Bruton’s tyrosine kinase (BTK) is known as a direct regulator of inflammasome, which is an intracellular target to therapeutically modulate innate immunity. Although there is great interest in developing small molecule-based drugs with BTK inhibition, there are only a few drugs available in the market, due to the difficulty of drug discovery and the potential side effects. To select suitable drug compounds to inhibit BTK signaling, molecular drug screening bioassay processes of single ginsenosides integrated with in silico molecular simulation were performed. The experimental results for the ginsenoside compositions (Rb2 and Rb3) exhibited showed that they effectively suppressed the activity of BTK expression in a rational agreement with molecular docking calculations of the compounds against the BTK binding site. They implemented a possible inhibiting effect of BTK signaling through increasing their molecular affinity for targeting BTK, enabling them to be useful in treating BTK-mediated diseases. Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase that plays a critical role in B-cell development, differentiation, and signaling through the induction of a signal transduction that is essential for cell survival and adaptive immunity ,2,3,4,5.However, when BTK is overexpressed by the abnormal signal transmission of BCR in B cells, the BCR signaling system undergoes excessive phosphorylation gradually and causes abnormal B-cell proliferation and pathological autoantibody formation, resulting in systemic erythematous lupus, cancers, rheumatoid arthritis, autoimmune diseases, B-cell malignancies, and inflammatory diseases ,5. In thCurrently, many pharmaceutical companies are struggling to develop BTK inhibitors. However, due to the difficulty of development and the potential side effects, there are only three approved drugs that inhibit BTK: Ibrutinib, Acalabrutinib, and Zanubrutinib. This suggests that the development of new drugs will have an incredible economic and industrial impact. More recently, BTK was recognized as a direct regulator of a major innate inflammatory machinery, the NLRP3  inflammasome, which is an intracellular target for therapeutically modulating innate immunity .Ginsenosides are dammarane-type triterpene saponins derived from ginseng, which have been used for a long time as a preventive supplement or health functional food to improve immunity and vitality . To dateGinseng ginsenosides are classified into three types according to the difference in their structure (location and quantity of sugar moiety) : protopaIn silico molecular docking is a computational method that rationally finds out the protein-ligand binding mode, which is as yet unknown, providing potential binding modes through calculations performed at the atomic level. If the binding mode is identified, it can greatly contribute to the study of protein function or the further development of new drugs that inhibit the function of disease markers ,13. VeryIn this context, by harnessing the drug screening techniques for single ginsenoside compounds with the molecular simulation method, we attempted to select suitable ginsenoside therapeutic candidates which could induce the therapeutic effect of anti-inflammation by inhibiting BTK expression and also verify their affinity to target BTK. For the identification of the BTK inhibitory activity of ginsenoside-derived candidate substances and the screening of them to show pharmacological efficacy, the BTK-inhibitory activity of ginsenoside compounds and their in vitro BTK inhibitory activity was measured as the inhibition rate (inhibition %) of the decreased phosphorylation rate compared to the BTK activity without ginsenoside treatment in a FRET assay. Firstly, through repeated experiments, the BTK concentration when the phosphorylation rate of tyrosine causing the FRET reaction was 20%–40% corresponded to the optimal concentration of BTK, 0.625 μg/mL, which corresponded to the 30.63% phosphorylation rate .50 levels that inhibited BTK activity were 61.05 μM, 9.27 μM, 50.96 μM, 104.30 μM, and 136.35 μM, correspondingly (IC50 of Ibrutinib: 0.63 nM).Thus, the 20 candidate substances derived from ginsenoside were treated with 0.625 μg/mL of purified BTK at concentrations of 25, 50, and 100 μg/mL, and the BTK inhibition activity was confirmed by the BTK inhibition % of the candidates through repeated experiments . AccordiIn spite of the promising pharmaceutical efficacies of the ginsenoside compounds, the therapeutic potential of ginsenosides is largely restricted by their lowering bioavailability when orally distributed (< 5%) due to their undesirable physicochemical properties, such as low water solubility, low biomembrane permeability, instability in the gastrointestinal fluid, and extensive body metabolism . Therefo50 value (61.05 μM), and the absorbance at 540 nm was increased by 0.271 compared to the control. The absorbance of Rh2 at 0.4 mM and 0.6 mM was increased by 0.037 and 0.111, respectively. Based on the above results, it was confirmed that the lower the concentration of Rh2 was, the better it was dissolved in the medium. Rb2 in the medium was transparent even at 0.2 mM, showing the lowest IC50 value (9.27 μM), and the absorbance at 540 nm was increased by 0.042 compared to the control group. The absorbance at 0.6 mM and 1.0 mM was decreased by 0.007 and 0.006, respectively, confirming that Rb2 is soluble in the medium even at a high concentrations. Rb3 at 0.9 mM was also transparent, showing a high IC50 value (50.96 μM), and the absorbance at 540 nm was increased by 0.047 compared to the control group. The absorbance of the 2.7 mM sample was reduced by 0.004 compared to the control, confirming that Rb3 is soluble in the medium even at a high concentration. Rb5 in the medium was transparent at 1.9 mM, showing a high IC50 value (104.30 μM), and the absorbance was increased by 0.032 compared to the control. The absorbance at 3.8 mM was reduced by 0.021 compared to the control, confirming that Rb5 is soluble in the medium even at a high concentration. The PPT at 1.7 mM was intensively turbid, showing the highest IC50 value (136.35 μM), and the absorbance was increased by 1.118 (over saturated OD) due to the murky solution compared to the control group. At 0.5 mM and 0.8 mM, the absorbances relating to concentrations of about 3 and 6 times the IC50 values were increased by 0.309 and 0.508, respectively, compared with the control group. This confirmed that the lower the PPT concentration, the better it was dissolved in the medium. PPD samples at 0.2 mM, 0.5 mM, 1.0 mM, and 1.5 mM were also turbid and showed spectra increased by 0.155, 0.442, 0.835, and 1.153, respectively, due to the turbidity-based OD interference. Overall, the compounds of Rb2, Rb3, and Rb5 were soluble in the medium at a high concentration, and Rh2 was soluble in the medium at a low concentration, while insoluble at a high concentration. Both PPT and PPD were poorly soluble in the medium at higher concentrations and were less soluble than other compounds at low concentrations, which is consistent with their hydrophobic chemical structures. Therefore, they are undesirable for use in further tests due to their poor solubility.Rh2 in the medium was slightly soluble at 1.2 mM, showing a high ICConsidering the above results of the sample preparations to be treated in cells, the specific concentrations of the ginsenoside compounds in the cellular assays were employed in further assays. Although BTK is almost constant in cells, the expression level of p-BTK varies depending on the degree of BTK activation and inhibition, and thus the expression level of p-BTK is based on the expression of BTK (p-BTK/BTK). By comparing the p-BTK/BTK ratio in the cells of the five ginsenoside candidates confirmed by ELISA, the five ginsenoside candidates  at a solubility-dependent concentration level exhibited substantial BTK inhibitory activity compared to that of Ibrutinib or the DMSO-treated group . Among tMoreover, by comparing the ratio of p-BTK/BTK based on the expression of p-BTK, BTK, and β-tubulin, the effect of inhibiting BTK against ginsenoside compounds in cells was confirmed. The BTK inhibitory efficacy of the drug candidates in Ramos cells was evaluated by Western blotting, as presented in Taken together, the five selected candidates exerted their efficacy primarily by blocking BTK-mediated signaling in the cellular environment. Yet, considering their solubility in water compared with the other compounds, it is assumed that Rb2 and Rb3 would be promising candidates in providing a pharmacological potential to enable the treatment of BTK-mediated diseases. Tumor growth in solid malignancies in the tumor environment might be also inhibited by the candidates. Moreover, since these compounds are derived from natural products, it is assumed that they are less toxic, less drug resistive, and have less probability of causing side effects, while the common side effects of Imbruvica include infection, anemia, neutropenia, thrombocytopenia, bruising, and cytopenia.A molecular docking simulation technique was exploited to predict the binding potential and binding mode of the ginsenosides over BTK domains, enabling the envisaging of how a compound may bind to a corresponding site when clusters form constant structures that frequently repeat at a specific site. Based on the combined form, the docking hit (genetic optimization for ligand docking (GOLD) fitness score) was calculated to estimate the bonding force that was obtained through the sum of energy, such as protein-ligand hydrogen bond energy , protein-ligand van der Waals (vdw) energy , ligand internal vdw energy , and ligand torsional strain energy .In general, the higher the docking score, the more energetic the interaction between the protein and the ligand is, representing that the compound fits well into the binding site within the protein. However, as the size of the calculated ligand increases, the number (degree of freedom) of the rotatable bonds increases, and thus it is difficult to effectively use this for conformation compared to relatively small ligands, reducing the accuracy of the docking result.The docking result of each ginsenoside was classified into one cluster of similar binding modes repeatedly appearing among 50 binding modes. Among them, the binding mode with the highest GOLD fitness score was selected as a representative (red colored), as demonstrated in The best way is to have an interpretation that matches the experimental results either way. Of these, the docking scores of Rb2 and Rb3 are 55.11 (cluster 1) and 68.90 (cluster 2), respectively, which are in good agreement with the selected final structures in the experimental section.The ginsenoside Ra1 exhibited the highest docking score of 73.92 in cluster 2, containing 7 out of 50 bonding modes. As shown in d) of various BTK constructs binding to Rb2 or Rb3 are required; this is under investigation in our group. The results indicate that these 3D-docking models with potential predictive ability could prospectively be used in diverse ligand