Bipartite binding interface recruiting HP1 to chromosomal passenger complex at inner centromeres.

Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.

Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells.

To develop an off-the-shelf therapeutic product for intervertebral disc (IVD) repair using nucleus pulposus cells (NPCs), it is beneficial to mitigate dimethyl sulfoxide (DMSO)-induced cytotoxicity caused by intracellular reactive oxygen species (ROS). Hyaluronic acid (HA) has been shown to protect chondrocytes against ROS. Therefore, we examined the potential of HA on mitigating DMSO-induced cytotoxicity for the enhancement of NPC therapy. Human NPC cryopreserved in DMSO solutions were thawed, mixed with equal amounts of EDTA-PBS (Group E) or HA (Group H), and incubated for 3-5 h. After incubation, DMSO was removed, and the cells were cultured for 5 days. Thereafter, we examined cell viability, cell proliferation rates, Tie2 positivity (a marker of NP progenitor cells), and the estimated numbers of Tie2 positive cells. Fluorescence intensity of DHE and MitoSOX staining, as indicators for oxidative stress, were evaluated by flow cytometry. Group H showed higher rates of cell proliferation and Tie2 expressing cells with a trend toward suppression of oxidative stress compared to Group E. Thus, HA treatment appears to suppress ROS induced by DMSO. These results highlight the ability of HA to maintain NPC functionalities, suggesting that mixing HA at the time of transplantation may be useful in the development of off-the-shelf NPC products.

Adolescent Idiopathic Scoliotic Deformity Correction Surgery Assisted by Smart Glasses Can Enhance Correction Outcomes and Accuracy and Also Improve Surgeon Fatigue.

OBJECTIVE: Augmented reality (AR) is becoming more common and slowly being integrated into the surgical field. With the continuous progression of navigation and visualization techniques, AR has great potential to improve surgical quality and safety. Nevertheless, the effects of AR on surgical outcomes and surgeons' well-being remains poorly studied. The present prospective controlled study aims to assess the effect of surgery assisted with AR smart glasses on adolescent idiopathic scoliosis (AIS) deformity correction outcomes and surgeon fatigue. METHODS: AIS patients scheduled for surgical deformity correction were prospectively recruited and assigned to standard or AR-supported surgery, using lightweight AR smart glasses. The demographic and clinical features were recorded. The pre- and postoperative spinal features, operative time, and blood loss were recorded and compared. Finally, the participating surgeons were asked to complete a questionnaire (e.g., visual analog scale for fatigue) to compare the effects of AR on their well-being. RESULTS: Our results have shown enhanced spinal deformity corrections with Cobb angle (-35.7° vs. -46.9°), thoracic kyphosis (8.1° vs. 11.6°), and vertebral rotation (-9.3° vs. -13.8°) changes favoring AR-supported surgery. Moreover, AR resulted in significantly lower violation rates per patient (7.5% vs. 6.6%; P = 0.023). Finally, the visual analog scale for fatigue scores consistently showed a significant reduction in fatigue (5.7 ± 1.7 vs. 3.3 ± 1.2; P < 0.001) and other fatigue classifiers for the surgeons after AR-supported surgery. CONCLUSIONS: Our controlled study has highlighted the enhanced spinal correction rates in AR-supported surgery and also improved surgeons' well-being and fatigue. These results endorse the adaptation of AR techniques to support AIS surgical correction.

Homing of vertebral-delivered mesenchymal stromal cells for degenerative intervertebral discs repair - an in vivo proof-of-concept study.

