Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts

Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by α-actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.

Groundwater Interacting at Depth With Hot Plastic Magma Triggers Phreatic Eruptions at Yugama Crater Lake of Kusatsu-Shirane Volcano (Japan)

Interpreting the triggering mechanisms for phreatic eruptions is a key to improving the hazard assessment of crater lakes. Yugama Crater Lake at Kusatsu-Shirane volcano, Japan, is the site of frequent phreatic eruptions with the recent eruptions in 1982–83, 1989, and 1996, as well as volcanic unrest, including earthquake swarms in 2014 and 2018. To understand the magma–hydrothermal interaction beneath Yugama Crater Lake, we analyzed lake waters from November 2005 to May 2021. From 2005 to 2012, Cl and SO4 concentrations decreased slowly, suggesting the development of a self-sealing zone surrounding the crystallizing magma. We focused on Ca, Al, and Si concentrations as representatives of the breach and dissolution of minerals comprising the self-sealing zone and the Mg/Cl ratio as an indicator for enhanced interaction between groundwater and hot plastic rock within the self-sealing zone. In 2006–2007, the Ca, Al, Si concentrations and the Mg/Cl ratio increased. No Cl and SO4 increase during this period suggests the self-sealing zone was leached by deep circulating groundwater rather than by magmatic fluids injection. After the 2014 earthquakes, Ca, Al, and Si increased again but were associated with a significant Cl increase and a pH decrease. We believe that the HCl-rich magmatic fluids breached the self-sealing zone, leading to fluids injection from the crystallizing magma to the Yugama crater. During this period, the Mg/Cl ratio did not increase, meaning that magmatic fluids ascending from the breached area of the self-sealing zone inhibited deep intrusion of groundwater into the hot plastic rock region. In 2018, magmatic fluids ascended through the self-sealing zone again with less intensity than in 2014. All eruptions since 1982 have been accompanied by a Mg/Cl ratio increase and a Cl decrease, whereas, when a significant HCl input occurs, as in 2014, no eruptions and no Mg/Cl ratio increase occurred. This demonstrates that the groundwater–hot plastic rock interaction, rather than the magmatic fluids input, played an essential role in triggering phreatic eruptions; i.e., phreatic eruptions can potentially occur without clear signs of fresh magma intrusions.

Stroke, spinal cord ischemia and upper limb ischemia in patients undergoing TEVAR with coverage of the left subclavian artery: a case series study

Background We performed routine spinal fluid drainage for patients who underwent TEVAR for thoracic aortic pathology together with left subclavian artery coverage, which was needed for achievement of a safe proximal sealing zone. We assessed the occurrence of spinal cord ischemia as well the rate of occurrence of other complications such as stroke, and upper limb ischemia. Results This was a case series study done between July 2014 and April 2020, in them all the left subclavian artery was covered to ensure a proximal safe seal zone. Routine spinal fluid drainage was done, keeping the spinal fluid pressure < 10–15 mmHg with catheter in place for 48 h. Data was obtained from twenty-three patients who underwent TEVAR for thoracic aortic dissection (73.91%), thoracic aortic aneurysm (21.74%), or ulcer (4.35%). Planning was based upon multi-slice computed tomographic angiography and covering the left subclavian was mandatory to achieve a proximal sealing zone. Technical success was achieved in 100% of cases. 4.35% of patients had three endograft, 56.52% had two endografts, 39.13% had one endograft. All patients lost their radial pulsations immediately after implantation, 8.70% developed post implantation syndrome(fever) that was managed conservatively, 4.35% developed stroke related to the anterior circulation, 4.35% developed signs of spinal cord ischemia. During the follow up, one patient died within 6 h after the procedure due to extensive myocardial infarction (patient was scheduled for CABG after our procedure). 17.40% developed upper limb symptoms that were tolerable and were managed conservatively. Conclusion By adopting routine spinal cord drainage and pressure monitoring, we can consider not to revascularize the left subclavian artery prior to TEVAR if it will be covered.