structure modification and optimization. Moreover, to better understand the molecular kinetic interaction, biophysical experiments to determine thermodynamic parameters such as the binding affinity  were obtained from MODNBIO Inc. . The Ibrutinib (PCI-32765)  was used as a control. Ramos cells  and the human Burkitt’s lymphoma cell line were cultured in suspension by Roswell Park Memorial Institute (RPMI) 1640 medium  with 10% fetal bovine serum (Gibco) at 37 °C in 5% CO6) were famished of serum for 1 h and treated with ginsenosides for 1 h, and then washed with phosphate-buffered saline , followed by stimulation with 1 μg/mL of goat F(ab’)2 anti-human IgM  for 10 min on ice. The cells were then lysed in a radioimmunoprecipitation assay (RIPA) buffer  and the proteins were separated by SDS-PAGE  and transferred to polyvinylidene fluoride membranes . The proteins were detected with anti-BTK Y223 , anti-BTK  and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH)  antibodies. The protein signals were monitored by chemiluminescence using Enhanced ChemiLuminescence (ECL) western blotting detection reagent (EMD Millipore), and the scanned blots were quantified using a Multi-Image analyzer . The phosphorylation % of each lane was determined using Multi-Gauge software (Fujifilm). The IC50 value was measured using control α-IgM+ set at 100% and control α-IgM− set at 0%.The Ramos cells . In addition, the change in absorbance at 540 nm compared to the control was measured by using the medium not treated with the ginsenoside compound as a control.In order to evaluate the BTK inhibitory activity of the ginsenoside compound, a kinase assay using a Z’-LYTE Kinase Assay Kit from Thermo Fisher Scientific was performed. A fluorescent microplate reader was used to measure the Fluorescence Resonance Energy Transfer (FRET) between coumarin and fluorescein dyes. The assay was optimized for BTK, as described in the manufacturer’s protocol. The BTK activity (%) was evaluated by measuring the ratio of the emission signal (445 nm/520 nm) according to the phosphorylation % of the substrate by BTK. When BTK is activated or inhibited, the phosphorylation % of BTK is increased or decreased accordingly, which can be confirmed by the amount of phosphorylated BTK in the cell. After treating the ginsenoside compound on the cells, the amount of BTK inhibition of the ginsenoside compound was compared and analyzed by the amount of phosphorylated BTK in the cell. Then, the cells were cultured with a ginsenoside compound at a concentration higher than the value using a biochemical kit, and the absorbance was measured at 450 nm through a cell-based phosphorylation enzyme-linked immunosorbent assay (ELISA) to measure the phospho-BTK (p-BTK). The BTK to GAPDH expression ratio was confirmed.4) were cultured in 96-well cell culture plates. After adding the compounds onto the cells, the absorbance was measured at 450 nm through a cell-based phosphorylation ELISA to confirm the expression of p-BTK, BTK, and GAPDH. All ginsenoside compounds, including a positive control group (Ibrutinib), were dissolved in DMSO. Free DMSO was used as a negative control group. The expression amount of p-BTK was compared and analyzed based on the expression of BTK and GAPDH to confirm the BTK inhibitory efficacy of the ginsenoside compounds in cells. The expression level of each p-BTK and the expression level of BTK were compared and analyzed using the ratios (p-BTK/GAPDH and BTK/GAPDH) based on the expression level of each β-tubulin. Namely, the p-BTK/BTK ratio means the (p-BTK/GAPDH)/(BTK/GAPDH) ratio, which is simply expressed as p-BTK/BTK.The Ramos cells (~1 × 106) were cultured in 6-well cell culture plates. Both the control and ginsenoside compounds were dissolved in DMSO. For the Western blotting, the α-IgM- groups and α-IgM+ groups in the presence of DMSO were used as the non-stimulation control and stimulation control, respectively, following by the quantitation of the expression ratio of p-BTK, BTK, and β-tubulin. The expression level of each p-BTK and the expression level of BTK were compared and analyzed using the ratios (p-BTK/β-tubulin and BTK/β-tubulin) based on the expression level of each β-tubulin. Namely, the p-BTK/BTK ratio means the (p-BTK/β-tubulin)/(BTK/β-tubulin) ratio, which is simply expressed as p-BTK/BTK.The Ramos cells (~3 × 10To perform molecular docking calculations between a ginsenoside compound and a BTK molecule, a sequential processing of precise three-dimensional conformation is required . By compDespite the numerous proven works on the pharmaceutical research into diverse types of ginsenosides derived from natural products, there are few reports on systematic methods of molecular drug screening for single ginsenosides to target a specific disease marker, BTK. In particular, the accumulated data are still insufficient for combined molecular bioassays and simulation modelling. Consequently, in this study, a high-throughput drug screening method for enhancing the disease selectivity of diverse ginsenosides was demonstrated, accompanied by a reliable, integrative approach with experimental biomolecular assays and molecular simulation techniques. Specially, the ginsenosides Rb2 and Rb3 presented the effective targetability of BTK, involving the possible therapeutic effects of anti-inflammation by inhibiting the BTK signaling through increasing the molecular affinity to target proteins such as NLRP3 inflammasome and BTK. Given the promising therapeutic potential of Rb2 and Rb3, further studies including in vivo systems are anticipated to reveal a detailed mode of action in multiple signaling pathways that is different from existing treatments. This will maximize the clinical potential of ginsenosides as a molecular target-specific natural medicine, thereby further benefiting global health.


To compare the safety and accuracy of the 3D-printed operation guide template technique and the free-hand technique in the placement of the S2-alar-iliac (S2AI) screw.We conducted a retrospective analysis of 47 patients undergoing S2AI screw placement in our hospital, divided into the 3D-printed operation guide template group and the free-hand screw placement group. The duration of single S2AI screw placement was documented in all patients. A postoperative CT scan was performed to assess screw placement effectiveness according to the distance from the screw tip to the breach of the cortical bone wall.In total, 42 screws were placed in the guide template group, with an average screw placement duration of 151.6 ± 44.8 s. Screw placement grading was as follows: 40 screws in grade 0, two in grade 1, and none in grades 2 and 3. This grading resulted in excellent and good rates of 95.2% and 100%, respectively. In total, 52 screws were placed in the free-hand group, with an average screw placement duration of 138.3 ± 45.9 s. Screw placement grading was as follows: 42 screws in grade 0, seven in grade 1, three in grade 2, and none in grade 3. This grading resulted in excellent, good and acceptable rates 80.8%, 94.2% and 100%, respectively. Screw placement duration did not significantly differ between the groups (p > 0.05). The excellent rate of screw placement was greater in the guide template group than in the free-hand group (p < 0.05), but the good and acceptable rates did not significantly differ between the two groups (p > 0.05).Both techniques can be applied to S2AI screw placement. The 3D-printed guide technique is superior to the free-hand technique in terms of safety and accuracy. Pelvic internal fixation is the key to treating adult spine deformity, severe pelvic inclination, and severe lumbar spondylolisthesis . Due to Due to the complexity of the pelvic structure, deviations in the position of the screw may cause irreparable harm. Amir et al.  conducteThe approaches of S2AI screw placement have mainly included the free-hand technique, intraoperative navigation, and robot-assisted screw placement –19. The We retrospectively analyzed the data of 47 patients treated with S2AI screws in our hospital from March 2015 to December 2018. Before surgery, X-ray and computed tomography (CT) scans were performed to exclude deformity and cancer of the sacrum and pelvis. There were 15 men and 32 women with an average age of 57.8 ± 8.4 years (range: 38–74 years). According to the screw placement technique, the patients were divided into the 3D-printed operation guide template group and the free-hand screw placement group.The time it took to insert each screw was recorded. All patients underwent CT examination after surgery, and the axial images were used to observe the results of screw placement. The evaluation of screw placement was based on the grading system proposed by Oh et al. . This grThe same surgical team performed all the surgeries in this study. This study was approved by the Ethics Committee of the 920 Hospital of Chinese People's Liberation Army Joint Logistics Support Force. All patients were informed and signed a surgical consent before surgery.Before surgery, CT scan of the lumbosacral spine was performed routinely to obtain the raw data in DICOM format, which were imported into Mimics 19.0 software  to reconstruct three dimensions model of the spine and pelvis. The MedCAD module was used to design the screw trajectory of the template. The screw trajectory was adjusted in the sagittal, coronal, and axial views . Next, a spherical probe was used to check the integrity of the screw trajectory, and a threading tap was used for tapping. According to the size of the screw trajectory measured before surgery, appropriate screws were selected for screw placement, and the screw placement duration was documented.In the free-hand screw placement, the point 1 mm lateral to the site 2–4 mm below the lower edge of the S1 posterior sacral foramina was selected as the entry point. A grinding drill was used to grind off part of the cortical bone around the entry point, and a pedicle drill was used to make the first approach. While making the approach, the anatomical landmarks were used to determine the tilt angles to the head and lateral side. In the vertical view of direct vision of the surgeon, the tilt angle toward the head for the screw placement was determined according to the projection of the reverse extension line of the screw trajectory in the coronal plane of the human body. That is, the pedicle drill was projected to the site from the lower edge of the superior articular process of the first sacrum to the first posterior sacral foramina on the contralateral side of the insertion point. The orthopedic drill was attached tightly to the upper edge of the superior spine ligament to determine the lateral-tilt angle. The pedicle drill advanced to the sacroiliac joint, and then an orthopedic hammer was used to strike the drill to pass through the joint. A spherical probe was used to explore the four walls and the depth of the bone trajectory. That the four walls were bony walls and the depth was 70–90 mm indicated the achievement of correct screw trajectory. Finally, tapping was performed along the