The vertebral body’s Hounsfield unit (HU) value can credibly reflect patients’ bone mineral density (BMD). Given that poor bone-screw integration initially triggers screw loosening and regional differences in BMD and strength in the vertebral body exist, HU in screw holding planes should better predict screw loosening. According to the stress shielding effect, the stress distribution changes in the fixation segment with BMD reduction should be related to screw loosening, but this has not been identified. We retrospectively collected the radiographic and demographic data of 56 patients treated by single-level oblique lumbar interbody fusion (OLIF) with anterior lateral single rod (ALSR) screw fixation. BMD was identified by measuring HU values in vertebral bodies and screw holding planes. Regression analyses identified independent risk factors for cranial and caudal screw loosening separately. Meanwhile, OLIF with ALSR fixation was numerically simulated; the elastic modulus of bony structures was adjusted to simulate different grades of BMD reduction. Stress distribution changes were judged by computing stress distribution in screws, bone-screw interfaces, and cancellous bones in the fixation segment. The results showed that HU reduction in vertebral bodies and screw holding planes were independent risk factors for screw loosening. The predictive performance of screw holding plane HU is better than the mean HU of vertebral bodies. Cranial screws suffer a higher risk of screw loosening, but HU was not significantly different between cranial and caudal sides. The poor BMD led to stress concentrations on both the screw and bone-screw interfaces. Biomechanical deterioration was more severe in the cranial screws than in the caudal screws. Additionally, lower stress can also be observed in fixation segments’ cancellous bone. Therefore, a higher proportion of ALSR load transmission triggers stress concentration on the screw and bone-screw interfaces in patients with poor BMD. This, together with decreased bony strength in the screw holding position, contributes to screw loosening in osteoporotic patients biomechanically. The trajectory optimization of ALSR screws based on preoperative HU measurement and regular anti-osteoporosis therapy may effectively reduce the risk of screw loosening. Anterior lateral single rod (ALSR) fixation can provide sufficient instant postoperative stability for oblique lumbar interbody fusion (OLIF) patients without the need for other surgical incisions . As a haTraditionally, the dual-energy X-ray absorptiometry is the gold standard for diagnosing osteoporosis. However, this imaging examination cannot eliminate pathological bone formation during lumbar degenerative diseases . This leads to an underestimation of the severity of osteoporosis in patients with lumbar degenerative diseases . The verPresently, the HU value of the vertebral body is defined by the average value of four planes, including the midsagittal plane, central transverse plane, and transverse planes close to the superior and inferior bony endplates  Bredow . We hypoAs above mentioned, surgeons believe that the decreased bony strength is the main reason for the increased risk of screw loosening in osteoporotic patients. Meanwhile, studies illustrated that stress concentration on the bone-screw interfaces and fixation screws would aggravate poor bone-screw integration and result in screw loosening . SpecifiIn this study, to verify these hypotheses, we investigated whether the HU in the screw holding plane is a better predictor during the judgment of screw loosening and investigated changes in the load transmission proportion between the vertebral body and ALSR screw system with BMD stepwise reduction. The prospectively collected radiographic and demographic data of OLIF patients fixed by ALSR were retrospectively reviewed. Changes in the stress distribution of the ALSR fixation segment were investigated by computing biomechanical changes in fixation screws, bone-screw interfaces, and cancellous bones of vertebral bodies in an anteriorly constructed and validated lumbosacral model. This study could provide theoretical guidance for understanding the screw loosening mechanism and feasible methods to reduce the risk of screw loosening.The ethics committees of West China Hospital approved the protocol of this study (2020-554). Informed consent was waived for this retrospective study. We retrospectively reviewed the radiographic and demographic data of OLIF patients with ALSR screw fixation from May 2017 to August 2019. Their age, sex, and body mass index (BMI) were recorded. A senior spine surgeon performed all operations. Screw types and sizes were identical in these patients. All screws were placed in a single attempt and penetrated the contralateral cortex.Patients who underwent single segment OLIF with ALSR screw fixation for patients with lumbar degenerative diseases, including spinal stenosis, grade 1 and grade 2 degenerative spondylolisthesis, and lumbar disc herniation, were included in this study. The exclusion criteria were as follows: 1) Patients with a history of lumbar surgery; 2) Patients with primary or metastatic spinal tumors, lumbar tuberculosis, rheumatic immune diseases, and secondary osteoporosis caused by medication or other metabolic diseases; 3) Patients with grade 3 and grade 4 degenerative spondylolisthesis or spondylolysis; 4) Patients who underwent lumbar revision surgery within the clinical follow-up period of 12 months for complications other than screw loosening; 5) Patients who underwent intraoperative screw replacement.Patients underwent lumbar computational tomography (CT) three times in the imaging center of West China Hospital, including 1 week before, 1 week after, and 1 year after OLIF surgery. The tube voltage was set to 120 kV, and all CT scan setting parameters were uniform in all enrolled patients . An expecortical shell, BEP, and osteophytes  angle restoration, and cage position could affect local transmission patterns, these values were regarded as covariables and were also been measured in this study . Disc heRadiographic and demographic indicators are presented as the mean ± standard deviation for continuous variables and the number (percentage) for categorical variables. We conducted statistical analyses in SPSS 23.0 software. The intraclass correlation efficiency (ICC) was computed to identify the repeatability of continuous variables (ICC ≥0.8 represents excellent reliability)  is a binary classification variable (which is from different patients and therefore fully independent); its classification is complete and exclusive (screw loosening or not). In the multivariate analysis, all factors had no significant collinearity, and there are no obvious outliers and strong influence points for all included parameter values. Univariate analyses of each potential risk factor were performed, and the variables that achieved a significance level of p < 0.1 were entered into multivariate analyses. Variables with p < 0.05 were considered independent risk factors in the multivariate analyses , these covariables have been included in regression analyses to judge the risk of screw loosening. Since these covariables did not differ significantly between the credible screw fixation group and the group of screw loosening and were not independent risk factors for screw loosening, we believe that not simulating these covariables will not affect the reliability of the numerical simulation results in this study. Therefore, when investigating the biomechanical significance of a particular variable, we believe researchers should eliminate the interference of covariables by reviewing radiographic and demographic data, and this may be a feasible method to optimize the credibility of FEA studies.in vitro studies could be reduced, and the computational stress values can make a good representation of real biomechanical situations.Simulation of OLIF with ALSR fixation was performed in a previously constructed and validated biomimetic lumbosacral FE model (L3-S1) . Bone stThe L4-L5 segment was selected to simulate ALSR fixed OLIF. Surgical simulations were performed based on a literature review and our surgical experience. In this process, lateral parts of the annulus, all of the nucleus, and CEPs were removed, and a polyether-ether-ketone (PEEK) OLIF cage (18 mm width and 50 mm length) filled with grafted bone was inserted into the interbody space . The lorThe three-dimensional model of the fixation screw was reversely constructed based on the outline of the screw used in ALSR fixation in our clinical practice. During the simulation of ALSR screw fixation, two titanium alloy screws were inserted into the L4-L5 vertebral bodies and penetrated the contralateral cortex. The axes of the screws in the transverse plane were parallel to the OLIF cage, whereas those in the coronal plane were parallel to the BEPs . Screw tin vitro measured values  with different sizes were set in different components of the FE model. Mesh refinement was set in structures with low thickness and large deformation  . To elimd values . When thalidated .cortical shell and cancellous bone were defined by anisotropic law. The annulus was assumed to be hypoelastic material, and the nucleus was set as a semifluid incompressible material  or in the screw holding plane (p = 0.707). Excellent consistency between vertebral bodies’ HU and HU of screw holding planes can be observed in cranial and caudal vertebral bodies and groups with credible screw fixation and screw loosening  with an average age of 56.57 ± 11.96 years treated by single segment OLIF with ALSR screw fixation were recorded. The interobserver and intraobserver results during the judgment of screw loosening were substantial, with Kappa values of 0.778 and 0.759, respectively. The reliability of continuous variable measurement was excellent, with ICCs of 0.867 and 0.835, respectively . The oveoosening . Althougoosening , 3.p = 0.033). The HU values in the credible screw fixation group  were significantly higher than those in the screw loosening group, whether the HU was measured by the mean value of vertebral bodies or in the screw holding plane . The variation tendency of HU changes was consistent with the cranial vertebral body. Considering that only the p values of vertebral body HU and screw holding plane HU reduction were <0.1 in the univariate logistic regression analysis, multivariate analysis was not performed. The reduction of vertebral bodies’ HU and screw holding planes’ HU were regarded as independent risk factors for screw loosening in the caudal vertebral body  were not significantly different between the credible screw fixation group and the screw loosening groups. These covariables were not independent risk factors for screw loosening on either the cranial or caudal sides.Concerning the caudal side, there were no significant age differences between patients with credible fixation and screw loosening  should be promoted in patients with lumbar screw fixation.Changes in the fusion segment’s stress distribution with BMD stepwise reduction could also provide a reasonable explanation for the clinical phenomenon observed in the radiographic review. Specifically, the screw loosening rate was higher in the cranial vertebral body, but HU values measured in vertebral bodies and screw holding planes, as significant risk factors for screw loosening, were not significantly different. As computed by the current numerical simulations, although the variation tendency of stress levels in bone-screw interfaces and cancellous bones were identical in both cranial and caudal vertebral bodies, the maximum equivalent stress step increased with BMD reduction except for the right axial rotation loading condition. In contrast, the caudal screw stress only increased under bending loading conditions. In other words, the maximum stress increased in the cranial screw but decreased in the caudal screw under flexion, extension, and left axial rotation loading conditions. Given the exact relation between the maximum stress increase and the increase in screw loosening risk, we believe the current computational result explains the higher screw loosening risk of the ALSR fixation system in the cranial vertebral body.To our knowledge, the most significant contribution of this study in methodology is the combination between the clinical review and numerical biomechanical simulations. Previously, these researches have been separately performed; clinical studies have observed a phenomenon without directly explaining its biomechanical mechanism; biomechanical studies have explored the potential mechanism of complications, but there is no clinical data to support this anticipation. Thus, the credibility of both types of studies is limited. In this study, the biomechanical mechanism of the observed clinical phenomenon has been directly investigated by corresponding numerical simulations. Combining these two parts is significant for better understanding a specific risk factor.Admittedly, the current study results should be interpreted within the context of the following-mentioned limitations. Specifically, larger sample sizes of clinical data with a longer follow-up period should be obtained, and morphological changes during BMD reduction should be simulated in FEA models. However, given that screw loosening commonly occurs in the early stage of postoperative follow-up, the construction strategy of models with poor BMD has been widely reported , especiaBoth vertebral bodies and “screw holding planes” HU can well predict screw loosening risk for OLIF patients with ALSR screw fixation. The predictive performance of screw holding plane HU is better than the mean HU of vertebral bodies. A higher proportion of ALSR load transmission triggers stress concentration on the screw and bone-screw interfaces in patients with poor BMD. This, together with decreased bony strength in the screw holding position, contributes to screw loosening in osteoporotic patients biomechanically. Therefore, the trajectory optimization of ALSR screws and regular anti-osteoporosis therapy may effectively reduce the risk of screw loosening.


