PD-1 deficiency aggravates spinal cord injury by regulating the reprogramming of NG2 glia and activating the NgR/RhoA/ROCK signaling pathway.

Spinal cord injury (SCI) is a devastating disorder and a leading cause of disability in adults worldwide. Multiple studies have reported the upregulation of programmed cell death 1 (PD-1) following SCI. However, the underlying mechanism of PD-1 deficiency in SCI is not well established. Therefore, we aimed to investigate the role and potential mechanism of PD-1 in SCI pathogenesis. PD-1 Knockout (KO) SCI mouse model was established, and PD-1 expression was evaluated in tissue samples by western blot assay. We then used a series of function gain-and-loss assays to determine the role of PD-1 in SCI pathogenesis. Moreover, mechanistic assays were performed to explore the association between PD-1, neuron-glia antigen-2 (NG2) glia cells, and miR-23b-5p and then investigated the involved signaling pathway. Results illustrated that PD-1 deficiency enhanced the inflammatory response, neuron loss, and functional impairment induced by SCI. We found that NG2 glia depletion aggravated inflammation, reduced neural survival, and suppressed locomotor recovery in murine SCI model. Further analysis indicated that NG2+ cells were increased in the spinal cord of SCI mice, and PD-1 deficiency increased the number of NG2+ cells by activating the Nogo receptor/ras homolog family member A/Rho kinase (NgR/RhoA/ROCK) signaling. Mechanistically, miR-23b-5p was identified as the negative regulator of PD-1 in NG2 glia. MiR-23b-5p deficiency reduced the expression of inflammatory cytokines, enhanced neural survival, and promoted locomotor recovery in SCI mice, which was counteracted by PD-1 deficiency. In conclusion, PD-1 deficiency exacerbates SCI in vivo by regulating reprogramming of NG2 glia and activating the NgR/RhoA/ROCK signaling.

Enhanced inhibition of protein disulfide isomerase and anti-thrombotic activity of a rutin derivative: rutin:Zn complex.

Rutin is a flavonoid that exists in plants and in commonly consumed foods. In recent years, rutin has been demonstrated to have anti-thrombotic efficacy through its inhibition of protein disulfide isomerase. However, the low aqueous solubility and high dose limit the therapeutic applications of rutin. In this study, we found that the chelation of zinc ions increased rutin aqueous solubility by 4-fold. More importantly, the thus-formed rutin:Zn complex inhibited PDI activity more potently than rutin itself. In a murine model with electric current-induced arterial thrombosis, the rutin:Zn complex slowed mouse arterial occlusion compared to rutin without increasing bleeding risk. Thus, the zinc chelation not only improved rutin aqueous solubility but achieved stronger inhibition of PDI. Furthermore, zinc chelation of a selected list of flavonoids containing the adjacent keto and phenoxy groups also increased their inhibition of PDI. Hence, our study provides a strategy to promote flavonoids' anti-thrombotic properties.

Diagnostic accuracy of cervical spine imaging to predict thoracic ossification of the posterior longitudinal ligament: Retrospective chart review.

PURPOSE: To analyze the significance of ossification index of cervical posterior longitudinal ligament as a risk factor for thoracic OPLL (ossification of the posterior longitudinal ligament) in patients with cervical OPLL. METHODS: We retrospectively analyzed the clinical data of cervical OPLL patients in Changzheng hospital, who received chest CT scans for screening of COVID-19, and included 87 patients into this study. According to the radiographic evidence, 87 patients were divided into CT group(cervical OPLL combined with thoracic OPLL）and C group(cervical OPLL group). We measured the cervical OS index (ossification index), and analyzed the relationship between thoracic OPLL and cervical OS index. RESULTS: There was no difference of age、sex、duration of symptoms、comorbidity between the 2 groups(P＞0.05). The mean cervical OS index was higher in the CT group than in the C group (8 ± 2 VS 3 ± 2，P＜0.001). CONCLUSIONS: Patients with cervical OS index ＞8 was considered as "high risk" of tandem OPLL, while with value ≤ 4 was considered as "low risk". Index between 5 and 8 were considered as "middle risk". This study demonstrated that the cervical OS index may be used as an indicator of thoracic OPLL in patients with cervical OPLL, with a high diagnostic accuracy.

Converting inert AlOOH into efficient electrocatalyst for oxygen evolution reaction via structural/electronic modulation.