screw trajectory, and then the appropriate S2AI screws were placed. The duration of screw placement was recorded. Intraoperative fluoroscopy of the pelvis  was performed to finally confirm the position of the screws.At the completion of the screw placement, spinal canal decompression, tuberculosis lesion removal, and scoliosis correction may be performed according to the patients' condition. We installed a connecting rod to complete the orthopedic fixation. The surgical wound was washed, one or two drainage tubes were routinely placed, and the instruments and gauzes were counted. The operative incision was closed in layers, and the procedure ended.2 test was used to compare the qualitative data between the two groups. Two tailed α = 0.05 was set as the test level.SPSS 21.0 software was used for data analysis. The Kolmogorov–Smirnov test was used to determine whether the quantitative data were normally distributed. The data with a normal distribution are expressed as In the guide template group, 21 sets of individual operation guide templates were designed. All patients underwent S2AI screw placement with assistance of the operation guide template. During all surgeries, the operation guide template fit the bone surface securely, and the operation went smoothly. The duration of single screw placement was 108–340 s, with average of 151.6 ± 44.8 s. Postoperative CT scans were performed to score the screw placement according to the screw grading system and showed 40 screw placements of grade 0, two of grade 1, none of grade 2, and none of grade 3. The excellent rate was 95.2%, and the good rate was 100%. Two of the screws passed through the sacroiliac joint and breached the medial cortex of the ilium.In the free-hand group, a total of 52 S2AI screws were designed for 26 patients. By referencing anatomical landmarks, we performed free-hand screw placement during each surgery. The duration for single screw placement was 75–243 s, with average of 138.3 ± 44.8 s. Postoperative CT scans were performed to score the screw placement according to the screw grading system and showed 42 screw placements of grade 0, seven of grade 1, three of grade 2, and none of grade 3. Breaching of the screw trajectory was observed in 10 screw placements. Eight of them were located at the medial iliac edge of the sacroiliac joint, and two were at the outer iliac edge of the sacroiliac joint. The excellent rate of screw placement was 80.8%, the good rate was 94.2%, and the acceptable rate was 100%.The diameter and length of the S2AI screw were 7.0–8.0 mm and 70–90 mm, respectively. Screw breaches from the trajectory were seen in some cases, but there were no related adverse events. There was no significant difference in the screw placement duration between the guide template group and the free-hand group (p > 0.05). The excellent rate of screw placement in the guide template group was greater than that in the free-hand group (p < 0.05), but the good rate and the acceptable rate of screw placement were not statistically significant between the two groups (p > 0.05), as shown in Table The patient was a 53-year-old woman with adult degenerative scoliosis Fig. a, b. BefA 38-year-old man was diagnosed with spondylolisthesis (L5) and spinal instability (Fig. The S2AI screw technique has many advantages, such as reduced trauma, fewer complications, and reliable fixation. Therefore, it has been rapidly developed and applied in spine and pelvic fixation surgery. However, important blood vessels, nerves, and other structures are adjacent to the S2AI screw trajectory, which can lead to disastrous consequences in the case of accidental injury. This study compared two screw placement methods for S2AI screws, namely, screw placement with a 3D-printed operation guide template and free-hand screw placement. After the operation, CT scans were performed to evaluate the two methods of screw placement according to whether the S2AI screw penetrated the screw trajectory and the degree of the screw trajectory.In free-hand screw placement group, the method we chose was different from that in previous studies –18. In tIn recent years, the 3D-printed operation guide template technique has been rapidly evolving in spine surgery. The advantages of the 3D-printed operation guide template include high accuracy, no impact of anatomical signs, low cost, and short production cycle , 25. TheIt should be noted that there are certain errors in the reconstruction of the spine and pelvis model, operation guide template design, 3D printing, and intraoperative application, which may impact the accuracy of screw placement. Therefore, the inner diameter of the guide navigation tube should be slightly larger than that of the drill bit. If a drill bit with a diameter of 2.5 mm is selected, the inner diameter of the navigation tube should be 2.7 mm. Before using the guide plate, it is necessary to fully expose the attachment surface of the guide plate, so that the guide plate can be fixed firmly, and slowly drill along the navigation tube to avoid a change in the screw trajectory caused by the instability of the guide plate or the sliding of the drill bit. Since the screw placement point is fixed after the guide plate is designed, the screw position of the lower lumbar segment and S1 vertebral body should be considered during the operation so that the screw placement point of the S2AI screw is in a straight line to facilitate fixation of the connecting rod. In addition, although the cost of 3D printing of surgical guide plate technology is decreased and the production cycle is short, the additional cost and time will still be increased. We usually need 1–2 h to design an operation guide plate and 3–5 h to print out the guide plate. Typically, it takes 2–3 days for preoperative planning from the design of the guide plate to the printing of the solid model and preoperative disinfection, which may increase the hospitalization time of patients. Thus, this technique is not suitable for emergency surgery.At present, 3D printing technology has achieved a satisfactory clinical effect in clinical application. Many large hospitals have digital medical centers according to the needs of clinicians to carry out preoperative planning to assist in the completion of various surgical operations. However, many local hospitals lack of relevant professional technology and equipment, which is also a reason for to further popularize the application of 3D printing technology.Both the 3D-printed operation guide template technique and the free-hand technique guided by anatomical landmarks have clinical application value in S2AI screw placement. The 3D-printed guide technique is superior to the free-hand technique in terms of safety and accuracy.


Here, we computed the levels of synchrony, dimensionality, and chaos of the inferior olive code by analyzing in vivo recordings of Purkinje cell complex spike activity in three different coupling conditions: carbenoxolone (gap junctions blocker), control, and picrotoxin (GABA-A receptor antagonist). To examine the effect of electrical coupling on dimensionality and chaotic dynamics, we first determined the physiological range of effective coupling strengths between inferior olive neurons in the three conditions using a combination of a biophysical network model of the inferior olive and a novel Bayesian model averaging approach. We found that effective coupling co-varied with synchrony and was inversely related to the dimensionality of inferior olive firing dynamics, as measured via a principal component analysis of the spike trains in each condition. Furthermore, for both the model and the data, we found an inverted U-shaped relationship between coupling strengths and complexity entropy, a measure of chaos for spiking neural data. These results are consistent with our hypothesis according to which electrical coupling regulates the dimensionality and the complexity in the inferior olive neurons in order to optimize both motor learning and control of high dimensional motor systems by the cerebellum.We previously proposed, on theoretical grounds, that the cerebellum must regulate the dimensionality of its neuronal activity during motor learning and control to cope with the low firing frequency of inferior olive neurons, which form one of two major inputs to the cerebellar cortex. Such dimensionality regulation is possible via modulation of electrical coupling through the gap junctions between inferior olive neurons by inhibitory GABAergic synapses. In addition, we previously showed in simulations that intermediate coupling strengths induce chaotic firing of inferior olive neurons and increase their information carrying capacity. However, there is no  in vivo recordings of spike activity in three different coupling conditions. We found that high coupling strengths induce synchronous firing and decrease the dimensionality of inferior olive firing dynamics. In contrast, intermediate coupling strengths lead to chaotic firing and increase the dimensionality of the firing dynamics. Thus, electrical coupling is a feasible mechanism to control dimensionality and chaotic firing of inferior olive neurons. In sum, our results provide insights into possible mechanisms underlying cerebellar function and, in general, a biologically plausible framework to control the dimensionality of neural coding.Computational theory suggests that the cerebellum must decrease the dimensionality of its neuronal activity to learn and control high dimensional motor systems effectively, while being constrained by the low firing frequency of inferior olive neurons, one of the two major source of input signals to the cerebellum. We previously proposed that the cerebellum adaptively controls the dimensionality of inferior olive firing by adjusting the level of synchrony and that such control is made possible by modulating the electrical coupling strength between inferior olive neurons. Here, we developed a novel method that uses a biophysical model of the inferior olive to accurately estimate the effective coupling strengths between inferior olive neurons from  The cerebellum plays important roles in motor learning and motor control, although how it performs these roles is still unclear. In particular, the role of the inferior olive (IO) continues to be debated. On one hand, there is evidence that the olivo-cerebellar system conveys error signals into the cerebellum –4 and inHowever, a fundamental and outstanding question that needs to be addressed by both theories is: how does the olivo-cerebellar system convey information, whether for learning or for controlling the high dimensional and nonlinear motor systems that generate movements, despite the low-firing rates of inferior olive neurons . Indeed, olivary neurons discharge at most one or two times during a typical movement . For motAn answer to this question is suggested by the unusual organization of the IO, in which neurons form the strongest electrically coupled neuronal network in the adult mammalian brain –29. Thisin silico computer simulations show that chaotic resonance occurs when