Successful cholinergic-noradrenergic pharmacotherapy for obstructive sleep apnea (OSA) is thought to be due to effects at the hypoglossal motor nucleus (HMN). Clinical efficacy varies with muscarinic-receptor (MR) subtype affinities. We hypothesized that oxybutynin (cholinergic agent in successful OSA pharmacotherapy) is an effective MR antagonist at the HMN and characterized its efficacy with other antagonists. We recorded tongue muscle activity of isoflurane anesthetized rats  in response to HMN microperfusion with MR antagonists with and without: (i) eserine-induced increased endogenous acetylcholine at the HMN and (ii) muscarine. Eserine-induced increased acetylcholine decreased tongue motor activity (p < 0.001) with lesser cholinergic suppression in females versus males (p = 0.017). Motor suppression was significantly attenuated by the MR antagonists atropine, oxybutynin, and omadacycline (MR2 antagonist), each p < 0.001, with similar residual activity between agents (p ≥ 0.089) suggesting similar efficacy at the HMN. Sex differences remained with atropine and oxybutynin (p < 0.001 to 0.05) but not omadacycline (p = 0.722). Muscarine at the HMN also decreased motor activity (p < 0.001) but this was not sex-specific (p = 0.849). These findings have translational relevance to antimuscarinic agents in OSA pharmacotherapy and understanding potential sex differences in HMN suppression with increased endogenous acetylcholine related to sparing nicotinic excitation. OSA remains undertreated due to poor compliance with continuous positive airway pressure, the leading therapy4.Obstructive sleep apnea (OSA) is a common and widespread disorder with serious clinical, social, and economic problems9, and a muscarinic receptor inhibitory mechanism that operates especially in rapid eye movement sleep10. Such findings identify the chief mechanisms controlling motor output to the pharyngeal muscle in sleep that is central to OSA in humans, and led to selection of cholinergic-noradrenergic drug combinations and reproducible improvements in OSA severity by pharmacotherapy15.Findings in animal studies identify an endogenous noradrenergic drive that tonically excites the hypoglossal motor nucleus (HMN) in wakefulness with this excitation withdrawn in sleep17 and accompanying editorials20, is that the agents are likely effective via their actions at the HMN. This assumption needs to be tested to pinpoint mechanism of action and stimulate identification of potentially more effective strategies including the targeting of specific pharmacological profiles: e.g., clinical efficacy varies with muscarinic receptor subtype affinities of the agents tested17.The common assumption or underlying rationale from those clinical studies22), omadacycline (recently FDA-approved agent and selective muscarinic receptor subtype 2 (M2) antagonist24), and atropine and/or scopolamine, broad spectrum muscarinic receptor antagonists with the latter also a component of effective OSA pharmacotherapy19. Acetylcholinesterase inhibition at the HMN to increase endogenous acetylcholine leads to muscarinic receptor-mediated inhibition of motor activity that dominates nicotinic receptor-mediated excitation27. Accordingly, this study also furthered an established protocol to use eserine-induced increased endogenous acetylcholine at the HMN to modulate muscarinic and nicotinic receptors, and muscarine at the HMN to target only muscarinic receptors25. Isoflurane was chosen as the inhalational anesthetic to enable characterization of tongue motor responses to controlled time-dependent sequential delivery of selected interventions into the HMN in vivo across 13 related protocols in male and female rats28.In the present study, respiratory muscle activities are recorded during microperfusion of the HMN in rats with oxybutynin  and females , with significant decreases with eserine compared to baseline artificial cerebrospinal fluid (ACSF) in both groups .There was a significant effect of the interventions on within-breath phasic tongue muscle activity in males  but a decrease occurred in male rats  during the second ACSF period . There were no significant effects on diaphragm amplitude observed in either males or females over the experiments .There was no effect on respiratory rate over the intervention periods in female rats  but not for Protocols 1.2a and 1.2c .Significant effects of the interventions on within-breath phasic tongue muscle activity were also identified in female rats for Protocols 1.2a-c, there was no effect of microperfusion of the muscarinic-receptor antagonist alone into the HMN on within-breath phasic tongue muscle activity of male  or female rats .In each of Protocols 1.2a-c show a general trend of a decline in respiratory rate over the course of the experiments for both male rats  and female rats . A small but statistically significant effect on diaphragm amplitude was observed in male rats in Protocols 1.2a and 1.2c .Comparison of Figs. As noted below, these effects on respiratory rate occurring during the experiments also similarly occur in the ACSF time controls Fig.  and as sProtocol 2. Group data are shown in Fig. Figure 2,24 = 0.90, p = 0.419, one-way ANOVA-RM, Fig. 2,10 = 0.90, p = 0.436, Fig. Analysis identified no significant effects of sequential microperfusion of ACSF into the HMN on within-breath tongue muscle activity of male . A significant change  in diaphragm amplitude was identified in one of the protocols in female rats  but not in the males .Consistent with previous protocols, compared to the baseline ACSF controls there was a statistically significant decline in respiratory rate observed over the course of the experiments for male rats , and effects that were sex specific , see symbols ‘*’ and ‘#’ for each of these intervention and sex effects respectively.Figure Within-breath phasic tongue muscle activity in the presence of eserine-increased acetylcholine at the HMN was significantly lower  compared to when each of atropine, oxybutynin and omadacycline were co-applied at the HMN in male and female rats , atropine co-applied with eserine (p < 0.001), and oxybutynin co-applied with eserine , but not for omadacycline co-applied with eserine (p = 0.722) or across the ACSF time controls (p = 0.795)—see the symbols ‘#’ in Fig. 4,92 = 2.17, p = 0.078 and F4,92 = 1.23, p = 0.305, respectively) or drug treatment .Any changes in respiratory rate and diaphragm amplitude over the course of the experiments were similar across all protocols . There were no effects on respiratory rate or diaphragm amplitude that were dependent on sex  and females , with significant decreases compared to baseline ACSF in both sexes (each p ≤ 0.008).Figure 2,20 = 2.19, p = 0.138, one-way ANOVA-RM) but a decrease occurred in male rats  during the second ACSF period . Figure 2,14 = 1.32, p = 0.229 and F2,20 = 0.72, p = 0.500, Fig. Figure Protocols 3.2a-d. Figures Figure Protocols 3.2a . These results identify that in the presence of these muscarinic receptor antagonists there was no inhibitory effect of muscarine per se on tongue motor activity in contrast to the significant inhibitory effects observed without the antagonists .Figure Within-breath phasic tongue muscle activity in the presence of muscarine at the HMN was significantly lower compared to when either atropine or scopolamine were co-applied at the HMN , i.e., indicating statistically significant marked antagonism of muscarinic receptor inhibition at the HMN by atropine and scopolamine respectively Fig. a.In contrast, the relative efficacy of oxybutynin  and omadacycline  in antagonising muscarinic receptor inhibition were reduced and not statistically different from activity in the presence of muscarine per se . This lack of sex-difference with muscarine was unlike the larger motor inhibitory responses in males compared to females in the presence of eserine-induced increased endogenous acetylcholine at the HMN (which would modulate both muscarinic and nicotinic receptors). Given this lack of sex-specific response to muscarine compared to eserine  were not performed in line with local animal care guidelines to reduce unnecessary experiments and animal numbers.There was no difference between males and females with muscarine at the HMN targeting only muscarinic receptors .As shown in Fig. 29. This assumption and rationale is largely based on previous animal studies: scopolamine-induced muscarinic receptor antagonism at the HMN leads to marked increases in tongue motor tone in sleep, especially rapid eye movement (REM) sleep, and restoration of physiological patterns and levels of activity in REM sleep that are typical of non-REM sleep10. The second major impetus was that effectiveness in improving human OSA severity and converting obstructive apneas to hypopneas or resolving apneas varies with muscarinic receptor subtype affinities of the agents administered17. For example, clinical studies using a variety of agents with different antimuscarinic properties  when combined with atomoxetine may themselves not change apnea hypopnea index but convert obstructive apneas to hypopneas and milder events14, unlike the more significant improvements with oxybutynin or scopolamine15. Overall, increased knowledge of cholinergic effects operating at the HMN, and their pharmacological manipulation, may translate to improvement or refinement of current pharmacological strategies and receptor subtype targets in future human studies and larger trials.The first major impetus for the present study was the common assumption and/or underlying rationale from previous human studies and their accompanying editorials that the antimuscarinic agents in OSA pharmacotherapy are exerting their effects, at least in some significant part, at the HMN31. Studies in rats have identified sex hormone effects on hypoglossal motor activity and the control of breathing34. Here we identify in rats that eserine-induced increased endogenous acetylcholine at the HMN in vivo elicits suppression of tongue motor activity, an effect that is sex dependent with lesser suppression in females versus males. Eserine was chosen given the physiological relevance of increasing endogenous acetylcholine to modulate all locally present nicotinic and muscarinic receptors and their subtypes. We identify that cholinergic-induced hypoglossal motor suppression was significantly attenuated by each of the selected FDA-approved muscarinic receptor antagonists at the HMN, including oxybutynin. Notably, oxybutynin was initially identified as a key component of effective OSA pharmacotherapy but with unknown effects at the HMN22. Notably also in the present study in rats, the residual tongue motor activity after atropine (broad spectrum muscarinic receptor antagonist), oxybutynin and omadacycline (M2 antagonist) at the HMN were statistically similar across agents in the presence of eserine-induced cholinergic inhibition via increased endogenous acetylcholine. This result suggests the capacity for similar efficacy at the HMN in modulating motor activity despite some differences in muscarinic subtype affinities and the sparing of nicotinic receptor-mediated excitation.Potential sex-dependent differences in HMN responses to cholinergic modulation were unknown in previous human, rodent, or other