Inadequate thickness of subcutaneous tissue, pectoralis muscle wasting, and/or a lack of availability of subpectoral space can become significant issues in patients with or requiring cardiovascular implantable electronic devices (CIEDs). This is particularly concerning but not exclusive in the elderly population, who may experience discomfort and hypersensitivity of the site as well as the potential for erosion and an increased risk of infection. Thus, the use of an alternative location, the axillary fossa, offers several advantages that make it a suitable option. Specifically, it usually has a preserved fat pad (even in thin patients); is unperturbed by arm movement; is not directly exposed to contact; is easily accessed with no significant compromise of neurovascular structures; and is near the conventional subclavicular sites, with enough lead length to reach in case of the need for generator replacement. Here, we present a series of five patients, including details of their anatomy and the implant techniques used. Two underwent device replacements, with one of them presenting with significant ongoing site discomfort and the other with extreme tissue thinning, respectively. Two patients with no significant fat layer or pectoral muscle wasting had new pacemakers implanted. Lastly, a biventricular implantable cardioverter-defibrillator generator was reimplanted in a younger patient who had issues with protrusion and discomfort in the setting of thin subcutaneous tissue and the subpectoral space being occupied by a large breast implant. In conclusion, the use of the axillary fossa as a new alternative CIED implantation site, using the proposed implant technique, appears feasible and safe and demonstrated excellent results related to patient comfort and adequate device cover in five cases. However, there are still patients who either have a thin/atrophic pectoralis muscle, which would allow for excessive device protrusion, or else they are already symptomatic while having the device placed at this site . We thus thought that the use of an alternative location, the axillary fossa, might be a viable alternative. This site has several advantages that make it a suitable option. First, it usually has a preserved fat pad, even in thin individuals. Additionally, it remains unperturbed by arm movement and is not directly exposed to contact. Furthermore, the site is easily accessible, with no significant compromise of neurovascular structures and is in close proximity to the conventional subclavicular sites, with enough lead length to reach in the event generator replacement is required. We have previously gained experience with the lateral chest wall for device placement primarily with subcutaneous implantable cardioverter-defibrillators (S-ICDs), yet the proposed new location of the axillary fossa, although lateral, is far more superiorly located, within reach of existing leads in the case of device replacement and with additional perceived advantages, as described below.The lack or inadequate thickness of subcutaneous tissue can arise as a significant problem in patients with or who require cardiovascular implantable electronic devices (CIEDs). This applies particularly but not exclusively in the elderly population, as they more often than not have tissue fragility and the presence of a thin subcutaneous fat layer, which can lead to pressure from the implanted device being placed on the subcutaneous tissue. With this, the potential for erosion and infection as well as local discomfort and hypersensitivity may arise..The anatomic boundaries of the axillary fossa space are somewhat triangular in shape. First, the serratus anterior muscle covers the rib cage. This is the anchoring site of the posterior aspect of the device (the back of the pocket). The lateral aspect is bordered by the teres major muscle and part of the latissimus dorsi. Medially located is the border of the pectoralis muscle. Anteriorly (covering the device’s front aspect) is the subcutaneous tissue. Superiorly, but not adjacent and not in contact with the device position, runs a major neurovascular package including the brachial plexus, axillary artery, and vein with major branches  and 1C.The device generator is then laterally located in the axillary fossa as opposed to anteriorly subpectorally, as shown in the chest radiograph We herein present five cases performed using the axillary fossa for implantation. Described are two pacemaker generator changeouts, two new pacemaker implantations, and a biventricular ICD generator reimplantation. All patients were female, with four being elderly with extreme subcutaneous and muscular tissue thinning and one being middle-aged with slim subcutaneous tissue and a developed pectoralis muscle lacking available submuscular space, respectively.The cases were performed in the electrophysiology laboratory and operating room. Conscious sedation was managed by the anesthesia service; one case (case 4) required general anesthesia.An 81-year-old white female presented to the clinic with a history of sick sinus syndrome, who had undergone an initial pacemaker implant in 1989 and several replacements—with the most recent being in 2016—all performed elsewhere. The patient came to our practice with complaints of significant discomfort at the pacemaker site occurring since her last replacement. The site was also aggravated by certain movements and arm positions, adversely affecting her quality of life and ability to sleep..The pacemaker site appeared well-healed, with no evidence of infection or inflammation. The suture line was preserved, but the overall subcutaneous tissue was extremely thin, with evidence of subpectoral placement of the device. The pacemaker generator protrusion was readily evident and palpable at the margin of the pectoral muscle (still submuscular) ® ADDRS1; Medtronic, Minneapolis, MN, USA) .The most common approach for this type of pocket revision would have been to move the device medially, keeping it in the subpectoral plane and possibly anchoring it to the rib periosteum to avoid migration. The concern with that approach, however, was that the patient’s arm was already extremely sensitized (having had prior interventions as well) and an adequate comfort level could not likely have been achieved via this option. Instead, we elected to use a novel alternative site: the axillary fossa. This location is exempt from being affected by any arm movement and seems neutral to any body position or postural change. The pacemaker generator was also replaced for a smaller footprint unit . Because of her poor subcutaneous and muscular tissue, we elected to implant in the axillary fossa .A 96-year-old white female requiring a new pacemaker implant for sick sinus syndrome and tachy-brady syndrome with symptomatic four-second pauses presented. She had readily evident poor subcutaneous and muscular tissue. A smaller-volume generator unit was used  and the atrial lead was capped. Again, due to extreme tissue thinning and site discomfort, we elected to implant in the axillary fossa .A 92-year-old white female with an initial pacemaker implant occurring in 2005 for sick sinus syndrome and generator replacement in 2010 presented. She eventually developed chronic atrial fibrillation with slow ventricular response. The device was at an elective replacement indicator with extreme tissue thinning and site discomfort. The pacemaker was downgraded to a single-chamber device  of 25%. Given the widening of the QRS with interventricular conduction delay and New York Heart Association functional class IV symptoms, she was upgraded to a biventricular pacemaker and ICD two months later. The patient had large subpectoral breast implants near where the device was implanted, creating a subcutaneous left infraclavicular area. Of note is that she had a slim/athletic body build with thin subcutaneous tissue. Her clinical condition dramatically improved postimplant, and after more than a year of medical therapy, her EF normalized and she was able to resume an active lifestyle. Her device site, however, continued to cause significant discomfort, affecting her quality of life and physical capacity.® model DTMA1QQ; Medtronic, Minneapolis, MN, USA) .After several requests for the removal of the device, it was decided that it would be turned off and subsequent follow-ups regarding her EF would be completed. After six months of maintaining a normal EF on no biventricular pacing, the generator was removed two years following the initial implant, at the patient’s request. The leads remained in place. Unfortunately, she subsequently developed progressive congestive heart failure with deterioration of her EF to 30% as well as worsening mitral regurgitation. She was admitted to the hospital and, after diuresis and stabilization of her condition, she underwent biventricular ICD reimplantation in the axillary fossa at two months after removal of the previous generator  requiring a pacemaker implant for tachy-brady syndrome with paroxysmal atrial fibrillation and a symptomatic 4.8-second conversion pause associated with recurrent syncope presented. She had minimal subcutaneous and muscular tissue as well as readily evident ICM protrusion and discomfort. The ICM was removed and a standard volume generator unit was implanted  in the axillary fossa.An 81-year-old white female with a history of syncope and insertable cardiac monitor (ICM) placement . Once the needle is through the skin of the axillary fossa, this can act as a good guideline of the incision site, ideally 3 cm to 4 cm in length at the center of the axillary fossa perpendicular to the midaxillary line  and 3. The incision should be taken down to the muscularis fascia of the serratus muscle and a subcutaneous pocket can be performed with essentially an inferior dissection, having a 1-cm to 2-cm area for superior dissection if needed. The pocket is irrigated with a copious amount of antibiotic solution. If, during the dissection of the pocket, the intercostobrachial nerve is encountered, it can be spared, but, if inadvertently compromised, there is little if any clinical consequence. Potential complaints of numbness of the upper medial aspect of the affected arm after surgery have been reported when accessing this area using a different approach .2A micropuncture needle (with aspirating syringe) is used to transect, in a tangential fashion, close to the rib cage, the pectoral muscle from the anchoring site of the leads to the axillary fossa .A micropuncture catheter is advanced over the wire, which can then subsequently be exchanged for a 0.35 typical J-guidewire. A 9-French (Fr) sheath and introducer are advanced over the wire through the pectoral muscle. At this point, the dilator is removed (leaving the J-wire in place), allowing for the advancement of a second J-guidewire. One of the J-wires is selected and the 9-Fr dilator and sheath are readvanced through the muscle to the fossa. The wire is then removed and the top valve/portion of the sheath is cut. One of the lead’s ends can be then advanced into the sheath, snugging the distal electrode into it. Now, the end of the sheath across the muscle into the fossa can be gently pulled, allowing the lead to pass through. The sheath is then reloaded over the dilator, which is used over the remaining wire to gain access to the second lead with the same technique. With both leads positioned across the pectoral fossa, the new pacemaker device is ready to be connected ® models 5620 and 5829 from Abbott Laboratories, Chicago, IL, USA and Medtronic® model AADRS1 from Medtronic, Minneapolis, MN, USA), yet, in the fourth case, a biventricular ICD with a rounded contour was able to be adequately seated in the space , while, in the fifth case, a standard pacemaker size was used .We chose the smaller-volume pacemaker generators available on the market . Dermabond™ /Steri-strips™  and