coupling is decreased, leading to a decrease in synchrony Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: It is an interesting study that provides new insights about how olivo-cerebellar loop processes information and how the timing and learning hypothesis of cerebellar function can be reconciled. However, I have some points that I would like you to address:1) During the whole study . It was not mentioned at all the contribution of excitatory synapses to the modulation of coupling strength in the IO . For example, I saw you mentioned the study of Turececk et al, 2014. However, you seem to bypass the main findings of that paper which is that the activation of NMDA receptors enhance electrical coupling in weakly coupled neurons in the IO. Taking those results into account, you could also say that the observed enhancement of electrical coupling during the application of PIX in rats is due to an enhancement of excitation rather than absence of inhibition. I think both introduction and discussion sections should be rephrased in such a way that also should be considered the possibility of the effect of excitatory synapses on coupling strength in the IO. This is very crucial to keep in mind because it is known the close proximity of excitatory and inhibitory terminals to the electrically coupled dendritic spines in the glomeruli .2) Figure 1, panel A: Do you think that the possible significant difference found between CBX and control conditions can be due to the possible direct actions of carbenoxolone on GABAa receptors .3) Figure 1, panel B: How can you explain the non-significant difference found in the gap junctional conductance between the control and PIX group?Reviewer #2: The inferior olive is crucial for setting the activity level of the cerebellum, affecting (motor) execution as well as (motor) learning. The authors hypothesize that adjusting the coupling strength between inferior olivary neurons changes the dimensionality of inferior olivary output. The authors integrate (previous) experimental data with computational modeling to demonstrate that their ideas are biologically plausible.Compared to other brain regions, neurons of the inferior olive (IO) have extraordinarily strong gap junctional coupling. This strong coupling has long been considered to be the basis of synchronization of IO output, and thus of complex spike firing by the postsynaptic Purkinje cells. However, it is now clear that the impact of coupling depends on the behavioral state of the animal and is strongly affected by anesthesia . In awake animals, synaptic input to the IO strongly limits the impact of gap junctional coupling within the IO . Indeed, the authors noticed this impact of synaptic input (lines 159-160), but largely ignored this observation during their study. While their idea – controlling the dimensionality by adjusting coupling strength within the IO – is interesting and appealing, the use of experimental data from anesthetized animals restricts the interpretational value of this study. Several studies, using predominantly optical recording techniques, but there are some electrophysiological studies as well, have published complex spike recordings of multiple Purkinje cells in awake animals and related to specific tasks. Relating the ideas of the current manuscript to (at least) one of the awake datasets would strengthen the study.In Fig. 3, the authors suggest – but do not show directly – that synchrony is linearly related to dimensionality. This seems an intuitive, if not trivial, result. It would help to show whether changes in synchrony that are physiologically relevant are indeed coupled to changes in dimensionality. This would demonstrate whether IO synchrony is indeed modulated during different phases of motor adaptation and learning, as suggested by the last paragraph of the Discussion.The authors present direct comparisons of biological data and modeling data . This comparison would benefit from providing more details . While the impact of the drugs on firing rate is obvious, more relevant measures like synchrony are not illustrated in a direct comparison.In short, the authors present an interesting hypothesis: that the degree of gap junctional coupling controls the dimensionality / entropy of the IO output via modulation of IO synchrony. Their data suggest that the amplitude of changes in gap junctional coupling required for having an impact on dimensionality / entropy are realistic given experimental data. However, the experimental data are recorded under anesthesia, which is known to affect IO synchrony, and variations in coupling are only linked to pharmacological intervention and not to behaviorally relevant changes, clearly limiting the relevance of this study in its current form.Reviewer #3: I apologize in advance if the review sounds harsh.The paper puts forward a story about the putative functional consequences of an adaptive modulation of gap junctional plasticity in the inferior olivein the course of learning. It extends a well-validated model of inferior olivary cells to a network in order to study the effect of gap junctional modulation on ‘dimensionality’ of the system.I have multiple problems with the paper in its current form, and I am afraid I cannot recommend publication in PLoS Comp. Bio. The argumentation in the paper is rather shallow and repetitive, mostly recapitulating earlier arguments but with scant new evidence.The whole introduction reads as a discussion of the functional consequences of an assumption. It heavily depends on a particular theoretical framework, that which upholsters the view that cerebellar function is to perform a basic form of supervised learning. It uses the word dimensionality profusely, but confusingly. It identifies reduction of dimensionality in the control problem with the reduction of dimensionality in synchronizing coupled oscillators, though the logics for this step are not spelled out. Furthermore, the authors argue here (and previously) that a reduction of dimensionality is a desirable end state for motor control, but that is not at all a given, and depends very much on the type of control problem .Major points:1. Heavy usage of abstract terms with vague and weak links to actual problems of motor control:- The use of the word ‘dimension’ is too loose. There seems to be a confused use of the term ‘reduction of dimensionality’, where the authors seem to assume that the reduction of dimensionality of an oscillatory system due to synchrony is akin to what happens in neural networks (such as in auto-encoders). This identification is highly problematic, because it is not clear which are the variables being encoded/decoded.- Substantial differences are expected in coupling conductances in real networks. Small perturbations would be sufficient to create mounting phase disparities in the cell populations, unless these perturbations are correlated.- In an oscillatory system driven by noise, the use of chaos as a functional property is contentious. The time scales where sensitivity to initial conditions would have a measurable effect are long with respect to motor behavior. More plausible sources of phase variability exist which are ‘transient’, rather than asymptotic, as required by the use of the term ‘chaos’.- Chaos in a slowly oscillating system would only be measurable in longer time scales (>>100ms) . But in long time scales the sensitive dependence of initial conditions will likely be swamped under the effects of (1) input noise (2) intrinsic plasticity (3) short term synaptic plasticity (4) changes of rectifying properties in gap junctions. In the absence of parameter space analysis- Many processes influence and drive synchrony, most importantly, input correlations. The level of synchrony induced by coupling is likely smaller than the correlations from GABAergic and Glutamatergic sources.- “Low dimension components of error signal” Usually the error signal is assumed to be a coarse representation of an organismic-level event.- Similarly, it is not always clear what the DoF that the paper is referring to. Motor synergies highly constrain the number of DoF in motor output systems. Only twitches and the like fit into ‘reduction of DoF’.2. Cerebellum as supervised learning machine: tacit acceptance of the theory that supervised learning in the cerebellum is driven primarily by the CF related LTD.- The idea is that reduction of coupling conductance and synchrony co-occurs with training in the system presumes that the main learning mechanism in the cerebellum is CS driven PF plasticity, i.e., supervised learning. Though this is a common belief, there are continued and substantial problems with the idea. Particularly as one attempts to create more complex control systems with more degrees of freedom in 3D space, the requirements for a motor error signal rise substantially. Though this is not the place for a full discussion of the problems with this assumption, given the number of years that the hypothesis had to prove its ability to explain high level control and acquisition, a more careful presentation of the theory is called for.- The paper uses the literature to justify the assumption that GABAergic sources reduce coupling between cells. In the paper mentioned, they have not controlled for the effects of inhibition in the measured reduction of coupling conductance. It remains highly plausible that reduction of coupling is simply a consequence of shunting of oscillations rather than a direct effect on the coupling values. Hence, adaptation of coupling is not induced by GABAergic sources, but simply a momentary reduction of oscillatory amplitude.