Garcinia indica and Garcinia cambogia. Recent studies have proven that garcinol has anti-inflammatory, anti-cancer, and anti-oxidant activities. The objective of this study was to evaluate the inhibitory effects of garcinol on the activities of the drug metabolizing cytochrome P450 (CYP) isozymes to predict potential herb-drug interactions with co-administered drugs. Garcinol was incubated with a mixture of rat liver microsomes and eight CYP probe substrate cocktail under optimized incubation conditions and the samples were analyzed using a validated method on LC-MS/MS. Garcinol showed strong inhibition with IC50 values of CYP1A2 (7.6 µM), CYP2C9 (8.0 µM), CYP2B6 (2.1 µM), CYP2D6 (9.5 µM), and CYP3A4 (5.1 µM), respectively, and moderate inhibition towards CYP2C19 (16.4 µM) and CYP2E1 (19.0 µM). Molecular docking studies were performed on garcinol against the active sites of CYP2B6 and CYP3A4 proteins. These results further confirmed that the inhibitory activity of garcinol occurred by occupying the active sites of these human CYPs and by making favorable interactions with its key residues. In-vivo CYP inhibition studies were carried out in Sprague-Dawley rats. These results suggest garcinol may cause herb-drug interactions, mediated by inhibition of CYPs involved in drug metabolism in-vivo by altering the pharmacokinetic parameters like AUC and Cmax in a clinically significant manner. Garcinol was found to upregulate the expression and activity of P-gp in western blotting study and P-gp inhibition study in-vivo. These findings give a clear understanding to predict potential herb-drug/drug-drug interactions of garcinol for safe clinical use in future.Garcinol is an active constituent of  Garcinia cambogia  and Garcinia indica (kokum) are small tropical trees grown in Africa, Asia (western ghats of India), and Pacific regions + ions were identified and quantified for Digoxin and IS as 781.4369 and 515.2442, respectively. Optimized MS operating parameters were as follows: drying gas 10 L/min, gas temperature 325 °C, nebulizer at 40 psig, fragmentor voltage of 140 V and skimmer at 65 V.50 values were determined by comparing each CYP isozyme activity in the presence of different concentrations of inhibitor (Garcinol) with the control incubations containing no inhibitor (Negative control). A plot of the logarithm of inhibitor concentration versus percentage of the activity remaining after inhibition was drawn from which IC50 values were calculated by nonlinear least-square regression analysis using graph-pad prism software (ver. 4.03).IC0-∞) or the last measured time (AUClast) and terminal half-life (t1/2), the peak plasma concentration (Cmax) and time to reach peak plasma concentration (Tmax).Pharmacokinetic parameters were calculated from the plasma concentration versus time curves of bupropion, hydroxyl bupropion, midazolam, and hydroxy midazolam obtained from each individual rat using a non-compartmental analysis using WinNonlin software. The parameters calculated directly from experimental data were total area under the plasma-concentration-time curve from zero to infinity . The statistically significant differences of the results were analyzed using one-way ANOVA and are considered to be statistically significant at a level of 50 values of 7.6, 2.1, 8.0, 16.4, 9.5, 19.0, and 5.1 µM, respectively, and no apparent inhibition on CYP2C8 as shown in 50 values of garcinol and the well-known inhibitors used for the eight CYP isoforms  are reported in 50 of any of the CYP isoform was observed.Garcinol displayed strong inhibitory effects on CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 with ICMolecular docking and binding energy calculation results are summarized in Additionally, a π-cation interaction (arene-cation interaction) by 3, 4-dihydroxyphenyl group of garcinol formed with the Lys100. Furthermore, several hydrophobic interactions were observed between the compound and the active site residues that stabilized the binding of the garcinol in the active site of CYP2B6 e.g., Ile101, Pro106, Phe108, Tyr111, Phe115, Ala116, and Ile370. Additionally, our molecular modeling studies also predicted that garcinol has reasonable affinity for CYP3A4. The binding model of the garcinol and CYP3A4 protein exposed that the compound showing two hydrogen bonding interactions with the key residues of the active site of CYP3A4  and also having comparable hydrophobic interactions with Phe57, Phe108, Met114, Ile120, Leu210, Leu211, Phe213, Phe241, Ile300, Ile301, Phe304, Ala305, Ile369, Ala370, Met371, Leu482, and Leu483. From this modeling study we observed that garcinol was showing more affinity towards CYP2B6 and also observed reasonable affinity towards CYP3A4. These molecular modeling studies suggest that the inhibitory activity of garcinol was established by occupying the active sites of CYP2B6 and CYP3A4 and by making favorable interactions with its key residues.0-t and Cmax of metabolites after co-administration with garcinol were comparable to those without garcinol and there was a significant change in the pharmacokinetic parameters of substrates and their metabolites formed with or without the administration of garcinol, demonstrating that the pharmacokinetics of bupropion and midazolam were affected by the presence of garcinol, which has potential to inhibit CYP enzymes. Plasma concentration vs. time plots were drawn using graph pad prism and are shown in The plasma concentrations of metabolites hydroxy bupropion (CYP2B6) and 1-hydroxy midazolam (CYP3A4) in the absence and presence of garcinol were calculated and the pharmacokinetic parameters are shown in p < 0.05), as shown in Rat brain and intestinal tissue homogenates were used to evaluate the potential of garcinol to alter the expression of P-gp in-vivo. There was a significant difference in the expression in brain and intestinal tissues between two groups, as shown in Effect of garcinol on the pharmacokinetics of digoxin was investigated in rat plasma. The plasma concentration-time profiles of digoxin administered singly or along with garcinol are shown in Herbal medicine (HM) has been administered along with other drugs by many people without any awareness of interactions, which intensifies the occurrence of herb–drug interactions, especially in combination therapies. Though HM is naturally-occurring, the complex nature of various phytomolecules may interact with conventional drugs. The main interactions are related to the transport and metabolism of one drug in the presence of other drugs. CYP enzymes are involved in the metabolism of most drugs and P-gp is the efflux transporter present in the brain, intestine, kidney, and liver . These e50 values 7.6, 2.1, 8.0, 16.4, 9.5, 19, and 5.1 µM, respectively and CYP2C8 was negligibly inhibited, as mentioned in results. Garcinol also exhibited inhibition of CYP2B6 and CYP3A4 in the rat plasma after oral administration. However, in a pre-incubation study using rat liver microsomes and NADPH, garcinol did not show shift of inhibition against tested CYP isoforms. In order, to assess the binding of garcinol towards different CYP isoforms, we performed molecular docking studies of garcinol and the respective known inhibitors at the active pocket of different CYP isoforms. We observed that garcinol was well lodged at the binding site of CYP3A4 and CYP2B6 with the binding energy of −34.634 and −32.432 Kcal/mol, respectively. This further confirmed the inhibitory potential of garcinol.The CYP inhibition potential of garcinol on eight CYP isoforms was evaluated by cocktail method. We observed that garcinol showed inhibition towards CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 with ICIn addition to the CYP inhibition studies, down-regulation and inhibition potential of garcinol towards P-gp transporter was evaluated. From western blotting study, it was observed that garcinol increased the expression of P-gp in brain and intestinal tissues. Effects of garcinol on the pharmacokinetics of P-gp substrate was investigated in-vivo with and without co- administration of garcinol. Digoxin, a P-gp substrate, was administered to rats orally. Intragastric co-administration of garcinol resulted in increase in the AUC of digoxin. In addition to showing direct inhibition or induction of P-gp, many P-gp modifiers also alters the expression of P-gp . In thisThe results of this study depict the CYP inhibition potential of garcinol, thus admonishing co-administration of CYP substrates with garcinol for various therapies. Also, garcinol did not inhibit P-gp, thus decreasing the bioavailability of P-gp substrates for its therapeutic activity. Further clinical studies are required to fully assess the safety of garcinol in terms of CYP2B6 and CYP3A4 inhibition.


This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley  TANK-binding kinase 1 (TBK1), a serine/threonine kinase that belongs to the noncanonical IκB kinase family, plays an essential role in the innate immune response to viral and bacterial pathogens by regulating the type I interferon–mediated T cell response . TBK1 coin vitro kinase assays, the ability of TBK1 to mediate these phosphorylation events under physiological conditions was not known  were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and Akt-T308 phosphorylation . The intMtorA/A) were stimulated with EGF. A marked reduction of Akt-S473 phosphorylation was observed in MtorA/A MEFs compared with WT MEFs (Mtor+/+). Using immunoprecipitation of Rictor to isolate the mTORC2 complex, TBK1 was observed to interact with mTORC2 and directly phosphorylate mTOR-S2159 to activate mTORC2 intrinsic kinase activity toward Akt-S473. TBK1 activity is increased by phosphorylation of S172 in its activation loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al. (MtorA/A and Mtor+/+ mice with poly(I:C). Spleen tissue isolated from MtorA/A mice showed diminished Akt-S473 phosphorylation. Therefore, the authors conclude that under both basal and activated states, the activation of Akt by TBK1 is mediated through mTORC2  and mTORC2, which are distinguished by the scaffolding proteins Raptor and Rictor, respectively . mTORC1 y et al.  found thh mTORC2  6).Figu.FiguMtoret al. . TheThe mechanism by which TBK1 regulates mTORC2 function has not been established. Although the kinase activity of TBK1 is required for Akt-S473 phosphorylation, neither phosphorylation of S172 in the activation loop of TBK1 nor phosphorylation of mTOR-S2159 was increased by growth factor stimulation in this study. Phosphorylation of S172 stabilizes the active confirmation of TBK1 and it is possible that additional uncharacterized phosphorylation sites could serve a similar function. Alternatively, the interaction of TBK1 with mTORC2 could impact TBK1 conformation, or multimerization, to enhance activity. Intracellular localization of mTORC2 could also be determined by TBK1 interaction, which could affect substrate availability. As little is known about the upstream regulation of mTORC2, the next acts should be elucidating further the mechanism of its activation by TBK1 to reveal novel approaches for targeting the mTORC2-Akt signaling pathway.The author declares that she has no conflicts of interest with the contents of this article.