Introduction: Cell transplantation shows promising results for intervertebral disc (IVD) repair, however, contemporary strategies present concerns regarding needle puncture damage, cell retention, and straining the limited nutrient availability. Mesenchymal stromal cell (MSC) homing is a natural mechanism of long-distance cellular migration to sites of damage and regeneration. Previous ex vivo studies have confirmed the potential of MSC to migrate over the endplate and enhance IVD-matrix production. In this study, we aimed to exploit this mechanism to engender IVD repair in a rat disc degeneration model. Methods: Female Sprague Dawley rats were subjected to coccygeal disc degeneration through nucleus pulposus (NP) aspiration. In part 1; MSC or saline was transplanted into the vertebrae neighboring healthy or degenerative IVD subjected to irradiation or left untouched, and the ability to maintain the IVD integrity for 2 and 4 weeks was assessed by disc height index (DHI) and histology. For part 2, ubiquitously GFP expressing MSC were transplanted either intradiscally or vertebrally, and regenerative outcomes were compared at days 1, 5, and 14 post-transplantation. Moreover, the homing potential from vertebrae to IVD of the GFP+ MSC was assessed through cryosection mediated immunohistochemistry. Results: Part 1 of the study revealed significantly improved maintenance of DHI for IVD vertebrally receiving MSC. Moreover, histological observations revealed a trend of IVD integrity maintenance. Part 2 of the study highlighted the enhanced DHI and matrix integrity for discs receiving MSC vertebrally compared with intradiscal injection. Moreover, GFP rates highlighted MSC migration and integration in the IVD at similar rates as the intradiscally treated cohort. Conclusion: Vertebrally transplanted MSC had a beneficial effect on the degenerative cascade in their neighboring IVD, and thus potentially present an alternative administration strategy. Further investigation will be needed to determine the long-term effects, elucidate the role of cellular homing versus paracrine signaling, and validate our observations on a large animal model.

Low Radiation Protocol for Intraoperative Robotic C-Arm Can Enhance Adolescent Idiopathic Scoliosis Deformity Correction Accuracy and Safety.

STUDY DESIGN: Retrospective case-series study. OBJECTIVES: To assess (1) low cone beam CT (CBCT) mediated intraoperative navigation to limit radiation exposure without compromising surgical accuracy, and (2) the potential of intraoperative C-arm CBCT navigation to augment pedicle screw (PS) placement accuracy in AIS surgery compared to pre-surgery CT-based planning. METHODS: The first part involved a prospective phantom study, comparing radiation doses for conventional CT, and standard (6sDCT) and a low dose (5sDCT) Artis Zeego®-imaging. Next, 5sDCT- and 6sDCT-navigation were compared on PS accuracy and radiation exposure during AIS correction. The final part compared surgical AIS deformity correction through intraoperative 5sDCT navigation to a matched cohort treated using conventional pre-surgery CT-scans for navigation. Outcome parameters included operation time, skin dose (SD), dose area product (DAP), intraoperative blood loss, postoperative complications, and PS deviation rates. RESULTS: The phantom study demonstrated a reduction in radiation for the 5sDCT protocol. Moreover, 5sDCT-imaged patients (n = 15) showed a significantly lower SD (-27.41%) and DAP (-30.92%), without compromising PS accuracy compared with 6sDCT-settings (n = 15). Finally, AIS correction through intraoperative CBCT C-arm navigation (n = 27) significantly reduced screw deviation rates (6.83% versus 10.75%, P = .016) without increasing operation times, compared with conventional CT (n = 37). CONCLUSIONS: Intraoperative navigation using a CBCT C-arm system improved the accuracy of PS insertion and reduced surgery time. Moreover, it reduced radiation exposure compared with conventional CT, which was further curtailed by adapting the low-dose 5sDCT protocol. In short, our study highlights the benefits of intraoperative CBCT navigation for PS placement in AIS surgery.

Somnolence and Dizziness During Mirogabalin Treatment in Patients With Neuropathic Pain Related to Lumbar Disease Who Switched From Pregabalin: A Retrospective Study.