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Computational Biologydata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: No: At least not in the current submission.Reviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Chris De ZeeuwGuest EditorPLOS Computational BiologyDaniele MarinazzoDeputy EditorPLOS Computational Biology***********************ploscompbiol@plos.org immediately:A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact [LINK]Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This new version of the manuscript has been improved substantially. You have answered all my questions and include my suggestions. Moreover, You have clarified some important concepts such as dimensionality which provides the reader with a better understating of the main message of this study.I only have minor remarks that I would like you to address:Legend of Figure 2: Should the CBX animal show ‘chaotic’ firing instead of ‘repetitive’ whereas the control animal should exhibit ‘repetitive’ firing and not ‘chaotic?.Figure 3: Would you expect a different relationship between dimensionality and synchrony if you would extract the coefficient of variation of adjacent interspike intervals (CV2) instead of the average firing frequency in order to calculate the dimensionality of complex spike activity?.Figure 3B: Can you explain why is there no significant difference between both the PIX and control group and CBX and control group?.Discussion: The last part of the discussion is redundant when it’s mentioned the role of synchrony at early and late stages of learning. It can be more concise.Reviewer #2: I would like to thank the Authors for taking the time to substantiate their manuscript.Synchrony within the inferior olive, and thereby between complex spikes of downstream Purkinje cells, is an abundant phenomenon. In this paper, the authors argue that gap junctional coupling between IO cells can affect synchrony and that this in turn affects the dimensionality of the olivary-cerebellar system. Intuïtively, there has to be a relation between synchrony and dimensionality: all cells firing in perfect synchrony minimize and all cells firing chaotically maximize entropy. In the first round of review, I mainly referred to the biological relevance of the current study. In response to my question regarding the relation between synchrony and dimensionality , the authors now demonstrate this relation in the new Fig. S7.I am afraid that my concerns were not fully addressed. I remarked that such a relation between synchrony and dimensionality is  trivial. I indicated that it would help to provide evidence that the synchrony varies under physiologically relevant situations to such a degree that changes in dimensionality become apparent. Related to this issue, I have also encouraged the inclusion of awake data.In response to the latter remark (on the inclusion of awake data) the authors have expanded the Discussion, giving a better representation of the value of the anesthetized data used for this study. I would accept this solution. However, the question remains whether "IO synchrony is indeed modulated durig different phases of motor adaptation and learning" (see my remarks during the previous round). I do not feel that the new Fig. S7 helps to address this.In other words, this study argues that IO dimensionality is related to IO synchrony (via gap junctions), and I agree with these conclusions, but leave me with the question to what extent this happens during real life. I would like to encourage the authors to address the expected variation in synchrony more explicitly.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Computational Biologydata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. 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For instructions see  9 Jun 2020AttachmentIO_2R_Reply.docxSubmitted filename: Click here for additional data file. 18 Jun 2020Dear Dr. Hoang,We are pleased to inform you that your manuscript 'Electrical coupling controls dimensionality and chaotic firing of inferior olive neurons' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from  During translation elongation, the ribosome moves along mRNA in a codon-by-codon manner while the mRNA is threaded through the mRNA channel of the small ribosomal subunit. In the bacterial ribosome, the mRNA channel accommodates 11 nucleotides downstream of the first (+1) nucleotide of the P-site codon . The traAlthough the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to pause or stall ribosome movement along mRNA. mRNA stem-loop structures can induce ribosome stalling that results in accumulation of truncated polypeptides  and no-g−1 PRF requires the presence of two signals in an mRNA: the heptanucleotide slippery sequence XXXYYYZ (where X and Z can be any nucleotide and Y is either A or U) and a downstream frameshift stimulating sequence (FSS). The FSS is an RNA hairpin or a pseudoknot . The sliIt remains puzzling why certain stem-loops including FSSs induce ribosome pausing in spite of the ribosome helicase activity. Slow unwinding of secondary structure, to which ribosome pausing is often attributed, is unlikely to account for the extent of translation inhibition induced by FSSs. Single-molecule experiments showed that translocation through three GC basepairs is only 2 to 3-fold slower than translocation along a single-stranded codon , indicatE. coli dnaX mRNA and the gag-pol transcript of HIV pause the ribosome. We asked whether these bacterial and viral mRNA sequences, which form stem-loops of similar lengths, act via a similar mechanism. In agreement with previous studies  and [3H]Glu-tRNAGlu (binds in −1 frame). Consistent with previous publications  are predominately in the NR (0.6 FRET) state -to-R (0.4 FRET) transition  state . After 1ansition . The traansition . Hence, The subsequent reverse transition from the R to the stable NR state (0.6 FRET lasting over 4 s) indicated translocation of mRNA and tRNA. In 64% of traces, the transition from R to NR was preceded by one or two short-lived excursions from R to NR, characteristic of pre-translocation ribosomes . This obLys in the 30 S P site in the absence of EF-G. In this complex, R and NR interconverted at rates of 0.2 sec−1 (R to NR)    and 0.6 NR to R) , respectNR to R) . Hence, bd and τtrl of dnaX_Slip mRNA programmed ribosomes were remarkably long with median values of 18.2 s and 10.7 s, respectively , rapid transition from NR to R was followed by rapid transition to the stable NR state (bd (3.8 s) and τtrl (1.5 s) for dnaX_Slip ΔFSS mRNA (Ribosome complexes assembled with dnaX mRNA lacking the FSS (dnaX_Slip ΔFSS mRNA) showed markedly different behavior in comparison with dnaX_Slip mRNA complexes. When EF-Tu•GTP•Lys-tRNANR state . In contNR state . Median FSS mRNA  were 5- FSS mRNA , our datFSS mRNA . In addiFSS mRNA . BecausednaX were shown to decrease frameshifting efficiency to low (≤5%) or undetectable levels  and UAC (Tyr) ‘non-slippery’ codons to create dnaX_NS (‘non-slippery’) mRNA . Mutatioe levels .Phe was added to ribosomes with the FSS positioned four nucleotides away from the entry of the mRNA channel, the ribosome population converted from a predominant NR  of ribosomes remained in the NR conformation  ribosomes in vitro and in vivo at frequencies comparable to those observed for HIV frameshifting in eukaryotic translation systems  that could also be reconstituted in the E. coli translation system  mRNA, in which the original UAC (Tyr) codon of HIV_NS mRNA was replaced with a GAG (Glu) codon . The resGlu  while riGlu . Thus, wTyr (or Glu-tRNAGlu in the case of ribosomes programmed with HIV_NS (GAG) mRNA). S6-cy5/L9-cy3 ribosomes, which were programmed with either dnaX_NS (UAC) or HIV_NS (UAC) mRNA and bound with P-site N-Ac-Phe-tRNAPhe  did not lead to an appreciable increase in the fraction of the R (0.4 FRET) conformation  was sufficient to decode an in-frame Glu (GAG) codon in ribosomes programmed with HIV_NS (GAG) ∆FSS mRNA as evident from the conversion of the ribosome population from the NR to R conformation  and NR (0.6 FRET) states at rates of 0.2–0.3 sec−1 (0.4 FRET to 0.6 FRET) and 0.7–0.8 sec−1, (0.6 FRET to 0.4 FRET), respectively, and spent 80% of time in the R conformation  inhibiting A-site tRNA binding, (ii) blocking the peptidyltransfer reaction after the binding of A-site tRNA or (iii) stabilizing the pre-translocation ribosome in the NR conformation. Pretranslocation-like S6-cy5/L9-cy3 ribosomes containing deacylated P-site tRNAormation . Hence, uromycin , indicatN-Ac-Met-tRNAMet were then incubated with EF-G•GTP, EF-Tu•GTP, Val-tRNAVal, Phe-tRNAPhe and Tyr-tRNATyr before loading ribosomes onto a nitrocellulose filter and washing away unbound aa-tRNA. The experiment was repeated three times with one of the three aminoacyl-tRNAs radio labeled, that is using [14C]Val-tRNAVal, [3H]Phe-tRNAPhe or [3H]Tyr-tRNATyr. Similar experiments were performed with ribosomes programmed with HIV_NS mRNA to measure binding of radio-labeled aa-tRNA during translation through three  consecutive codons.To further test whether FSSs positioned near the entry of the mRNA channel inhibit tRNA binding, we used a filter-binding assay to measure binding of radio-labeled aa-tRNA during translation through four  consecutive codons along the dnaX_NS mRNA. The ribosomes containing P-site 3H]Tyr-tRNATyr was considerably diminished while the incorporation of Val and Phe into the polypeptide chain were only mildly inhibited compared to levels measured in ribosomes programmed with dnaX_NS ∆FSS mRNA lacking the FSS  or 13 nucleotides (HIV_NS13-nt linker mRNA), respectively. Extending the P-site-FSS spacer from 11 to 12 nucleotides increased the apparent pseudo first order rate of [3H]Tyr-tRNATyr binding to the A site of the ribosome from 0.1 to 0.4 min−1  codon and the FSS was extended from 11 (HIV_NS mRNA) to 12  mRNA  codon in the A site  mRNA  and boune A site  and 7.Maximum-likelihood classification of a 640,261-particle data set revealed predominant ribosome states that contained strong density in both the P and A sites, which we interpreted as P-site tRNA and the FSS hairpin, respectively  . Two claN-Ac-Phe-tRNAPhe and an in-frame UUC codon. This is consistent with smFRET data showing that in the absence of the slippery sequence, dnaX and HIV FSSs do not promote frameshifting  NS mRNA between GAG and the FSS), which we further refer to as the linker sequence, to occupy the ribosomal A site . To exclThe local resolution of the A site allows to distinguish the major and minor grooves of the A-form RNA, but individual basepair locations cannot be determined, suggesting that the FSS hairpin samples an ensemble of conformations. We were able to build a plausible pseudoatomic model of the linker and hairpin based on resolved density features and previously solved structures. We used a published NMR model of the HIV FSS hairpin , which iConformational heterogeneity of the hairpin observed in our cryo-EM structures is likely important for the hairpin’s mechanism of action. To allow FSS hairpin entry into the A site, the mRNA linker has to be released from the mRNA channel in the 30S subunit. Dynamic occupancy of the A site by the FSS hairpin may allow an incoming tRNA to eventually overcome the translational block that the hairpin imposes on the ribosome. Binding of tRNA likely stabilizes the A-site codon, allowing the linker to reestablish its position in the mRNA channel and the hairpin to bind next to the channel entry, restoring an elongation ribosome state.E. coli dnaX and HIV mRNAs induce ribosome pausing. Although the sequences of these mRNA elements are different, they appear to act via the same mechanism due to similar hairpin structures. We found that when positioned 11–12 nucleotides downstream of the P-site codon, the FSSs perturb translation elongation through two parallel pathways: (i) inhibiting tRNA binding to the A site of the ribosome and (ii) inhibiting ribosome translocation  derived from phage T4 gene 32 mRNA . CrystaldnaX and HIV mRNAs involves P-site tRNA slippage and frameshifting during translocation of two tRNAs, that is pathways (i) and (iii)  slippage of the single P-site tRNA when the A site remains vacant, (ii) frameshifting of both A-site and P-site tRNAs during aa-tRNA accommodation to the A site, and (iii) slippage of A- and P-site tRNAs during translocation . A numbend (iii) . Partitind (iii) . Our obsOur findings of hairpin competition with tRNA expose a novel mechanism that stem-loop FSSs in retroviruses, including HIV, likely employ to regulate viral gene expression and expand the viral proteome via mRNA frameshifting. Likewise, transient binding of FSS stem-loops to the A site of the ribosome may mediate frameshifting in insertion sequences of the IS1-IS3 family of bacterial transposable elements  and orf fMet, tRNAMet, tRNAPhe, tRNAVal, tRNATyr, tRNALys, and tRNAGlu  were aminoacylated as previously described  with 0.8 mg/mL glucose oxidase, 0.625% glucose, 1.5 mM 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic (Trolox) and 0.4 μg/mL catalase. smFRET data were acquired with 100 ms time resolution.Sequences encoding dnaX and HIV mRNAs were cloned by directional cloning downstream of T7 promoter in pSP64 plasmid vector (Promega Co). Model mRNAs  were genDI) and Cy5 acceptor (AI), from which apparent FRET efficiency  was calculated:IDL software (ITT) was used to extract flourescence intensities of Cy3 donor  at 37°C for 5 min. For the mixture of all li tRNAs , 30-  was delivered at 0.4 mL/min speed by a syringe pump (J-Kem Scientific) after 10 s of imaging.To prepare dnaX_Slip mRNA-programmed ribosomes that contained e P site , N-Ac-VaN-Ac-[3H]Phe-tRNAPhe in polyamine buffer at 37°C for 15 min followed by 10 min incubation with 1 mM puromycin. The puromycin reaction was terminated by diluting the ribosome samples using MgSO4-saturated 0.3 M sodium acetate (pH 5.3), and the N-Ac-[3H]-Phe-puromycin was extracted ethyl acetate.0.6 μM 70S ribosomes were incubated with 1.2 μM dnaX_NS mRNA and 1.2 μM 14C]Val-tRNAVal, [3H]Phe-tRNAPhe, [3H]Tyr-tRNATyr, [3H]Glu-tRNAGlu and N-Ac-[3H]Tyr-tRNATyr as indicated in figure legends) similarly to smFRET experiments described above. Ribosome complexes were applied to a nitrocellulose filter (MiliporeSigma), which was subsequently washed with 500 μl (for complexes programmed with dnaX mRNA) or 800 μl (for complexes programmed with HIV mRNA) of ice-cold polyamine buffer containing 20 mM Mg2+ to remove unbound tRNAs. 20 mM Mg2+ concentration was used to stabilize ribosome complexes under non-equilibrium conditions.The filter-binding assay was performed as previously described  with minN-Ac-Val-tRNAVal in polyamine buffer at 37°C for 15 min. The ribosomes were then incubated with 4 μM EF-G•GTP, 10 μM EF-Tu•GTP, 2.4 μM Lys-tRNALys, 1.2 μM Arg-tRNAArg (binds in 0 frame) and 1.2 μM [3H]Glu-tRNAGlu (binds in - one frame) at 37°C for 6 min. Incorporation of [3H]Glu into the ribosome was measured by filter-binding assay as described above. Frameshifting efficiency (ribosome A-site occupancy by [3H]Glu-tRNAGlu) was normalized by the P-site occupancy of N-Ac-[3H]Glu-tRNAGlu non-enzymatically bound to the ribosome programmed with dnaX_Slip ΔFSS mRNA.0.6 μM 70S ribosomes were incubated with 1.2 μM dnaX_Slip mRNA and 1.2 μM N-Ac-Phe-tRNAPhe and 0.8 μM HIV_NS (GAG) mRNA in polyamine buffer.The 70S ribosomes re-associated from 30S and 50S subunits were purified using sucrose gradient. 0.4 μM 70S ribosomes were bound with 0.7 μM C-flat grids  were glow-discharged for 30 s in a PELCO glow-discharge unit at 15 mA. 3 μl of the 70S•HIV FSS-mRNA complex at 250 nM concentration were applied to the grid and incubated for 30 s before vitrification using an FEI Vitrobot Mark IV (ThermoFisher). The grids were blotted for 3 s using blotting force 3 at 4°C and ~90% humidity, plunged in liquid ethane, and stored in liquid nitrogen.2. The super-resolution pixel size at the specimen level was 0.5115 Å. All movies were saved dark-corrected.A dataset was collected using SerialEM  on a Tit2 pixel boxes.Gain and dark references were calculated using the method described by Phe in the P-site. Particles for this class were extracted and aligned with increasing resolution limits to 5 Å. Finally, CTF refinement to 4 Å resolution without alignment and a step size of 50 Å was run and the final reconstructions were calculated using a beam-tilt corrected particle stack yielding final resolutions of 3.4 Å and 3.3 Å (Fourier Shell Correlation = 0.143).Extracted particles were aligned to an unpublished reference volume using a global search in the resolution range from 8 to 300 Å , omitting EF-Tu and the A-site tRNA. An NMR structure of the HIV-1 frameshifting element (PDB ID 1PJY) was used as the starting model for the hairpin and to generate secondary-structure restraints. Missing parts of the mRNA were built manually and the geometry was regularized in phenix.geometry_minimization before refinement. The A-site finger was modeled using nucleotides 873–904 from PDB ID 5KPS where the A-site finger is well-ordered. Protein secondary structure restraints were generated in Phenix  and addInitially, the ribosomal subunits, tRNA and the hairpin were separately fitted into the cryo-EM, using Chimera, followed by manual adjustments in Coot (version 0.9-pre) . The str In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor The reviewers have opted to remain anonymous.Thank you for submitting your article "mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:E. coli components, and was shown to occur with a different A-site codon/TC pair. Filter binding assays of TC loading were used to support the conclusion that FSSs inhibit A-site tRNA binding only when positioned near the opening of the mRNA entry channel. They go on to present a cryoEM structure of a 70S ribosome bound to a variant of the HIV PRF sequence in which the slippery codons were replaced by UUC-GAG codons with Phe-tRNA in the P site. Interestingly, the FSS occupies the A-site, providing a structural explanation for its ability to inhibit TC binding to the A-site. This structure differs from one presented recently for the dnaX PRF sequence in which the FSS was found just outside the entry channel opening. They propose a model in which either of these locations of the FSS sequence can interfere with A-site binding, and the empty A site would allow frameshifting through one of two proposed mechanisms for frameshifting involving slippage of the single P-site tRNA when the A-site is vacant.This paper probes the molecular mechanism of structured frameshifting stimulatory sequences (FSSs) in stimulating -1 programmed ribosome frameshifting (PRF) on the bacterial dnaX slippery site. Single-molecule FRET assays that monitor the intersubunit rotation of the large and small subunits to determine the rate of EFTu-GTP-tRNA TC binding to the A site and then EF-G stimulated translocation show that positioning of the FSS in the mRNA entry channel slows down both reactions. The effect on translocation rates had been seen before, but the slowing of A-site binding of the TC in the zero frame appears to be a novel finding. This effect was dependent on positioning of the FSS at the entry channel. The same was demonstrated for the FSS of the HIV -1 PRF site that could be reconstituted in vitro with Essential revisions:It is necessary to revise the interpretation of the results and include the appropriate alternative possibilities and the qualifications described in reviewer #2's major comments. Owing to the COVID-19 pandemic, it is not required that the control experiments requested by reviewer #1 be conducted, but additional text relating to the missing data should be included.Reviewer #1:The work is well executed and interdisciplinary, and generally well described, and the results provide significant insights into the molecular mechanism of frameshift stimulation by FSS elements.The authors need to include the binding of labeled Phe-tRNA or Val-tRNA as controls for the experiments in Figure 6—figure supplement 1B.Reviewer #2:E. coli translation system using -1 PRF signals derived from a prokaryotic (dnaX) and eukaryotic (HIV-1) sources. Note that these -1 PRF signals are atypical: they are representative of only a few known -1 PRF signals in which the Frameshift Stimulating Signal (FSS) is a stem loop as opposed to an RNA pseudoknot. This may or may not be germane. Three orthogonal approaches are employed: smFRET, classical tRNA binding biochemistry, and cryo-EM. The methodologies are very strong and cutting edge, and the data all convincingly point to slippage by a single peptidyl-tRNA while the A-site is empty and when the ribosome is in an unrotated state. The data also indicate that this is because the FSS occludes aa-tRNA binding to the A-site.In this manuscript, the authors attack a longstanding and interesting problem in the field of translational control, programmed -1 ribosomal frameshifting. The technical basis for this work involves an in vitro E. coli, see work from the Brakier-Gingras group), this is "non-canonical. Indeed, the more commonly accepted "simultaneous slippage model" proposed by the Varumus lab in 1988 posited that slippage occurs with a ribosome in which both the 0-frame A- and P-sites slip over the slippery site. However, as described below, that paper did indicate that ~30% of -1 PRF was due to single tRNA slippage.The data presented here also indicate a non-simultaneous slippage mechanism, i.e. a 1 tRNA slip event. While it has been shown that such slips can happen  state. Here, the data indicate that ribosomes are in the non-rotated state. Is the difference because frameshifting was decoupled from FSS induced pausing?Qin et al., 2014; Chen et al., 2014. Additionally, the Rodnina lab's recent work supports this view as well.This not irresolvable. Earlier studies from many labs support the hypothesis that there are at least three different kinetic pathways to frameshifting: 1) slippage of the peptidyl-tRNA with an empty A-site, 2) simultaneous slippage of two tRNAs during accommodation, and 3) simultaneous slippage as they translocate out of the slippery site.It is possible that different experimental designs favor one pathway over another. This one for example seems to favor pathway 1. Mutants of eEF1A (and presumably of EF-Tu) illuminate pathway 2. And the setups used by the Rodnina, Giedroc and Puglisi labs seem to favor number 3.E. coli) strongly favored pathway 1. Work from the Dinman lab, using frameshift signals in yeast, presented data supporting all three. That group also did an interesting set of simulations describing how each of the three pathways can be favored by changing various kinetic parameters.Indeed, if we look at Jacks and Varmus 1988 paper, they show evidence supporting all three pathways . Their mass spec analysis of the frameshift fusion peptide show that 70% of the products include the 2 zero-frame amino acids (supportive of pathways 2 and/or 3), and that 30% have the zero-frame P site amino acid and the -1 frame A-site encoded amino acid (supporting pathway 2). Work from the Brakier-Gingras lab  may be swing the balance to favor one pathway over another.It is possible that this particular experimental setup, where the frameshift signal is extremely close to the start codon, may be influencing the preferred pathway. Earlier genetic studies showed that putting frameshift signals close to start codons inhibited -1 PRF. More recent work from the Djuranovic lab has identified a short translational ramp after initiation that affects protein synthesis. It may also be an artifact of the translational