such studies to our knowledge. It is known that there is a sex difference in human OSA prevalence, with increased incidence in males, and female incidence increasing post menopause10. It is currently unknown if oxybutynin, or other agents with more specific M2 affinities such as omadacycline, is (are) similarly capable of preventing cholinergic inhibition of the HMN in REM sleep in animal studies. Omadacycline was only used here as a recently FDA-approved agent23 for mechanistic interrogation of M2 receptor targets24, although it is acknowledged that other agents could also have been selected. Such proof-of-concept may stimulate testing of agents with central activity and M2 specificity with a view to potential activation of pharyngeal motor activity in sleep in combination with noradrenergic reuptake inhibitors. However, it is recognized that both atomoxetine and oxybutynin, for example, can increase cardiovascular activity although minimal changes were observed in OSA patients: 2 beats per minute in heart rate and no change in blood pressure11. Such concerns will need to be followed clinically with cholinergic-noradrenergic pharmacotherapy for OSA and effects may vary, being more favourable with some combinations than others depending on receptor subtype-targeting profiles and length of treatment, with relatively short duration published studies in humans to date.Despite the significant attenuation of cholinergic inhibition in the presence of each of these antimuscarinic agents at the HMN in the present study, the sex differences remained with atropine and oxybutynin  but not with omadacycline . In contrast, the primary muscarinic receptor-mediated inhibition elicited by muscarine at the HMN was not sex dependent. The similar efficacy of oxybutynin and omadacycline also occurred in the presence of muscarine at the HMN across the range of doses tested. In the presence of muscarine at the HMN, scopolamine is capable of full blockade of cholinergic inhibition Fig.  and capa40. The net effects of cholinergic modulation of the HMN on motor output to the tongue musculature is complex, with muscarinic receptor-mediated inhibition dominating nicotinic receptor-mediated excitation in rodents in vivo25 and in vitro27. Initial immunohistochemical and in-situ hybridization studies indicated strong abundance of proteins or mRNAs for the M2 receptor on brainstem motoneurons with relatively little for M1, M3, M4 and M541. Subsequent immunohistochemical studies focussing on the HMN indicate the presence of M1, M2 and M5 at the HMN in neonatal rats42. Muscarinic receptor mediated inhibition of motoneurons occurs via two major mechanisms: (i) pre-synaptic muscarinic inhibition via M2 receptors produces dis-facilitation of spinal43 and hypoglossal motoneurons27, and (ii) post-synaptic M2 receptor activation inhibits motoneurons via induction of inwardly rectifying potassium current45. Both such effects likely explain, at least in great part, previous10 and current effects of cholinergic agents at the HMN in rats in vivo. M2 receptors are the most highly expressed at the rodent HMN and the major pre-motor inputs to the HMN46. Notably, pre-synaptic M2 receptors inhibit excitatory glutamate inputs to hypoglossal motoneurons27 that mediate transmission of excitatory respiratory drive48. The dominant muscarinic receptor-mediated inhibition operating at the adult rat HMN appears to overcome any muscarinic (non-M2) excitatory effects as identified for neonatal hypoglossal motoneurons39.Cholinergic influences at the HMN include a combination of muscarinic and nicotinic receptor-mediated effects15 should stimulate future work to combine a full molecular identification of each of the muscarinic receptor subtypes with their pre and/or post-synaptic locations at the HMN, with identified effects on known inputs  and full characterization of the pharmacological profiles of the applied antimuscarinic agents. Such work is needed to further define and refine targeted pharmacotherapeutic approaches to optimize modulation of HMN output to the tongue musculature for OSA pharmacotherapy46. It is important to note, however, that some component of the positive responses to antimuscarinic drugs used clinically could be due to effects at the HMN and/or elsewhere, the latter including upstream effects altering other factors that improve sleep apnea 46. Those upstream effects could involve muscarinic receptor subtypes targeted by the antimuscarinic agents used clinically but those subtypes may not be present at the HMN. The contribution of the present study is to characterize effects of the selected agents at the HMN.Overall, the present findings and positive responses to antimuscarinic agents in OSA pharmacotherapy in patient populationsIncreased endogenous acetylcholine at the HMN in the presence of muscarinic receptor antagonists would leave nicotinic excitation intact. Differences in nicotinic receptor function between males and females may therefore contribute to the lesser motor suppression observed after co-application of atropine or oxybutynin in the presence of eserine-induced cholinergic inhibition via increased endogenous acetylcholine. By itself, however, such a difference would not explain the lack of sex difference with the M2 antagonist omadacycline also in the presence of eserine-induced endogenous acetylcholine. Sex differences in excitatory muscarinic (non-M2) receptors may, therefore, also contribute.alone to OSA patients does not improve OSA severity, and likewise with noradrenergic reuptake inhibitors alone: both agents need to be applied together for successful OSA pharmacotherapy11. This requirement for combination therapy may be based on physiological precedent and known mechanisms. Studies across natural sleep–wake states in rats identify an endogenous noradrenergic (α1 receptor-mediated) drive to the HMN that contributes to tongue motor activity in wakefulness, with this contribution withdrawn in non-REM sleep and further reduced to a minimum in REM sleep9. In contrast, the contribution of muscarinic receptor-mediated inhibition of hypoglossal motor output to the tongue musculature in rats is strongest in REM sleep but also present, to a lesser degree, in non-REM sleep10. As such, although the relative net contributions of noradrenergic excitation and muscarinic inhibition to net hypoglossal motor output may vary inversely to each other in states of non-REM and REM sleep, modulation of both would be expected to result in higher net activity than either alone across the sleeping period and, if applicable from rats to humans, explain why the agents need to be applied together for successful OSA pharmacotherapy11.Administration of antimuscarinic agents 51. Nevertheless, the improvements were variable, likely reflecting the multi-factorial nature of OSA and the varying contributions of the four major endotypes operating within and between patients: pharyngeal anatomy, pharyngeal dilator muscle activity, arousal threshold and loop gain55. Given the consistent findings from the present study  motor activity was stable throughout the experiments  tongue muscle activity in non-REM with larger effects in REM sleep, as well as increased tonic motor activity in phasic REM sleep10. Nicotinic receptor agonism at the HMN in rats via microperfusion of dimethyl-4-phenylpiperazinium iodide (DMPP) increases tonic as well as respiratory-related tongue motor activity25 but tonic activity was never elicited by any intervention in the present study  the intermediate reticular nucleus (IRt) in the lateral medulla, and (ii) the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei in the ponsTogether, the results of the present study identify differential pharmacological and sex-specific effects of select antimuscarinic agents at the HMN in rats in vivo. The results have translational relevance to understanding current and potential future directions for antimuscarinic OSA pharmacotherapy. Sex differences in HMN suppression with increased endogenous acetylcholine at the HMN but not muscarine may relate to the sparing of accompanying nicotinic excitation.74. The inclusion criteria for a technically proficient experiment identified above were met for 121 males , range) and 60 females  yielding the population of 181 subjects studied across the 13 separate protocols.Experiments were performed on a total of 256 Wistar rats  of which data from 181 subjects were included for analysis (70.7% success rate). The inclusion criteria for the data from an individual animal to be included in the analysis required that each of the following be met: (i) a stable 30-min baseline control period following insertion of the microdialysis probe into the HMN, (ii) a positive motor activation response to microperfusion of 5-hydroxytryptamine (5-HT) into the HMN at the end of the experiment , and (iii) positioning of the microdialysis probe membrane within the HMN as confirmed by histology. All procedures conformed to the recommendations of the Canadian Council on Animal Care, and the protocols were approved by the University of Toronto Animal Care Committee. The studies and their reporting followed the ARRIVE guidelinesAnimals were first anesthetized in an induction chamber using 3.5% isoflurane for approximately five minutes followed by 2.5% for another 10 min. Following induction of general anesthesia as judged by the abolition of pedal withdrawal reflex to paw pinch, the animals were placed on a heating pad for the experiments for warming for the duration of the experiments . The rats breathed a 1:1 mixture of air and oxygen, and general anesthesia was maintained with isoflurane  delivered via a snout mask  for the remainder of the experiment.To record diaphragm electromyogram (EMG) activity, bipolar stainless-steel electrodes  were sutured onto the right side of the costal diaphragm via an abdominal approach. Two stainless-steel needle electrodes  were also inserted bilaterally into the tongue musculature via a per-oral approach to record EMG activity of the tongue musculature. The rats were positioned in a stereotaxic apparatus  using blunt ear bars and the skull leveled. Two electroencephalogram (EEG) electrodes attached to small screws were implanted over the frontal-parietal cortex to measure EEG activity.For local administration of pharmacological agents into the HMN, microdialysis probes  were used. The probes were connected to fluorinated ethylene propylene (FEP) Teflon tubing  which in turn were connected to 1.0 mL syringes via a zero dead-space switch . The syringes were mounted on a syringe pump  that regulated the flow of the perfusion medium via a controller .2, 1 mM MgSO4, 25 mM NaHCO3, and 30 mM glucose with a pH value of 7.41 ± 0.0, 7.31–7.525. The microdialysis probes were placed through a small hole drilled at the junction of interparietal and occipital bones at the following midline coordinates: 14.1 ± 0.02 mm (13.4–15.0 mm) posterior to bregma, and 9.51 ± 0.03 mm (8.2–10.6 mm) ventral to bregma. A brief burst of tongue muscle activity  was observed once the probe penetrated the HMN, with no effects observed on diaphragm activity or respiratory rate. This response is expected and typical, and is used as a preliminary confirmation of probe placement into the HMN prior