Sealing Mechanism Based on Force Chain and Experimental Investigation of Sealing Fractures

Lost circulation often occurs in fractured formations, which was a main technological problem during drilling. Conventional lost circulation material (LCM) was often used to form a plugging zone to prevent fluid loss during drilling. The formed seal was a granular material system composed of LCMs. This paper presented the physical mechanism of the force chain within the plugging zone. The seal performance is related to the properties of LCMs. A device for testing seal performance of LCMs with long fracture was developed. The effects of LCM performance on seal integrity were investigated using a plugging device with long fracture. The results showed that the wide particle size distribution (PSD) of LCMs tended to form a strong force chain network structure within the sealing zone. Increasing the stiffness and roughness of LCMs resulted in higher breaking pressure. The addition of fiber with high length-diameter ratio could improve the shear strength of the sealing zone and form a strong force chain network structure, and it can reduce fluid loss.

L-Plastin Phosphorylation: Possible Regulation by a TNFR1 Signaling Cascade in Osteoclasts

Tumor necrosis factor-alpha (TNF-α) signaling regulates phosphorylation of L-plastin, which is involved in forming the nascent sealing zone, a precursor zone for the matured sealing ring. This study aimed to illustrate the molecular mechanisms of L-plastin phosphorylation and the subsequent formation of the nascent sealing zone in osteoclasts treated with TNF-α. Here, we report that anti-TNF-receptor 1, inhibitors of signaling proteins (Src, PI3-K, Rho, and Rho-kinase), and siRNA of TRAF-6 attenuated the phosphorylation of LPL and filamentous actin content significantly in the presence of TNF-α. An inhibitor of integrin αvβ3, PKC, or PKA did not inhibit TNF-α-induced L-plastin phosphorylation. Inhibitors of Src and PI3-K and not Rho or Rho-kinase reduced tyrosine phosphorylation of TRAF-6, suggesting that Src and PI3-K regulate TRAF-6 phosphorylation, and Rho and Rho-kinase are downstream of TRAF-6 regulation. Osteoclasts expressing constitutively active or kinase-defective Src proteins were used to determine the role of Src on L-plastin phosphorylation; similarly, the effect of Rho was confirmed by transducing TAT-fused constitutively active (V14) or dominant-negative (N19) Rho proteins into osteoclasts. Pull-down analysis with glutathione S-transferase-fused SH2 and SH3 domains of Src and PI3-K demonstrated coprecipitation of L-plastin and TRAF-6 with the SH3 and SH2 domains of the PI3-K and Src proteins. However, the actual order of the interaction of proteins requires further elucidation; a comprehensive screening should corroborate the initial findings of protein interactions via the SH2/SH3 domains. Ultimately, inhibition of the interaction of proteins with SH2/SH3 could reduce L-plastin phosphorylation and affect NSZ formation and bone resorption in conditions that display osteoclast activation and bone loss.

ELMO1 signaling is a promoter of osteoclast function and bone loss

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important ‘signaling node’ in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the ‘ELMO1 interactome’ in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.

The Mechanism Switching the Osteoclast From Short to Long Duration Bone Resorption