system that is used: Lastly, one has to question the assumption that substitution of non-slippery codons for slippery ones in the "slippery site" does not affect the molecular mechanism underlying -1 PRF. I think that this should not be taken as a matter of faith.In sum, I think that this manuscript is technically robust and technologically state-of-the-art. They have certainly illuminated some aspect of -1 PRF, but it has certainly not solved all of the questions. Indeed, it raises many more (which is what good science does). My concern is that the authors knowledge of the -1 PRF literature is not as deep as it could be, and that as a result their interpretation of the data is not properly contextualized. Reviewer #1:The work is well executed and interdisciplinary, and generally well described, and the results provide significant insights into the molecular mechanism of frameshift stimulation by FSS elements.The authors need to include the binding of labeled Phe-tRNA or Val-tRNA as controls for the experiments in Figure 6—figure supplement 1B.Phe to the A site is not affected when the dnaX FSS is 15 nucleotides downstream of the P site. Since our labs are shut down due to the COVID-19 crisis, we cannot do the requested additional filter-binding measurements of non-enzymatic binding of N-acetyl-Phe-tRNAPhe.smFRET and filter-binding experiments  indicate that EF-Tu-dependent binding of Phe-tRNAReviewer #2:E. coli, see work from the Brakier-Gingras group), this is "non-canonical. Indeed, the more commonly accepted "simultaneous slippage model" proposed by the Varumus lab in 1988 posited that slippage occurs with a ribosome in which both the 0-frame A- and P-sites slip over the slippery site. However, as described below, that paper did indicate that ~30% of -1 PRF was due to single tRNA slippage.The data presented here also indicate a non-simultaneous slippage mechanism, i.e. a 1 tRNA slip event. While it has been shown that such slips can happen  state. Here, the data indicate that ribosomes are in the non-rotated state. Is the difference because frameshifting was decoupled from FSS induced pausing?[…]In sum, I think that this manuscript is technically robust and technologically state-of-the-art. They have certainly illuminated some aspect of -1 PRF, but it has certainly not solved all of the questions. Indeed, it raises many more (which is what good science does). My concern is that the authors knowledge of the -1 PRF literature is not as deep as it could be, and that as a result their interpretation of the data is not properly contextualized.We thank the reviewer for these insightful comments and for appreciating that our work is technically robust.Presence of multiple pathways of -1PRF in our data. We agree that the three mechanisms of -1PRF have been considered in the field: (i) slippage of the single P-site tRNA when the A site remains vacant, (ii) frameshifting of both A-site and P-site tRNAs during aa-tRNA accommodation to the A site, and (iii) slippage of A- and P-site tRNAs during translocation . Multiple lines of evidence indicate that these pathways could be active at the same time. Our data also support this idea.Our smFRET experiments performed with dnaX_Slip mRNA showed FSS-induced ribosome pausing in both non-rotated and rotated states , indicating that the FSS inhibits both A-site tRNA binding and translocation. The ribosome pausing in the rotated state is consistent with the work of the Puglisi and Rodnina labs. As suggested by the reviewer and discussed in our manuscript, our observation that FSSs induce ribosome pausing in both non-rotated and rotated states is consistent with single tRNA slippage (i) and translocation (iii) frameshifting pathways, respectively. Because pausing in the non-rotated state caused by inhibition of tRNA binding is a novel observation, we focused the rest of our study on this.In the original manuscript, we tried to streamline our discussion of -1 PRF mechanisms because our work is mainly focused on one aspect of -1 PRF – mechanisms by which frameshift-inducing stem-loops slow down the ribosome. Although we cited many of the works mentioned by the reviewer in the previous version of manuscript, we agree that discussion of -1 PRF mechanisms in the manuscript should be expanded. We added a paragraph to Discussion to address the reviewer’s concerns:“Our findings provide new insights into the mechanisms of -1PRF stimulated by stem-loop FSSs. […] It is not clear whether pseudoknot FSSs also utilize both pathways or A-site inhibition/P-site tRNA slippage is unique to stem-loop FSSs”.E. coli ribosomes. Hence, RNA hairpin competition with tRNA binding is unlikely an artifact of using E. coli ribosomes with the stem-loop from eukaryotic virus. Nevertheless, we agree with the reviewer that depending on experimental conditions (primarily tRNA abundance as indicated by works of Rondina’s group), one frameshifting pathway may be favored over the other.Experimental conditions may favor one -1PRF pathway over the other. We find that FSSs of both bacterial (dnaX) and eukaryotic virus (HIV) origins inhibit A-site binding in Ribosome pausing in the presence of non-slippery codons. We observe that the dnaX FSS inhibits A-site tRNA binding in the context of both slippery and non-slippery sequences. Hence, our discovery of mRNA stem-loop binding to the A site may have broader implications for translation regulation not limited to -1 PRF.


In this study, we evaluate sea surface pCO2 measurements collected over a 6‐year period along a fixed cruise track in the Canada Basin. The measurements show that mean pCO2 levels are significantly higher during low ice years. The pCO2 increase is likely driven by ocean surface heating and uptake of atmospheric CO2 with large interannual variability in the contributions of these processes. These findings suggest that increased ice‐free periods will further increase sea surface pCO2, reducing the Canada Basin's current role as a net sink of atmospheric CO2.Less than three decades ago only a small fraction of the Arctic Ocean (AO) was ice free and then only for short periods. The ice cover kept sea surface  The Arctic Ocean is losing ice cover at a rapid rate2, increasing sea surface CO2 levelsThe loss of ice cover is leading to heating and uptake of atmospheric CO2 will likely reduce the uptake of atmospheric CO2 in the futureContinued increase of sea surface CO St‐Laurent during 2012–2017. No pCO2 measurements were made in 2015. The starting dates for the five ~4 week cruises were 6 August 2012, 3 August 2013, 25 September 2014, 24 September 2016, and 8 September 2017. The pCO2 was recorded using an infrared‐gas equilibrator system  located in the ship's lab. The instrument uses an infrared analyzer  and gas phase equilibrator  as described in Hales et al. (2 gas standards and a zero CO2 gas sample. Temperature was measured in the equilibrator and at the seawater intake (9 m depth), assumed equal to sea surface temperature (SST), as discussed below. The infrared analyzer CO2 mole fraction was corrected to SST and converted to pCO2 using 100% humidity at SST and local barometric pressure  and the pCO2 was discarded during these periods. A flow meter was installed in 2016 to detect periods of low flow rate. The pCO2 uncertainty is estimated to be ±5 μatm based on the reproducibility of the standards and baseline zero. CTD stations showed that the seawater intake was sometimes within the halocline , and this was also evident from the salinity and temperature variability recorded by the ship's thermosalinograph. These conditions were found mostly on the continental shelf during 2012 and 2013 due to high Mackenzie River outflow. This analysis focuses on the Canada Basin bounded by 155–130°W and 72–82°N where CTD stations consistently found that mixed layer depths were greater than the ship intake depth.Underway sea surface s et al. . The equ−3  Earth System Research Laboratory (ESRL) (https://www.esrl.noaa.gov). Sea ice concentration with daily, 12 km resolution was obtained from the French Research Institute for Exploration of the Sea (IFREMER) (http://cersat.ifremer.fr/oceanography-fromspace/our-domains-of-research/sea-ice) that provides data collected by the satellite‐based Special Sensor Microwave Imager (SSM/I) and processed by the National Snow and Ice Data Center (https://nsidc.org).Air temperature, wind speed, wind direction, and barometric pressure were recorded by the ship's weather system. Mixed layer depths, defined as the depth where the density difference from the surface first exceeds 0.25 kg m2.2pCO2 levels are related to interannual variability in ice concentration and to evaluate processes that might control pCO2 under low (or no) ice conditions. To facilitate analysis of the spatially and temporally disparate shipboard pCO2 and other data, Canada Basin data were gridded by identifying 20 × 20 km grid areas that contain data, and those data were then averaged as described in Evans et al.  for each grid cell. DOR is the day when ice concentration dropped below 15% in any grid cell  mixed layer mass balance , Fgasex is the air‐sea CO2 flux , MLD is the mixed layer depth, and ρ is seawater density. Warming (increasing SST) is accounted for in the equilibrium calculation as described below. Fgasex was calculated usingk is the gas transfer velocity, K0 is the CO2 solubility, ΔpCO2 is the pCO2 difference between the sea surface and atmosphere, and f is the fraction of open water . Fgasex is negative when there is a net uptake of CO2 by the ocean from the atmosphere. We used the wind speed relationship in Wanninkhof  was calculated as opposed to wind speed averages because short‐term (<daily) variability in the winds is retained leading to higher gas transfer rates during periods of greater wind speed variability . We used values from Ji et al.  in the CO2 equilibrium program CO2sys  calculations used the Mehrbach et al. . Sea surface pCO2 was also highly variable spatially and interannually. Open water in the Canada Basin typically had higher pCO2 levels than pCO2 recorded in ice‐covered areas; for example, compare 2012 (low ice) and 2014 (more ice) in Figure The shipboard pCO2 is higher and closer to atmospheric saturation during years of low ice concentration and exceeded atmospheric pCO2 , MLD, wind speed, and NCP  differs significantly, as do data from the late September cruises (2014 and 2017). These differences are also evident in the pCO2 data in Figure The gridded sea surface y Figure . Each yepCO2 with increasing DSR and that these processes are highly variable from year to year . Also, employing DSR as a time variable in the model assumes that no air‐sea exchange or warming occurred when sea ice concentration was greater than 15% which is clearly not true  over the range of NCP reported in Ji et al. (2/argon method integrates NCP over the residence time of O2 in the mixed layer (10–30 days)  (doi:10.18739/A2R785P3X).The data are available through the U.S. National Science Foundation (NSF) Arctic Data Center (Supporting Information S1Click here for additional data file.