to subsequent determination by histology after the experiment76.Prior to insertion into the brain, the microdialysis probes were initially perfused with distilled water for 10 min, and subsequently with ACSF at a flow rate of 2.1μL/min to document continuous flow and no blockage. The composition of the ACSF was 125 mM NaCl, 3 mM KCl, 2 mM CaClFollowing insertion of the microdialysis probe, ACSF was microperfused into the HMN. The signals were monitored closely until at least a 30-min period of stable tongue and diaphragm EMG activities, respiratory rate and EEG activity were observed before any experimental interventions were performed. This period was designated as the baseline control condition for each experiment. The specific pharmacological agents of interest (see separate protocols below) were then microperfused into the HMN at a flow rate of 2.1 μL/min and a lag time of 5.4 min. The lag time was taken as the duration for the perfusion medium to reach the tip of the microdialysis probe following a switch between drugs and was calculated using the known flow rate and volume of the tubing.The electrophysiological signals were amplified and filtered . The bandpass filters for the EEG and EMG signals were 1–100 Hz and 100–1000 Hz, respectively. The moving-time averaged signals of the tongue and diaphragm signals were also obtained with a time constant of 100 ms . The electrocardiogram artifact was removed from the diaphragm EMG using an electronic blanker . Using a CED 1401 data acquisition system, the raw analog signals were digitized at a sample rate of 1984 Hz and stored for analysis .Each protocol consisted of three 30-min intervention periods  during which agents of interest were microperfused into the HMN. The first intervention in all protocols was the baseline control condition with microperfusion of ACSF into the HMN.Protocol 1.1 was performed to quantify the net inhibitory effects of increasing endogenous levels of acetylcholine on tongue motor activity by local microperfusion into the HMN of 100 μM eserine : 324.39 g/mol, catalogue number: 0622, Tocris)25. Experiments were performed in nine male rats  and 10 females . The sequence for this protocol consisted of: (i) ACSF 1; (ii) ACSF 2 (to be consistent with the timeline of Protocol 1.2 where select muscarinic receptor antagonists are microperfused into the HMN); (iii) 100 μM eserine.27.Notably, application of acetylcholinesterase inhibitors to the HMN to increase endogenous acetylcholine levels leads to muscarinic receptor-mediated inhibition of genioglossus activity that dominates nicotinic receptor-mediated excitationProtocols 1.2a-c were performed to quantify the relative efficacy of three muscarinic receptor antagonists at the HMN in reducing tongue motor inhibition by eserine-induced increased acetylcholine.77; (iii) 10 μM atropine co-applied with 100 μM eserine.Experiments were performed in nine male rats  and 10 females . This protocol consisted of: (i) ACSF; (ii) 10 μM atropine, a muscarinic receptor antagonist  with high affinity for all subtypes21 and a component of effective OSA pharmacotherapy22.Experiments were performed in nine male rats  and 10 females . This protocol consisted of (i) ACSF; (ii) 100 μM oxybutynin ; (iii) 100 μM oxybutynin co-applied with 100 μM eserine. Oxybutynin is a muscarinic receptor antagonist with affinities across the range of muscarinic receptor subtypes24.Experiments were performed in 13 male rats  and 13 females . This protocol consisted of: (i) ACSF; (ii) 100 μM omadacycline ; (iii) 100 μM omadacycline co-applied with 100 μM eserine. Omadacycline is a recently FDA-approved agent and a selective M2 receptor antagonistProtocol 2, repeated switches to microperfusion of ACSF into the HMN  were performed over the same time course as the drug interventions in Protocols 1.1 and 1.2a-c. These experiments were performed to determine if there were any effects of time per se on respiratory motor activities independent of the drug interventions. Experiments were performed in 13 male rats  and six females . This protocol consisted of three repeated ACSF interventions at the HMN, i.e., ACSF 1, ACSF 2, and ACSF 3.In Protocol 3.1 was performed to quantify the effect of muscarinic receptor stimulation on tongue motor activity by the local microperfusion into the HMN of muscarine 25. Experiments were performed in eight male rats  and 11 females . This protocol consisted of a sequential order of microperfusion into the HMN of: (i) ACSF 1 ; (ii) ACSF 2 ; (iii) 100 μM muscarine.Protocols 3.2a-d were performed to quantify the relative efficacy of four muscarinic receptor antagonists at the HMN in reducing muscarine-induced tongue motor inhibition. Given the lack of sex-specific responses to muscarine compared to eserine (see “3.2a-d below) were not performed in both sexes , in line with local animal care guidelines to reduce unnecessary experiments and animal numbers.ine see “” the subExperiments were performed in eight male rats . This protocol consisted of a sequential order of microperfusion into the HMN of: (i) ACSF; (ii) 10 μM atropine; (iii) 10 μM atropine co-applied with 100 μM muscarine.10; (iii) 1 mM scopolamine co-applied with 100 μM muscarine.Experiments were performed in 10 male rats . The sequence for this protocol consisted of: (i) ACSF; (ii) 1 mM scopolamine For this protocol one set of experiments was performed with 10 μM oxybutynin in seven male rats , and another set with 100 μM oxybutynin in a separate group of seven male rats . This protocol consisted of (i) ACSF; (ii) 10 μM or 100 μM oxybutynin; (iii) 10 μM or 100 μM oxybutynin co-applied with 100 μM muscarine.Three sets of experiments were performed with (i) 10 μM omadacycline in 10 male rats , (ii) 100 μM omadacycline in 10 male rats , and (iii) 1 mM omadacycline in eight male rats . This protocol consisted of (i) ACSF; (ii) 10 μM or 100 μM or 1 mM omadacycline; (iii) 10 μM or 100 μM or 1 mM omadacycline co-applied with 100 μM muscarine.78. A positive response was defined as (at least) a doubling of the tongue EMG signal. Tongue EMG response to 5-HT occurred within 14 min and 45 s ± 31 s of switching to 5-HT microperfusion medium, consistent with previous studies78.At the end of each experiment in each protocol, 10 mM 5-HT  was microperfused into the HMN as a positive control to confirm the responsivity of the HMN and its ability to respond to the pharmacological interventions3COOH, 1 M NACH3COO, 50% ethanol). A bright field microscope attached to a charge-coupled device (CCD) camera  was used to capture images using the Infinity Capture software . The captured images of stained sections were cross referenced to a standard stereotaxic atlas of the rat brain79 to identify the anatomical placement of the microdialysis probes.At the end of the experiments, the rats were overdosed with 5% isoflurane for 20 min. The heart was then perfused with 30 mL of 0.9% saline and 30 mL of 10% formalin. The brainstem was removed and fixed in 10% formalin for a minimum of 24 h at room temperature for subsequent histological processing. Three days prior to tissue sectioning, the brainstem was transferred into a 30% sucrose solution for cryoprotection and stored at − 4 °C. Sectioning of the brainstem was performed in − 20 °C at 50 μm thickness using a cryostat . The histological sections were then mounted on plus microscope slides , sealed with Cytoseal 280 , and stained with neutral red dye  for each breath, quantified in volts, tabulated in a spreadsheet and matched to the intervention at the HMN80. The tongue EMG signals were quantified as tonic activity  and within-breath phasic activity (peak inspiratory activity minus tonic activity). Mean diaphragm EMG amplitudes  and respiratory rates were also calculated80.Signals recorded during the last minute of each intervention at the HMN were used for data analysisSigmaplot . To test for differences between multiple interventions in each animal, a one-way analysis of variance with repeated measures (ANOVA-RM) was performed followed by Dunnett’s test for post hoc comparisons. If data were not normally distributed, a one-way ANOVA on ranks was performed followed by Dunnett’s test for post hoc comparisons. For comparisons between protocols a one-way ANOVA was performed on normalized data. Two-way ANOVAs were used to identify sex effects on the responses to the interventions, with Holm–Sidak tests for pairwise post hoc comparisons. Unpaired t-tests were also performed as appropriate where indicated. For all tests, differences were considered significant if the null hypothesis was rejected at p ≤ 0.05.Analyses were performed using


In recent decades, Greenland's peripheral glaciers have experienced large‐scale mass loss, resulting in a substantial contribution to sea level rise. While their total area of Greenland ice cover is relatively small (4%), their mass loss is disproportionally large compared to the Greenland ice sheet. Satellite altimetry from Ice, Cloud, and land Elevation Satellite (ICESat) and ICESat‐2 shows that mass loss from Greenland's peripheral glaciers increased from 27.2 ± 6.2 Gt/yr (February 2003–October 2009) to 42.3 ± 6.2 Gt/yr (October 2018–December 2021). These relatively small glaciers now constitute 11 ± 2% of Greenland's ice loss and contribute to global sea level rise. In the period October 2018–December 2021, mass loss increased by a factor of four for peripheral glaciers in North Greenland. While peripheral glacier mass loss is widespread, we also observe a complex regional pattern where increases in precipitation at high altitudes have partially counteracted increases in melt at low altitude. Greenland peripheral glacier mass loss increased from 27.2 Gt/yr (February 2003–October 2009) to 42.3 Gt/yr (October 2018–December 2021Mass loss increased by a factor of four for peripheral glaciers in North Greenland during February 2003–December 2021Enhanced precipitation in northeast Greenland during October 2018–December 2021 resulted in ice thickening at higher elevations Ice loss estimates from satellite gravimetry, which cannot separate the peripheral glaciers from the contiguous ice sheet, are often blended with altimetry and mass‐budget ice loss estimates that only sample the contiguous ice sheet. Satellite gravimetry‐based community assessments of Greenland's recent sea level rise contribution note that they effectively employ “Greenland ice loss” as being synonymous with “Greenland ice sheet ice loss” . The most recent laser altimetry estimates of Greenland peripheral glacier ice loss pertain to the 2003–2009 Ice, Cloud, and land Elevation Satellite (ICESat) observational period. These estimates range between 28 ± 12 and 44 ± 18 Gt/yr of ice loss, depending on which glaciers are classified as “peripheral” . We correct for GIA using the GNET‐GIA empirical model of Khan et al. . For eacg et al.  for elasg et al.  with a rg et al. .2.2https://nsidc.org/data/atl06  Release 004 retrieved from 2.3We fill the gap between ICESat and ICESat‐2 satellite missions by merging elevations from both missions. We estimate elevation changes for Greenland's peripheral glaciers by performing a crossover analysis between ICESat and ICESat‐2 data. We use all available ICESat data points from October 2008 to October 2009 and ICESat‐2 data