Efficient and stable water-splitting electrocatalysts play a key role to obtain green and clean hydrogen energy. However, only a few kinds of materials display an intrinsically good performance towards water splitting. It is significant but challengeable to effectively improve the catalytic activity of inert or less active catalysts for water splitting. Herein, we present a structural/electronic modulation strategy to convert inert AlOOH nanorods into catalytic nanosheets for oxygen evolution reaction (OER) via ball milling, plasma etching and Co doping. Compared to inert AlOOH, the modulated AlOOH delivers much better OER performance with a low overpotential of 400 mV at 10 mA cm-2 and a very low Tafel slope of 52 mV dec-1, even lower than commercial OER catalyst RuO2. Significant performance enhancement is attributed to the electronic and structural modulation. The electronic structure is effectively improved by Co doping, ball milling-induced shear strain, plasma etching-caused rich vacancies; abrupt morphology/microstructure change from nanorod to nanoparticle to nanosheet, as well as rich defects caused by ball milling and plasma etching, can significantly increase active sites; the free energy change of the potential determining step of modulated AlOOH decreases from 2.93 eV to 1.70 eV, suggesting a smaller overpotential is needed to drive the OER processes. This strategy can be extended to improve the electrocatalytic performance for other materials with inert or less catalytic activity.

Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b' domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b' domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 µM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b' substrate-binding pocket and the a' domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b' domain is a critical domain for inhibitor development. Besides, we also report an innovative high-throughput screening method for the rapid discovery of PDI b' targeted inhibitors.

Flavonoids as Protein Disulfide Isomerase Inhibitors: Key Molecular and Structural Features for the Interaction.

Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b' domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

Cyclic Tensile Stress to Rat Thoracolumbar Ligamentum Flavum Inducing the Ossification of Ligamentum Flavum: An In Vivo Experimental Study.

STUDY DESIGN: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), radiological, and histological analyses of the rat ossification of ligamentum flavum (OLF) induced by cyclic tensile stress. OBJECTIVE: The aim of this study was to induce the OLF using cyclic tensile stress to rat thoracolumbar ligamentum flavum, and to investigate the possible molecular mechanism of tension-induced OLF. SUMMARY OF BACKGROUND DATA: Tensile stress has been considered as an important factor leading to the OLF. So far, however, no OLF induced by tension has been reported. METHODS: Forty rats were randomly divided into five equal groups. For control groups, the blank and anesthesia groups were not subjected to tension. For experimental groups, the 4-, 8-, and 12-week groups were subjected to cyclic tensile stress of ligamentum flavum after abdominal anesthesia for 4 weeks, 8 weeks, and 12 weeks, respectively, using an original stress apparatus for rats. The radiological and morphological changes of rat spine, as well as the protein and mRNA expressions of CD44, bone morphogenetic protein-2 (BMP-2), integrin ß3, collagen protein type I (COL1), osteopontin (OPN), runt-related transcription factor 2 (RUNX-2), and vascular endothelial growth factor (VEGF), were concerned. RESULTS: The micro-CT showed OLF in the 4-, 8-, and 12-week group. The axial maximum occupied area of ossifications was 1.42 mm2, 3.35 mm2, and 7.28 mm2, respectively. In histopathology, chondrocytes proliferated in the experimental model; woven bone arose in the 8- and 12-week groups, and was more noticeable in the 12-week group. According to western blot and RT-PCR, the expressions of seven osteogenesis-related molecules were all increased in three experimental groups. CONCLUSION: Cyclic tensile stress to the ligamentum flavum in rats can induce the OLF, and the longer the duration, the more visible the osteogenesis. The upregulation and synergism of osteogenesis-related molecules may contribute to the OLF induced by tensile stress.Level of Evidence: N/A.

Osthole enhances the bone mass of senile osteoporosis and stimulates the expression of osteoprotegerin by activating ß-catenin signaling.

INTRODUCTION: Osthole has a potential therapeutic application for anti-osteoporosis. The present study verified whether osthole downregulates osteoclastogenesis via targeting OPG. METHODS: In vivo, 12-month-old male mice were utilized to evaluate the effect of osthole on bone mass. In vitro, bone marrow stem cells (BMSCs) were isolated and extracted from 3-month-old OPG-/- mice and the littermates of OPG+/+ mice. Calvaria osteoblasts were extracted from 3-day-old C57BL/6J mice or 3-day-old OPG-/- mice and the littermates of OPG+/+ mice. RESULTS: Osthole significantly increased the gene and protein levels of OPG in primary BMSCs in a dose-dependent manner. The deletion of the OPG gene did not affect ß-catenin expression. The deletion of the ß-catenin gene inhibited OPG expression in BMSCs, indicating that osthole stimulates the expression of OPG via activation of ß-catenin signaling. CONCLUSION: Osthole attenuates osteoclast formation by stimulating the activation of ß-catenin-OPG signaling and could be a potential drug for the senile osteoporosis.

Prevalence of Ossification of Posterior Longitudinal Ligament in Patients With Degenerative Cervical Myelopathy: Cervical Spine 3D CT Observations in 7210 Cases.