an occlusive dressing were applied.The device and leads for all cases were placed in an envelope made of a bioresorbable extracellular matrix (ECM) that was hydrated and embedded in 1 g of vancomycin or 80 mg of gentamicin antibiotic solution for infection prevention. This material conforms to the pacemaker generator to stabilize the can and leads, after being anchored to the muscle to prevent migration. Additionally, the ECM remodels and revascularizes over time, providing another layer of tissue, which, in these cases, was thought to help with patient comfort (CanGaroo. The patient in case 4 did experience left upper arm swelling thought to be due to heart failure (associated with edema of the lower extremities as well as a negative ultrasound for thrombosis) and transient numbness of the middle aspect of the arm postoperatively with minimal residual at two weeks after surgery and no pain or functional mobility impairment.In all patients, the procedures were well-tolerated and the devices were well-seated, with adequate device function including defibrillation threshold testing in the biventricular ICD case. There were no vascular or other significant complications and no infections. After an initial period of one week to two weeks of common wound-healing-related soreness, all patients had unobstructed, full range of motion on the ipsilateral arm Between two weeks and three months of follow-up, there were no infections or complaints of discomfort at the site. Cases 1 and 4 experienced significant improvement of their preexisting symptoms.We present an initial experience of a new cardiac device implantation site at the axillary fossa with a limited number of patients, yet the foundation of the technique and anatomy are very predictable and reproducible. Although this technique was conceived for use in elderly patients with minimal subcutaneous and/or thin muscular tissue, we showed that it could also be applied in other settings for cosmetic or comfort reasons and in cases when the subpectoral space is not available.The axilla appears as a protected space subject to no direct contact with other body structures or external elements like undergarments, other clothing, and seat belts, which present as the most common sources of discomfort or wound stress in the typically used infraclavicular space. Different from the lateral chest wall (a site used commonly for S-ICD implantation), the axillary space is not subject to direct pressure while sleeping or lying on that site. Specifically, the axillary fossa site lacks significant movement and is unaffected by surrounding muscle groups. As a result, the leads’ motion or torqueing stress is minimal.Possible complications to consider related to this approach other than those related to standard device implant in the subclavicular region with subcutaneous or subpectoral pockets may include injury of the intercostobrachial nerve, resulting in numbness of the upper medial/internal aspect of the related arm, which could be temporary or permanent, without affecting the motor function of the limb. Although unlikely to be compromised, there is also a major neurovascular package, including the brachial plexus and axillary artery and vein, with their branches in the superior aspect of the intended axillary fossa pocket area that should be avoided.A best effort is always made to match the patient’s body profile to the generator can using the smallest-volume pacemaker and the softest rounded-contour biventricular ICD shape to fit in the available space. Not all devices are suitable for all patients, and assessments must be performed on an individual basis. Further experience regarding long-term patient comfort and other unforeseen interactions would be needed.Of note, the use of intracardiac transcatheter devices  could certainly overcome some of these issues, yet, at present, the device may not be optimally used in patients with preserved atrioventricular synchrony or may add an additional procedural risk in patients with existing adequately functioning leads.The presented experience and description of this novel approach in a group of patients without many other options does not pretend to be claiming a position as a new standard in practice but rather should be taken as a subject of scrutiny by other operators who may decide to try it when warranted.Here, the axillary fossa is proposed as a new alternative pacemaker and ICD implantation site in patients with thin subcutaneous tissue, pectoralis muscle wasting, a lack of availability of the subpectoral space, and/or who are experiencing discomfort and hypersensitivity at the conventional implant site. The technique, at least in the short-term, appears to be feasible and safe and to demonstrate excellent results with regard to patient comfort and adequacy of device cover.


Anemarrhena asphodeloides Bunge (Asparagaceae), a commonly used Chinese traditional paediatric medicine. Anemarsaponin BII is one of the most active saponins isolated from  This study investigates the effects of anemarsaponin BII on the activity of CYP450s to provide more guidance for the clinical use of anemarsaponin BII. Using various diagnostic substrates, the effects of a fixed concentration of anemarsaponin BII (100 μM) on the activity of eight main isoforms of CYP450s  was first studied with pooled human liver microsomes (HLMs). Then, dose-dependent  and time-dependent  experiments were performed to obtain corresponding kinetic parameters.50 values of 13.67, 16.26 and 19.72 μM. Anemarsaponin BII acted as a non-competitive inhibitor of CYP3A4 with the IK value of 6.72 μM and competitive inhibitors of CYP2D6 and 2E1 with the IK values of 8.26 and 9.82 μM, respectively. Additionally, the inhibition of CYP3A4 was revealed to be time-dependent with the IK value of 4.88 μM and the inactK value of 0.053/min. Anemarsaponin BII showed significant inhibitory effects on the activity of CYP3A4, 2D6 and 2E1 with the ICin vivo experiments are needed to validate the potential drug–drug interactions. The inhibitory effect of anemarsaponin BII on the activity of CYP3A4, 2D6 and 2E1 indicated the potential drug–drug interaction between anemarsaponin BII and drugs metabolized by these CYP450s. Further  Cytochrome P450 enzymes (CYP450s) comprised a superfamily of hemoproteins that participate in various oxidative reactions and play an essential role in the metabolism of xenobiotics (Manikandan and Nagini Anemarrhena asphodeloides Bunge (Asparagaceae) is traditional medicine in China and many other Asian countries that has been widely administrated for thousands of years. Anemarrhena asphodeloides was reported to possess anti-inflammatory, immune-stimulating and anti-neuroinflammatory effects , NADP+, phenacetin (≥98%), acetaminophen (≥98%), 4-hydroxymephenytoin (≥98%), 7-hydroxycoumarin (≥98%), 4′-hydroxydiclofenac (≥98%), sulfaphenazole (≥98%), quinidine (≥98%), tranylcypromine (≥98%), chlorzoxazone (≥98%), 6-hydroxychlorzoxazone (≥98%), paclitaxel (≥98%), 6β-hydroxytestosterone (≥98%), clomethiazole (≥98%) and furafylline (≥98%) were obtained from Sigma Chemical Co. Montelukast (≥98%) was obtained from Beijing Aleznova Pharmaceutical . Coumarin (≥98%), diclofenac (≥98%), dextromethorphan (≥98%), and ketoconazole (≥98%) were purchased from ICN Biomedicals. Pooled HLMs were purchased from BD Biosciences Discovery Labware. All other reagents and solvents were of analytical reagent grade.Anemarsaponin BII (≥98%) and testosterone (≥98%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . +, 10 mM glucose-6-phosphate, 1 U/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), the appropriate concentration of HLMs, a corresponding probe substrate and anemarsaponin BII (or positive inhibitor for different probe reactions). The HADPH-generating system was added before the incubation to preincubate for 3 min at 37 °C. The reaction was terminated with 100 μL acetonitrile (10% trichloroacetic acid for CYP2A6). Then, the mixture was centrifuged at 12,000 rpm for 10 min, and an aliquot (50 μL) of supernatant was transferred for HPLC analysis with the help of the Agilent 1260 series instrument with DAD and FLD detector according to previous studies. The HPLC conditions for analysis of corresponding metabolites of CYP isoforms are summarized in The activity of CYP450s was evaluated by the marker reactions with specific substrates in human liver microsomes (HLMs). The reaction conditions are summarized in Incubations were performed in triplicate, and results were represented with the mean value. The final microsomal protein concentration and incubation times for the different probe reactions are summarized in 50). Additionally, 0–50 μM anemarsaponin BII was incubated with various concentration of probe substrates  to obtain the values of IK.The effect of anemarsaponin BII on the activity of CYPs was evaluated in pooled human liver microsomes according to previous studies  was performed in the presence of 0–50 μM anemarsaponin BII for 0, 5, 10, 15 and 30 min.The effect of incubation time on the inhibition of anemarsaponin BII was evaluated with 20 μM anemarsaponin BII. HLMs were preincubated with anemarsaponin BII and the NADPH-generating system for 30 min at 37 °C. Then, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates, of which final concentrations were approximate to maxV and mK. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (IK) values were calculated using non-linear regression according to the following equation:I is the concentration of the compound, IK is the inhibition constant, S is the concentration of the substrate, and mK is the substrate concentration at half the maximum velocity (maxV) of the reaction. The mechanism of the inhibition was inspected using the Lineweaver–Burk plots and the enzyme inhibition models. The data were obtained from triplicate experiments and the data comparison was performed using Student’s t-test and performed using IBM SPSS statistics 20 (SPSS Inc.).The enzyme kinetic parameters for the probe reaction were estimated from the best fit line, using least-squares linear regression of the inverse substrate concentration versus the inverse velocity (Lineweaver–Burk plots), and the mean values were used to calculate 50 values of 13.67, 16.26 and 19.27 μM . Additionally, the values of 53 min−1 .This study characterized the effects of anemarsaponin BII on the activity of eight isoforms of CYP450 enzymes  in human liver microsomes, which are commonly involved in the biotransformation of clinical drugs.4, but also regulates the metabolism of the majority of clinical drugs  of anemarsaponin BII reached to 239.5 ng/mL with the half-life (t1/2) of 2.7 h (Li et al. A. asphodeloide extracts containing 171.61 mg/kg anemarsaponin BII. The maxC of anemarsaponin BII was obtained as 271.22 ng/mL with the t1/2 of 3.21 h (Tang et al. 50 values of CYP3A4, 2D6, and 2E1. Therefore, the in vivo effect of anemarsaponin BII on the activity of CYPs needs further validation.Previous studies on the A. asphodeloide, a widely used herb in paediatric (Kim et al. P-gp is an important protein that related to the transport of various drugs and UDP-glucuronosyltransferases were considered to mediate the biotransformation of several xenobiotics, of which the activity also induced changes in the plasma concentration of drugs (Liu Anemarsaponin BII is the major component of rugs Liu , 2019a. Taken together, anemarsaponin BII acted as a non-competitive inhibitor of CYP3A4 and competitive inhibitors of CYP2D6 and 2E1. Moreover, the inhibition of CYP3A4 was affected by the concentration of anemarsaponin BII and the incubation time, while the inhibition of CYP2D6 and 2E1 was only affected by the concentration of anemarsaponin BII. These results indicated the potential drug–drug interaction between anemarsaponin BII and drugs metabolized by CYP3A4, 2D6, and 2E1, which needs further studies to validate.


This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca Moreover, Cx36 is essential for the primary and secondary rod pathways in the retina8.In the central nervous system, electrical synapses directly connect the cytoplasm of neighboring neurons and provide a means for fast signal transmission. Electrical synapses are formed by gap junctions, which are assembled from connexin molecules. Among these proteins, Connexin36 (Cx36) is the most abundant connexin isoform in neurons of the mammalian central nervous system. It is strongly expressed in the olfactory bulb12. In the mammalian retina, for example, coupling in AII amacrine cells is increased by calcium influx through extra-synaptic NMDA receptors, subsequent activation of Ca2+/calmodulin-dependent kinase II , and phosphorylation of Cx369.Recent evidence shows that gap junction channels are not simple intercellular channels but undergo complex, activity-dependent regulation2+ changes. Among these proteins, several kinases were shown to directly or indirectly modulate Cx36-containing gap junctions, e.g., protein kinase A16 (PKA) and CaMKII17, with different CaMKII isoforms presumably regulating Cx36 in different neuronal cell types13. Accordingly, the protein sequence of mouse  Cx36 contains several consensus motifs for PKA, CaMKII and other kinases18. Serine 315 (S315) constitutes a CaMKII phosphorylation site11, which is located close to the C-terminal end of the protein and thereby in immediate vicinity of the PDZ binding domain formed by the last four amino acids  Cx35  dependent manner.Thus, our data provide evidence that MUPP1 (via PDZ 10) and 14–3–3 proteins γ and η bind differentially to the C-terminal tail of maCx35: MUPP1 increases its binding when S298 24 showed that phosphorylation of connexins may alter the internalization rate and affect gap junction size. Therefore, we tested in cultured cells whether the phosphorylation of S315 has an influence on the size of gap junctions. As we have successfully used mmCx36 in a mammalian expression system to measure gap junction volume25 and sequences for mouse and perch connexins are conserved with respect to the phosphorylated serine and PDZ binding domain . This suggests that phosphorylation of mmCx36 S315 does not affect gap junction size in HEK293 cells. However, when we compared gap junction volume between mmCx36 and a truncated mutant (Cx36 S318 ter), we found clusters to be significantly smaller in the truncated mutant , consistent with an earlier report20.Earlier studiesRattus norwegicus, rn) in HEK293 cells. Again, we used the wild-type and phosphomimetic mutant (S315D) and cotransfected it with MUPP1, which was fused to GFP  fails to form gap junctions in transfected HeLa cells, the same construct assembled into gap junctions in HEK293 cells, suggesting that the assembly machinery differs between cell lines.As an earlier reportons Fig. c–e and c26. To stimulate endogenous CaMKII activity and consequently phosphorylation of mmCx36, we treated transfected HeLa cells with 100 µM glutamate for 15 min  in a microarray. SAP102 was also added due to its similarity to PSD95, sharing with it three PDZ domains, an SH3 domain and a C-terminal guanylate kinase domain. For both proteins, phosphorylation of S315 in mmCx36 led to a decrease in binding of the PDZ domain  and FLAG-tagged PSD95. As expected, PSD95 showed virtually no colocalization with Cx36  failed to reproduce this result, presumably because the aspartate residue is not a good substitution for a phosphorylated serine in this case. Also, activation of CaMKII via endogenously expressed glutamate receptors did not lead to detectable differences in the association of mmCx36 and its scaffolds.11 and the PDZ-dependent association with scaffolding proteins20. As CaMKII-mediated phosphorylation was shown to be linked to NMDA receptor activation11, this switch may provide a substrate for activity-driven plasticity at Cx36-containing electrical synapses.Here, we provide evidence that phosphorylation of a serine residue in the C-terminal tail of Cx36/Cx35 may serve as a molecular switch to regulate the binding of different scaffolding proteins to Cx36/35. This concept connects two well-known mechanisms that were shown to regulate Cx36-containing gap junctions: phosphorylation by CaMKII28. However, the Cx36/35 sequence does not contain a classical consensus 14–3–3 binding site29. Still, earlier studies reported a similar switching mechanism in viral31 but also mammalian proteins32, such as the receptor tyrosine kinase ERBB4 and the inward rectifier potassium channel IRK1 (Kir2.1): phosphorylation of a PDZ binding motif decreases PDZ binding activity and mediates interaction with 14–3–3 proteins31. This suggests that this switch is a rather general means to regulate protein–protein interactions.Interestingly, the phosphorylation-induced switch seems to act in two different directions: it increases binding to 14–3–3 proteins and decreases binding to PDZ 10 of MUPP1. Most likely, two different mechanisms are at work: (1) the binding to MUPP1 is likely mediated via the PDZ binding site at the very C-terminal end of Cx36/35 and this binding affinity changes when S315/298 is phosphorylated. (2) The binding to 14–3–3 proteins may be direct, via the phosphorylated S315/298, as was reported for Cx43Our results are based on a protein microarray approach, which resulted in several candidate proteins potentially affected by Cx36/35 S315/298 phosphorylation. Pull-down experiments were used to test some of the candidate proteins for direct interaction which confirmed the binding interactions, but only confirmed the phosphorylation-dependent switch for MUPP1 and 14–3–3 proteins (γ and η isoforms).24. However, there may be several explanations for the absence of an effect on gap junction size: First, the phosphomimetic serine-to-aspartate mutation—although also introducing a negative charge, just like phosphorylation—might not be similar enough to the real phosphorylation because phosphate groups contain an additional negatively charged oxygen atom and differ in their ionic shell from negatively charged amino acid residues33. Thus, phosphate groups create a different chemical landscape34 and may be more potent in disrupting protein–protein interactions. In line with this, an earlier study reported that also 14–3–3 proteins do not bind to phosphomimetic threonine-to-aspartate mutations30. Lack of efficacy for the phosphomimetic mutation may also explain why binding of MUPP1 to the PDZ binding domain of Cx36 was not affected in the S315D mutants in HEK293 cells, whereas it was when maCx35 peptides were used that carried a phosphorylation at S298 (Cx35 S298 phos).Co-expression studies in HEK293 cells were employed to test whether the phosphorylation-dependent interaction with MUPP1 affects the size of mmCx36-containing gap junctions. This was not the case; introduction of a phosphomimetic mutation (S315D) did not affect the volume of Cx36-containing gap junctions in HEK293 cells. This was somewhat surprising because introduction of a phosphomimetic mutation at Cx43 S373 affects gap junction formationSecond, phosphorylation of S315/298 may only moderately affect PDZ binding affinity: our microarray data revealed some residual binding of MUPP1-PDZ-10 to S298 phos, which might also explain the persistent colocalization between MUPP1 and the phosphomimetic mmCx36 mutant, which we observed in the cell culture system. We obtained similar results for PDZ2 of PSD95 and SAP102: association clearly depended on the PDZ binding domain in the C-terminal tail but phosphorylation only decreased the binding and did not entirely prevent it.17, by influencing the orientation of intracellular domains in the Cx36 protein. Here, the interaction with 14–3–3 proteins may come into play, which we found to be stronger when S298 was phosphorylated in Cx35. 14–3–3 proteins are able to bind two phosphorylated residues simultaneously35. Thus, it is tempting to speculate that 14–3–3 proteins might simultaneously interact with two phosphorylated amino acids within different domains of Cx36/35, forming an intramolecular bridge and thereby affecting Cx36/35 gating.Finally, phosphorylation of S315 may not affect gap junction size but channel gating36 showed that less than 5% of all connexons present in a gap junction plaque contribute to gap junction conductance, and only this small fraction of active channels needs to be phosphorylated to enhance coupling. Thus, it seems likely that confocal microscopy is not sensitive enough to detect these subtle changes. Furthermore, a change in the binding affinity of a small fraction of gap junction channels for PDZ scaffolds may free those channels to engage in conformational changes, including those potentially induced by 14–3–3 protein binding, that regulate coupling, while maintaining the important associations of the PDZ scaffolds with the plaque as a whole.Our attempts to use endogenous phosphorylation of S315 also failed to demonstrate a detectable phosphorylation-driven switch in association of Cx36 with PDZ scaffolds Fig. . It is iPhosphorylation in gap junction proteins is very common and serves different functions. Here, we provide evidence for a molecular mechanism that allows the Cx36/35 protein to recruit different interaction partners to the synapse in response to activity-related calcium entry and subsequent activation of CaMKII. This again confirms the notion that electrical synapses share many properties with their chemical counterparts when it comes to synaptic plasticity.Purified GST fusion proteins . After expression, cells were lysed with lysozyme (1 mg/ml) and insoluble material was separated by centrifugation at 17,000 rpm for 40 min. The supernatant was applied to a glutathione sepharose column and incubated overnight on a rotating platform at 4 °C. Non-bound material was removed at the next day and the column was washed several times with binding buffer . Proteins were eluted with binding buffer, supplemented with 10 mM reduced glutathione. For purification of His-tagged proteins, supernatants were applied to a nickel column. After flow-through of non-bound