points from October 2018 to April 2019. Our elevation changes of dh point measurements span between 9.0 and 10.5 years. We estimate elevation change rates based on dh differences and apply corrections for elastic uplift, GIA, and firn compaction  is ∼18% smaller than the 88,083 km2 of Citterio and Ahlstrøm . Given that Greenland's peripheral glaciers comprise 4% of land ice by area (Pedersen et al., 4.2Peripheral glacier mass loss in North Greenland has effectively quadrupled over the observational period, from 6.7 ± 1.6 Gt/yr during the February 2003–October 2009 ICESat period to 26.1 ± 1.4 Gt/yr during the October 2018–December 2021 ICESat‐2 period. The large mass loss rate during the ICESat‐2 period is strongly influenced by the major melt event in 2019 (Sasgen et al., Figures The North Greenland peripheral glacier mass loss during the October 2018–December 2021 period is clearly influenced by the extreme 2019 melt season. This extreme summer melt appears to have been caused by two compounding factors: relatively low winter snowfall, which preconditioned ice for an earlier and longer melt season, and relatively stable summer high‐pressure systems, with enhanced melting through increased northward advection of midlatitude air masses (Hanna et al., 5We present the first comprehensive laser altimetry‐based assessment of ice loss from all of Greenland's land ice, including both peripheral glaciers and the Greenland ice sheet, spanning the period February 2003–December 2021. While peripheral glaciers comprise only a small portion of Greenland's land ice area, mass loss is increasing faster in relation to the ice sheet. In North Greenland especially, peripheral glacier mass loss has increased fourfold over the satellite observation period. In recent years, this area of glaciers has contributed to approximately 10% (Table Greenland's peripheral glaciers contain an estimated total ice volume of 4.7 ± 0.7 cm of sea level equivalent (Huss & Farinotti, The trends and variability in Greenland's peripheral glacier mass loss that we document are clearly complex in both space and time. Better scientific understanding of the presatellite era mass balance of Greenland's peripheral glacier population would place contemporary mass loss rates into a better temporal context. At present, geodetic estimates of Greenland's peripheral glacier mass balance are a missing component of the 20th century global sea level budget.Supporting Information S1Click here for additional data file.


West Antarctica has experienced dramatic ice losses contributing to global sea-level rise in recent decades, particularly from Pine Island and Thwaites glaciers. Although these ice losses manifest an ongoing Marine Ice Sheet Instability, projections of their future rate are confounded by limited observations along West Antarctica’s coastal perimeter with respect to how the pace of retreat can be modulated by variations in climate forcing. Here, we derive a comprehensive, 12-year record of glacier retreat around West Antarctica’s Pacific-facing margin and compare this dataset to contemporaneous estimates of ice flow, mass loss, the state of the Southern Ocean and the atmosphere. Between 2003 and 2015, rates of glacier retreat and acceleration were extensive along the Bellingshausen Sea coastline, but slowed along the Amundsen Sea. We attribute this to an interdecadal suppression of westerly winds in the Amundsen Sea, which reduced warm water inflow to the Amundsen Sea Embayment. Our results provide direct observations that the pace, magnitude and extent of ice destabilization around West Antarctica vary by location, with the Amundsen Sea response most sensitive to interdecadal atmosphere-ocean variability. Thus, model projections accounting for regionally resolved ice-ocean-atmosphere interactions will be important for predicting accurately the short-term evolution of the Antarctic Ice Sheet. Systematic satellite, ocean and atmosphere records show the pace and extent of melting in West Antarctica vary by location, with glaciers flowing to the Amundsen Sea most sensitive to atmosphere‒ocean variability atop a marine ice-sheet instability. Marked and accelerating losses from Pine Island and Thwaites glaciers into the Amundsen Sea, especially, may signify the onset of an irreversible ice-sheet collapse, with important implications for global sea-level rise4. These phenomena have been ascribed both to event-based atmospheric forcing, such as the possible 1940s El-Niño-triggered ungrounding of Pine Island Glacier Ice Shelf5, and a long-term trend in forcing over the 20th Century6. Previous modelling of Thwaites Glacier’s evolution has signalled that “bed topography controls the pattern of grounding-line retreat, while oceanic thermal forcing impacts the rate of grounding-line retreat”7, but to date, observations of this relationship are lacking beyond the Amundsen Sea Embayment. Several studies have shown that much of West Antarctica’s Pacific coast has experienced ice loss over the satellite era, evinced by ice-flow acceleration9, ice thinning11 and retreat of the grounding line13. However, these previous studies have not been performed over consistent timescales all along West Antarctica’s Pacific-facing margin, limiting our ability to evaluate how atmospheric and oceanic forcing regulate ice-sheet losses.Three decades of continuous Earth observation have shown significant ice loss from West Antarctica in response to ocean-driven ice-shelf melt and consequent thinning and recession of inland iceHere, we present a comprehensive analysis of glaciological change across the Pacific margin of West Antarctica for the period 2003-2015. We quantify changes in grounding-line position along 94% of the ~3500-km coastal margin, and compare these records to contemporaneous changes in ice-surface velocity and ice-sheet mass loss. We then assess the correspondence of the observed glaciological changes with outputs from a suite of atmosphere and ocean reanalysis datasets.18, which we have augmented to derive a complete record of grounding-line migration rate change along the entire Pacific margin of West Antarctica  occurred within Eltanin Bay  observations, over the periods 2003-2008 and 2010-2015 , Thwaites (84 m yr−1), Pope (126 m yr−1) and Smith/Kohler (107 m yr−1) basins, while along Getz Ice Shelf, grounding-line retreat slowed by 24 m yr−1 on average , the observed slowdown in grounding-line retreat was largely associated with a decrease in the pace of ice-flow acceleration, including at and inland of the Pine Island, Thwaites and Smith glaciers' grounding lines  and later (2010–2015) periods analysed, ECMWF ERA5 reanalysis data (Methods) reveal that there was a significant decrease in mean sea-level pressure  to the coast, and hence the intensification of opposing easterlies that we observe implies suppressed CDW access to the Amundsen margin in 2010-2015. No region-wide CDW records exist spanning 2003-2015, but Ekman vertical velocity  reduced significantly (implying less upwelling) near the entrance of Pine Island Trough East  also occurred near the mouth of the tributary Pine Island Trough West.Previous work−1) detected around the entrance of the deep continental-shelf-bisecting Belgica Trough , sea-level pressure in the region of the Amundsen Sea Low is lower24. Increasingly positive SAM + conditions between 2003 and 2015 entclass1pt{minimaentclass1pt{minima44.Our results provide spatially extensive and systematic observations that the glaciological evolution of West Antarctica’s Pacific-facing margin between 2003 and 2015 was controlled by the variability with which CDW accessed glacier fronts. Ice margins fringing the Ross Sea changed little reflecting a general barrier to CDW ingress; ice response along the Amundsen Sea was highly sensitive to CDW fluctuations driven by inter-decadal variability in the position of the Amundsen Sea Low; while grounding lines meeting the Bellingshausen Sea retreated progressively in response to pervasive and persistent CDW forcing less susceptible to atmospheric forcing. Our data are now available to underpin observationally-constrained modelling experiments to elucidate and predict short-term atmosphere-ocean-induced variable rates of West Antarctic ice loss superimposed onto the longer-term projected retreat trend45, hence our focus on this time period. Other studies have mapped different proxies for the grounding line since 2015 using independent techniques, including altimetry and machine learning applied to InSAR observations47. However, reconciling grounding-line location proxies identified by different techniques is subject to bias; hence we chose not to extend our study period beyond 2015.An analysis of glaciological, atmospheric and oceanic changes along the entire Pacific-facing margin of West Antarctica must be underpinned by systematic records. Prior to this study, we generated grounding-line location records between 2003 and 2015 along West Antarctica’s Marie Byrd Land coast using optical satellite-based techniques48. For this study, we primarily used optical satellite imagery due to its superior temporal and spatial coverage over the full period 2003–2015 compared with observations from other sensors.While no satellite-based technique can directly image the grounding line, various proxies can be used depending on the sensors available for a given timeframeIb48), building upon previous studies that have shown that migration of 49. We also tracked the seaward limit of grounded glaciers draining directly to the ocean (i.e. those without an ice shelf). We used imagery acquired by the Enhanced Thematic Mapper  and Operational Land Imager  sensors on-board Landsat 7 and Landsat 8, respectively, in our analysis . Grounding lines were mapped using all available cloud-free satellite imagery acquired during the height of austral summertime , and represent the mean summertime grounding-line location for each year. Epochal rates of net grounding-line migration  binned into 30 km segments along the coast were then calculated using recently documented techniques45 , and these derived values were subsequently used to ascertain the overall between-epoch uncertainty, Positional uncertainties associated with our Landsat-derived grounding lines, calculated on a per-segment basis and derived as a function of grounded-ice boundary classification and sensor-specific geometric errors, are estimated to range between 32 and 500 m along the coast  records of the ice-sheet-shelf limit of tidal flexure, Point F12 , Sentinel-1a/b16 (2016) and COSMO-Skymed14  observations, and from coarser-resolution speckle-tracking applied to TerraSAR-X images15  where no publicly available DInSAR observations exist  and ~1500 m (TerraSAR-X)50, we estimate combined DInSAR-DInSAR, DInSAR-TerraSAR-X and TerraSAR-X-TerraSAR-X sensor-based epochal uncertainties to be 141, 1503 and 2121 m, respectively  between c. 2003–2008 and c. 2010–2015 , and offers near-complete spatial coverage of coastal West Antarctica as derived from the error-weighted average of all Landsat 8 image-pair velocity fields that have a centre date falling within 20158. For years 2003, 2008 and 2010, where spatial coverage of ice velocity is more limited, we generated stacked grids of ITS_LIVE53 and