Left atrial appendage (LAA) thrombus in patients with atrial fibrillation is usually detected by transesophageal echocardiography (TEE). Intracardiac echocardiography (ICE) can be a suitable alternative to detect thrombosis. However, the effectiveness of the two methods for detecting LAA thrombus is still unclear, we performed a meta‐analysis that compared ICE versus TEE for LAA thrombosis.We searched PubMed, Cochrane Library, and Embase for published abstracts and manuscripts on June 1, 2020. The analysis was performed using RevMan 5.3, STATA 15, and Meta‐Disc 1.4.p > .05).Eight studies consists of 1108 patients (TEE = 558 vs. ICE = 550) were included. The average sensitivity of ICE and TEE to diagnose LAA thrombus is 1.0 (95% CI: 0.91–1.00) versus 0.68 (95% CI: 0.49–0.83), and specificity of ICE and TEE to diagnosis of LAA thrombus is 1.0 (95% CI: 0.99–1.00) versus 0.98 (95% CI: 0.96–0.99). The AUC of ICE and TEE is 0.9846 (SEAUC = 0.0196) and 0.9655 (SEAUC = 0.0401), and the Q* statistics is 0.9462 (SEQ* = 0.0406) and 0.9127 (SEQ * = 0.0616), respectively. Z test was performed on Q* statistics (Z = 0.45, The ICE and TEE have similar diagnostic efficacy for LAA thrombosis, but the ICE has higher sensitivity. Compared with TEE, ICE may be more advantages and prospects for clinical application. The proportion of AF is as high as 8% over 75 years of age.Intracardiac echocardiography (ICE) has been increasingly applied to probe the structure of the left atrium and LAA. Currently, the two methods are unclear about the real events of detecting LAA thrombus. Therefore, we performed a meta‐analysis.22.1After searching PubMed, Cochrane Library, and Embase by using keywords: atrial fibrillation, transesophageal echocardiography, Intracardiac echocardiography, and thrombosis from their inception on June 1, 2020. we found that studies only be published in English language.2.2The eligibility criteria for our meta‐analysis including: (a) Studies are prospectively or retrospectively including TEE and ICE. (b) The clinical result is the gold standard which results in an uneventful AF ablation. (c) The studies had to provide sufficient information to construct the 2 × 2 contingency table, that is, false and true positives and negatives were provided. If not directly given, get it from the corresponding author via email. (d) TEE and ICE inspection interval is less than 48 h and the definition of thrombosis consistent or similar, and the anticoagulation standard is consistent.Exclusion criteria: (a) Data cannot be accurately extracted, and it cannot be obtained from the corresponding author. (b) Animal experiments and review literature. (c) Articles with undetectable thrombus.2.3Two independent reviewers screened the documents according to the pre‐established inclusion and exclusion criteria and including the documents according to the QUADAS‐2  evaluation criteria.2.4Using Q test to detect whether there is heterogeneity, and using I2 to estimate the size of the heterogeneity, and then selecting the appropriate statistical analysis model for subsequent meta‐analysisρ of true positive rate and false‐positive rate, and analyzing whether there is an threshold effect. Once Q statistic does not prove the heterogeneity is not necessary to look for the Spearman correlation coefficient. Deeks linear regression will be used to assess whether the included studies had publication bias. The statistical software for this article is Review Manager 5.3, STATA 15, and Meta‐Disc 1.4, p < .05 is considered statistically significant.We tabulated true positives, false negatives, false positives, and true negatives in patients with LAA thrombus in ICE and TEE. The random‐effects model was used to calculate the average sensitivity, specificity, likelihood ratio, and 95% confidence interval (CI) of TEE and ICE respectively. The Mose's constant linear model was used to fit the SROC curve, and the diagnostic odds ratio (DOR), the area under the curve (AUC), and Q * statistics were used to evaluate the accuracy of the diagnostic tests ICE versus TEE in the diagnosis of LAA thrombus.33.1A total of 368 articles were found, 336 articles were excluded from reading titles and abstracts, and 30 articles were initially included Figure . After f3.2We have applied DOR as the effect size to analyze the heterogeneity of ICE and TEE, respectively. The Q test proves that Cochran‐Q is 1.75 and 6.15 respectively. That means heterogeneity between studies is small.The sensitivity and specificity of the forest plots are shown in Figure p > .05), and there is no statistical difference between ICE and TEE.The SROC curve of ICE and TEE to diagnose LAA thrombosis is shown in Figure . The AUC3.3Sensitivity analysis is performed by reducing one article at a time to assess the impact of a study on the meta‐analysis. There is no difference in results after excluding each article.3.4p < .05, and we found publication bias. The Deeks linear regression of TEE shows p > .05, and no bias was found. ICE publication offset may comes from the search scope which is limited to the published research, and the search for the unpublished research is not performed. .We used Deeks to evaluate publication bias for included studies, as shown in Figure . ICE's D4ICE and TEE mainly evaluating LAA through the following methods: (a) measurement of LAA length, width, and cross‐sectional area; (b) evaluation for thrombus; (c) evaluation for spontaneous echo contrast (SEC), Spontaneous echo contrast (SEC) indicates blood stasis in cardiac chambers and major vessels, and is a known precursor of thrombus formation. TEE plays a pivotal role in detecting and grading SEC in the left atrial (LA) cavity. Assessing LA SEC can identify patients at increased risk for thromboembolic events; (d) measurement of ejection velocities of the appendages by pulsed‐wave Doppler. When be diagnosed as thrombus, TEE and ICE measured similarly low pulsed‐wave Doppler velocities of the LAA (≤20 cm/s). ICE detected a moderate or greater degree of SEC, and had an appendage ejection Doppler velocity ≤20 cm/s as measured by TEE.p < .0001).ICE and TEE have their advantages and limitations. TEE would cause more discomfort for patients, requiring fasting and drinking, and damage to the esophagus. Maltagliati et al. found that TEE has artifacts in LAA, Artifacts were identified in 11 controls (37%); no thrombi were detected during surgical left appendage inspection in these cases. Therefore, TEE could lead to false positive diagnosis of LAA thrombus.p > .05), and there is no statistical difference between ICE and TEE. It shows that ICE is not significantly better than TEE for LAA thrombus identification ability. The main reasons that why we thought ICE is better than TEE are: (a) TEE probe obviously can be positioned only in the esophagus, whereas the ICE probe can be placed in various sites inside the cardiac chambers, and more conducive to understanding the real situation of LAA thrombus and structure. (b) TEE requires good cooperation from the patients to better observe LAA, and poor synergism may leads to negative results. (c) ICE can reach the left atrium, which is more conducive to find the thrombus hidden between the pectinate muscles. (d) ICE can reduce the number of contrast agents and radiation. The heterogeneity among the studies included in this article is relatively small.This article conducted a meta‐analysis of the eight included studies, compared with the diagnostic efficacy of ICE and TEE for LAA thrombus by combining diagnostic effect amounts and SROC curves. Finally, the credibility of this meta‐analysis was evaluated by sensitivity analysis and test publication bias. The results be combined with DOR of ICE and TEE were 872.70 and 89.46, respectively, which suggested that both of them have a significant correlation with LAA thrombus, and the correlation of ICE is higher. The SROC curve shows that the AUC of ICE and TEE are 0.9846 (SEAUC = 0.0196) and 0.9655 (SEAUC = 0.0401), and the Q* statistics are 0.9462 (SEQ* = 0.0406) and 0.9127 (SEQ* = 0.0616), respectively. Z test was performed on Q* statistics (Z = 0.45, 5The number of cases reported in the relevant literature retrieved literature is not large enough, and more randomized controlled trials are needed to verify the reliability of the results. The incidence of LAA thrombotic events is low, which may impacts the interpretability of the meta‐analysis and its ability to detecting differences. Four studies were performed in a retrospective fashion, which might become a limitation of this meta‐analysis. The retrieved literature is not comprehensive. The search scope is limited to the published research, some gray documents in other language studies may be missed. TEE is usually performed prior to ICE. Therefore, in these studies, the ICE operators were blinded to the TEE results. Maybe the 1.0 sensitivity is because the ICE reader already had the TEE results.6In summary, ICE and TEE have similar diagnostic efficacy on LAA thrombosis, but ICE has higher sensitivity. TEE is a choice for those with financial difficulties. ICE may be more appropriate for patients who require transseptal puncture, which has advantages over TEE and has clinical application prospects.The authors declare no conflicts of interest.Appendix S1: Supporting information.Click here for additional data file.


