Replantation of lamina spinous process ligament complex and miniature titanium plate shaping internal fixation in the treatment of tumors in the spinal canal.

OBJECTIVE: Purpose This study aims to explore the clinical efficacy of laminospinous process ligament complex reimplantation combined with mini-titanium plate fixation in the treatment of thoracolumbar intraspinal tumors. METHODS: A retrospective analysis was performed on 43 cases of intraspinal tumors treated with thoracolumbar intraspinal tumor resection from August 2018 to March 2021, and 27 cases underwent laminospinous process ligament complex reimplantation combined with micro titanium plate shaping. Fixation (laminar replantation group), and 16 patients underwent laminectomy combined with pedicle screw internal fixation (laminectomy group). The operation time, blood loss, drainage tube removal time, cerebrospinal fluid leakage, spinal instability, and the incidence of secondary spinal stenosis were compared between the two groups. The pain VAS score, ODI score, and modified Macnab at the last follow-up were compared between the two groups. And the laminar fusion rate of the laminoplasty group was measured. RESULTS: Both groups successfully completed the surgery and obtained complete follow-up. The incidence of cerebrospinal fluid leakage and secondary spinal canal stenosis in the laminectomy group was lower than that in the laminectomy group, and the difference was statistically significant (P < 0.05). There was no statistically significant difference in the incidence of spinal instability between the two groups (P > 0.05). The operation time and intraoperative blood loss in the laminectomy group were less than those in the laminectomy group, and the drainage tube removal time was earlier than that in the laminectomy group. The difference was statistically significant (P < 0.05). At the final follow-up, there was no statistically significant difference in the pain VAS score, ODI score, and modified Macnab between the two groups (P > 0.05), but they were all significantly improved compared with preoperative ones. Fusion evaluation was conducted on the laminoplasty group. Two years after surgery, the fusion rate was 97.56% (40/41). CONCLUSIONS: The application of laminospinous process ligament complex reimplantation combined with mini titanium plate fixation during thoracolumbar intraspinal tumor resection can effectively reconstruct the spinal canal and posterior column structure, reduce the incidence of cerebrospinal fluid leakage and secondary spinal stenosis. The laminar fusion rate is high.

One-hole split endoscopy technique versus unilateral biportal endoscopy technique for L5-S1 lumbar disk herniation: analysis of clinical and radiologic outcomes.

BACKGROUND: Lumbar disk herniation (LDH) is one of the most common diseases of the spine, especially occurring in L4-5 and L5-S1 intervertebral disks, and surgery is a choice when conservative treatment is ineffective. The purpose of this study is to investigate the clinical efficacy and radiologic outcomes of one-hole split endoscopy (OSE) technique versus unilateral biportal endoscopy (UBE) technique in the treatment of L5-S1 lumbar disk herniation (LDH). METHODS: A total of 133 patients of a single center surgically treated for L5-S1 LDH between 2019 and 2021 were retrospectively included in this study, of which 70 were treated by UBE technique and the rest were treated by OSE technique. Hospitalization time, operative time, intraoperative blood loss, fluoroscopy times, incision length and related complications were recorded. Bone resection area (BRA), articular process resection rate, range of motion (ROM), sagittal translation (ST), disk height (DH), Visual Analog Score (VAS), Oswestry Disability Index (ODI) and Macnab criteria were used to evaluated the clinical efficacy. RESULTS: There was no statistically significant difference in hospitalization time or fluoroscopy times between the two groups. The operation time was shorter in the UBE group than that in the OSE group; however, the incision length was longer. Intraoperative blood loss and BRA were larger in the UBE group than in the OSE group. There was no significant difference in ROM, ST, DH, or postoperative facet resection rate between the two groups. There was no significant difference in ROM, ST, or postoperative facet resection rate compared with preoperative indicators in each group, but there was a significant difference in DH among distinct groups. At any time point, the lower back and leg VAS and ODI in each group were significantly improved compared to those before the operation, with no significant difference between the two groups. There was one case of dural tear in the UBE group. One case of transient hypoesthesia occurred in each of the two groups. The excellent-good rates of the UBE group and the OSE group were 88.6% and 90.5%, respectively. CONCLUSION: The OSE technique is an effective minimally invasive surgical option as well as the UBE technique in the treatment of L5-S1 LDH.

One-hole split endoscope technique for migrated lumbar disc herniation: a single-centre, retrospective study of a novel technique.

