourteen centuries. In the WDB, the centuries-long delay in the retreat of grounded ice was partly due to rapid aggradational stacking of an unusually large volume of grounding-zone-wedge sediment as ice-stream discharge accelerated following ice-shelf collapse. This new deglacial reconstruction shows that ongoing changes to ice shelves may trigger complex dynamics whose consequences are realized only after a significant lag. In the present interglacial climate, ice shelves are melting from their upper3 and lower surfaces7. The current rapid rate of thinning reduces the buttressing effect of ice shelves8. The collapse of the Larsen Ice Shelf B on the Antarctic Peninsula in 20029  are an important part of the cryosphere because they slow the offshore flow of grounded ice. Hence, the presence/absence of an ice shelf greatly influences the balance between ice-volume accumulation and ablation029 Fig.  triggerein situ foraminifera from sub-ice-shelf and grounding-line-proximal sediments to constrain the time elapsed between a paleo-ice-shelf breakup and the subsequent major grounding-line retreat in the WDB of eastern Ross Sea. The development of this retreat chronology specifically relied on a detailed stratigraphic framework that was presented in two recent studies21.Here, we present radiocarbon dates of 26. Broad and wide bathymetric troughs and banks on the outer continental shelves demonstrate that several fast-flowing erosive ice streams formerly drained the AISs  and a steeply seaward-dipping GZW foreset . In other words, GZWs are essentially subaqueous moraines deposited at the marine termination of the ice stream. Identifying and mapping GZW morphology on seismic profiles and bathymetric data is crucial to accurately reconstruct AIS retreat because these features represent the former locations occupied by grounded ice and thus provide unequivocal evidence that ice streams became stationary, or momentarily re-advanced, during their overall retreat. Early deglacial reconstructions were routinely based on reconnaissance level surveys of the major trough basins24. The physical reconstructions have continually been improved as more marine data have been acquired. These improved stratigraphic frameworks are important because they highlight the locations from which a chronology of ice-sheet and ice-shelf retreat from the outer continental shelves can best be obtained.During the Last Glacial Maximum (LGM), i.e., from 24,000 to 19,000 years ago, mass balance had shifted to positive and the extent of ice flow from East and West Antarctica expanded towards the Antarctic continental shelf edge Fig. 21–26. BrISs Fig. . In RossISs Fig. . The sub2) multibeam swath bathymetry survey, three regional dip-oriented seismic reflection profiles from the center of the WDB and a transect of strategically-located core during expedition NBP1502B  had advanced to the continental shelf edge as evidence by megascale glacial lineations (MSGLs). A backstepping succession of seven GZWs that partly bury the earlier-formed MSGLs marks the initial retreat of the groundling line from the continental shelf edge. This overlapping stack (GZW1 through 7) indicates that the grounding line paused multiple times as the ice stream vacated the WDB  record the time-transgressive retreat of both grounded and floating ice. The correlation of the geophysical and geological data permitted a much more detailed reconstruction of WAIS retreat18  and a large ice shelf reformed over the middle continental shelf , which were almost barren of calcareous foraminifera. In contrast, in all underlying sediments on the outer continental shelf, the total foraminiferal abundances were variable but strongly dominated by calcareous foraminifera, reaching in some intervals up to 40 specimens per gram of dry sediment. Taxonomic composition, although limited to less than twenty-five species in total, was variable22. The calcareous assemblage was always dominated by Globocassidulina and/or Trifarina, accompanied by Cibicides, Ehrenbergina, Astrononion, Epistominella, and other benthic genera, in addition to the planktonic Neogloboquadrina pachyderma sinistral. Preservation of calcareous foraminifera was variable but mostly ranged from fair to pristine. Some broken or etched tests were encountered but well-preserved, complete, transparent tests with no sediment infill were dominant in the most fossiliferous intervals. Some examples of pristine preservation with intact fragile spines, no signs of wall dissolution, and no secondary filling of very fine pores were present  were collected from all kasten cores (KC) at intervals of 5 to 10 cm for foraminiferal analyses