SPAST, which encodes SPASTIN, a microtubule-severing protein, are considered the most common cause of hereditary spastic paraparesis (HSP). In some cases, patients with HSP also manifest cognitive impairment. In addition, mice with Spastin depletion exhibit working and associative memory deficits and reduced AMPAR levels. However, the exact effect and molecular mechanism of Spastin on AMPARs trafficking has remained unclear. Here, we report that Spastin interacts with AMPAR, and phosphorylation of Spastin enhances its interaction with AMPAR subunit GluA2. Further study shows that phosphorylation of Spastin can increase AMPAR GluA2 surface expression and the amplitude and frequency of miniature excitatory synaptic currents (mEPSC) in cultured hippocampal neurons. Moreover, phosphorylation of Spastin at Ser210 is crucial for GluA2 surface expression. Phosphorylation of Spastin K353A, which obliterates microtubule-severing activity, also promotes AMPAR GluA2 subunit trafficking to the surface and increases the amplitude and frequency of mEPSCs in cultured neurons. Taken together, our data demonstrate that Spastin phosphorylation promotes the surface delivery of the AMPAR GluA2 subunit independent of microtubule dynamics.Synaptic plasticity is essential for cognitive functions such as learning and memory. One of the mechanisms involved in synaptic plasticity is the dynamic delivery of AMPA receptors (AMPARs) in and out of synapses. Mutations of  AMPA-type glutamate receptors (AMPARs) are heterotetrameric assemblies of four highly homologous subunits, GluA1–4, that are highly enriched at synapses  is a heterogeneous group of genetic neurodegenerative disorders, characterized by distinct lower limb spasticity and weakness , however, later studies reported that patients with SPG4-HSP also exhibit cognitive impairment  rats purchased from the Experimental Animal Center of Sun Yat-sen University. We conducted the animal experiments in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals produced by the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at Jinan University, China. All efforts were taken to minimize the suffering and the number of animals used.Green fluorescent protein (GFP)-Spastin, mCherry-Spastin, and Glutathione-S-transferase (GST)-Spastin constructs were described previously , different constructs were transfected into neurons using the calcium-phosphate method. All experiments were performed after transfection for 48 h.Standard hippocampal neuron culture and transfection were performed as described in our previous study . After transfection for 48 h, COS1 cells were fixed for performing fluorescence immunostaining. HEK293T cells were harvested for performing immunoprecipitation.COS1 cells and HEK293T cells were cultured in a 5% CO E. coli (Invitrogen). The GST-fusion proteins expression was performed as described previously : 147 KCl, 5 Na2-phosphocreatine, 2 EGTA, 10 HEPES, 2 MgATP and 0.3 Na2GTP. Recordings were performed in voltage clamp mode, at a holding potential of −70 mV, using a Multiclamp 700 B amplifier  and Clampex 10.5 software . Series resistance below 30 MΩ was monitored during recordings. Signals were sampled at 10 kHz, filtered at 1 kHz and MiniAnalysis software was used for analyzing signals .After culturing hippocampal neurons for 13 DIV, they were transfected with GFP, GFP-Spastin, and other constructs. After transfection for 48 h, electrophysiology assays were performed as per a previously described method . All data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s To investigate the relationships between Spastin and AMPAR, we first constructed GST-Spastin plasmid and purified the protein . Then GSvia immunofluorescence staining of nonpermeabilized neurons with anti-GluA2 antibodies. As shown in To further clarify the effects of the interaction between Spastin and GluA2 on AMPAR levels, we monitored surface AMPAR levels Does the increased surface expression of AMPAR reflect in synaptic function? To determine whether the synaptic functional level of neurons overexpressing Spastin and its phosphorylation were changed, whole-cell patch clamp recordings were performed to measure mEPSCs levels in target neurons. As shown in After clarifying the relationship between Spastin phosphorylation and AMPAR transport, we asked which of the four phosphorylation sites played an important role during this process. To answer this question, we first generated a series of mutants with Ser210, Ser233, Thr271, and Ser562 replaced with alanine  or aspartic acid  to mimic dephosphorylated or phosphorylated Spastin, respectively. Next, we examined the level of GluA2 surface expression in HEK293T cells stably expressing the GluA2 subunit of AMPAR. HEK293T cells stably expressing the GluA subunits make it easier for us to observe the effects of AMPAR binding proteins on AMPAR trafficking  were generated. Then, immunofluorescence staining and electrophysiology assessments were performed to explore the effect of Spastin S210 phosphorylation on the trafficking of AMPAR after Spastin K353A, Spastin K353A/S210A, and Spastin K353A/S210D were transfected into the hippocampal neurons. As shown in The present study demonstrated that Spastin interacted with AMPAR and phosphorylation of Spastin enhanced its interaction with AMPAR subunit GluA2. Further immunostaining and electrophysiology experiments showed that phosphorylation of Spastin increased the surface expression of AMPAR GluA2 subunits and synaptic function. Additionally, our study further clarified that it was the Ser210 site phosphorylation of Spastin that contributed to the surface delivery of AMPAR and this process was not dependent on microtubule dynamics.First, our results provide evidence that Spastin acts as a regulatory protein on postsynaptic AMPA receptors. In our previous study, we reported that Spastin promotes dendrite outgrowth  and dephosphomimetic mutants (Spastin QmA) of Spastin. After clarifying the role of Spastin phosphorylation in AMPAR transportation, we generated a series of mutants, with Ser210, Ser233, Thr271, and Ser562 replaced with alanine  or aspartic acid , respectively, to mimic dephosphorylated or phosphorylated Spastin and determine which site played a key role in this process. We found that like the “quadruple phosphomutants,” the “single phosphomutants” at S210 of Spastin could promote the GluA2 surface expression and the amplitude and frequency of mEPSCs. Other phosphorylation sites of Spastin did not show any effect. Thus, S210 phosphorylation appears to be crucial for Spastin in AMPAR trafficking. Simultaneously, S210 phosphorylation also has been reported to play a role in neurite outgrowth and branching (Li et al., Finally, our study clarified whether microtubule motility was involved in Spastin phosphorylation-mediated AMPAR trafficking. Previous studies have revealed that loss of Spastin can cause abnormalities in the stability of the neuronal microtubule cytoskeleton and lead to synaptic growth and neurotransmission defects (Trotta et al., SPAST are the most common cause of HSP (Kara et al., in vivo. In addition, since the upstream kinase of Spastin S210 phosphorylation is unknown, we relied on overexpression of various constructs in primary hippocampal neurons. Pharmacological manipulations will shed light on this physiological process once the upstream kinases are identified. In addition, our previous studies have confirmed spastin phosphorylation at site S210 in rat brain tissue, although it is unclear whether the Spastin S210 phosphorylation level is reduced in the brain tissue of patients with SPG4-HSP. Nevertheless, Spastin phosphorylation is beneficial for enhancing AMPA receptor function, which may be a potential target for improving cognitive dysfunction in patients with SPG4-HSP in the future.Mutations in In summary, this study reveals a novel role of Spastin on AMPAR trafficking. It provides the first evidence that Spastin interacts with AMPAR and demonstrated that their phosphorylation-dependent interactions rather than microtubule dynamics are required for GluA2 surface delivery. Finally, we also verified that it is Ser210 site phosphorylation of Spastin that contributed to GluA2 AMPAR trafficking. Our study provides important evidence on the role of Spastin phosphorylation in AMPAR trafficking, which will advance our understanding of cognitive dysfunction in HSP.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Jinan University.JifZ and GG conceived and designed the study. JifZ wrote the article. LC, HW, and SC performed most of the experiments and analyzed the data. JL, JiaZ, and JW helped with pulldown and Co-IP experiments. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This observation allowed us to create inhibitor–substrate fusions that achieved inhibition at low nanomolar concentration. We conclude that inhibitor–substrate cooperativity can be exploited for the design of fibril growth inhibitors.In amyloid fibril elongation, soluble growth substrate binds to the fibril-end and converts into the fibril conformation. This process is targeted by inhibitors that block fibril-ends. Here, we investigated how the elongation of α-synuclein (αS) fibrils, which are associated with Parkinson's disease and other synucleinopathies, is inhibited by αS variants with a preformed hairpin in the critical N-terminal region comprising residues 36–57. The inhibitory efficiency is strongly dependent on the specific position of the hairpin. We find that the inhibitor and substrate concentration dependencies can be analyzed with models of competitive enzyme inhibition. Remarkably, the growth substrate,  Amyloid fibril elongation of α-synuclein can be described with the Michaelis–Menten model, where α-synuclein monomer plays a dual role by serving as growth substrate as well as supporting the competitive inhibitor CC48 in blocking fibril ends. The amyloid state is thought to be a generic state that all proteins can adopt and is associated with several diseases, especially neurodegenerative ones.2–5A growing number of proteins have been shown to undergo an autocatalytic aggregation reaction where soluble polypeptide chains convert to insoluble 1D quasi-crystals exhibiting cross-β conformation.6–9In this paper we focus on the protein