STUDY DESIGN: Multicenter retrospective study. OBJECTIVES: To investigate adverse events (AEs) in patients with neuropathic pain related to lumbar disease who switched to mirogabalin from pregabalin. METHODS: This study surveyed the records of 82 patients with peripheral neuropathic leg pain related to lumbar disease who switched to mirogabalin from pregabalin. We evaluated AEs associated with pregabalin and mirogabalin, the continuation rate of mirogabalin, and the pain-relieving effect at 4 weeks after switching from pregabalin to mirogabalin. We compared patients who switched due to lack of efficacy (LoE group) and patients who switched due to AEs (AE group). RESULTS: The incidence rates of somnolence and dizziness with pregabalin were 12.2% and 14.6%, respectively, while the incidence rates with mirogabalin were reduced to 7.3% for somnolence and 4.9% for dizziness. The incidence of AEs with pregabalin was significantly higher in the AE group (LoE group: 11.1%, AE group 100%), especially for somnolence (LoE group: 3.2%, AE group: 47.1%) and dizziness (LoE group: 4.8%, AE: 52.9%). After switching, the incidences of AEs with mirogabalin were not significantly different between the 2 groups (LoE group: 15.9%, AE group: 23.5%), including for somnolence (LoE group: 7.9%, AE group: 5.9%) and dizziness (LoE group: 4.8%, AE group: 5.9%). There were no significant differences in continuation rate of mirogabalin or the pain-relieving effect between groups. CONCLUSIONS: The patients who experience somnolence and dizziness with pregabalin might be able to continue safely receiving treatment for their pain by switching to mirogabalin.

N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production.

Background: Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long-term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off-the-shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO-induced cytotoxicity. Furthermore, we examined the potential of antioxidant N-acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. Methods: NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. Results: DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose- and time-dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9-fold and 3.6-fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose-dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO-induced oxidative stress and supported maintenance of cell viability. Conclusions: This study demonstrated dose- and time-dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO-induced oxidative stress in the freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 µM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites.

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinome-wide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin ß1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

HP1-Assisted Aurora B Kinase Activity Prevents Chromosome Segregation Errors.

Incorrect attachment of kinetochore microtubules is the leading cause of chromosome missegregation in cancers. The highly conserved chromosomal passenger complex (CPC), containing mitotic kinase Aurora B as a catalytic subunit, ensures faithful chromosome segregation through destabilizing incorrect microtubule attachments and promoting biorientation of chromosomes on the mitotic spindle. It is unknown whether CPC dysfunction affects chromosome segregation fidelity in cancers and, if so, how. Here, we show that heterochromatin protein 1 (HP1) is an essential CPC component required for full Aurora B activity. HP1 binding to the CPC becomes particularly important when Aurora B phosphorylates kinetochore targets to eliminate erroneous microtubule attachments. Remarkably, a reduced proportion of HP1 bound to CPC is widespread in cancers, which causes an impairment in Aurora B activity. These results indicate that HP1 is an essential modulator for CPC function and identify a molecular basis for chromosome segregation errors in cancer cells.

Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2.

The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K](2-5) as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

Emi2 mediates meiotic MII arrest by competitively inhibiting the binding of Ube2S to the APC/C.

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Emi2, a direct inhibitor of the APC/C ubiquitin ligase. Two different ubiquitin-conjugating enzymes, UbcH10 and Ube2S, work with the APC/C to target APC/C substrates for degradation. However, their possible roles and regulations in unfertilized/fertilized eggs are not known. Here we use Xenopus egg extracts to show that both UbcH10 and Ube2S are required for rapid cyclin B degradation at fertilization, when APC/C binding of Ube2S, but not of UbcH10, increases several fold, coincidently with (SCF(ß-TrCP)-dependent) Emi2 degradation. Interestingly, before fertilization, Emi2 directly inhibits APC/C-Ube2S binding via the C-terminal tail, but on fertilization, its degradation allows the binding mediated by the Ube2S C-terminal tail. Significantly, Emi2 and Ube2S bind commonly to the APC/C catalytic subunit APC10 via their similar C-terminal tails. Thus, Emi2 competitively inhibits APC/C-Ube2S binding before fertilization, while its degradation on fertilization relieves the inhibition for APC/C activation.

Dynamic regulation of Emi2 by Emi2-bound Cdk1/Plk1/CK1 and PP2A-B56 in meiotic arrest of Xenopus eggs.

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56ß/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.