Detailed knowledge of Holocene climate and glaciers dynamics is essential for sustainable development in warming mountain regions. Yet information about Holocene glacier coverage in the Alps before the Little Ice Age stems mostly from studying advances of glacier tongues at lower elevations. Here we present a new approach to reconstructing past glacier low stands and ice-free conditions by assessing and dating the oldest ice preserved at high elevations. A previously unexplored ice dome at Weißseespitze summit (3500 m), near where the “Tyrolean Iceman” was found, offers almost ideal conditions for preserving the original ice formed at the site. The glaciological settings and state-of-the-art micro-radiocarbon age constraints indicate that the summit has been glaciated for about 5900 years. In combination with known maximum ages of other high Alpine glaciers, we present evidence for an elevation gradient of neoglaciation onset. It reveals that in the Alps only the highest elevation sites remained ice-covered throughout the Holocene. Just before the life of the Iceman, high Alpine summits were emerging from nearly ice-free conditions, during the start of a Mid-Holocene neoglaciation. We demonstrate that, under specific circumstances, the old ice at the base of high Alpine glaciers is a sensitive archive of glacier change. However, under current melt rates the archive at Weißseespitze and at similar locations will be lost within the next two decades. A major scientific question is whether this process of deglaciation is unprecedented within the Holocene. If we want to assess the pace of future deglaciation in the Alps, it is essential to understand past glacier dynamics and their relation to changes in climate2. We already have comprehensive information about the maximum extent of Alpine glaciers from the investigation of moraine positions and ages4. Yet comparatively little is known so far about the times of minimum ice cover or ice-free conditions at high-elevation. Indeed, only the dating of wood fossil emerged from ice melt has made it possible to constrain glacier retreat, but at lower elevation6. Complementary information to findings from glacier tongues can come from glaciers at high elevations7, where the ice is frozen to bedrock. Despite the strong retreat since the Little Ice Age (LIA), access to the oldest ice near bedrock is still complicated for high-elevation areas and usually requires drilling glacier ice cores.The majority of the glacier ice volume in the Alps may disappear within the next two centuries8. In the Eastern Alps and at elevations below 4000 m, such records are sparse, with only one ice core record obtained from an already partially temperate site at Ortler9 and at the temperate Silvretta glacier10. Still, it generally holds that if basal ice temperatures are below the melting point, the so-called “cold ice” remains frozen to bedrock. If it moves at all, it does so by slow internal deformation, in contrast to temperate ice (i.e. ice at around 0 °C), which is able to slide along the bedrock11. Even though its remaining thickness is only around 10 m, millennial-old stagnant cold ice has recently been found at the base of an Alpine summit glacier12. Constraining the maximum age of the stagnant ice near bedrock can indicate past ice-free periods, followed by neoglaciation13. Hence this age information alone contains important information on paleoclimatic conditions.Most previous ice core research in the Alps was aimed at continuous stratigraphic climate records. This limited it to glaciers where there is usually no melting on the surface throughout the year. Few suitable drilling sites exist as they are mostly confined to above 4000 m altitude, e.g. locations in the Western Alps7) and the “Tyrolean Iceman” in the Eastern Alps14. Schnidejoch pass is easily blocked by glacier advances, making the artifact findings a sensitive indicator of past glacier minima. Radiocarbon dating of artifacts indicates three phases of minimal ice extent, suitable for crossing the pass. The earliest phase was around 6.8–6.3 ka cal, a second phase from 5.7 to 4.9 ka cal, and an adjacent third period from 4.9 to 4.2 ka cal15. The Tyrolean Iceman mummy emerged from a small ice field, vanished since, at Tisenjoch, a saddle located at 3210 m. Radiocarbon dating of the mummy indicated that the Iceman lived roughly 5.1–5.3 ka cal16. The well-preserved state of the corpse and of artifacts suggests that they had been conserved in frozen conditions. The ice field at Tisenjoch must therefore have been present during several known periods of glacier retreat, such as the Roman and Medieval warm phases17. Unfortunately, after the discovery of the Iceman, pollen analyses were only conducted on the surrounding ice18. With the arrival of modern radiometric ice-dating techniques based on 14C-dating of the water-insoluble organic carbon fraction of carbonaceous aerosols embedded in the ice matrix, it is now possible to constrain the age of the glacier ice itself20. Dating the ice with today’s radiometric techniques could have told us if the Iceman had in fact died in a mostly ice-free environment, or if he fell into a crevasse on a glacier-covered Tisenjoch.Until recently, only human artifact findings were discussed in connection with low stands of high-elevation glaciers in the Alps; at Schnidejoch pass  marks the highest point of Gepatschferner in the Austrian Alps. It is a unique site: 1) located only 12 km from the Iceman location, it offers a potential surrogate to investigate the local glacier conditions during the lifetime of the Iceman; (2) it has a dome-shaped glacier geometry, which is an extremely rare feature in the Alps  and hydrogen (δD) ratios of a range typical for the seasonal variation in snow at this altitude, with no deviation from the meteoric water line  ka cal  at about 4.5 m depth to (5.884 ± 0.739) ka cal just above the bed  ka cal and has been glaciated continuously ever since. This implies that even the WSS summit location at 3500 m altitude was ice-free during an interval prior to (5.9 ± 0.7) ka. In the context of other regional climate evidence, this finding is consistent with the glacier length reconstructions of Gepatschferner, which document a distinct minimal extent starting around 5.9 ka23 is also observed in Austrian stalagmite records, indicating the onset of a cooling period around 5.9 ka24. In the Eastern Alps, tree-ring-dated subfossil wood remains indicate that several advances occurred between 5.9 and 5.5 ka at three different glaciers in the Alps: Unteraar, Pasterze, Tschierva25. The former two have much longer response times than Tschierva, and generally fluctuations of glacier tongues deviate from mass balances at summit glaciers because the terminus position also depends on ice dynamics and because wind erosion affects net accumulation at summits. Nonetheless, the general picture agrees well. Around 5.9 ka, there was a limited advance at Tschierva glacier, reaching the 2000 ice extent and ending the preceding warm period25. During those warm periods, the tree line was up to 165 m above the 1980 tree line in Kaunertal, in ultimate vicinity to WSS26. A chironomid record obtained at Schwarzsee in nearby Oetztal suggests that roughly between 5.2 and 4.5 ka cal a climate transition occurred, with a distinct cooling trend in summer temperatures, which prevailed until the end of the LIA27.The end of the so-called “Holocene Climatic Optimum”28. Although the effective altitude of the glaciers leading to the fluvioglacial deposits is not closely defined, it must have been significantly lower than the WSS summit. In this regional context, the findings from WSS fit remarkably well into a general picture of regional warm conditions ending around 5.9 ka at high altitude. This was followed by a period of glacier advance that started around the lifetime of the Iceman, with a delayed onset of glaciation on lower elevation summits.This is consistent with other regional evidence provided by the Oberfernau bog sediments (Buntes Moor): a radiocarbon-dated layer marks the end of peat growth during a warm phase Fig. . From ab29, this may not be true of high Alpine glaciers. At high-elevation and summit locations, glaciers are typically very limited in size and heavily exposed to wind erosion as a main constraint on net accumulation there8. Instead, the dependence on altitude found here suggests a primary connection to mean annual temperature, and its lapse rate, for neoglaciation at these sites. In a simplistic view, a cooling trend would lead to snow accumulation, prolonged positive mass balance and subsequent ice formation on highest elevation sites earlier than on lower elevation, as the ablation rate is smaller at the former. Vice versa, warm periods might lead to increased ablation and prolonged negative mass balance on lower sites, leaving higher glaciers unchanged in geometry, as is the case today for the highest summits of the Western Alps30.The findings from WSS are integrated in an overview of already existing maximum age constraints Table , in orde14C dating indicates a glacier formation starting earlier at the higher altitude of around 3860 m, namely around (6.7 ± 0.4) ka cal9. For glaciers above 4000 m, maximum age constraints were obtained from ice cores drilled at Colle Gnifetti  and Col du Dôme . Both glaciers are in the highest region of the glacier’s accumulation zone and feature a saddle-shaped geometry. As opposed to the ice dome at WSS, the saddle geometry affects the interpretation of the ice core dating in relation to glacier maximum age constraints. At Colle Gnifetti, for two ice cores drilled close to the saddle point, radiocarbon dates from the deepest layers point to ice older than 11.5 ka cal31. In contrast, in an ice core drilled at a flank position, a basal age of only (4.1 ± 0.2) ka cal was obtained20. Likewise, for an ice core drilled at a flank position at Col du Dôme, the basal age was constrained recently to about (5.0  ± 0.6) ka cal32.Within the region of WSS, maximum glacier age constraints were obtained from a deep ice core drilled at Ortler summit glacier: aerosol-based micro-Two non-exclusive interpretations may explain the age difference between saddle and flank locations. Due to ice flow at flank positions, the deepest ice layer originating from the neoglaciation may have been severely thinned, making it difficult to adequately resolve its age in a bulk sample. Moreover, the original neoglaciation may have started as a small ice patch around the saddle and not reached the flanks until a further build-up of the glacier was completed. Notably, if taken at face value as the maximum glacier age, the radiocarbon dates from the saddle positions at Colle Gnifetti are a good fit with the elevation gradient of neoglaciation revealed at sites below 4000 m . The total surface elevation change since 1969 exceeds 10 m, based on digital elevation models (Table 00 MHz. T36. Cores were packed on site into insulating boxes and transported via cold-storage transport to the cold-room facilities (− 20 °C) of the Institute of Environmental Physics, Heidelberg University. The visual stratigraphy of the ice cores showed white, bubble-rich ice layers, occasionally interrupted by cm-thick clear layers of refrozen meltwater. The lowermost 30 cm of the cores showed small rocks incorporated into the ice, a clear sign of bedrock proximity. This is consistent with scratch marks on the cutters of the core barrel after the last drilling run to bedrock. No further ice could be recovered, providing clear evidence that bedrock had been reached.Two cores were drilled at the same location on WSS down to bed using a 2″ electro-mechanical drill18O and δD, respectively12. Calculating ordinary linear regression of δD against δ18O yields a slope of 7.5 at a correlation coefficient of r = 0.99  were prepared from the top, bottom and an intermediate section. The samples were shipped with cold-storage transport to Seibersdorf Laboratories, Seibersdorf, Austria, where tritium was measured after electrolytic enrichment, which was necessary because of the low concentrations  detector at the Laboratory for the Analysis of Radiocarbon with AMS (LARA Laboratory) of the University of Bern. The Sunset instrument is coupled to a gas introduction interface system (GIS), which in turn is coupled to a MIni radioCArbon DAting System . The system allows online 14C measurements of the carbonaceous fractions separated by the Sunset. 14C ages were calibrated using OxCal software with the Northern Hemisphere  calibration curve39. The micro-radiocarbon dating results are summarized in the Table A total of six samples selected for micro-radiocarbon dating (55–70 cm in length) was shipped via cold-storage transport to the Paul Scherrer Institute (PSI). There, samples were rinsed with ultrapure water to remove the potentially contaminated outer layer. Around 20–30% of the ice mass was lost in this step. Samples were melted at room temperature in 1L containers  and filtered with prebaked (5 h at 800 °C) quartz-fiber filters. The Water Insoluble Organic Carbon (WIOC) and Elemental Carbon (EC) fractions were separated using a commercial combustion system  with a recently developed thermal-optical method Swiss_4S). The resulting COS. The re