MEaSUREs52-derived velocity mosaics falling within ± 2 years (for c. 2008 and c. 2010) and ± 4 years (for c. 2003) of our grounding line observations , we first calculated the median standard error  sensor radiometric quality issues inherent in optical-image feature-tracking53, all of which act to limit observation densities leading to potentially biased standard error values. In this instance, formal propagation of biased ITS_LIVE-derived errors with SAR-based estimates would also yield overestimated combined errors relative to the maximum values observed in each input data stack. For these reasons, we double-weighted all SAR-based observations in our calculation of median standard error and, following the metadata associated with the various velocity datasets utilised53, consider all derived uncertainty estimates generated in this study to be a sensitive indicator of relative data quality (i.e. the amount of observations in each data stack) rather than absolute error.Uncertainties in our velocity and acceleration grids  were combined and used to determine total mass-loss trends over the observational period .We examined Antarctic Ice Sheet mass-balance trends derived from Bayesian hierarchical modelling of all existing laser- and radar-altimeter, GRACE and in-situ GPS records acquired over the ice sheet between 2003 and 20151 , and detailed information on the ensemble estimates, calculation techniques and uncertainty quantifications associated with this dataset can be found in ref. For comparison with the trends outlined above, we additionally examined contemporaneous mass-balance trend estimates as derived from the 2016 Ice sheet Mass Balance Intercomparison Exercise (IMBIE)56, which is considered to provide the most accurate depiction of climatic conditions around Antarctica58. The ERA5 data archive was accessed free of charge at: https://cds.climate.copernicus.eu/. The data shown in Fig. Our mean sea-level pressure , and f (s−1), the Coriolis parameter at latitude φ given Earth’s angular velocity, ω (7.292 × 10−5 rad s−1). In this study, positive wE denotes Ekman upwelling. Numerous studies have detailed the close correspondence between Ekman upwelling at the continental-shelf break, on-shelf flooding of CDW, and near-contemporaneous glacier change along this and other regions of Antarctica62, thereby justifying its use in this study.In the absence of high spatial-and temporal-resolution in-situ ocean observations offshore of West Antarctica, we also examined for anomalous changes in Ekman vertical velocity, }wE Fig.  and 6, w65; for example, it does not factor in ocean surface current and sea-ice motion-related interactions that several studies suggest can influence on-shelf Ekman velocities and ocean heat content65. Except for the strong near-shore downwelling signals exhibited near the Getz and Dotson ice shelves  index presented in Supplementary Fig. 69. This dataset is freely available at: https://psl.noaa.gov/data/correlation/oni.data.The monthly El Niño-Southern Oscillation timeseries presented in Supplementary Fig. 70 (ORAS5), the UK Met Office’s Forecast Ocean Assimilation Model-Global Seasonal Forecasting Service 5 version 1372 (FOAM-GloSea5v13), the Euro-Mediterranean Center on Climate Change’s (CMCC) C-GLORS05 reanalysis73 and Mercator Océan’s Global Ocean Reanalysis and Simulations 2 version 474 (GLORYS2v4). All products are posted on a common horizontal and vertical grid , and are based on the Nucleus for European Modelling of the Ocean (NEMO) model75. Each reanalysis product is also constrained by quality-controlled ocean observations provided by the UK Met Office’s EN4 global objective analysis76  or the Coriolis Ocean database for Reanalysis77 (for GLORYS2v4), and are published together as the European Copernicus Marine Environment Monitoring Service’s (CMEMS) Global Ocean Ensemble Physics Reanalysis product78 (GREP). GREP was chosen for use in this study due to its potentially more realistic, ensemble-based representation of the state of the Southern Ocean at any one time rather than, for example, the use of a single reanalysis product which may be subject to unknown bias. GREP is publicly available at https://marine.copernicus.eu/, and further information on this product, its ensemble reanalysis members and their data assimilation strategies can be found in ref. To corroborate our Ekman vertical velocity observations, we also examined corresponding changes in the vertical hydrography of the Southern Ocean fringing West Antarctica using the ensemble median of four independent, eddy-permitting ocean reanalysis products: the ECMWF’s Ocean Reanalysis System 528, we identify CDW as having an ocean potential temperature of between 1–2 °C and a minimum salinity of 34.62 PSU.To examine for changes in CDW upwelling offshore of West Antarctica, we calculated monthly median ocean potential temperature (81. In the Southern Ocean, the observational data assimilated into these reanalyses are also sparse77, but the generally close correspondence between the results we present in Fig. 30 suggests that the reanalyses perform well.We acknowledge that all four of the ocean reanalyses products described above are based on similar NEMO configurations which are not parameterised to account for, for example, sub-ice shelf cavity circulation and the presence of icebergs, both of which may induce structural biasesSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Dear EditorsIt came to our attention that in several places of the manuscript, the name of the S122 phosphorylation site in TFEB protein has been misspelled as S112. The errors and their proposed corrections are indicated below (corrections are needed in 13 places). We apologize for the inconvenience; this correction bears no impact on the meaning of the text.Life Sci Alliance 5(7): e202101239. doi: 10.26508/lsa.202101239. PMID: 35354596; PMCID: PMC8967991.Article: Wróbel M, Cendrowski J, Szymańska E, Grębowicz-Maciukiewicz M, Budick-Harmelin N, Macias M, Szybińska A, Mazur M, Kolmus K, Goryca K, Dąbrowska M, Paziewska A, Mikula M, Miączyńska M (2022 Mar 30) ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway. In the AbstractWhere it reads:S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation.However, we discovered that this activation occurs due to the inhibition of Rag GTPase–dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at It should read:S122. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S122 phosphorylation and prevented TFEB/TFE3 activation.However, we discovered that this activation occurs due to the inhibition of Rag GTPase–dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at In the Results sectionWhere it reads:S112 without having a broad effect on mTORC1 signalingESCRT-I deficiency reduces TFEB phosphorylation at 2+-dependent dephosphorylation, we investigated whether the induction of TFEB/TFE3 in ESCRT-I depleted cells occurs via regulation of mTORC1 kinase signaling. Having observed reduced phosphorylation of S112 that is a direct mTORC1 target , we investigated whether it occurs because of inhibition of general mTORC1 signaling, which would point to a broad starvation response in ESCRT-I–deficient cells . To this end, we compared the regulation of TFEB S112 phosphorylation to that of other described mTORC1 kinase targets , in control or ESCRT-I–depleted cells, under normal growth conditions (EMEM full medium) or upon nutrient deprivation (EBSS medium). In control cells, the phosphorylation signals of all tested targets were easily detected upon EMEM, and their levels were clearly reduced upon EBSS , verifying that all of these phosphorylations are under constant activation in RKO cells. Importantly, the reduction of S112 phosphorylation due to depletion of Tsg101 or Vps28 was as strong as observed for control cells upon EBSS . However, ESCRT-I deficiency did not inhibit phosphorylations of other tested mTORC1 targets . Reassuringly, we also observed reduced phosphorylation of TFEB at S112 but not of Ulk1, S6K, or 4E-BP1 in cells lacking ESCRT-I because of CRISPR/Cas9–mediated Tsg101 depletion . Consistent with no effect on the canonical mTORC1 targets, we found that ESCRT-I depletion did not alter the association of the mTOR protein with LAMP1-positive structures  that is required for general mTORC1 signaling , either upon normal growth conditions or when this association was reduced due to nutrient deprivation. Hence, we discovered a specific response to ESCRT-I deficiency that involves reduced S112 phosphorylation of TFEB, independent of canonical mTORC1 signaling. This pointed to the inhibition of a recently identified mTORC1 pathway that specifically regulates MiT-TFE factors .After excluding the involvement of endolysosomal cholesterol accumulation or CaIt should read:S122 without having a broad effect on mTORC1 signalingESCRT-I deficiency reduces TFEB phosphorylation at 2+-dependent dephosphorylation, we investigated whether the induction of TFEB/TFE3 in ESCRT-I depleted cells occurs via regulation of mTORC1 kinase signaling. Having observed reduced phosphorylation of S122 that is a direct mTORC1 target , we investigated whether it occurs because of inhibition of general mTORC1 signaling, which would point to a broad starvation response in ESCRT-I–deficient cells . To this end, we compared the regulation of TFEB S122 phosphorylation to that of other described mTORC1 kinase targets , in control or ESCRT-I–depleted cells, under normal growth conditions (EMEM full medium) or upon nutrient deprivation (EBSS medium). In control cells, the phosphorylation signals of all tested targets were easily detected upon EMEM, and their levels were clearly reduced upon EBSS , verifying that all of these phosphorylations are under constant activation in RKO cells. Importantly, the reduction of S122 phosphorylation due to depletion of Tsg101 or Vps28 was as strong as observed for control cells upon EBSS . However, ESCRT-I deficiency did not inhibit phosphorylations of other tested mTORC1 targets . Reassuringly, we also observed reduced phosphorylation of TFEB at S122 but not of Ulk1, S6K, or 4E-BP1 in cells lacking ESCRT-I because of CRISPR/Cas9–mediated Tsg101 depletion . Consistent with no effect on the canonical mTORC1 targets, we found that ESCRT-I depletion did not alter the association of the mTOR protein with LAMP1-positive structures  that is required for general mTORC1 signaling , either upon normal growth conditions or when this association was reduced due to nutrient deprivation. Hence, we discovered a specific response to ESCRT-I deficiency that involves reduced S122 phosphorylation of TFEB, independent of canonical mTORC1 signaling. This pointed to the inhibition of a recently identified mTORC1 pathway that specifically regulates MiT-TFE factors .After excluding the involvement of endolysosomal cholesterol accumulation or CaWhere it reads:S112 phosphorylation , although levels of Ulk1 or S6K phosphorylations remained unaffected.Importantly, the inhibition of TFEB/TFE3 nuclear translocation by the active RagC mutant, in control and Tsg101-depleted cells, was associated with a strong induction of TFEB It should read:S122 phosphorylation , although levels of Ulk1 or S6K phosphorylations remained unaffected.Importantly, the inhibition of TFEB/TFE3 nuclear translocation by the active RagC mutant, in control and Tsg101-depleted cells, was associated with a strong induction of TFEB In the legend for Fig 7Where it reads:Ser112 and the indicated canonical mTORC1 targets in cells lacking ESCRT-I (transfected with siTsg101#2 or siVps28#1) as compared with control cells  at 48 h post transfection (48 hpt) upon culture in regular medium (EMEM) or nutrient-deficient medium (EBSS) for 2 h.