material, the column was washed with washing buffer . Captured proteins were eluted with elution buffer .Recombinant proteins/protein domains Table  were exp25.For peptide pull-downs (n = 2–4), 100 µl of µMACS streptavidin beads (Miltenyi Biotec) were mixed with 0.25 µg of maCx35 peptides and equal amounts of purified PDZ domains/proteins for each condition in phosphate buffered saline (PBS) with 0.05% P20 (P20 is 20% Tween). The samples were incubated on ice for an hour and applied to a magnetic column for isolation. After several washes, adsorbed proteins were eluted with pre-heated (95 °C) elution buffer, containing 50 mM Tris HCl (pH 6.8), 50 mM dithiothreitol, 1% SDS, 1 mM EDTA, 0.005% bromophenol blue, 10% glycerol. SDS-PAGE (10% gels) and western blot analysis were performed as previously described37. Monomeric eGFP-CaMKIIa fusion construct was a gift of Dr. M. Neal Waxham 38. HEK293 and HeLa cells were plated at a density of 5 × 105 cells in a Petri dish (6 cm diameter), including 12 mm coverslips, in 5 ml Dulbecco’s Modified Eagle Medium. Cells were transfected with 0.6–1.4 µg/ml DNA using Lipofectamine (Life Technologies), 24 h after seeding. All transfections were done as triplicates and independently performed at least twice.Three different mmCx36 constructs  were cloned into the pRK5 vector . The PDZ domain 10 of rnMUPP1 was cloned into the PetM11 vector. All constructs were sequenced for accuracy. PSD95-FLAG in pcDNA5/FRT/TO was a gift from Dr. Wei-dong Yao . This clone was initially generated by Zhang et al.2). Afterwards, HeLa cells were fixed in 2% PFA in PBS for 15 min and prepared for confocal scans.To stimulate endogenous CaMKII activity, transfected HeLa cells were treated with serum-free media containing 1 mM glycine and 100 µM glutamate for 15 min in a cell culture incubator  and incubated in primary antibodies . Stacks were deconvolved with Huygens Essential deconvolution software  and further processed in Fiji . After background subtraction and histogram normalization, stacks were thresholded using the automated Otsu threshold. Gap junction clusters were detected with the 3D Simple Segmentation plugin and their volume was measured using the 3D Manager plugin in Fiji. Data were tested for statistical differences using the Mann–Whitney test in Prism 6 (GraphPad Software), at an alpha level of 0.05.The volume of Cx36-containing gap junctions between adjacent HEK293 cells was quantified as describedmeasure function in Fiji. A linear region of interest (2 µm in length) was placed in a gap junction and the average intensity along this ROI was measured for each channel. The average intensity ratio of both channels in each experimental condition was used as a measure of PSD95/Cx36 association.The intensity ratio of mmCx36 and PSD95 located at gap junctions was calculated using the Images of co-transfected cells are shown as single scans. Single transfected cells are presented as a maximum intensity projections of 12 sections (z-distance: 0.2 µm). Contrast and brightness were adjusted for presentation purposes in Fiji.Supplementary Information


R2 = 0.99). The peak stress in screws with realistic or simplified design correlated well (R2 = 0.82) but simplified models underestimated the peak stress. In conclusion, the results showed that the diameter of the screw has a major role on the mechanics of the screw-vertebral structure for each patient. Simplified screws can be used to estimate the mechanical properties of the implanted vertebrae, but the systematic underestimation of the peak stress should be considered when interpreting the results from the FE analyses.Pedicle screw fixation is extensively performed to treat spine injuries or diseases and it is common for thoracolumbar fractures. Post-operative complications may arise from this surgery leading to back pain or revisions. Finite element (FE) models could be used to predict the outcomes of surgeries but should be verified when both simplified and realistic designs of screws are used. The aim of this study was to generate patient-specific Computed Tomography (CT)-based FE models of human vertebrae with two pedicle screws, verify the models, and use them to evaluate the effect of the screws’ size and geometry on the mechanical properties of the screws-vertebra structure. FE models of the lumbar vertebra implanted with two pedicle screws were created from anonymized CT-scans of three patients. Compressive loads were applied to the head of the screws. The mesh size was optimized for realistic and simplified geometry of the screws with a mesh refinement study. Finally, the optimal mesh size was used to evaluate the sensitivity of the model to changes in screw’s size (diameter and length) and geometry . For both simplified and realistic models, element sizes of 0.6 mm in the screw and 1.0 mm in the bone allowed to obtain relative differences of approximately 5% or lower. Changes in screw’s length resulted in 4–10% differences in maximum deflection, 1–6% differences in peak stress in the screws, 10–22% differences in mean strain in the bone around the screw; changes in screw’s diameter resulted in 28–36% differences in maximum deflection, 6–27% differences in peak stress in the screws, and 30–47% differences in mean strain in the bone around the screw. The maximum deflection predicted with realistic or simplified screws correlated very well ( In the lumbar spine, pedicle screw fixation is the most widespread technique to achieve spinal fusion and stabilization . In 2008Despite the extensive use of pedicle screws in the current clinical practice, screw loosening and screw breakage are recurring mechanical complications of spinal fixation that can bring to a revision surgery in about 6% of cases . For thiThe aim of this study was to verify and evaluate the sensitivity of subject specific FE models of the vertebra with two pedicle screws for different sizes of the implant and in case of realistic and simplified geometry of the screw.Anonymized CT-scans of the thoracolumbar spine of three patients were collected. One vertebra per patient was segmented, converted to a FE model, virtually implanted with pedicle screws, and vertical loads were applied to the head of the screws, perpendicular to their axis. A mesh refinement study for realistic or simplified geometry of the screws was performed to choose the optimal mesh size that was used to evaluate the sensitivity of the model to changes in screw’s size (diameter and length) and geometry . An overview of the study is presented in Three anonymized clinical pre-operative CT-scans of the thoracolumbar spine of three patients were analyzed. The scans were previously acquired at the University Hospital Centre (CHU) of Poitiers (France) and transferred only after anonymization (CHU86-RECH-R2020-02-01). These patients were treated with a posterior pedicle screw fixation for different reasons: two patients reported a vertebral fracture at L1 (Patients #1 and #3), one patient had osteoarthritis (Patients #2). The scanning parameters are reported in The pedicle widths and the distances between the approximated insertion points and the anterior wall of the vertebral body were measured in a cross-section corresponding to the approximated insertion points and including the longitudinal axes of the screws. From these measurements, and based on the advice of an experienced surgeon, it was concluded that the size of the vertebrae was ideal for the insertion of pedicle screws with diameter (D) equal to 6.5 mm and length (L) equal to 45 mm. The shape of the vertebrae was reconstructed by manual image segmentation of the CT cross-sections  . The res® SpaceClaim Release 20.2 . Through a reverse engineering process (“SkinSurface” command), a 3D solid model of each vertebra was reconstructed. The surface at the bottom of the model, representative of the inferior endplate, was used to apply the boundary conditions.The segmented vertebrae were exported as surface meshes (STL) and imported in the 3D modeling software Ansys® S4® Element MIS Monoaxial) was simulated. The realistic geometry of the implant was imported as STP file. Nine different sizes of pedicle screws available on the market were tested including D equal to 5.5, 6.5, or 7.5 mm and L equal to 45, 50, or 55 mm. Nine simplified screws with a smooth conic body without the thread were also generated to evaluate how the thread affected the loading distribution and deformation within the vertebra-screws construct. The solid model of the simplified screws was obtained from each of the nine realistic screws as following. The head of the screw until the end of the junction with the conic feature where the thread begins, and the last portion of the screw after the end of the thread were kept from the original realistic design. The two circular exposed sections were then connected with a conic surface  were virtually inserted at the pedicles by a Boolean subtraction. The insertion point was determined by following medical guidelines  for meshing. The vertebra and the screws were meshed separately with tetrahedral quadratic elements (T10). For the vertebra, a uniform meshing algorithm was used so that the CT-scan grid was sampled uniformly during the definition of material properties of the bone. The element size was defined based on a mesh convergence study (see section “Generation of the FE Model”). A bonded contact was considered at the interface between the screws and the vertebra.Each vertebra-screws construct was imported in AnsysQCT), using a scale factor to convert the physical units to g/cm3. This assumption was considered acceptable for the goal of this study, which is focused on the verification and sensitivity analysis of the models. From the BMD equivalent density, the apparent density (ρApp) was obtained through Eq. 1  :boneν = 0.3  was used to solve the analysis. A workstation with processor model Intel(R) Xeon(R) CPU E5-2690 v3, 2.60 GHz was used. The analysis was run in parallel processing on 4 CPU Cores.The model was loaded with a quasi-static uniformly distributed force of 200 N (100 N per screw) applied to the head of the screw in a direction perpendicular to the longitudinal