α-synuclein (αS) which is believed to play a central role in the pathology of Parkinson's Disease (PD). In PD, αS is found in insoluble inclusions, termed Lewy bodies, where it is thought to predominantly inhabit the amyloid state.10–13 This complexity can make interpretation of experimental data exceedingly complicated.10,14 However, all of these reaction steps are amenable to modulation by ligands, affording a range of therapeutic opportunities that target different sites on distinct species on the aggregation pathway.15 Importantly, molecules that are able to interact with specific sites/species can also provide insight into the mechanism of amyloid formation.16Amyloid fibril formation is a multi-step reaction that minimally includes primary nucleation and elongation, but commonly involves additional reactions including secondary nucleation, fragmentation, and competition from so-called off-pathway reactions.17 During elongation, a free αS monomer, which in its free state is intrinsically disordered,18 (i) absorbs onto the fibril-end and (ii) converts into the specific structure of the templating fibril.12 This is reminiscent of enzyme kinetics, and elongation can be treated as a two-step enzymatic reaction, in which fibril-end and monomer serve as catalyst and substrate, respectively.19–21Here we will focus on elongation of fibrils which is the most frequent process in amyloid formation. Elongation of αS fibrils can be studied in isolation using specific solution conditions.22–25e.g., by aiming to dock complementary β-strands onto the open β-sheets at the fibril-ends, to prevent the catalytic site from guiding the conformational conversion of further monomers. However, understanding how monomers and inhibitors get incorporated at fibril-ends is still a subject of active research.12 We have previously reported that a double cysteine αS mutant containing the amino acid exchanges G41C and V48C, here denoted CC48, inhibits the elongation of wild-type (WT) αS fibrils.26 This double exchange introduces an intramolecular disulfide bond that is important for inhibitory activity of CC48. The positions of the two cysteines were chosen to promote the formation of a β-hairpin motif in the region spanning residues 36–57, a region we previously observed to be in complex with an αS monomer-binding protein, the β-wrapin AS69 .34 As elongation is a bimolecular reaction, the initial rates, r, should be directly proportional to available fibril-ends and initial WT αS monomer concentration. The initial rates were extracted by fitting linear curves to the initial slopes as shown in We performed elongation assays by incubating 2.5 μM preformed and sonicated fibrils (seeds) with WT monomer. Our specific choice of elongation reaction conditions were tested by measuring the rate of elongation using Thioflavin T (Tht) fluorescence over time , as Tht Our first experimental goal was to gauge the dependency of the inhibitory activity of CC48 on the precise position of the disulfide bond. In addition to CC48, we therefore generated a set of double cysteine αS variants by systematically mutating residues 49 through 52 into cysteines while keeping the other end of the disulfide fixed at position 41 . All mutr, were extracted and divided by the initial rate when no CC48 was present r0  . Rationalisation of this observation has to take into consideration that αS fibrils consist of two protofilaments, in which αS subunits are staggered with respect to their neighbours in the other protofilament (schematically depicted in 35–40 Binding of CC48 to the fibril-end might alter the protofilament interface, disfavouring addition of another WT monomer. Once a WT monomer attaches to FI nonetheless, a structurally different binding site with high affinity for an additional WT monomer is created. While the kinetic data does not provide structural information on the different fibril-end complexes, it indicates that at least two WT monomers cooperate with the CC48 inhibitor to form a stabilized blocked state that is incompatible with fibril elongation.According to the obtained equilibrium constants, binding of WT monomer to FIM is much more favourable than to FI (The cooperation of CC48 with WT monomers in inhibition suggests that an improved inhibitor could be designed by combining CC48 and WT in fusion constructs. As formation of the FIM complex from FI and M was the least favoured step on the inhibition path, IM fusion constructs consisting of one CC48 and one WT unit might show increased inhibitory activity by bypassing this step. We recombinantly expressed dimeric constructs of WT and CC48 separated by flexible linkers as shown schematically in i.e., two orders of magnitude less than what was needed for CC48 alone. The heterodimeric constructs were also more inhibitory than the homodimeric ones, showing that it is in fact the particular combination of CC48 and WT that blocks fibril elongation most efficiently. The WT–WT dimer was almost as inhibitory as CC48 alone, a result that is in agreement with what has been observed for similar constructs.24,49 The CC48–CC48 dimer also exhibited strongly increased inhibition compared to CC48, which could be an avidity effect. As expected, the heterodimeric construct exhibited much less monomer dependency than what was observed for CC48 alone (i.e. CC48, now carries its own co-inhibitor, i.e. the WT, in the heterodimeric fusion constructs.In addition to two heterodimeric constructs WT–CC48 and CC48–WT that differ by the order of WT and CC48 with respect to the linker, we also constructed two homodimers WT–WT and CC48–CC48. The relative initial rates of elongation assays in the presence of these dimers are shown in 48 alone . This is50 of 11 ± 1 nM. This compares favourably to previously reported elongation inhibitors based on αS fusions. These inhibitors were based on different design principles, namely transport of steric bulk to the fibril-end or direct linkage of two αS subunits at different positions within the αS sequence, and reached IC50 values of 300 nM,23,50 or 22 nM.24 In one of these approaches, the function of a fused WT monomer is to serve as a fibril-end-binding domain that brings the fused inhibitor domain close to the second protofilament, with the inhibitor acting as steric bulk that impedes incorporation of further WT monomers.23,50 While this approach is related to the current study with regard to the fusion of a WT monomer domain to an inhibitor domain, there are crucial differences: First, CC48 forms an inhibiting FI complex without requiring fusion to a WT monomer. Second, WT monomer, i.e., the unmodified substrate of the elongation reaction, stabilizes the CC48-FI state without requiring fusion to an inhibitor domain. Third, the WT monomer concentration dependency of the steric bulk fusions is different from those of CC48 and the CC48–WT dimers,23 indicating a different mechanism of inhibition. Nevertheless, all these approaches show that modified versions of αS can block fibril-ends, with the potency determined by the nature of the fused proteins as well as the type of linkage.At a WT monomer concentration of 25 μM, the WT–CC48 fusion showed an IC51,52 which were subsequently also reported for several other amyloid proteins.53–56 These stop states were suggested to be due to docking of the WT monomer on the fibril-end in a templating-incompetent conformation.52,54,56 Thus, the inhibitory efficiency of CC48 might be an enhanced representation of a property that is already inherent to WT monomers. Possibly in a similar vein, certain types of post-translational modified αS might inhibit fibril elongation by establishing templating-incompetent fibril-ends, which could for example explain the inhibitory activity reported for dityrosine-modified αS.49Binding of CC48 to the fibril-end creates a templating-incompetent state with an efficiency that is highly dependent on the specific disulfide fusion . Can WT 47,48 Here, we showed that modification of αS by introduction of a hairpin in a critical N-terminal region results in an inhibitor of fibril elongation, whose efficiency is strongly dependent on the precise position of the hairpin. Our data demonstrates that the efficiency of such fibril-end blocking inhibitors may be dramatically enhanced by linkage to WT monomer, as WT monomer is capable of stabilizing the blocked fibril-end state. As a consequence of the catalytic nature of fibril formation,21 we find that inhibition of fibril elongation can be analysed along the lines of enzyme inhibition. However, the specific architecture of the fibril-end can lead to atypical inhibitor properties. We observed here that the substrate of the fibril elongation reaction can contribute to inhibition by stabilizing the enzyme–inhibitor complex.Exploitation of the principle of self-recognition has proven fruitful for the design of amyloid formation inhibitors.E. D. A. and W. H. designed the experiments. E. D. A., V. B., and M. M. W. performed the experiments. E. D. A., V. B., and W. H. analyzed the data. E. D. A. and W. H. wrote the manuscript. All authors commented on the manuscript.There are no conflicts to declare.SC-011-D0SC04051G-s001