BACKGROUND: Lumbar disc herniation (LDH) is one of the most common diseases of the spine, and migrated LDH is a more serious type, associated with nerve root function injury or abnormality. Regarding the increasing surgery adoption of treating migrated LDH, we aimed to investigate the clinical efficacy and safety of discectomy with a novel technique-one-hole split endoscope (OSE) technique. METHODS: This was a retrospective analysis of migrated LDH treated between December 2020 and September 2021. Hospitalization time, operative duration, intraoperative blood loss, number of fluoroscopy exposures, incision length, postoperative facet preservation rate, number of excellent-good cases, lower back and leg visual analogue score (VAS), Oswestry Disability Index (ODI) and surgical complications were compared between high-grade migration group (82 cases) and low-grade migration group (148 cases). The Macnab criteria was used to evaluate the clinical outcome. The Shapiro‒Wilk test was used to test measurement data, and the χ2 test was used to test counting data. RESULTS: There was no significant difference in hospitalization time, operative duration, intraoperative blood loss, number of fluoroscopy exposures, incision length or postoperative facet preservation rate between the two groups by independent sample t test or nonparametric test. At any time point, the lower back and leg VAS and ODI of the two groups were significantly improved compared to those before the operation, but there was no significant difference between the two groups at the same time point by two-way repeated measures ANOVA. There were two cases of postoperative nerve root stimulation symptoms in the high-grade migration group and three cases in the low-grade migration group. There was one patient reoperated in the high-grade migration group. There was no significant difference in number of excellent-good cases between the two groups. The overall excellent-good rate was 89.6%. CONCLUSION: The OSE technique has the advantages of less trauma, faster recovery, complete removal of the nucleus pulposus and a satisfactory early clinical efficacy in the treatment of migrated LDH.

[Early-effectiveness of unilateral biportal endoscopic laminectomy in treatment of two-level lumbar spinal stenosis].

Objective: To analyze the early effectiveness of unilateral biportal endoscopy (UBE) laminectomy in the treatment of two-level lumbar spinal stenosis (LSS). Methods: The clinical data of 98 patients with two-level LSS treated with UBE between September 2020 and December 2021 were retrospectively analyzed. There were 53 males and 45 females with an average age of 59.9 years (range, 32-79 years). Among them, there were 56 cases of mixed spinal stenosis, 23 cases of central spinal canal stenosis, and 19 cases of nerve root canal stenosis. The duration of symptoms was 1.5- 10 years, with an average of 5.4 years. The operative segments were L 2, 3 and L 3, 4 in 2 cases, L 3, 4 and L 4, 5 in 29 cases, L 4, 5 and L 5, S 1 in 67 cases. All patients had different degrees of low back pain, among of which 76 cases were with unilateral lower extremity symptoms and 22 cases were with bilateral lower extremity symptoms. There were 29 cases of bilateral decompression in both segments, 63 cases of unilateral decompression in both segments, and 6 cases of unilateral decompression and bilateral decompression of each segment. The operation time, intraoperative blood loss, total incision length, hospitalization stay, ambulation time, and related complications were recorded. Visual analogue scale (VAS) score was used to assess the low back and leg pain before operation and at 3 days, 3 months after operation, and at last follow-up. The Oswestry disability index (ODI) was used to evaluate the functional recovery of lumbar spine before operation and at 3 months and last follow-up after operation. Modified MacNab criteria was used to evaluate clinical outcomes at last follow-up. Imaging examinations were performed before and after operation to measure the preservation rate of articular process, modified Pfirrmann scale, disc height (DH), lumbar lordosis angle (LLA), and cross-sectional area of the canal (CAC), and the CAC improvement rate was calculated. Results: All patients underwent surgery successfully. The operation time was (106.7±25.1) minutes, the intraoperative blood loss was (67.7±14.2) mL, and the total incision length was (3.2±0.4) cm. The hospitalization stay was 8 (7, 9) days, and the ambulation time was 3 (3, 4) days. All the wounds healed by first intention. Dural tear occurred in 1 case during operation, and mild headache occurred in 1 case after operation. All patients were followed up 13-28 months with an average of 19.3 months, and there was no recurrence or reoperation during the follow-up. At last follow-up, the preservation rate of articular process was 84.7%±7.3%. The modified Pfirrmann scale and DH were significantly different from those before operation ( P<0.05), while the LLA was not significantly different from that before operation ( P=0.050). The CAC significantly improved ( P<0.05), and the CAC improvement rate was 108.1%±17.8%. The VAS scores of low back pain and leg pain and ODI at each time point after operation significantly improved when compared with those before operation, and the differences between each time points were significant ( P<0.05). According to the modified MacNab criteria, 63 cases were excellent, 25 cases were good, and 10 cases were fair, with an excellent and good rate of 89.8%. Conclusion: UBE laminectomy is a safe and effective technique with little trauma and fast recovery for two-level LSS and the early effectiveness is satisfactory.

Anterior Cervical Discectomy and Fusion Using Zero-P System for Treatment of Cervical Spondylosis: A Meta-Analysis.