axis of the screw and perpendicular to the superior endplate, toward the caudal direction . The forvo study . Howevervo study . ANSYS® D = 6.5 mm, L = 45 mm) as advised by surgeons was tested for verification purposes. A mesh convergence study was conducted to estimate the optimal mesh size. The element size was changed separately in the bone and pedicle screws. Six maximum element sizes were tested for the screws between 0.4 and 1.2 mm while keeping the element size in the bone constant and equal to 1 mm. A maximum element size larger than 1.2 mm resulted in an inaccurate discretization of the circular cavity of the screw’s body; the inferior boundary was considered at 0.4 mm based on the dimension of the smaller thread in the realistic screw  . The lowThe computational time needed to solve the models with different element sizes is reported in The following metrics were considered for the different mesh sizes:•dmax) of the head of the screw calculated as the magnitude of the displacement vector .The maximum total deflection (•VMσ) in the screws  for the finest mesh. For the coarser meshes the VMσ was evaluated in the same coordinates, using the element shape functions to interpolate nodal values. Since the peak VMσ in the screws always occurred in a node on the external surface, for coarser meshes the coordinates of that node could fall outside the screw. To avoid this issue the outputs of the models were compared in a point within the volume of the screw at a distance equal to 0.05 mm from the point with peak VMσ.The peak Von Mises stress (•p3ε) in the bone  for the finest mesh. For the coarser meshes the p3ε was evaluated in the same coordinates, using the element shape functions to interpolate nodal values. Some peaks were excluded from the analysis because their location was either close to the boundary conditions of the model, or in geometric sharp corners (for example close to the cuspid at the insertion point or close to the tip of screws), or in an area on the external surface of the vertebra potentially affected by segmentation problems . In these cases, the next peak was considered.The peak Minimum Principal Strain . The following parameters were calculated for the three patients:Once the optimal mesh size was chosen for the bone and the screws, the influence of the diameter and length of screws on the stability of the simulated structure, for both the realistic and simplified models, was evaluated. The diameter and length of the left and right screws were changed simultaneously. The effect of changing the size of the screws was estimated with respect to the structural and local parameters estimated for the optimal screw size (•dmax) of the head of the screw calculated as the magnitude of the displacement vector .The maximum total deflection (•VMσ) in the screws . Some peaks were excluded from the analysis because their location was close to the sharp corner of the bone geometry generated by the Boolean subtraction at the screw insertion point. This happened only for Patient #1, for a screw diameter of 5.5 mm. In this case the next peak, at a distance higher than five element sizes from the sharp corner, was considered in the analysis.The peak Von Mises stress (•p3ε) in the bone . This value was calculated within a Region of Interest (ROI) defined at the screw-bone interface with a shape similar to the smooth conic body of simplified screws. The ROI was coaxial with the longitudinal axis of the screw and had a diameter equal to two times the diameter of the screw. Therefore, the dimensions of the ROI were scaled to each screw size. The same ROI was used for both simplified and realistic models.The mean Minimum Principal Strain (VMσ in the screws and p3ε in the bone (Patient #2), the frequency plots for p3ε for three screw sizes  were compared for models with simplified and realistic screw geometries.For the patient characterized by the highest relative differences in dmax and VMσ from the models with simplified and realistic screw geometry. The Slope, Intercept and coefficient of determination (R2) were calculated for each linear regression.Linear regression analyses were performed between the predictions of dmax for both simplified and realistic screw models was lower than 0.1% (screw) and 0.5% (bone) for each tested element size  and between 25 and 29% (right screws) were found. Since this patient had the largest pedicle among patients, models with screws with D = 5.5 mm were more sensitive to local changes in material properties. Generally, lower percentage differences in peak stress were found for the realistic screws compared to those obtained from simplified models. The percentage differences presented overall similar trends for the three patients. Also, the peak VMσ in the screw was higher in realistic models compared to those with simplified screws. For a fixed length, the VMσ increased for lower diameters; for a fixed diameter, the VMσ decreased for longer screws. However, in some cases with realistic screws, this behavior was not observed probably due to differences in local mechanical properties of bone adjacent to screws among models with different screw sizes.The diameter had higher impact on peak c models . In factp3ε more than the length    . A good  = 0.82) . Neverthp3ε was highly influenced by the combination of screw geometry  and the distribution of Young’s modulus in the bone, whereas the distribution of values of p3ε within a ROI around the screw was similar for simplified and realistic design of screws, with only a localized increase of strain for a few elements in the realistic screw models . This was due to the fact that areas of concentration of stress occurred close to the thread, which may play a larger role compared to the diameter of the screw. However, the VMσ distribution over the screws was similar among the different mesh sizes for both realistic and simplified models, showing that the stress pattern is not much influenced by the element size.As expected, in this study percentage relative differences in peak VMσ in the screws and on the mean p3ε in the bone than the length of the screw. This shows that for mono-cortical screws the anchorage in the pedicle, which mainly consists of cortical bone, is more important compared to the anchorage within the vertebral body, which is mainly composed of trabecular bone. Therefore, finding the compromise between the largest diameter of the screws by avoiding iatrogenic fractures is crucial to provide a good anchorage on the cortical bone, which results in lower micromotions at the screw-vertebra interface and better distribution of stress, thus preventing post-operative complications. Our results are in line with most experimental and numerical studies in the literature, that showed the predominant effect of the diameter of the screws compared to their length . This finding suggests that geometrical differences between the two designs of screws and local differences of material properties around the screw between the two models do not influence the overall stiffness of the model. VMσ, due to the lack of stress raisers considered in the realistic design. These differences could also be amplified by local heterogeneity in the Elastic modulus of bone elements. In fact, the distribution of Young’s modulus in the bone had a strong influence on the peak p3ε, whereas the distribution of strain around the screw-bone interface was similar for simplified and realistic models. This finding highlights the importance of the choice of modeling the screw’s geometry realistically or to use a simplified model depending on the application.Overall, the predictions of the simplified models correlated well with the predictions from the realistic ones, especially for the global structural properties . CS and TG were employed by company Aesculap (Germany). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TAp63γ is an important transcription factor. We previously reported that peptidyl‐prolyl isomerase (PPI) Pin1 directly binds to TAp63γ protein and identified that serine 12 (S12) in the transactivation domain (TAD) of TAp63γ is required for regulation of its transcriptional activity. In the present study, we report that Pin1 stimulates transcriptional and pro‐apoptotic activities of TAp63γ; this Pin1‐mediated stimulation may depend on phosphorylation of S12 mediated by JNK1 and results in striking activation of TAp63γ. JNK1 represses transactivity of TAp63γ in cells without abundant Pin1 proteins and enhances it in the presence of sufficient levels of Pin1. Collectively, our data suggest a novel mechanism for regulation of TAp63γ transactivity: TAp63γ with unphosphorylated S12 is moderately active, phosphorylation at this residue (pS12) makes it hypoactive, and Pin1 binds to the pS12‐P13 motif and makes TAp63γ hyperactive. Our findings will aid in the elucidation of the mechanism underlying modulation of TAp63γ.The  12) in the transactivation domain of TAp63γ is unphosphorylated, TAp63γ is moderately active to transactivate its downstream genes. Phosphorylation at this residue (pS12) can be mediated by kinase JNK1, leading to a repression of TAp63γ transactivity; isomerization of the pS12‐P13 motif can be mediated by Pin1, which makes TAp63γ hyperactive.When the serine 12 residue (S AalanineCL‐PARP1cleaved PARP1CoIPco‐immunoprecipitationIBimmunoblottingIPimmunoprecipitationMTT3‐‐2,5‐diphenyl‐tetrazolium bromidePprolinePPIpeptidyl‐prolyl isomerasepSphosphorylated serineSserineSAMsterile alpha motifTthreonineTADtransactivation domainTIDtransinhibition domainYtyrosinep63 gene belongs to the p53 family and encodes at least 10 isoforms, which can be classified into TA and ∆N isotypes (TAp63 and ∆Np63 proteins) according to their differences at the N termini. TAp63s contain the full transactivation domain (TAD) at the N termini, while ∆Np63 isotypes have an incomplete TAD with a weaker transactivity. After transcription, both TA and ∆N isotypes can be spliced into mRNAs with different 3’ termini, generating at least 5 different C termini, α, β, γ, δ, and ε. Among them, the γ types miss the sterile alpha motif (SAM) and the transinhibition domain (TID) at their C termini