GBM thickness was measured at 20 separate locations in the biopsy specimen and plasma levels of cystatin C and creatinine were retrieved from health records. A modified two‐pore model was used to simulate the effects of a thicker GBM on glomerular water and solute transport. The mean age of the patients was 52 years, and 38% were women. The mean eGFRcystatin C/eGFRcreatinine‐ratio was 74% in DKD compared to 98% in MCD (p < 0.001). Average GBM thickness was strongly inversely correlated to the eGFRcystatin C/eGFRcreatinine‐ratio . Two‐pore modeling predicted a eGFRcystatin C/eGFRcreatinine‐ratio of 78% in DKD. We provide clinical and theoretical evidence suggesting that thickening of the glomerular filter, increasing the diffusion length of cystatin C, lowers the eGFRcystatin C/eGFRcreatinine‐ratio in DKD.Diabetic kidney disease (DKD) is a leading cause of end‐stage renal disease and renal replacement therapy worldwide. A pathophysiological hallmark of DKD is glomerular basal membrane (GBM) thickening, whereas this feature is absent in minimal change disease (MCD). According to fundamental transport physiological principles, a thicker GBM will impede the diffusion of middle‐molecules such as cystatin C, potentially leading to a lower estimated GFR (eGFR) from cystatin C compared to that of creatinine. Here we test the hypothesis that thickening of the glomerular filter leads to an increased diffusion length, and lower clearance, of cystatin C. Twenty‐nine patients with a kidney biopsy diagnosis of either DKD ( cystatin C/eGFRcreatinine‐ratio and glomerular basement membrane thickness in diabetic kidney disease. The results imply that a reduced eGFRcystatin C/eGFRcreatinine‐ratio could be a possible biomarker for diabetic nephropathy. Diabetes and chronic kidney disease is rapidly increasing globally. Here we found a correlation between eGFR In fact, while most of the plasma content of cystatin C and other small plasma proteins are catabolized in the renal tubules  is defined as a difference in estimated GFR from cystatin C versus creatinine , and han Grubb, . Small pn Grubb, . Thus, wn Grubb,  which isA decreased pore size is not the only possible mechanism leading to SPS and/or reduced estimated GFR from cystatin C versus creatinine, but could potentially result also from a thicker glomerular barrier leading to an increased diffusion length for middle molecules like cystatin C. One of the earliest structural alterations in DKD, preceding microproteinuria, is thickening of the glomerular basal membrane (GBM) . We assessed 579 entries in the Örestad Registry between August 2013 and August 2017. Inclusion criteria were biopsy diagnosis of DKD or MCD and estimated GFR measurement using both cystatin C and creatinine within 2 weeks of the time of biopsy. All patients were assessed for medication including angiotensin‐converting enzyme inhibitors/angiotensin‐receptor blockers and corticosteroids. Exclusion criteria were signs of other pathologies in the biopsy specimen, for example, Ig A nephropathy or hypertensive nephrosclerosis. We identified 12 patients with MCD and 17 patients with DKD who fulfilled the inclusion criteria and none of the exclusion criteria. The Research Ethics Board at Lund University approved the study (Dnr 2017/568). Patient data and samples were treated anonymously in all statistical analyses.All patients referred for a kidney biopsy in Skåne, southern part of Sweden, are registered in a local data repository called the 2.2Plasma concentrations of creatinine and cystatin C were collected from health records. Cystatin C was determined by a particle‐enhanced immunoturbidimetric method using a reference material traceable to the international cystatin C calibrator  and the plasma protein concentration Cpr(y) as functions of the relative position y  along the glomerular capillary, can be determined by use of the conservation of mass equationsThree pathophysiological changes will affect glomerular solute and water transport in DKD: (1) GBM hypertrophy, (2) glomerular hypertension, and (3) hyperfiltration (single nephron). We simulated these changes by (1) lowering the filtration coefficient (LpA) and solute diffusion capacity , (2) increasing the glomerular hydraulic pressure gradient to achieve, and (3) a single nephron GFR (SNGFR) of 90 nL/min. If the glomerular filtration coefficient, hydraulic pressure gradient, SNGFR, and (afferent) oncotic pressure are known, then the single nephron plasma flow profile Re‐arranging these equations gives the well‐known  is the local filtration flux  multiplied by the ultrafiltration coefficient (LpA).Above, GC) was calculated using the Landis–Pappenheimer equation, (Π=2.1Cpr+0.16Cpr2+0.009Cpr3), assuming a total plasma protein concentration of Cpr(0) = 6.7 g/dl corresponding to a plasma colloid osmotic pressure of ~24 mmHg. In the human glomerulus, the capsular hydrostatic pressure (PBS) has been estimated to be between 15 and 20 mmHg  0 mmHg. In order to achieve an effective filtration pressure (EFP) in the normal state of ~10 mmHg, we assumed a glomerular hydraulic pressure gradient of 40 mmHg  of 55–60 mmHg. The pressure drop along the glomerular capillary network was set to 1 mmHg  of 3.7 nm and a large pore radius (rL) of 11.0 nm  due to glomerular filtration  is given byJs(y), is calculated fromCi(y) is the solute concentration at position y along the glomerular capillary; fS and fL are the fractional volume flows; WS and WL are the convective hindrance factors  are calculated fromActual measurements of glomerular sieving coefficients for small proteins like cystatin C across the human glomerular filter are scarce in the literature. Perhaps the most reliable estimations are those obtained in children with Fanconi syndrome , αS and αL denote the fractional hydraulic conductance of the small and large pore system, respectively , occurring chiefly via glomerular filtration:cysC is the glomerular sieving coefficient of cystatin C. A small pore system with a 3.7‐nm pore radius implies a sieving coefficient for cystatin C of ~0.87. For an adult male having a GFR of 120 ml/min, this translates to a cystatin C concentration of about 0.69 mg/L.Solute diffusion capacities PS2.4Core biopsies were first put in 4% formaldehyde and thereafter embedded in paraffin. Cases were sectioned in two levels, each 1.5 µm thick, and stained for hematoxylin and eosin staining, Alcian blue periodic acid‐schiff, Trichrome staining, elastin van Gieson staining, periodic Schiff‐methenamine silver, alkaline Congo red, and each case were re‐evaluated by light microscopy. Samples for electron microscopy (EM) were put in 4% formaldehyde and embedded in (plastic) and thereafter sectioned in 60 nm thick sections. Transmission EM was performed for each case with a transmission microscope (TECNAI FEJ Company) as well as photo documented in JPEG‐format by a digital camera (VELETA). One glomerulus per case with at least one full segment was examined with EM. It is standard in clinical practice to examine 1 or 2 glomeruli for EM while the rest is embedded in paraffin for light microscopy. Each case was evaluated regarding basal membranes, foot processes, and mesangium and were found to correspond to the diagnosis of diabetic nephropathy or minimal change nephropathy. Each case showed homogenous thickness of basal membranes of segments without any focal irregularities or deposits. For each glomeruli, 20 measurements points were taken in a representative loop. GBM thickness was assessed through ultrastructural morphometry on enlarged electron micrographs as the arithmetic mean of 20 orthogonal intercepts across the GBM. Electron micrographs were available in 8 patients with MCD and in 11 patients with DKD.2.5cystatin C/eGFRcreatinine‐ratio assuming a SD of 0.16 (Cohen's d = 1.25). Moreover, a sample size of 19 patients had 87% power to detect a true correlation coefficient of 0.6 or better between GBM‐thickness and eGFRcystatin C/eGFRcreatinine‐ratio, at a significance level of p < 0.05. A post‐hoc ANCOVA was applied using the model (ratio ~eGFR + diagnosis) using type III SS. We assumed an α‐level of 0.05 and a β‐level of 0.20 unless otherwise specified. p‐values are denoted: * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. All statistical calculations were performed using R for macOS .Values are presented as average (IQR) unless otherwise specified. Differences between groups were assessed using a Wilcoxon signed rank test. Monte Carlo‐based power analysis  was conducted, applying Wilcoxon tests between fixed unequal sample sizes of 17 and 12 patients, showing that the study had 89% power to detect a true difference of 0.2 in eGFR33.1n = 17) or MCD (n = 12). Baseline characteristics and eGFR estimations are shown in Table cystatin C/eGFRcreatinine which was lower  in DKD patients compared with MCD patients. Furthermore, four patients with DKD also fulfilled the criteria for SPS, defined as eGFRcystatin C <60% eGFRcreatinine whereas none of the MCD patients fulfilled this criteria (the lowest ratio in the MCD group was ~76%). Average estimated GFR (eGFR), calculated as the arithmetic average of eGFRcystatin C and eGFRcreatinine, was different in the groups, which is expected since GFR is usually much less affected in MCD .We first assessed estimated GFR (eGFR) from cystatin C and creatinine in two groups of patients with a biopsy diagnosis of either DKD n = 7 or MCD 3.2cystatin C/eGFRcreatinine‐ratio and GBM thickness (nm). The scatterplot  (p < 0.01). The slope coefficient for mean GBM‐thickness was −0.04% nm−1 so the eGFRcystatin C/eGFRcreatinine‐ratio was apparently reduced by ~4% for every 100 nm increased thickness. As shown in Figure cystatin C/eGFRcreatinine we used linear correlation between weight versus eGFRcystatin C/eGFRcreatinine‐ratio and body mass index (BMI) versus eGFRcystatin C/eGFRcreatinine‐ratio, but found no significant association.Linear regression was performed to investigate the theoretically predicted relationship between the eGFRt Figure  indeed s3.3cystatin C/eGFRcreatinine ratio. Glomerular hypertension and GBM hypertrophy evoked marked reductions in the clearance of cystatin C , glomerular hypertension is assumed necessary to maintain the same GFR and the EFP will increase linearly with the thickness of the barrier, thus for a 600 nm barrier (dashed line) the EFP is 20 mmHg. This thickening of the barrier will also double the diffusion distance for cystatin C, effectively lowering its fractional clearance to 0.74 (ClCC ≈89 ml/min/1.73 m2) for the 600 nm barrier and to 0.67 for the 900‐nm barrier (ClCC ≈80 ml/min/1.73 m2). Assuming a constant production of cystatin C, these latter scenarios will cause the serum concentration of cystatin C  to increase so that the estimated GFR using, for example, the CAPA equation . This had the effect of reducing the eGFR Figures  and 3. P3.4In Figure 4cystatin C/eGFRcreatinine and basal membrane thickness. According to theory, during conditions of glomerular vascular wall thickening, there should be a measurable difference in the eGFR measured using cystatin C and the actual GFR  under the assumption that their plasma concentrations are determined chiefly by glomerular filtration. In line with our present results, a decreased diffusional clearance of mid‐sized molecules has been found in experimental and clinical studies of DKD  but many of them