STUDY DESIGN: A cross-sectional study. OBJECTIVE: The aim of this study was to investigate the prevalence of ossification of posterior longitudinal ligament (OPLL) in patients with degenerative cervical myelopathy (DCM). SUMMARY OF BACKGROUND DATA: OPLL of the cervical spine is one of the main entities of DCM in Asian populations. However, few studies have reported the prevalence of cervical OPLL in DCM patients. METHODS: A total of 7210 DCM patients (4546 males and 2664 females; mean age: 54 years) who underwent cervical spine three-dimensional computed tomography (3D-CT) at the Shanghai Changzheng Hospital between January 2012 and December 2016 were included in this study. Demographic data including age, sex, height, body weight, body mass index (BMI), concomitant diabetes mellitus (DM), and hypertension were recorded. The imaging diagnosis criterion for OPLL was thickness >2 mm on axial imaging. RESULTS: The overall prevalence of OPLL in the 7210 DCM patients was 18.22%, including 19.73% in males and 15.65% in females, with a significant difference between the two groups (P < 0.001). The prevalence of OPLL in DM and hypertensive patients was significantly higher than that in non-DM and normotensive patients (24.16% vs. 18.76% and 22.26% vs. 17.91%, both P < 0.001). Comparison by age and BMI showed that the prevalence of OPLL was the highest in the 70- to 79-year age group (21.91%) and obesity group (26.51%), respectively. CONCLUSION: This CT-based study revealed that the overall prevalence of OPLL in DCM patients was 18.22%. Furthermore, old age, male sex, comorbid hypertension or DM, and high BMI were risk factors for cervical OPLL. Given its high prevalence, CT examination is suggested to identify possible OPLL in DCM patients. LEVEL OF EVIDENCE: 2.

Long Non-coding RNA XIST May Influence Cervical Ossification of the Posterior Longitudinal Ligament Through Regulation of miR-17-5P/AHNAK/BMP2 Signaling Pathway.

Long non-coding RNAs (lncRNAs) play an important role in the development of bone-related diseases. This study was conducted to investigate the role and mechanism of lncRNA X inactive specific transcript (XIST) in the occurrence of cervical ossification of the posterior longitudinal ligament (OPLL). Here, primary human ligament fibroblasts cells (LFCs) were isolated from 30 cases of OPLL and 30 normal cervical posterior longitudinal ligament (non-OPLL) tissues to perform the qPCR and Western blot assay. We found that the mRNA level of lncRNA XIST was significantly increased in OPLL LFCs compared to non-OPLL LFCs. By bioinformatics analysis, we found that lncRNA XIST has four binding sites for miR-17-5p and found that the mRNA level of miR-17-5p was also significantly decreased in OPLL LFCs compared to non-OPLL LFCs. Since AHNAK is the target gene of miR-17-5p, we further found that the expression of AHNAK was significantly reduced in non-OPLL LFCs after being transfected with miR-17-5p mimic. The qPCR results showed that the mRNA expressions of BMP2 and Runx2 were significantly decreased. After being transfected with lncRNA XIST siRNA in the non-OPLL LFCs, the mRNA levels of lncRNA XIST, AHNAK, BMP2, and Runx2 were significantly decreased and the phosphorylated protein of Smad1/5/8 was reduced. After being cultured by mechanical vibration, the mRNA levels of lncRNA XIST, AHNAK, BMP2, Runx2, COL1, OC, OPN, and Phospho1 were significantly increased, but the mRNA expression of miR-17-5p was significantly decreased. The expression of phosphorylated Smad1/5/8 protein was also significantly increased. Together, this study was the first to determine that XIST gene inhibition plays an important role in the occurrence of cervical OPLL, through the mechanism of regulation of miR-17-5P/AHNAK/BMP2 signaling pathway. Thus, XIST may be a potential target that could be modulated for the treatment of cervical OPLL.

Reconstructing spinal dura-like tissue using electrospun poly(lactide-co-glycolide) membranes and dermal fibroblasts to seamlessly repair spinal dural defects in goats.