compared with the α isoform of p63 proteins [The proteins , 3. TAp6proteins , 5, 6. Tproteins , 9, 10. proteins , 12 and proteins . These Tproteins , 14, reaproteins . Accordiproteins . It has proteins . Recentlproteins . As a shproteins , 20, 21.Due to their key roles in cell cycle control, both expression levels and activities of p63 proteins are tightly regulated in cells . Accordi2 incubator. Transient transfection was performed with Entranster™‐H4000 , and total amounts of plasmid DNA were balanced with corresponding vectors for each transfection. Constructs of pcDNA3.1‐HA‐TAp63γ, pcDNA3.1‐HA‐TAp63γ(S12A), pcDNA3.1‐Pin1, pcDNA3.1‐Pin1(W34A), and pcDNA3.1‐JNK1 were previously described [Saos‐2, Hela, and H1299 cells were cultured in Modified McCoy's 5a Medium (BI) supplemented with 10% FBS (BI) and 1% penicillin G/streptomycin  at 37 °C in a humidified 5% COescribed , 27, 29.Immunoprecipitation (IP) and immunoblotting (IB) analyses were performed as previously described , 27. AntLuciferase assays were performed as described previously , 27. Saot‐test was used for comparison between two groups. P < 0.05 was considered statistically significant. All the error bars indicate SD.All experiments were carried out in triplicate. Two‐tailed In a previous study, we performed a pull‐down experiment and found that TAp63γ protein forms a complex with PPI Pin1; mutation on tryptophan 34 to alanine (W34A) in Pin1, which was reported to disrupt the binding of this isomerase to its substrates, significantly impairs its physical interaction with TAp63γ . To conf12) is crucial to transactivity of TAp63γ [12 is followed by a proline residue (P13), composing a putative Pin1 modification site [12‐P13 site is involved in Pin1‐mediated stimulation of TAp63γ  motif in the TAD of TAp63γ can strikingly activate its transcriptional activity .These results suggest that JNK1‐mediated phosphorylation of TAp63γ at serine 12 can repress its transactivity, in the absence of abundant Pin1 ; in cells rich in Pin1 , the peptidyl‐prolyl isomerization of this phosphoserine‐proline (pSp63 gene encodes multiple transcription factors [12) in the TAD is critical to regulation of TAp63γ transactivity [12 and the adjacent residue, proline 13 (P13), compose a potential Pin1‐binding site, which is supposed to lose the putative interaction by S12A mutation. We find that S12A mutant TAp63γ cannot be stimulated by Pin1  mediated by IKKβ or JNK1 can repress its activity; in the presence of Pin1, isomerization of this pS12‐P13 motif makes TAp63γ hyperactive. Our data are helpful to elucidate the regulation of TAp63γ, which is an important transcription factor in tumorigenesis and germline quality control, as well as a potential therapeutic target against p53‐altered tumors [Sactivity , 27. In d tumors , 16.The authors declare no conflict of interest.CL devised the hypothesis. XF, WH, KH, HC, LC, and SF designed and performed the experiments. W H and CL analyzed the data and wrote the manuscript.


Ice exhibits extraordinary structural variety in its polymorphic structures. The existence of a new form of diversity in ice polymorphism has recently been debated in both experimental and theoretical studies, questioning whether hydrogen-disordered ice can transform into multiple hydrogen-ordered phases, contrary to the known one-to-one correspondence between disordered ice and its ordered phase. Here, we report a high-pressure phase, ice XIX, which is a second hydrogen-partially-ordered phase of ice VI. We demonstrate that disordered ice undergoes different manners of hydrogen ordering, which are thermodynamically controlled by pressure in the case of ice VI. Such multiplicity can appear in all disordered ice, and it widely provides a research approach to deepen our knowledge, for example of the crucial issues of ice: the centrosymmetry of hydrogen-ordered configurations and potentially induced (anti-)ferroelectricity. Ultimately, this research opens up the possibility of completing the phase diagram of ice. Water ice exhibits several hydrogen-ordered and disordered phases and it’s unclear if a disordered phase can transform into only one ordered phase. Here, the authors identify a partially hydrogen-ordered phase at high pressure, ice XIX, as the second hydrogen-ordered phase of ice VI beside ice XV. This extraordinary polymorphism makes ice unique with its universality and has inspired many studies in the wide-ranging fields of material science and Earth and planetary science. The structural variety of ice arises from the geometric flexibility of hydrogen bonds and hydrogen order-disorder phase transition2, and hydrogen ordering in ice structure also induces significant changes in the dynamic/static properties of ice along with the phase transition, such as immobilisation of molecular rotation3 and ferro- or antiferroelectrically aligned molecular structures6. A prominent unsolved question7 concerning the structural diversity induced by hydrogen ordering is whether a hydrogen-disordered phase of ice transforms into only one hydrogen-ordered phase, as inferred from the currently known phase diagram of ice, although the energies of its possible hydrogen configurations are close because of the inherent geometric frustration of the ice lattice12. Recent experiments on a high-pressure hydrogen-disordered phase, ice VI, revealed an unknown hydrogen-ordered form (β-XV13) besides the known ordered phase, ice XV14. Although the unknown ordered form would be a counterexample of the question, it has not been clarified whether β-XV is a distinct crystalline phase to be assigned as a new Roman numeral label due to lack of experimental evidence17. Herein we report a second hydrogen-ordered phase for ice VI, ice XIX, unambiguously demonstrated by in-situ dielectric and neutron diffraction measurements under high pressure. The phase boundary between ice VI and ice XIX shows that ice VI contracts upon hydrogen ordering, which thermodynamically stabilizes ice XIX in the higher-pressure region compared to ice XV because of the smaller volume of ice XIX than ice XV18. The pressure-induced multiplicity of hydrogen-ordered phases, also theoretically suggested in other ice polymorphs11, can induce (or control) the different manners of hydrogen ordering of ice. Thus, this study demonstrates a hitherto undiscovered polymorphism of ice.To date, more than 20 crystalline and amorphous phases of ice have been reportedComprehensive observation of hydrogen ordering in ice VI was conducted by dielectric experiments in the pressure range 0.88–2.2 GPa. Ice VI was initially obtained at room temperature and its dielectric properties were determined in both cooling and heating runs in the temperature range 100–150 K, using a newly developed pressure cell , ice XV is in the lower pressure region and the hydrogen-ordered phase in the higher-pressure region is a new phase, ice XIX, which has a smaller volume than ice VI and also ice XV. The appearance of ice XIX is governed by the PV term in the Gibbs energy expression, because the volume contraction thermodynamically stabilises ice XIX compared to ice XV. In this context, the phase boundary between ice XV and XIX would be like close to vertical unlike horizontal suggested in the previous study13 if their entropy difference is enough small, because ice XV has a larger volume than ice XIX  upon hydrogen ordering, probably due to the small volume contraction.A transition from ice VI to ice XIX was also observed in the neutron diffraction experiments, as appearance of new peaks due to symmetry lowering Fig. . Some ofing Fig. ; this teGPa Fig.  showed tP42/nmc, considering the primitive unit cell of ice XIX. Among them, thirteen space groups, having Pc, P21, P2, P1, which are the lower-symmetry space groups of Pca21, Pcc2, P21/a and P21/c, selected as the best candidates for ice XIX based on their fitting 8. The Pcc2 structural models are the most plausible for the space group of ice XIX, based on the structure refinements. Considering the suggested space group of ice XV, 14 or Pmmn8, centrosymmetry of hydrogen configurations is the most significance difference in hydrogen configuration between ice XIX and ice XV. In particular, Pcc2 suggests a pyroelectric structure as well as ice XI and its polar direction is along the c axis. Although further investigations, such as a single-crystal neutron diffraction experiment, are necessary to precisely determine the hydrogen configurations, centrosymmetry will be an intriguing point in structural studies of ice XV and XIX. In addition, the Pmmn structure model suggested for ice XV by Komatsu et al8. The space groups (Pcc2) indicate a possibility that there exists another fully hydrogen-ordered phase of ice VI instead of the partially hydrogen-ordered ice XIX considering the third law of thermodynamics. It is noted that Gasser et al. also came to the identical conclusion from their neutron diffraction measurements using decompressed samples at about the same time as we did. They referred our structural analysis and reported that the best fitting was obtained by the Pcc2 structural models, the same as our results20. They reported volume contraction of the hydrogen ordering from ice VI to XIX at ambient pressure. This result supports the phase diagram of ice VI and its hydrogen ordered phases clarified in this study.For the structure analysis of ice XIX, we considered candidates of its space group based on the group–subgroup relationship between ice VI and XIX, in addition to the experimentally confirmed reflection conditions. There are 36 subgroups for the space group of ice VI, 11 and the currently known phase diagram of ice, the low-temperature region of the phase diagram (below approx. 