are catabolized in other tissues and regulated via other mechanisms making it unclear what effect shrunken pores would have on their plasma concentrations. It is well established that GFR is an important determinant of plasma cardiac troponin T (cTnT) levels  in mmol/ml. From the equation above, it can be seen that the diffusion capacity represents the maximal possible absolute diffusive clearance of a solute, being approached as the concentration on one side of the barrier is approaching zero. Mathematically, PS is the product of the diffusion coefficient D and the effective surface area A of the membrane divided by the membrane thickness Δx (i.e. PS = D × A/Δx), which means that PS will be reduced if the thickness, or diffusion length, Δx, is increased. Due to their small size, solutes like creatinine, urea, glucose et cetera have a great capacity to be transported via diffusion over the GFB since they have a very large diffusion capacity. Yet, diffusion is a process that requires a concentration gradient; and since small solutes <1.0 nm are not sieved at all by the GFB Δc = 0 they will be transported entirely via convection and their clearance will equal GFR. For mid‐sized solutes 1.0–3.0 nm, there is a small but important concentration difference across the renal filter, leading to a considerable part of their clearance being due to diffusion due to the fact that the diffusion capacity is still very high (>> higher than GFR) for these solutes. With increasing solute sizes >3.0 nm the diffusion capacity rapidly drops to low values (<< lower than GFR), and the diffusive clearance becomes negligible.Here PS is the diffusion capacity in ml/min, 4.1A validated registry for renal biopsy in which GFR was estimated from both creatinine and cystatin C is a strength of this study. The ultrastructural morphometry of the thickness of the GBM was evaluated by an experienced pathologist on enlarged electron micrographs as the arithmetic mean of 20 orthogonal intercepts across the GBM.4.2cystatin C/eGFRcreatinine ratio and GBM thickness, were statistically significant. It would also be an advantage having more patients with early DKD. Sweden has a homogenous racial distribution which limits our conclusions to this population. We also used a very simplistic pore model for glomerular filtration during DKD, assuming that the hydraulic conductance (LpA) and solute diffusion capacities (PS) are inversely proportional to the thickness of the GBM. It is likely that the actual changes in these parameters are more complex. However, the lack of more accurate measurements of these parameters in patients with (or without) DKD, indicates that a more sophisticated approach is difficult to justify at the moment.This study has several limitations. A major limitation is the low number of patients in both groups. However, the differences between the two groups, regarding eGFRcystatin C/eGFRcreatinine.In conclusion DKD develops insidiously over several years, well before any clinical manifestations  are evident, and once microproteinuria is present, structural lesions are often considerably advanced. Caramori et al.  analyzedcystatin C/eGFRcreatinine may also be helpful in diagnosing other cases of CKD in early phases. In this context, it should be pointed out that kidney diseases may result in both thickening and thinning of the GBM.In light of our current findings, further research should explore the possibility that plasma levels of mid‐sized molecules like cystatin C could act as biomarkers for detecting DKD, allowing early treatment. The ratio eGFRAll the authors declare no conflict of interest.CAÖ and AC designed the study and collected data. CAÖ performed mathematical modeling. CAÖ and AC analyzed the data. CAÖ made the figures. ML analyzed kidney biopsies. CAÖ, AG, and AC drafted the manuscript. All authors contributed to, and approved the final version of the manuscript.


Sea ice is one of the main loads acting on a wind turbine tower in areas prone to icing, and this threatens safe working life of the wind turbine tower. In our study, a simplified calculated model of ice, wind turbine tower, and water dynamic interaction under earthquake action was proposed, which could avoid to solve a large number of nonlinear equations. Then, the seismic behaviour of the wind turbine tower with and without the influence of sea ice was investigated, and we found that the influence of the greater mass of the sea ice on the seismic response of a wind turbine tower should be considered when the wind turbine tower is designed in an area with thick ice. With the influence of the most unfavourable ice mass, the deformation and energy dissipation capacity of the wind turbine tower are decreased, and the wall thickness or stiffening rib thickness should be increased to improve the seismic performance and ductility of the wind turbine tower; the shear force and bending moment increased significantly on the wind turbine tower, and the shear force changes at the bottom of the wind turbine tower and position of action of the sea ice: attention should be paid to the wind turbine tower design at these positions. Finally, we conducted the shaking table test, and verified the rationality of our proposed simplified model. Code for design of high-rising structures (GB50135-2019) {x¨}Mjk is the mass at the action point of the sea ice on the tower, and the dynamic equilibrium equation for Mjk is as shown in Eq , maximum payload (5000 kg), and the dimensions of the shaking table (1.5 m×1.5 m). This experiment was designed to simulate the seismic dynamic interaction between the ice and structure to verify the accuracy of the simplified calculation model of water-sea ice–wind turbine tower affected by earthquake based on the added mass method, so a large water tank was fabricated and fixed onto the shaking table to hold water and ice. The dimensions of the water tank are 2.0 m (length)×1.3 m (width)×1.5 m (height). The height of the prototype structure is 80 m, and the upper structure mass is 265,585 kg. Subject to the size of the test equipment, test water boundary conditions are limited, and the similarity coefficient is λl = 1:50 to simulate the infinite boundary conditions pertaining to the water. The model height is 1.6 m, and the base of the model is welded to steel plate of the tank, as shown in The experiment was conducted on a shaking table simulation test system (ES-15) including a: shaking table controller, shaking table, and hydraulic pump. The main technical parameters of the shaking table test equipment include its rated working frequency (100 Hz), maximum acceleration . The compressive strength of the paraffin wax was measured by use of the small-specimen method, as shown in Eq :σcu=PfπThe compression of the sample was recorded at 0.2 kN increments, and the compressive strength of each sample was calculated according to the crushing load .The pressure and corresponding displacement were recorded every 0.2 kN and converted into stress and strain, as shown in In the initial stage of the compression test, the ice sample is in its elastic working stage, and the elastic modulus is as listed in As shown in To measure the dynamic response of the model, four acceleration sensors were placed along the model height, and two displacement sensors were placed at the top and bottom of the model . The diaThe Tianjin seismic wave and Kobe seismic wave were selected as the input load to simulate seismic action: the Tianjin wave is an artificial earthquake, and the Kobe seismic wave is an actual seismic record. At the same time, sinusoidal waves were used to study the effect of different frequencies of vibration on the model, and the test conditions with, and without, the influences of ice are summarised in The Tianjin seismic wave was recorded during the Tangshan earthquake in 1976 in China, and the Kobe seismic wave was recorded in the Kobe earthquake in 1995 in Japan. The parameters of the two seismic waves are listed in In the experiment, the dynamic characteristics of the model with, and without, the influence of ice was measured by hammer decay model test and sine-wave sweep test, respectively: first order vibration frequencies of the models were obtained .As shown in The acceleration and displacement of the model were calculated when the sinusoidal input frequencies were 1 Hz and 3 Hz are shown in Figs As shown in Figs To verify the accuracy of our proposed simplified calculation model, we compared the test results and the calculated results using the proposed simplified calculation model. First, we compared the natural frequencies .As shown in As shown in Figs Finally, we compared the calculated peak acceleration and peak displacement along the model height, and comparisons with test results are shown in Figs As shown in Figs In the present study we take a monopile wind turbine tower in ice and water as the research object. The wind turbine tower is location in an active seismic zone along the east side of the south yellow sea shelf and it is close to the coast of Jiangsu province in China. The mechanical parameters of the wind turbine tower are designed according to specifications, and site soil parameters and sea ice parameters are obtained according to field tests, so the calculation results can be generalized to other turbines.