(A) Western blots showing levels of phosphorylation of TFEB at It should read:Ser122 and the indicated canonical mTORC1 targets in cells lacking ESCRT-I (transfected with siTsg101#2 or siVps28#1) as compared with control cells  at 48 h post transfection (48 hpt) upon culture in regular medium (EMEM) or nutrient-deficient medium (EBSS) for 2 h.(A) Western blots showing levels of phosphorylation of TFEB at Where it reads:Ser112 of TFEB measured by densitometry analysis of Western blotting bands, including those shown above.(G) Western blots showing levels of phosphorylation of the indicated proteins as well as total levels of TFEB, Tsg101, and HA-GST-RagC in ESCRT-I–depleted (siTsg101#2) or control (siCtrl#1) cells at 48 hpt with ectopic expression of the HA-GST-RagC protein (WT or S75L). Vinculin was used as a loading control. The bottom graph shows phosphorylation levels of It should read:Ser122 of TFEB measured by densitometry analysis of Western blotting bands, including those shown above.(G) Western blots showing levels of phosphorylation of the indicated proteins as well as total levels of TFEB, Tsg101, and HA-GST-RagC in ESCRT-I–depleted (siTsg101#2) or control (siCtrl#1) cells at 48 hpt with ectopic expression of the HA-GST-RagC protein (WT or S75L). Vinculin was used as a loading control. The bottom graph shows phosphorylation levels of In the legend for Fig S7Where it reads:S112 phosphorylation and activates TFEB/TFE3 factors independently of general mTORC1 signaling.ESCRT-I deficiency inhibits TFEB Ser112 and phosphorylations of the indicated canonical mTORC1 targets(A) Representative Western blots showing levels of phosphorylation of TFEB at It should read:S122 phosphorylation and activates TFEB/TFE3 factors independently of general mTORC1 signaling.ESCRT-I deficiency inhibits TFEB Ser122 and phosphorylations of the indicated canonical mTORC1 targets(A) Representative Western blots showing levels of phosphorylation of TFEB at


Introduction: Sufficient screw hold is an indispensable requirement for successful spinal fusion, but pedicle screw loosening is a highly prevalent burden. The aim of this study was to quantify the contribution of the pedicle and corpus region in relation to bone quality and loading amplitude of pedicle screws with traditional trajectories.Methods: After CT examination to classify bone quality, 14 pedicle screws were inserted into seven L5. Subsequently, Micro-CT images were acquired to analyze the screw’s location and the vertebrae were split in the midsagittal plane and horizontally along the screw’s axis to allow imprint tests with 6 mm long sections of the pedicle screws in a caudal direction perpendicular to the screw’s surface. Force-displacement curves in combination with the micro-CT data were used to reconstruct the resistance of the pedicle and corpus region at different loading amplitudes.Results: Bone quality was classified as normal in three specimens, as moderate in two and as bad in two specimens, resulting in six, four, and four pedicle screws per group. The screw length in the pedicle region in relation to the inserted screw length was measured at an average of 63%, 62%, and 52% for the three groups, respectively. At a calculated 100 N axial load acting on the whole pedicle screw, the pedicle region contributed an average of 55%, 58%, and 58% resistance for the normal, moderate, and bad bone quality specimens, respectively. With 500 N load, these values were measured at 59%, 63%, and 73% and with 1000 N load, they were quantified at 71%, 75%, and 81%.Conclusion: At lower loading amplitudes, the contribution of the pedicle and corpus region on pedicle screw hold are largely balanced and independent of bone quality. With increasing loading amplitudes, the contribution of the pedicle region increases disproportionally, and this increase is even more pronounced in situations with reduced bone quality. These results demonstrate the importance of the pedicle region for screw hold, especially for reduced bone quality. Dorsal pedicle screw instrumentation is one of the main techniques used for spinal fusion and spinal stabilization . PedicleTo reduce the rate of pedicle screw loosening, alternative trajectories have been proposed, such as the cortical bone trajectory (CBT)  or screwTo quantify screw hold, axial load-to-failure pullout tests , differeThe aim of this study was to qualitatively analyze the local support along traditional pedicle screw trajectories and to quantify the contribution of the pedicle and corpus region in relation to bone quality and loading amplitude.Seven L5 vertebrae of fresh frozen human cadavers were used for this study after approval by the local ethics authorities (BASEC Nr. 2017–00874). Clinical CT-scans  were acquired to exclude fractures and to identify anatomical particularities, such as lumbosacral transitional anomalies. The acquisition parameters included a slice thickness of 1 mm, pixel dimensions of 0.3516 × 0.3516 mm, a peak voltage of 90–140 kVp, an x-ray tube current of 34–72 mA, and exposure times of 1,000–1,232 ms. The convolution kernel Br60s was used. To correct for the effect of different peak voltages, the grey values were approximately corrected to values corresponding to 120 kVp by using the reference values provided by the work of After thawing, the vertebrae were skeletonized, and the pedicle screw entry points were prepared with a bone rongeur . The pedμCT scans of all 14 specimens were acquired  with a voxel size of 35.43 μm. The picture planes of the reconstructions were oriented to align with the screw’s axis using dedicated software (Skyscan 1176 control software) to guarantee the same orientation of the specimens in both the µCT-scans and later biomechanical testing.To achieve reliable specimen fixation and adequate load distribution over a large surface area during testing, Polymethyl methacrylate (PMMA) potting  was used. After wrapping the specimens in plastic foil to prevent potting intrusion , the taiThe projected surface area of the two imprint probes  was measThe screw-imprint measurements were only assigned to the corpus region when they were fully localized in the corpus region . The mis2 (screw diameter of 5 mm and implanted screw length of 40 mm), the screw-bone interface would fail at a total loading amplitude of around 480 N. With the variability in trabecular bone strength ranging from 0.6 to 7.8 MPa  correspond to a maximal stress of 21.3 MPa for the tip piece and 16.8 MPa for the center piece.The projected surface area of the 6 mm long screw tip piece was measured at 16.42 mmFor a qualitative analysis of the results, the parasagittal micro-CT scans in plane with the screw trajectories are plotted in The implanted screw length was measured at an average of 42 mm, 36 mm, and 41 mm for the group with normal, moderate, and bad bone quality. The screw length in the pedicle region compared to the total implanted screw length was measured at averages of 63%, 62%, and 52%, which relates to an average of 68%, 68%, and 55% of the projected screw area being localized in the pedicle region for the three bone quality groups, respectively  2. At a Pedicle screw loosening poses a relevant complication risk after posterior instrumentation of the lumbar spine. Consequently, the aim of this study was to qualitatively analyze the local support of the vertebral body along traditional pedicle screw trajectories in the caudal direction, and to quantify the contribution of the pedicle and corpus region in relation to bone quality and loading amplitude.The overlays of the stress-displacement plots with the parasagittal micro-CT reconstructions along the screw trajectories  illustraThe overall shapes of the load-deflection curves can be grouped into roughly three types of behavior: curves with a rather constant stress absorbance (plateau phase) throughout a large portion of cancellous bone after an initial ramping phase, curves with a slowly progressive stress absorbance in cancellous bone after a similar initial ramping phase and finally, curves with a rather sharp increase in stress absorbance until the end of the imprint test. The first two curve types are typically found in the corpus region and the third type is primarily seen in the pedicle region. Interestingly, in situations with a large enough gap between the screw and the cortical shell, a similar plateau phase can also be seen in measurements of the pedicle region . The stress values at the plateau phase are assumed to be closely related to the local ultimate strength because further displacement can only be achieved through local failure of the trabecular bone structures. Therefore, the specimens with a large plateau phase in the corpus region provide a rather straight-forward method to compare the stress resistance of trabecular bone in relation to bone quality. In specimens with normal bone quality, the plateau phase varies in the region of roughly 5 MPa, while in specimens with bad bone quality the plateau phase ranges at values from 1 to 3 MPa. Although general trend of lower stress absorbance in situations with reduced bone quality can clearly be seen in the presented results, some notable exceptions must be discussed: For example, the corpus region of the left side of specimen #6 shows high local bone density (as seen in the micro-CT image) and, despite the overall bone quality being labeled as bad in this specimen, the local stress resistance in this area is surprisingly good with values exceeding 10 MPa . In contIn the final 1–2 mm of the imprint tests, a rather steep increase in stress absorbance can be seen in virtually all measurements, independent of the location along the screw trajectory and largely unaffected by the previous shape of the load-deflection curve. This rather sharp increase in resistance is interpreted as the result of (trabecular) bone material compaction against the stable cortical shell or vertebral endplate, as well as the known increase of bone mineral density towards the cortical shell in the pedicle region . In situTo compare the contribution of the corpus and the pedicle region quantitatively, the behavior at the measured locations was used to interpolate the missing locations along the screw, which allowed us to virtually displace the whole screw caudally and compute the relative contribution of the corpus and pedicle region, the mean stresses at the screw-bone interface of these two regions, as well as the screw’s displacement.In the situation of low loading amplitudes (100 N acting on the whole screw), no failure at the screw-bone interface is expected and differences in cortical bone stiffness can be assumed to be the primary factor for the local differences in screw support. In this situation, the contribution of the pedicle and corpus region are largely balanced, with the pedicle region