directly after onboard visual core description. Care was taken to not include abrupt facies transitions in a single sample. The jumbo piston cores (JPCs) were cut and sampled using a pair of 50 cm3 sediment plugs in July 2015 after their arrival at the Antarctic Marine Geology Research Facility in Tallahassee, Florida. Sediment samples were collected every 10-cm using 2 cm thick core intervals. Samples directly below and above each facies transition were processed using the methods outlined below. These samples did not always yield enough biogenic carbonate in pristine condition for radiocarbon analysis. In these instances, preliminary foraminiferal abundances from Majewski et al.22 were used as a reference for sample selection.Sediment samples  in vacuo. Glass ampoules of CO2 gas were sent to Woods Hole Oceanographic Institute to be analyzed for 14C on the National Ocean Sciences Accelerator Mass Spectrometer. One sample of G. biora was split into two equal masses and replicate ages were within 50 14C yr of one another. These data were calibrated to calendar year BP using MARINE13 calibration curve in CALIB 7.130 with a marine reservoir effect of 1,300 ± 100 yr  established for Antarctic marine carbonates31. All foraminiferal radiocarbon ages are in cal kyr BP as the median age ± the age range representing the 95% confidence interval as per radiocarbon reporting conventions32. All data generated or analyzed during this study are included in this published article.Foraminiferal samples were sent to the University of South Florida College of Marine Science to be converted to CO22. Where possible, we used monospecific samples comprised of benthic G. biora and T. earlandi (and in one instance planktonic N. pachyderma) that were the most numerous and fairly massive . The largest uncertainty, from the smallest sample mass, was 512 years. Eleven dates were obtained from sub-ice-shelf sediments that accumulated on the outer continental shelf prior to the ice-shelf collapse. Only two of the median calendar ages from the sub-ice-shelf unit on the outer continental shelf are out of stratigraphic order. Given the possible range of calibrated ages represented by the median dates, however, the ages are considered to be in good stratigraphic order. The oldest date from the base of sub-ice-shelf sediments (in KC9) is 14.7 ± 0.4 cal kyr BP. The youngest date from the top of the sub-ice-shelf sediments (in KC8) is 12.3 ± 0.3 cal kyr BP. To better constrain the range of timing for sub-ice-shelf sedimentation on the outer continental shelf, two samples from KC12, comprised of mixed benthic species G. biora and T. earlandi, were radiocarbon dated. These ages are 13.5 ± 0.2 cal kyr BP at 38 cm depth and 13.8 ± 0.3 cal kyr BP at 97 cm depth. These data essentially overlap with the monospecific age 14.0 ± 0.3 at 53 cm and therefore also represent viable radiocarbon ages for sub-ice-shelf sedimentation on the outer continental shelf prior to ice-shelf collapse. No radiocarbon dates were obtained for the ice-shelf-breakup unit. Only one radiocarbon date, 0.4 ± 0.2 cal kyr BP, was obtained from the upper part of the open-marine sediment on the outer continental shelf at KC03.We obtained a total of eighteen foraminiferal radiocarbon ages Table . SeventeG. biora) that were isolated from diamict sediments that accumulated on GZW7 foreset on the outer continental shelf. The ages come from three core sites that are separated by 50 km. In the one core with multiple dates, i.e., KC05, the calendar ages are in stratigraphic order. The age uncertainties range from 281 to 416 years. The calendar ages range from 12.4 ± 0.3 cal kyr BP to 11.5 ± 0.3 cal kyr BP. No radiocarbon dates were obtained for open-marine sediments that overly the GZW7 diamict because those sediments contained only a few calcareous foraminifera. The new radiocarbon dates  to progradational/aggradational  suggest that the grounding-line position on the WDB outer continental shelf was maintained for eight centuries after the ice shelf collapse. Fully considering the calibration-induced age ranges, the grounding-line position persisted for a minimum of 200 years and a maximum of 1400 years after ice shelf breakup. In a geologic context, even our maximum estimate could be considered an instantaneous response. As concerns human-relevant time scales, an important point here is that the ongoing changes to ice shelves may trigger complex dynamics whose consequences are realized only after a significant lag. The land-based glaciers that were formerly fronted by the Larsen Ice Shelf B experienced immediate acceleration following the ice-shelf collapse in 2002 however, there