σy of the material is 345 MPa, its modulus of elasticity E is 2.06×1011 Pa and Poisson’s ratio γ is 0.3. The tower outer diameter is 3.07 m to 4.5 m, and the wall thickness is 18 mm to 50 mm. The compressive strength of sea ice was taken as 2.12 MPa. The mass of the ice is 900kg, the damping of the ice is 0.04, and the stiffness of the spring is 2.2Gpa. To adopt the proposed simplified calculation model, the masses of the structure were determined as shown in The sea water depth is 15 m, the tower height is 60 m, and the tower height above bedrock is 20 m. According to the investigation report, the ice is at this thickness 0.6m for a long time in the year. The monopile wind turbine tower is located in a Class-II site, and the seismic fortification intensity is 8 degrees. The tower body design adopts a variable-section and variable-wall-thickness cylindrical structure; the material of the tower and the tower above the ground is Q345C steel. The yield strength Standard Specification for Highway Bridges [E/100.The stress-strain relation of the steel adopts the bilinear model recommended in Japanese  Bridges , as showCode for Seismic Design of Buildings (GB 50011–2010) [The basic intensity of the region of the wind turbine is 8 degrees, and the site is II. According to the 11–2010)  of ChinaAccording to the Japanese code , near-fiThe soil deposits are obtained through the borehole in the site which was 40-meter depth and consisted of nine layers. Parameters of soil in this study are obtained by in-situ and laboratory tests, i.e., dynamic triaxial test and the parameters are presented in p-y curve method is used to simulate the pile-soil interaction. The horizontal displacement is one of the main indicators in wind turbine tower design [In our study, r design ; therefoIn aseismic design of wind turbine tower, the curvature of the tower was conveniently used in analysis of damage to the tower , so the 8 kg, the maximum curvature of the tower bottom under different water depths will exceed the ultimate curvature of the tower bottom section with the increase in the mass of sea ice, which means that the wind turbine is damaged. The sea ice mass, when the wind turbine tower is destroyed, decreases with increasing depth of water. For example, the curvature at the bottom of the wind turbine tower reaches a maximum when the sea ice mass is 5×107 kg in water depths of 5 m and 10 m, and the curvature at the bottom of the tower reaches a maximum when the sea ice mass is 1×107 kg in a water depth of 15 m, and the curvature at the bottom of the tower reaches the maximum when the sea ice mass is 5×106 kg in water with a depth of 20 m. Under T2-II-1 earthquake action, the maximum curvature of the tower bottom changed significantly as the mass of sea ice increased. The least favourable curvature at the tower bottom appears when the sea ice mass ranges from 1×107 kg to 5×107 kg. The sea ice mass corresponding to the least favourable curvature of the tower bottom increased with the depth of the water. For example, the curvature at the bottom of the tower reaches a maximum when the sea ice mass is 5×107 kg in water with a depth of 5 m, and the curvature at the bottom of the tower reaches a maximum when the sea ice mass is 1×107 kg in water depths of 10 m, 15 m, and 20 m. Consequently, the sea ice mass exerts a significant influence on the maximum curvature of the bottom of the tower, and the least favourable curvature of the tower bottom mainly appears in the range from 5×106 kg to 5×107 kg under different types of seismic wave action, implying that a 100-m ice-plate becomes consolidated around the tower of the wind turbine tower. The effect of sea ice on the maximum curvature of the tower bottom of wind turbine tower is different in different depths of water. The sea ice mass corresponding to the maximum curvature of the tower bottom decreased with increasing depth of water under near-field earthquake (T1-II-1) excitation.As shown in The thickness of the sea ice around the wind turbine tower changes with both season and temperature, so the ice thicknesses h are set to 0.2 m, 0.4 m, 0.6 m, 0.8 m, 1.0 m, 1.2 m, 1.4 m, and 1.6 m, and the water depth is set to 15 m. Changes of the maximum curvature at the tower bottom, as influenced by different ice thicknesses, are shown in 6 kg, and the maximum curvature began to change significantly with the further increase of the mass of sea ice under different seismic loads. Under T1-II-1 earthquake action, the maximum curvature is much larger than the ultimate curvature when the ice mass ranges from 0 kg to 1×108 kg. Under T2-II-1 earthquake action, the maximum curvature increases with the increase of ice thickness in addition to the ice thickness of 0.8 m. Therefore, the influence of the greater mass of the sea ice on the seismic response of a wind turbine tower should be considered when the wind turbine tower is designed in an area prone to development of thick ice.As shown in 7 kg, and we set the sea mass and water depth to 1×107 kg and 20 m, respectively. Then, the flexural moment and curvature hysteresis curves of the tower bottom were investigated (From the above analysis, we found that the least favourable mass of sea ice is 1×10stigated .As shown in Seismic responses of wind turbine tower surrounded by sea ice can increase several times compared to those in the absence of sea ice, especially the seismic response of a wind turbine tower at point of connection. We investigated the maximum horizontal displacement, bending moment, and shearing force along the wind turbine tower height with and without influence of the sea ice, as shown in As shown in A simplified model of the water-ice-structure dynamic interaction was proposed to investigate the effect of the sea ice on the seismic behaviour of a wind turbine tower, and the novelties of the study are as follows:A simplified seismic response analysis model of a wind turbine tower, ice, and water under earthquake load was established, and this obviated the need to solve complicated non-linear equations and reduced the computational burden to a significant extent.6 kg; therefore, the wall thickness or stiffening rib thickness should be increased to improve the seismic performance and ductility of the wind turbine tower.The energy dissipation capacity of the wind turbine tower was decreased by the effects of the sea ice, and the mass of sea ice exerted a significant influence on the curvature at the wind turbine power bottom, which could reflect the plastic deformation of the wind turbine tower. The curvature began to change significantly under different earthquake loads when the ice mass exceeded 1×10The maximum bending moment and shear force increased significantly at the bottom of the wind turbine tower and the action point of the sea ice, thus, in the seismic design of such wind turbine towers, it was deemed necessary to conduct anti-seismic design check-calculations on the wind turbine tower bottom and the action point of the sea ice. According to the design principle of the similarity ratio, paraffin ice was used to simulate sea ice and shaking table tests were conducted to verify the accuracy of the calculated results using our proposed simplified model: the calculated results were within accepted tolerances.


Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro‐specific 2′,3′‐cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.The structure of life's first genetic polymer is a question of intense ongoing debate. The “RNA world theory” suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high‐yielding, stereo‐, regio‐ and furanosyl‐selective prebiotic synthesis of threo‐cytidine  erythro‐diastereomer enriching any potential pool of TNA monomers.TNA cytidine, a candidate precursor to RNA cytidine at the origin of life, is synthesized from prebiotic precursors via a high‐yielding, regio‐ and stereo‐selective, protecting‐group free synthesis utilizing an efficient photochemical anomerization. Phosphorylation also enables selective photodestruction of the contaminating  Accordingly, we set out to investigate the protecting‐group‐free, prebiotic synthesis of threo‐cytidine 3 by UV‐light driven photoanomerization TAO and EAO, can be formed in high yields (>90 %) from the reaction of 2AO with glycolaldehyde 5 ,[6 and 7 .Previous work has established that cyanamide e HC3N),43, 44N,43, 46 and 7 to their corresponding β‐thiocytidines 9 and 10 in formamide  we observed conversion of β‐threo‐thiocytidine 9 (25 mm) to α‐threo‐thiocytidine 8 (46 %). After 1.5 days irradiation α‐threo‐thiocytidine 8 (51 %) was observed to be the major product . Spiking with an authentic standard of 8 (See Supplementary Information for synthesis of authentic 8) confirmed the effective photochemical anomerization.After 1 day of irradiation with UV‐light (254 nm) in water at pH 7 with Hthreo‐ and erythro‐ thiocytidine 9 and 10 was observed to occur at a similar rate  and 11 (42 %) after 1 day. Maximum conversion was observed after 2 days . Conversely, when 8 and 11 were irradiated (254 nm) in neutral water for 3 days with hydrogen sulfide we observed significant formation of 2‐thiocytosine 12 from both α‐isomers, but interestingly little reversion to the β‐anomers .Concomitant photoanomerization of e Figure , to furn8 to the TNA nucleoside, α‐threo‐cytidine 3, in quantitative yield . Interestingly, however, cis‐1′,2′‐isomers 9 and 10 were observed to rapidly cyclize to reform their corresponding anhydronucleoside 6 and 7  cyclization to 2,2′‐thioanhydronucleotides.threo‐isomer (i.e. TNA cytidine) by selective destruction of the erythro‐isomer of thiocytidine.Encouraged by the anomerization of tetrose thiocytidines, we next considered the role and timing of phosphorylation in the sequence towards TNA. Phosphorylation of prebiotic nucleosides has been demonstrated in the dry state with ureaerythro‐thiocytidine 10 underwent highly selective urea‐mediated phosphorylation,[13 as the major product . We next irradiated a mixture of erythro‐cyclic phosphate 13 and threo‐9; after 3 days 8 was the major nucleoside (47 %), whilst 13 was observed to degrade in only 1 day  by sample spiking with α‐threo‐thiocytidine 8 . However, the high temperature required for urea‐mediated phosphorylation resulted in some conversion of 9 back to anhydrocytidine 6 and concomitant phosphorylation and thermal rearrangement to yield photostable α‐erythro‐cytidine‐2′,3′‐cyclic phosphate 14 .β‐rylation,23, 458 can be photochemically selected. However, this selection is only feasible in the thiocytidine intermediates, as β‐erythro‐cytidines, such as 14, are photostable. A milder phosphorylation strategy would be required to further enhance the observed selection in the intermediate thiocytidines and avoid conversion of 9 to 14.trans‐disposition of hydroxy groups in cytidine 3 means that phosphorylation cannot yield a 2′,3′‐phosphate, whereas the cis‐disposition of hydroxy groups in 11 (and its cytidine congener 15) mean that 2′,3′‐phosphate formation is inevitable in erythro‐nucleotides. Therefore, the sluggish chemical reactivity of 2′,3′‐cyclic phosphates[3 can be chemically activated to polymerisation  should be addressed to the authors.SupplementaryClick here for additional data file.