150 K) is a frontier region for exploring undiscovered ways of hydrogen-ordering in ice, which would greatly change the phase diagram of ice. It is additionally noteworthy that the unit cell size of ice XIX allows many possible hydrogen-ordered configurations (1964 symmetry-independent configurations), such that an exhaustive theoretical analysis for the all configurations is difficult. However, such a wide variety of hydrogen-ordered configurations and their stability evaluations might be a good benchmark for modern theoretical trials toward modelling biochemical and environmental processes with a large number of water molecules, such as using topological graph invariant theory21, combining oriented graph theory and density functional calculations, which can evaluate the energy stability of a large number of water-molecule arrangements. To the best of our knowledge, this is the first report in a hydrogen-bonded material for which different hydrogen-ordered configurations are realised depending on the pressure, although electric field is a known effective parameter to control ferro- and antiferroelectric structures of organic compounds23. This newly discovered coupling between hydrogen bond and pressure will extensively develop an research field of the pressure-controllability of hydrogen-ordered configurations. Furthermore, by combining high-electric field with high-pressure, this multi-extreme condition is expected to provide more various types of hydrogen ordering and physical properties ferroelectricity) to hydrogen-bonded materials. Very recent technical development of neutron diffraction experiment24 will encourage the intriguing exploration in P-T-E phase diagram focusing on the multiplicity of hydrogen-ordered phases.This study demonstrates the existence of multiple hydrogen-ordered phases for a hydrogen-disordered phase and clarifies the effectiveness of applying pressure to induce phase competition among the hydrogen-ordered phases. Based on previous theoretical studies−2 M) to accelerate the hydrogen ordering of ice VI12, and dielectric experiments were conducted on DCl-doped D2O ice VI (DCl concentration: 10−2 M) following the same experiment procedure as that for HCl-doped ice VI , and ice VI was prepared through solid–solid phase transitions, ice III → V → VI, to obtain a fine powder sample . We used the initial structural parameters with the ice VI structural model, based on the neutron diffraction pattern obtained at 1.6 GPa and 80 K. It was noted that site occupancies of hydrogen atoms were initially varied in the refinements with fixed atomic positions of hydrogen. χ2 values of the structure models are plotted in Supplementary Fig. χ2 < 9. In the first step of structure refinements, the site occupancies of hydrogen atoms were fitted one by one, and subsequently fitted together as variables. Since the first step has arbitrariness in its fitting order (e.g. α → β → ··· and β → α → ···), we conducted structure refinements in several ways for each model by changing the fitting order cyclically, such as α → β → ··· and β → γ → ···. However, the fitting results are almost independent of the order. The space group χ2 values of Pcc2 were comparable considering the dispersion of their refinement results, and those structural models were considered the most plausible for ice XIX. Atomic fractional coordinates and O-D bond lengths for ice XIX using the two models are shown in Supplementary Tables Initial candidates of ice XIX were determined based on the group–subgroup relationship between ice VI and XIX, using the SUBGROUPS program opened on the Bilbao crystallographic serverSupplementary InformationPeer Review File


Water availability for Native Americans in the southwestern United States during periods of prolonged droughts is poorly understood as regional hydroclimate records are scant or contradicting. Here, we show that radiocarbon-dated charcoal recovered from an ice deposit accumulated in Cave 29, western New Mexico, provide unambiguous evidence for five drought events that impacted the Ancestral Puebloan society between ~ AD 150 and 950. The presence of abundant charred material in this cave indicates that they periodically obtained drinking water by using fire to melt cave ice, and sheds light on one of many human–environment interactions in the Southwest in a context when climate change forced growing Ancestral Puebloan populations to exploit water resources in unexpected locations. The melting of cave ice under current climate conditions is both uncovering and threatening a fragile source of paleoenvironmental and archaeological evidence of human adaptations to a seemingly marginal environment. The type of water resource, as well as its accessibility and reliability, strongly influenced settlement and subsistence strategies, agricultural intensification, demographic trends, and migration from the end of the Late Archaic through the early Pueblo periods (AD 100–900) in the Zuni-Acoma area of the Little Colorado River drainage5. The type of water resource and often its context also can have ritual significance to bring rain, for the preparation of medicine and for ceremony. Water from certain contexts such as springs and caves engendered ritual responses involving emblematic connections through pilgrimages and ceremonial activities to obtain the water8. Archeological evidence suggests periodic altitudinal dislocations trending toward increased population growth and dispersal through upland environments11. The trend increases gradually from the pre-ceramic (early Formative Basketmaker) through early Pueblo periods, and even more dramatically after AD 850–90011. Temporal correlations between population movements in the Zuni-Acoma area from AD 100–950 and shifts in climate have been proposed, but the range and nature of short- and long-term environmental variability that potentially had an impact on the residential mobility of early Puebloans is poorly understood due to limited paleoenvironmental records14. Of the climate proxy records used to assess past precipitation and temperature in the southern Colorado Plateau, tree rings are among the most important16. They provide some of the most detailed studies on severe droughts and link Puebloan site occupation with periods of higher moisture14. Here, we contribute to this line of research by documenting five drought events over an 800-year period using well-dated charcoal fragments preserved in an ice core recovered from a lava tube in the El Malpais National Monument  has some of the best known and most conspicuous archeological sites in North America  congelation ice deposit that once filled most of the cave’s deepest section , except for its middle passage where a massive rafted boulder and breakdown fills almost the entire volume creating a short (5 m) narrow constriction  returned an age of AD 1097 ± 30. Two other pieces of charred pine tree needles (CF-1 and CF-2) collected from the cave floor at 15 and 17 m from the coring site  fragment of corrugated pottery was exposed as ice melting accelerated over the past decade . In the Southwest, the effects of El Niño are synergistically enhanced during highly positive phases of Pacific Decadal Oscillation 15. Thus, it is possible that a combination of multi-year La Niña-related winter precipitation deficits amplified by a negative state of PDO, and variation in summer monsoon patterns could better explain some of the persistent drought periods over the Southwest28. In fact, this scenario is consistent with results that associate the Medieval Warm Period drought (AD 800–1300) with persistent La Niña-like conditions and negative PDO30.The timing of all charcoal horizons Fig. a overlapLMA Fig. b. They a31 and later recognized in the tree-ring record just west of ELMA16. This event is coincident with a significant positive temperature anomaly in the Northern Hemisphere and a period of high solar irradiance32. The SCD slowed down noticeably the growth of stalagmites from caves in the Guadalupe Mountains  sediment δ18O record35. A series of other sites across the central Great Basin and the midcontinental US also document a period of aridity between ~ AD 20–30036. Although it has been claimed SCD may have been equal if not more severe than the Medieval megadrought31, the total amount of charcoal particles found in our ice core corresponding to this interval is very small. This does not necessarily imply the drought was less severe than others in the record, but it likely shows that there was less human activity in this cave and perhaps more in adjacent lava tubes.The age of the first charcoal horizon (AD 167) falls within the so-called Second Century Drought , with one of the drought events that occurred between AD 350 and 400 in west-central New Mexico16. These lower two charcoal and soot-rich layers correspond with early agricultural adaptations of the Basketmaker II (BM II) period, which in the Southwest is associated with the shift from foraging and residential mobility to agricultural dependence, the development of pithouse villages and seasonal if not year-round sedentism37. Although few in ELMA, BM II sites are well-documented regionally in the Zuni area38  in the Summerville bristlecone record31, it is tempting to associate the presence of the lower two horizons with micro-charcoal pieces in our core with anthropogenic ice melting practices, rather than wildfires. It is conceivable that the Ancestral Puebloan, who probably knocked down (by hand or with ground stone implements) ice stalactites and stalagmites accumulated near the cave entrance and carried them outside, moved to melt ice deeper in the cave when the need for water increased. Alternatively, the ice could have receded due to continual usage. Altogether, this supports our interpretation that minor BM II communities having temporary or seasonal camps nearby Cave 29 conducted small-scale melting ice activities for securing water for drinking and/or ceremonial purposes over these time periods. It is also expected that the water resources hidden in some of the lava tubes throughout ELMA were known to the Ancestral Puebloans who used a well-established network of trails that date back to prehistory  to reach to these resources  in the ice core dated at ~ AD 829 ± 29. This marks the first indisputable, large-scale melting activities beyond the narrow median section of the cave.The upper 30 cm of our ice record spans the time period from AD 700 to ~ AD 950. It includes three charcoal horizons Fig. , all of 17. The combustion event occurring ~ AD 829 could indicate an increased aridity event that forced