The current models of osteoclastic bone resorption focus on immobile osteoclasts sitting on the bone surface and drilling a pit into the bone matrix. It recently appeared that many osteoclasts also enlarge their pit by moving across the bone surface while resorbing. Drilling a pit thus represents only the start of a resorption event of much larger amplitude. This prolonged resorption activity significantly contributes to pathological bone destruction, but the mechanism whereby the osteoclast engages in this process does not have an answer within the standard bone resorption models. Herein, we review observations that lead to envision how prolonged resorption is possible through simultaneous resorption and migration. According to the standard pit model, the “sealing zone” which surrounds the ruffled border (i.e., the actual resorption apparatus), “anchors” the ruffled border against the bone surface to be resorbed. Herein, we highlight that continuation of resorption demands that the sealing zone “glides” inside the cavity. Thereby, the sealing zone emerges as the structure responsible for orienting and displacing the ruffled border, e.g., directing resorption against the cavity wall. Importantly, sealing zone displacement stringently requires thorough collagen removal from the cavity wall - which renders strong cathepsin K collagenolysis indispensable for engagement of osteoclasts in cavity-enlargement. Furthermore, the sealing zone is associated with generation of new ruffled border at the leading edge, thereby allowing the ruffled border to move ahead. The sealing zone and ruffled border displacements are coordinated with the migration of the cell body, shown to be under control of lamellipodia at the leading edge and of the release of resorption products at the rear. We propose that bone resorption demands more attention to osteoclastic models integrating resorption and migration activities into just one cell phenotype.

Effect of technological voids on swelling behaviour of compacted bentonite–claystone mixture

The effect of technological voids on the swelling pressure of compacted MX80 bentonite–Callovo-Oxfordian (COx) claystone mixture was investigated by simulating the technological voids with a pre-defined space between the compacted disks of the mixture. Both axial and radial swelling pressures were monitored. After the tests, the microstructure of samples at different positions was investigated using mercury intrusion porosimetry (MIP), together with the determination of dry density and water content. Results showed that two main processes, filling and homogenization, occurred during soil hydration. In the filling process, the initial technological voids were gradually filled and the axial swelling pressure tended to increase. In the homogenization process, the samples had a sealing zone and a swelling zone. The sealing zone was characterized by a lower dry density than the expected final dry density while the swelling zone was characterized by a larger one. From the MIP results, the sealing zone showed larger inaccessible-pore, medium-pore, and large-pore void ratios and a lower small-pore void ratio than the swelling zone. Over time, the medium and large pores in the sealing zone were compressed, while the small pores in the swelling zone decreased. The stabilized axial swelling pressure followed a unique relationship with the expected final dry density. Moreover, the swelling pressure anisotropy was found to decrease as the technological voids increased.

Exclusion of complex aortic aneurysm with chimney endovascular aortic repair is applicable in a minority of patients treated with fenestrated endografts

OBJECTIVES The Medtronic Endurant II stent graft has recently received Conformité Européenne (CE) approval for the use in chimney endovascular aortic repair (ChEVAR) for the treatment for juxtarenal aortic aneurysms. The aim of this study was to assess the percentage of patients treated by fenestrated endovascular repair who would have been alternatively suitable for the treatment by the CE approved Medtronic ChEVAR. METHODS Preoperative computed tomography scans of 100 patients who underwent fenestrated endovascular aortic repair (FEVAR) between April 2013 and February 2017 were retrospectively assessed for the applicability of the ChEVAR technique according to the Medtronic instructions for use. Eligibility criteria included an aortic neck diameter of 19–30 mm, a minimum infrarenal neck length of 2 mm, a total proximal sealing zone of at least 15 mm, thrombus in the aortic neck in ˂25% of the circumference, and maximum aortic angulations of 60° in the infrarenal, 45° in the suprarenal segment and ˂45° above the superior mesenteric artery. RESULTS According to CE-approved inclusion criteria, 19 individuals (19%) would have been eligible for ChEVAR. In 81 patients, at least 1 measure was found outside instructions for use: (i) excluding factor was detected in 26 patients, (ii) incongruous measures in 28 patients and in 27 patients, 3–5 measures were outside the instructions for use. The most frequently identified excluding factor was an insufficient infrarenal neck at ˂2 mm length (n = 63; 63%). CONCLUSIONS Patients with juxta- or pararenal aneurysm treated by FEVAR are in 19% of the cases alternatively suitable for the treatment by ChEVAR within CE-approved instructions for use. While ChEVAR is suitable in many emergency cases, FEVAR offers a broader applicability in an elective setting.