providing an average of 55%–58% for all three bone quality groups . InteresWith intermediate loading amplitudes (500 N acting on the whole screw), local failure can occur, and therefore ultimate strength of the bone can be assumed to play a more important role compared to the previously discussed loading situation. While a small increase in the support provided by the pedicle region can be observed in specimens with normal and moderate bone quality, this increase is much more accentuated in specimens with bad bone quality . In addiIn the high loading amplitude situation (1000 N acting on the screw), the trends observed at the intermediate loading situation are emphasized and the relative contribution of the pedicle region is further increased to 71%, 75%, and 81%. This can be explained by the stress resistance of the pedicle region being larger than the stress resistance of the corpus region in this loading scenario. This constellation stands in contrast to the situation with 100 N loading and highlights the importance of the pedicle region for such  high loading amplitudes. The load redistribution can further be seen in the changes of the local stresses from 500 N to 1000 N. At the corpus region, only a relatively small increase of the local stress can be seen (averaging at +35%), while the average increase in the pedicle region is +140%.This work has experienced several limitations. First, the chosen loading condition is a gross simplification of the complex loading conditions that can occur during activities of daily living and represents just one specific loading scenario in which the pedicle screw is loaded perpendicular to its axis in a caudal direction. Additional loading conditions, such as bending or pull-out forces, are not represented and the consequence of different compliance along the screw axis is not included. Furthermore, simple load-to-failure tests were performed and parameters such as fatigue failure or biological adaptation such as the bone remodeling are not considered. The method that was used to interpolate the measurement data to the whole screw is associated with some uncertainty and could interfere with the results. Furthermore, screw insertion depth and with that the distribution of the projected screw surface in the corpus and pedicle region were not standardized, and therefore some variability exists between specimens . Because3 (Replica screw insertion and bandsaw cutting of the specimens could have induced some damage to the vertebral bodies, which could have in turn affected the measurements. Visually, there were no signs of relevant bone damage due to the specimen preparation. Because any potential damage would be affecting all measurement locations to a similar degree, the effect on the results should be minimal. To achieve reliable specimen fixation, PMMA-potting was used, which can generate elevated temperatures due to the exothermic nature of the chemical reaction . While t3 , which w3 . This is3 . TherefoUnidirectional imprint tests of pedicle screw sections perpendicular to the screw surface have been performed to analyze the resistance along the pedicle screws following the traditional trajectory. At low loading amplitudes, the trabecular bone of the corpus region appears to provide slightly more support than the trabecular bone of the pedicle region. This observation could be the result of trabecular bone anisotropy, which could be used to further optimize screw trajectories and implant designs. At higher loading amplitudes, and especially in specimens with reduced bone quality, the contribution of the pedicle region becomes predominant, which can be ascribed to the increasing support of the cortical shell after some screw displacement towards the cortex.To reduce the risk of screw loosening, it could be beneficial to limit the available subsidence distance until adequate resistance is met. To achieve this goal, the distance to the cortical bone could be reduced by placing the pedicle screw closer to the cortex, by selecting a larger screw diameter, or potentially by augmenting the pedicle with bone cement.


Ficus benghalensis is one of the potential medicinal plants which is used locally for the treatment of various ailments such as diabetes, antiasthmatic, and wound healing. To provide a scientific background to these folklores, the current study was designed to evaluate the extract and isolated compound against various enzymes such as ureases, tyrosinase, and phosphodiesterase. The methanolic extract and carpachromene demonstrated a significant urease inhibition effect with maximum percent inhibition of 72.09 and 92.87%, respectively. Regarding the tyrosinase inhibition, the percent antagonist effect of carpachromene and the methanolic extract was 84.80 and 70.98%, respectively. The phosphodiesterase was also significantly antagonized by crude extract and carpachromene with a maximum percent inhibition of 82.98% and 89.54%, respectively. The docking study demonstrated that the carpachromene fits well into the active site of all three enzymes with significant interactions. Carpachromene might possess the potential to inhibit all three enzymes and can effectively treat different diseases associated with the hyperactivity of these enzymes. In conclusion, the crude extract and carpachromene exhibit significant urease, tyrosinase, and phosphodiesterase inhibitory activity which might be used against various diseases. In conclusion, the crude extract and carpachromene exhibit significant urease, tyrosinase, and phosphodiesterase inhibitory activity which might be used against diabetes and bronchoconstriction. Further, the current study provides scientific backup to the folklore (antidiabetic and antiasthmatic) of Ficus benghalensis. Biodiversity is approved with the remedial plant's permanent resource, which provides mankind with a loaded source of drugs . More thFicus benghalensis is an evergreen higher plant. Ficus benghalensis is a member of the family Moraceae  × 100.The samples to be tested were performed as that extract or carpachromene (0.2 rocedure . The perμl of Tris hydrochloric acid buffer with a pH of 8.8 and 20 μl of magnesium acetate (30 mM) to 96-well plates. The above mixture was supplemented with extract or carpachromene (0.2 μg/ml) and 15 μl (1 μl/well) of PDE-1 as the final concentration of 0.000742 U/well, and the mixture was incubated in ELISA at a temperature of 37°C for a period of 30 min. In this assay, magnesium acetate was mixed as PDE-1 cofactor. After the above treatment, 60 μl of the substrate in the form of 0.33 mM bis (p-nitrophenyl) was added. The inhibitory potential of samples was monitored at 410 nm on a microplate reader for 30 min duration. The phosphodiesterase (PDE) inhibitory activity of the extracts and carpachromene was compared with EDTA (standard). The inhibitory activity of substances is determined in the form of % inhibition. It is calculated by using the following formula:The samples to be tested were subjected to a phosphodiesterase (PDE) assay . The ass50 values.The EZ-fit enzyme kinetics software from Perrella Scientific  was used to calculate the ICMolecular docking was performed to study the binding mode of the carpachromene in the active site of urease, tyrosinase, and phosphodiesterase. The crystal structures of tyrosinase urease (PDB ID: 4H9M), an oxy form of tyrosinase (PDB ID: 1WX2), and phosphodiesterase 1B (PDB ID: 5W6E) were taken from the Protein Data Bank. The MOE (2016) software was used to carry out molecular docking studies. The structure of the target was prepared in MOE as well . The eneFicus benghalensis. ESI-MS exhibited m/z 337 (M+H) demonstrating the molecular formula C20H16O5. 1HNMR  δH; 1.48 , 5.77 , 6.53 , 6.55 , 6.80 , 6.92 , 7.90  183.1 (C-4), 164.3 (C-7), 162.1 (C-5), 158.8 (C-2) 156.8 (C-4/), 155.7 (C-9), 128.9 (C-2//), 128.7 (C-2/), 121.4 (C-1/) 115.8 , 114.9 (C-1//), 105.1 (C-6), 104. 8 (C-10), 102.8 (C-3), 95.2 (C-8), 77.9 (C-3//), 27.8 (C-4//), 27.8 (C-5///), respectively. The structure of compound 1  while ethyl acetate (79.12%). Methanol and carpachromene demonstrated 79.12, 82.98, and 89.54% effects, respectively.The samples to be tested were also effective against phosphodiesterase as shown in The isolated compound was docked to urease, tyrosinase, and phosphodiesterase, to estimate the best binding pose. The docking score  and bonding interaction pattern (hydrogen) were used to investigate the docked complexes. Molecular docking analysis revealed that the docking score of phosphodiesterase and isolated compound-complex was −6.732 followed by urease-compound-complex −6.531. The docking score of tyrosinase and compound-complex was −6.215. Ficus benghalensis against different biological enzymes. The results of urease enzyme indicated that methanolic extract and carpachromene showed promising inhibition. The urease is one of the notorious enzymes for the induction of H. pylori, urinary tract infection, and even hepatic encephalitis [Ficus benghalensis is locally used against live inflammation and urinary tract infection [H. pylori is treated with multiple antibiotics for longer times which are associated with side effects and poor patient compliance. To avoid polypharmacy and improved compliance, the search for new, effective, and safe compounds is very essential. Therefore, the further mechanistic study is of utmost essential up to clinical trials. The significant antagonistic effect of methanolic extract and carpachromene against tyrosinase indicates the cosmeceutical effect of these samples. The tyrosinase inhibitors such as kojic acid, azelaic acid, and hyderquin are topical whiting and antipigment dosage form [Ficus benghalensis against urease, tyrosinase, and phosphodiesterase was evaluated by molecular docking, the docking results are in agreement with the experimental activity, the compound docked well in the active site of urease forming three hydrogen bonds, and the best interaction of the compound with the target protein reflects the best inhibitory activity of compound toward the target protein, compared to urease and phosphodiesterase compound forming less hydrogen bond interaction with tyrosinase. Overall, the isolated compound formed significant interactions with all the target enzymes and has the potential to combat the disease associated with this enzyme.The use of plant-based remedies is increasing among the world population due to easy availability and affordability , 29. Thephalitis . The signfection , 33. So age form . In the age form , 35. Theage form , vasodilIn conclusion, the crude extract and isolated compound showed significant urease, tyrosinase, and phosphodiesterase inhibitory potential. Furthermore, the molecular docking studies also confirmed the binding mode of the isolated compound in the active site of urease, tyrosinase, and phosphodiesterase. The docking study shows that the isolated compound fits well into the active site of all three enzymes and makes strong bonds with all three enzymes. Our compound might possess the potential to inhibit all three enzymes and effectively treat different diseases associated with these enzymes. The identified inhibitors can act as lead compounds for the discovery of new treatments for the diseases associated with the disorder of said enzyme.