OBJECTIVE: The current study aimed to explore the efficacy of Zero profile intervertebral fusion system (Zero-P) and traditional anterior plate cage system (PC) in the treatment of cervical spondylotic myelopathy (CSM). Further, the present study evaluated effects of the treatments on medical security, height of intervertebral disc, adjacent-level ossification development (ALOD), and adjacent segmentation disease (ASD) through a systematic retrospective analysis. METHODS: Studies on Zero-P system and traditional anterior plate cage system for ACDF in the treatment of CSM were searched in PubMed, Web of Science, Ovid, Embase, and Cochrane Library databases. Two independent researchers screened articles, extracted data, and evaluated the quality of the articles based on the inclusion and exclusion criteria of the current study. RevMan5.3 software was used for meta-analysis following the guidelines of Cochrane collaboration network. Cervical curvature, interbody fusion rate, preoperative and postoperative disc height index (DHI), fusion cage sinking rate, postoperative dysphagia, ASD, ALOD, and loosening of screw were compared between the two groups. RESULTS: A total of 17 literatures were included in the present study, including 6 randomized controlled trials and 11 observational studies. The studies comprised a total of 1204 patients with CSM, including 605 patients in the Zero-P system group (Zero-P group) and 599 patients in the traditional animal plate cage group (PC group). Results of this meta-analysis showed that postoperative dysphagia [OR = 0.40, CI (0.28, 95% 0.58), P < 0.00001], ALOD [OR = 0.09, CI (0.02, 95% 0.39), P = 0.001], ASD [OR = 0.42, CI (0.20, 95% 0.86), P = 0.02], and screw loosening [OR = 0.20, CI (0.08, 95% 0.52), P = 0.0009] of the Zero-P group were significantly lower compared with the PC group. On the other hand, preoperative cervical curvature [WMD = -0.23, CI (-1.38, 95% 0.92), P = 0.69], postoperative cervical curvature [WMD = -0.38, CI (-1.77, 95% 1.01), P = 0.59], cage sinking rate [OR = 1.41, CI [0.52, 95% 3.82], P = 0.50], intervertebral fusion rate [OR = 0.76, CI (0.27, 95% 2.48), P = 0.38], preoperative DHI [WMD = -0.04, CI (-0.14, 95% 0.22), P = 0.65], and postoperative DHI [WMD = 0.06, CI (-0.22, 95% 0.34), P = 0.675] were not significantly different between the two groups. CONCLUSION: It was evident that the Zero-P system used in ACDF is superior compared with the traditional anterior plate cage system in postoperative dysphagia, avoiding ALOD, ASD, and screw loosening.

THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma.

Increasing studies have demonstrated that dysfunction of long noncoding RNAs (lncRNAs) plays critical roles in the development of human cancers. THAP9-AS1 has been reported to be dysregulated and associated with tumor progression in some cancers. However, the function and mechanism of THAP9-AS1 in osteosarcoma (OS) remain unclear. In the present study, we found that the expression of THAP9-AS1 was significantly upregulated in OS tissues and associated with the advanced stage of tumors and poor prognosis of patients. Blast comparison results showed that the SOCS3 promoter region and THAP9-AS1 had base complementary pairing binding sites. The interactions between THAP9-AS1, DNA methyltransferases (DNMTs), and SOCS3 were assessed by RIP and ChIP assays. The results of methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) validated that THAP9-AS1 enhanced the methylation level of the SOCS3 promoter. The mRNA levels of SOCS3 in OS cells could be reversed by the demethylation agent 5-aza-2'-deoxycytidine. The mRNA expression of SOCS3 was downregulated in OS tissues and negatively correlated with THAP9-AS1 expression in tumors. Moreover, the western blot and immunofluorescence (IF) assay data showed that THAP9-AS1 activated the JAK2/STAT3 signaling pathway by upregulating p-JAK2 and p-STAT3 and the nuclear translocation of p-STAT3. Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion and inhibited apoptosis, and this phenomenon could be reversed by SOCS3. Introduction of the JAK/STAT inhibitor AG490 partially abolished the stimulative effect of THAP9-AS1 on cellular processes. In addition, THAP9-AS1 decreased oxidative stress by reducing reactive oxygen species (ROS) and enhancing the mitochondrial membrane potential of OS cells via the SOCS3/JAK2/STAT3 pathway. Stable overexpression of THAP9-AS1 contributed to tumor growth and metastasis in vivo. In total, our findings suggested that upregulation of THAP9-AS1 might recruit DNMTs to epigenetically inhibit SOCS3, thereby activating the JAK2/STAT3 signaling pathway and oncogenesis of OS. These results provide novel insights for the understanding of OS progression.

Exosomal MATN3 of Urine-Derived Stem Cells Ameliorates Intervertebral Disc Degeneration by Antisenescence Effects and Promotes NPC Proliferation and ECM Synthesis by Activating TGF-ß.