the Pueblo I people in the ELMA region to melt more cave ice over a longer period of time. The presence of this charcoal layer alone, however, cannot be taken as evidence of a larger community in the region. This is because surface site frequency around Cave 29 remains relatively low and no long-term residential sites have been documented in the area, except for small clusters of stacked basalt rock enclosures around the cave entrance, possibly associated with hunting  or other short-term resource-focused occupations, including water collection18.These two charcoal layers fall within the P I period, when ELMA saw modest increases in population characterized by dispersed settlement and continued use of pithouse structures along with early surface architecture including pueblos of masonry and jacal17 at Cave 29 and two other surface sites nearby, which is dominated by ceramics, mostly jar sherds dating to the P II period when the use of the lava fields and flow margins increased significantly11. In addition, four other 14C-dated charcoal samples, one from the uppermost horizon in the ice core (AD 933), one from inside the Cibola sherd exposed on top of the ice deposit (AD 1097), and two from the cave floor deposit  to undertake sustained exploitation of ice in Cave 29). This is revealed by the artifact assemblage documented by Powers and OrcuttWe interpret the lack of charcoal younger than AD 950 in the ice core, but its abundance on the cave floor to suggest that the uppermost part of the cored ice deposit melted during the Medieval era megadrought. The age of the charcoal residue collected from the interior of the ceramic sherd, AD 1097, indicates a Pueblo II period ice melting activity. The in-situ discovery of the sherd, its position in relation to the top of the ice deposit, and the presence of charcoal residue coating its interior surface provides insights on the method employed by pre-contact Puebloan people (since the P I period) to obtain water from the cave ice.43. It could be that this fire event involved a small group of Ancestral Puebloans hunters or travelers of the P IV period on pilgrimage to collect water at Cave 29 or stopping at the cave in transit between regions along the trail routes across the lava fields.The last documented use of fire to melt ice in Cave 29 comes from well preserved charred material scattered around and between breakdown blocks some 27 m from the end of the gallery. The radiocarbon age suggests the combustion episode dates to ~ AD 1424 and is coincident with a hydrologic drought that struck the Southwest as revealed by extended low flows in the Colorado River BasinWe speculate that during the Little Ice Age (~ AD 1500–1850) the gallery beyond the mid narrow passage was again sealed with ice since no younger charred material was found so far. It is plausible that during this period new ice accumulated throughout the deeper part of the cave, including in the proximity of the coring site. This is probably when the Cibola corrugated sherd with charcoal residue from the P II period was completely covered by ice. The accelerated melting of ice documented over the past decade, exposed the sherd.19. Corroborating this information with the total absence of charcoal between the entrance and narrow median section of Cave 29  and millimeter-size angular charred particles  to capture water perhaps from larger and smokier unattended combustions. Smaller fires would allow better control of the melting and collection process providing cleaner water in smaller quantities than unattended larger fires that produce water at a faster rate with greater potential for contamination from charcoal and ash. Both scenarios point toward ways to capture melt water from perennial cave ice.An interesting question regarding the use of fire by Ancestral Puebloans, is how they handled the smoke in the rear part of the cave, knowing that the summer air circulation model within Cave 29 implies no exchange with surface. However, hot air and smoke emitted during combustion events in the inner part of the cave will always rise and generate an outward flow along the ceiling18. While not direct proof, the use of grooved or troughed wood to direct water supports the scenario of tending small fires and collecting water while in the cave.The presence of pots or even bowl-shaped sherds (including the corrugated Cibola Gray Ware fragment next to the coring site) could have served used as a platform to support small fires or coals and allow for water collection while melting ice. The curvature of the sherd acted as a container for the heat generated by fire or coals that in turn warmed the sherd and transferred that heat melting the ice, which was collected in vessels or from the cave floor. The melt water may have been directed to hand-held ceramic or pitch-coated basketry receptacles on the cave floor using a variety of methods such as branches or timbers cut along their axis. A timber with a trough-like groove cut along its axis is documented in another lava tube cave in ELMA, and though it likely dates to the historic era, its purpose appears to be associated with melting ice18. Windes18 and others have noted the distinct east–west trail that passes by Cave 29 and through the stacked basalt enclosure site near the cave entry. This is one among a network of trails that crisscross ELMA’s lava fields including the Acoma-Zuni trail, a well-known travel corridor that bisects the northern half of ELMA. Many of these trails link caves with caves and/or pre-contact pueblo sites along the flow margins and in kipukas in the lava flows17 . Such trails may have conducted Ancestral Puebloans the considerable distance from residential bases on the east and north sides of the lava fields to Cave 29 and other ice caves during pilgrimages to collect melt ice water, as travelers moving between the Acoma and Zuni areas or hunters seeking game at the cave or near it18.Surface archeological sites in the vicinity of the cave are dispersed and contain little evidence of year-round occupation tending instead to low diversity of features and material culture and lacking substantial architecture and middens. Population centers throughout ELMA’s Puebloan sequence lie along the margins of the lava fields 25 km east of Cave 29 and in the Cerro de Jaspe’ area 8 km northeast of the cave18. Ethnographic studies in the Cibola region have identified cave ice as a target resource for ritual activity, pilgrimage and for medicine societies seeking pure water from the ice for ceremonial and medicinal use7.An estimated 1.000.000 L of water would have been available from the ice in Cave 29 making it a reliable and long-term water source for the area that could have supported opportunistic as well as frequent utilization of the resource. Knowledge of the location and reliability of this water resource is suggested by the clusters of small, stacked basalt structures near the cave (and near other caves and crevasses where ice forms in ELMA’s lava flows), suggesting that the location could have served as a stopover for travelers and hunters or as an anchor point for logistical foragers targeting seasonally available resources in the area7. For example, lava tube caves were significant destinations for religious pilgrimages and use of these areas to help keep abreast of what plant, animal, and mineral resources were available18. Since Cave 29 sits on well-established trail routes crisscrossing ELMA, it is possible that the melted ice could also have been used during trading missions6 or accessed by migrants passing by the lava tube. Regardless, the timing of the charcoal fragments preserved within the cave ice deposit discussed here sheds light on one of many human–environment interactions documented in ELMA in a context highly susceptible to the influence of climate change. It also highlights the importance of an increasingly threatened source of paleoenvironmental and archeological evidence. The melting of cave ice under current climate conditions will result in the loss of ancient climate data. As such, this resource must be further examined in the near future before it completely vanishes.This study robustly characterizes five drought periods, during which Ancestral Puebloans entered Cave 29 and other lava tubes to harvest ice. We suggest that prior to Pueblo I people who left behind recognizable artifacts within ELMA, small groups of Basketmaker II and III utilized the cave seasonally for melt water for domestic purposes, while hunting or exploiting other resources but lived in communities in low land valleys of the Zuni area. Based on a variety of evidence for burning in archeological context, the fire-aided ice melting activities carried out during P I-III increased notably throughout the Medieval Warm Period. Physical modifications to the floor between the cave entrance and the narrow mid-passage are testament to water procurement activities at many times in the human history of the cave. The seasonal melting of ice near cave entrances leaves temporary shallow pools of water that would have been accessible to Ancestral Puebloans who surely would have taken advantage of this readily available water for drinking. When ice was absent or retreated in warmer, dryer periods and/or was harvested in this part of the lava tube, Ancestral Puebloans would have worked their way to the back of the cave, melting ice and leaving charcoal and ash deposits along the way. The process may have taken decades and perhaps repeated many times depending on the ice volume fluctuation. They certainly used the water from melted ice for drinking, but ceremonial and medicinal use cannot be excluded, since ice was considered purer and more desirable for harvesting for such purposes3 ice block in Cave 29 once filled-up much of the large gallery beyond a constriction that occurs halfway between the entrance and cave’s farthest point  were sent to the RoAMS Laboratory at the National Institute for Physics and Nuclear Engineering in Romania, for radiocarbon dating using a 1 MV Tandetron accelerator mass spectrometer were senSupplementary Information.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.