has of yet been no significant retreat in the grounding line. In the case of the WDB, a centuries-long lag necessitates sediment aggradation from a high sediment flux. Sediment flux to the grounding line depends on several factors including the ice-sheet drainage area46 but there is no sedimentologic evidence that a second catastrophic breakup of the reformed ice shelf that covered the middle continental shelf. Grounded ice eventually retreated past Roosevelt Island at 3.2 kyr BP47.In the WDB, the subsequent 200-km retreat of the grounded line toward Roosevelt Island constituted an abrupt additional 15% contraction in the extent of grounded ice. A large-area ice shelf reformed over the middle continental shelf Fig. . Compounet al., in review). This is less than, but a significant portion of, the minimum amount of lag between ice shelf breakup and grounding line retreat that our foraminifera dates constrain. Given that the reservoir age of warmer water masses  is related to far field processes that create it in the north Atlantic as well as local processes that pull it onto the continental shelf, it is difficult to postulate the potential for a large difference in reservoir age during deglaciation compared to what is observed today. Indeed, there is evidence of a slowdown of North Atlantic Deep Water formation, which would potentially have increased reservoir ages during the LGM48. However, the overall properties of Southern Ocean water masses also involve more local processes related to the Antarctic Circumpolar Current and shelf-slope exchange. These are less well constrained, limiting how much we can forecast differences in the reservoir age of subsurface, potentially warm water masses that could have driven the ice dynamics preserved in WDB sediments.Thus far, all consideration of uncertainty of the timing of paleo-BIS deglaciation has incorporated analytical uncertainty  and calibration uncertainty (larger). Certainly, the sub-ice-shelf melting, if comparable to today, could possibly have been driven at least in part by a water mass with an older age (greater reservoir age). Deep-sea stylasterid hydrocorals record radiocarbon age differences during Circumpolar Deep Water (CDW) incursion of about 100–200 years  all predisposed the paleo-BIS to its subsequent rapid major (200 km) retreat to Roosevelt Island at 11.5 ± 0.3 cal kyr BP (Fig. 50.The only previous study of the WAIS retreat from the eastern Ross Sea outer continental shelf relied on bulk acid-insoluble organic matter isolated from deglacial sediment BP Fig. . The neg55. The WDB ice-shelf collapse may have been synchronous with an increase in iceberg rafted debris flux in Scotia Sea referred to as AID2 by Weber et al.53. Their composite records of ice-rafted debris from the Scotia Sea permits the possibility that either pulsed ice-shelf collapse, subsequent accelerated ice-stream discharge or abrupt grounding-line retreat may have ultimately caused the increased iceberg rafting fluxes53.A review of the literature on Antarctic margins indicates that many ice streams experienced backstepping retreats of floating and grounded ice, but, given uncertainties in calibrations for different dating strategies, it is not yet possible to definitely say whether the WDB ice-shelf collapse and grounding-line shifts were synchronous with ice-sheet changes recorded elsewhere57. The new detailed reconstruction from the WDB shows that a paleo ice-shelf collapse triggered complex dynamics whose consequences were not fully realized until after a lag of at least two centuries. Even in isolation, the ice volumes returned to the global ocean from the WDB in the time elapsed since the paleo-ice-shelf collapse at 12.3 ± 0.2 cal kyr BP and subsequent 200-km grounding-line retreat at 11.5 ± 0.3 cal kyr BP were relevant to coastal settings in terms of the resulting sea-level rise and coastal transgressions it would have produced. As concerns the potential for future ice-shelf collapse in the present-day interglacial, it is worth noting that many ice-stream systems are now grounded on the foredeepened inner continental shelves that is presumably underlain by either basement rock or other less erodible substrate. Hence, a transition to unbuttressed flow of those modern ice streams might be less able to maintain a grounding position by accelerated deposition of sediment within the grounding zone.Calving-front and grounding-line shifts are significant measures of climate change. The geological perspective provided by these results are important to climate models that seek to predict dynamic grounding-line and calving response to either external and/or internal forcing