Many neuro- and spinal surgeries involving access to the underlying nervous tissue will cause defect of spinal dural mater, further resulting in cerebrospinal fluid leakage. The current work was thus aimed to develop a package which included two layers of novel electrospun membranes, dermal fibroblasts and mussel adhesive protein for repairing spinal dural defect. The inner layer is electrospun fibrous poly(lactide-co-glycolide) membrane with oriented microstructure (O-poly(lactide-co-glycolide)), which was used as a substrate to anchor dermal fibroblasts as seed cells to reconstitute dura-like tissue via tissue engineering technique. The outer layer is chitosan-coated electrospun nonwoven poly(lactide-co-glycolide) membrane (poly(lactide-co-glycolide)-chitosan). During surgery, the inner reconstituted tissue layer was first used to directly cover dura defects, while the outer layer was placed onwards with its marginal area tightly immobilized to the surrounding normal spinal dura aided by mussel adhesive protein. Efficacy of the current design was verified in goats with spinal dural defects (0.6 cm × 0.5 cm) in lumbar. It was shown that seamless and quick sealing of the defect area with the implants was realized by mussel adhesive protein. Guided tissue growth and regeneration in the defects of goats were observed when they were repaired by the current package. Effective cerebrospinal fluid containment and anti-adhesion of the regenerated tissue to the surrounding tissue could be achieved in the current animal model. Hence, it could be ascertained that the current package could be a favorite choice for surgeries involving spinal dural defects.

Longer plate-to-disc distance prevents adjacent-level ossification development but does not influence adjacent-segment degeneration.

STUDY DESIGN: Retrospective case-control study. OBJECTIVE: To clarify the association between plate-to-disc distance (PDD) and adjacent-level ossification development (ALOD) and adjacent segment degeneration (ASD). SUMMARY OF BACKGROUND DATA: Anterior cervical discectomy and fusion with plating provides higher fusion rate and improved alignment but has been reported to result in ALOD and ASD. METHODS: We retrospectively reviewed 218 patients with solid fusion after anterior cervical arthrodesis with plating at our institution between January 2004 and December 2010. PDD was measured on postoperative lateral radiographs for each adjacent disc space and used to assign patients to 1 of 3 groups: group L, long PDD (>5 mm); group S, short PDD (0 mm ≤PDD ≤5 mm); and group N, PDD (<0 mm, disk space encroachment). Mean follow-up was 5.3 years. Incidences of cranial and caudal ALOD and ASD with and without symptoms were compared among groups. Severity of caudal ossification was not measured in 30 patients because bony overlap precluded adequate visualization of the caudal level. RESULTS: Ossification developed in 134 (61%) of 218 cranial adjacent disc spaces and 45 (24%) of 188 caudal adjacent disc spaces (P < 0.01). Mean cranial PDD was shorter than mean caudal PDD (P < 0.01). Ossification rate was higher in group N than in group S at the cranial adjacent disc spaces (100% vs. 66%, P < 0.01). Incidences of both cranial and caudal ALOD were significantly higher in group S than in group L (66% vs. 31%, P < 0.01; and 31% vs. 13%, P < 0.01, respectively). No significant differences in symptomatic and asymptomatic ASD were seen among groups. CONCLUSION: Longer PDD does not decrease the incidence of ASD but it can prevent ALOD. We now attempt to place anterior cervical plates of 5 mm or greater from adjacent disc spaces. LEVEL OF EVIDENCE: 3.

Posterior hybrid technique for ossification of the posterior longitudinal ligament associated with segmental instability in the cervical spine.

STUDY DESIGN: Retrospective case series. OBJECTIVE: To discuss the indications for a posterior hybrid technique for ossification of the posterior longitudinal ligament (OPLL) associated with segmental instability in the cervical spine and evaluate its effectiveness and safety. SUMMARY OF BACKGROUND DATA: Dynamic factors have been shown to play an important role in the progression of ossification and OPLL myelopathy. Laminoplasty has been widely used to treat cervical OPLL, but progressive kyphosis and progression of ossified lesions are often detected in long-term follow-up. METHODS: Fifteen patients were treated by a posterior hybrid technique including laminoplasty and lateral mass screw fixation at unstable levels. Preoperatively, the extent and type of OPLL, spinal cord compression, and presence of high-intensity zones were investigated by x-ray, computed tomography, and magnetic resonance imaging. Segmental instability in the cervical spine was investigated by dynamic x-ray. Postoperatively, clinical outcomes were evaluated with the Japanese Orthopedic Association scoring system and visual analog scale scores for neck pain. Radiologic results included cervical alignment and progression of OPLL. RESULTS: A total of 17 intervertebral levels in 15 patients (11 mixed-type and 4 continuous-type OPLL) had segmental instability, which was consistent with the presence of high-intensity zone levels in 10 (66.7%) patients. Neurological function as evaluated by the Japanese Orthopedic Association scores was significantly improved 6 months postoperatively and well maintained 4 years postoperatively. Neck pain was significantly improved 4 years postoperatively. No patients developed progressive kyphosis or progression of ossified lesions during the follow-up. Only 1 patient developed unilateral C5 palsy and completely recovered 2 months later. CONCLUSIONS: This hybrid posterior technique seems to be effective and safe in the treatment of selected patients with OPLL associated with segmental instability. The potential benefits of this technique include a stable environment for spinal cord recovery and prevention of progressive kyphosis and OPLL.