AR) antagonist picrotoxin and the glycine receptor antagonist brucine rapidly dephosphorylated eEF2. Conversely, potentiation of GABAARs or inhibition of AMPA receptors (AMPARs) induced rapid phosphorylation of eEF2 in both the hippocampus and forebrain of mice. Chemogenetic suppression of hippocampal principal neuron activity promoted eEF2 phosphorylation. Novel context exploration and acute restraint stress rapidly modified the phosphorylation status of hippocampal eEF2. Furthermore, the hippocampal eEF2 phosphorylation levels under basal conditions were reduced in mice exhibiting epilepsy and abnormally enhanced excitability in CA3 pyramidal neurons. Collectively, the results indicated that eEF2 phosphorylation status is sensitive to neural activity and the ratio of phosphorylated eEF2 to total eEF2 could be a molecular signature for estimating neural activity in a specific brain area.Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABAThe online version contains supplementary material available at 10.1186/s13041-021-00852-0. Neural activity markers provide opportunities to estimate circuit activity, and immediate early genes (IEGs), such as c- markers , 2. Howe markers . In addiAR antagonist pentylenetetrazol or reduction of interneuron density resulted in dephosphorylation of eukaryotic elongation factor 2 (eEF2) in the mouse hippocampus [We previously observed that administration of the GABApocampus . eEF2 ispocampus . eEF2 kipocampus . Reportepocampus . Furtherpocampus . HoweverAR agonist muscimol and the antagonist picrotoxin on eEF2 phosphorylation were examined. Intraperitoneal (i.p.) injection of muscimol (1.5 mg/kg) and picrotoxin (2 mg/kg) produced opposite effects on eEF2 phosphorylation in both the hippocampus and forebrain without affecting total eEF2 levels  designer receptors exclusively activated by designer drugs (DREADDs) were unilaterally expressed in the hippocampus of CaMKIIα-Cre mice Fig. e. Strong−/− mice that exhibit reduced p-eEF2/eEF2 ratio [−/− mice (Fig. We hypothesized that eEF2 phosphorylation status is regulated during physiological brain activity of living animals. Indeed, p-eEF2 level in the hippocampus was reduced and then significantly enhanced during the 1-h novel context exploration (Fig. F2 ratio , the hipCollectively, the present study shows that the phosphorylation status of eEF2 is sensitive to drug-induced synaptic modification, chemogenetic suppression of neuronal activity, environmental stimuli, and the resting activity of the specific brain area, and indicates that the p-eEF2/eEF2 ratio can be a molecular index to estimate neural activity in the brain. As multiple signaling mechanisms regulate eEF2K activity in neurons , 8, howeAdditional file 1. Materials and methods, figures, and discussion.


Salvage total hip arthroplasty (THA) may be required to manage femoral neck fracture implant failures, avascular necrosis and secondary hip arthritis. Cannulated cancellous screws can become stripped or incarcerated during the initial implantation and pose hardware removal issues.We present a novel technique for safe screw removal in a 62-year-old female patient with a painful right hip. She had undergone cancellous screw fixation for a fracture of the neck of femur ten years ago. There was avascular necrosis with screw cut out leading to secondary hip arthritis necessitating THA. Intra-operatively cannulated cancellous screw along the inferior femoral neck region was incarcerated. After posterior dislocation of the head, the neck was osteotomised, and the screw threads were exposed for possible extraction. However, the thickened femoral neck region with solid cortical bone prevented the screw disengagement in either direction. The screw along the femoral trochanter region was cut with a Harrington cutter and the remaining screw disengaged with careful removal of bony spicules and controlled anticlockwise rotations, to remove the screw in around fifteen minutes. Arthroplasty could be completed uneventfully thereafter.We could remove the screw while avoiding an iatrogenic fracture along the calcar region and excessive bone loss along the screw track. The femoral canal remained uncompromised. The anticipation of a difficult implant removal with a thorough understanding of the devices and techniques, is an invaluable asset to the operating surgeon. With a simple tool and novel technique in a difficult situation, we can save on operating time and minimise complications. Fractures of the femoral neck are difficult to manage. Management techniques by surgical fixation are associated with complications. Avascular necrosis of femoral head, implant failure or screw cut out may lead to secondary arthritis. Recommended treatment option with salvage total hip arthroplasty (THA) gives good functional outcomes.1. Cannulated cancellous screws commonly used for fixation can become incarcerated or stripped during the process of initial open-reduction and internal fixation or at time of implant removal2. An uneventful removal of screw entails short operative time. However, one may have to adapt tricks and tips for better management in difficult situations of screw breakage, stripping or incarceration3. Care must be taken to avoid excessive bone loss and iatrogenic fractures during attempts at removal.Removal of hardware may pose a problem in itselfWe report a case of salvage THA in a 62-year-old female patient with secondary arthritis. An inferiorly placed incarcerated cannulated cancellous screw removal was achieved with a simple novel trick devised per-operatively with a common orthopaedic tool for a successful screw removal and subsequent arthroplasty.A 62-year-old female patient presented with a painful right hip for the last three months. She had no relief with conservative measures. She had a reduction and fixation for a right-sided fracture neck of the femur with two cannulated cancellous screws ten years back. Radiographically there was a loss of femoral head sphericity and superior femoral head cut out with progression to avascular necrosis and secondary hip arthritis . No signThe patient was operated under a combined spinal and epidural anaesthesia with the affected right hip placed up in lateral position. A posterior approach was used, and the external rotators were tagged. Both cancellous screws were easily identified. The superiorly placed screw with a washer was removed in a routine fashion with a cannulated cancellous screwdriver. The inferior screw was backed out for a distance but was incarcerated, stuck at the region of the distal-most screw thread. Repeated forceful attempts to disengage resulted in the rounding of the screw head. Since a hip arthroplasty was planned, the femoral head was dislocated posteriorly to save on operating time. A neck osteotomy was done at the level based on pre-operative templating. Piecemeal removal of the head was done, and screw threads were exposed . HoweverExtra screw length along the femoral trochanter side was cut with a Harrington cutter . The rem3. Excessive biomechanical and/or shearing stress during repetitive attempts to remove screws during hip arthroplasty may lead to iatrogenic fractures. Systemic osteoporosis and local bone stock reduction due to stress protection are predisposing factors for iatrogenic fractures1. The fracture may compound the pre-existing pathology, and there may be a need for revision of plans with an extended surgery time. A revised plan may require a resurfacing or a mini-stem option1. With the technique adopted, we could avoid an iatrogenic fracture and excessive bone loss along the screw track. The femoral canal remained uncompromised during the manoeuvre. Obesity may predispose to additional management challenges.Salvage THA following a proximal femoral fixation failure is a challenging procedure. Hardware removal before THA is associated with high rates of complications2. Titanium screws have a greater risk of screw head tip stripping and incarceration during removal due to increased osteointegration. Accurate identification of the screw size and manufacturer from conventional radiographs is usually not possible. Implant logs are often unavailable, and screw mismatch with a screwdriver is a major concern3.Screw breakage, screw retention, infection, and prolonged operative time are significant problems associated with cannulated cancellous screw removal3. Multiple techniques for cannulated screw removal have been described. Synthes broken screw set’s conical reverse-cutting male-threaded tap is a useful device3. Use of a spinal needle to locate a cannulated screw tip and subsequent removal of a buried screw is an alternate technique4. A set of sterile Allen keys of different diameters and lengths may be a possible solution5. Steinman pin stacked with cement is another option5. We used a commonly available orthopaedic tool, the Harrington cutter, and quickly resolved the difficult situation. The aim was to conduct the procedure safely in a short operating time with an eventual good functional result.Extensive bone loss secondary to the use of osteotomes, burrs, or intraosseous drilling should be avoidedA cancellous screw may be incarcerated due to bony ingrowths or abnormal bending. Shear forces at femoral neck probably remodelled the bony trabeculae pattern leading to incarceration. An inferiorly placed cancellous screw should alarm the surgeon for a difficult operative removal.Implant removal continues to pose challenges for even an experienced surgeon. An understanding of an additional tool and technique which may be used for hardware removal is invaluable for the safe conduction of a difficult salvage THA.