OBJECTIVE: Low back pain (LBP) is one of the top three causes of disability in developed countries, and intervertebral disc degeneration (IDD) is a major contributor to LBP. In the process of IDD, there is a gradual decrease in nucleus pulposus cells (NPCs) and extracellular matrix (ECM). Exosomes are important exocrine mediators of stem cells that can act directly on cells for tissue repair and regeneration. In this study, we determined the antisenescence, cell proliferation promotion, and ECM modulation effects of human urine-derived stem cell (USC) exosomes (USC-exos) on degenerated intervertebral discs and explored the underlying mechanism. METHODS AND MATERIALS: USCs were identified by multipotent differentiation and flow cytometry for mesenchymal stem cell- (MSC-) specific surface protein markers. USC-exos were isolated from the conditioned medium of USCs by ultracentrifugation and then analyzed by transmission electron microscopy (TEM), particle size analysis, and western blotting (WB) for exosome marker proteins. The effects of USC-exos on NPC proliferation and ECM synthesis were assessed by Cell Counting Kit-8 (CCK-8), WB, and immunofluorescence (IF) analyses. The protein differences between normal and degenerative intervertebral discs were mined, and the temporal and spatial variations in matrilin-3 (MATN3) content were determined by WB and IF in the intervertebral disc tissues. The candidate molecules that mediated the function of USC-exos were screened out and confirmed by multiple assays. Meanwhile, the mechanism underlying the candidate protein in USC-exos-induced cell proliferation and regulation of ECM synthesis promoting the activities of NPCs was explored. In addition, the effects of USC-exos on ameliorating intervertebral disc degeneration (IVD) in mice were examined by assessing computed tomography (CT), magnetic resonance imaging (MRI), and histological analyses. RESULTS: The flow cytometry results showed that USCs were positive for CD29, CD44, and CD73, which are USC surface-specific markers, but negative for CD34 and CD45. In addition, USCs showed osteogenic, adipogenic, and chondrogenic differentiation potential. USC-exos exhibited a cup-shaped morphology, with a mean diameter of 49.7 ± 7.3 nm, and were positive for CD63 and TSG101 and negative for calnexin. USC-exos could promote NPC proliferation and ECM synthesis. The protein content of the matrilin family was significantly reduced in degenerative intervertebral discs, and the decrease in MATN3 was the most significant. USC-exos were found to be rich in MATN3 protein, and exosomal MATN3 was required for USC-exos-induced promotion of NPC proliferation and ECM synthesis, as well as alleviation of intervertebral disc degeneration in IVD rats. In addition, the effects of MATN3 in USC-exos were demonstrated to be achieved by activating TGF-ß, which elevated the phosphorylation level of SMAD and AKT. CONCLUSIONS: Our study suggests that reduced MATN3 can be considered a characteristic of intervertebral disc degeneration. USC-exos may represent a potentially effective agent for alleviating intervertebral disc degeneration by promoting NPC proliferation and ECM synthesis by transferring the MATN3 protein.

Elevated lymphotoxin-α (TNFß) is associated with intervertebral disc degeneration.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFß, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB. RESULTS: The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 µg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels. CONCLUSIONS: This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway.

BACKGROUND: MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, highly expressed in diverse cancers and was involved in cancer molecular pathogenesis. However, its deliverance profile and biological function in osteosarcoma (OS) remain unclear. METHODS: The expression of MNAT1 in OS was detected by western blot (WB) and immunohistochemistry (IHC). The potential relationship between MNAT1 molecular level expression and OS clinical expectations were analyzed according to tissues microarray (TMA). Proliferation potential of OS cells was evaluated in vitro based on CCK8 and OS cells colony formation assays, while OS cells transwell and in situ tissue source wound healing assays were employed to analyze the OS cells invasion and migration ability in vitro. A nude mouse xenograft model was used to detect tumor growth in vivo. In addition, ordinary bioinformatics analysis and experimental correlation verification were performed to investigate the underlying regulation mechanism of OS by MNAT1. RESULTS: In this research, we found and confirmed that MNAT1 was markedly over-expressed in OS tissue derived in situ, also, highly MNAT1 expression was closely associated with bad clinical expectations. Functional studies had shown that MNAT1 silencing could weaken the invasion, migration and proliferation of OS cells in vitro, and inhibit OS tumor growth in vivo. Mechanism study indicated that MNAT1 contributed to the progression of OS via the PI3K/Akt/mTOR pathway. We further verified that the MNAT1 was required in the regulation of OS chemo-sensitivity to cisplatin (DDP). CONCLUSIONS: Taken together, the data of the present study demonstrate a novel molecular mechanism of MNAT1 involved in the formation of DDP resistance of OS cells.

Exosomes Derived from Human Urine-Derived Stem Cells Inhibit Intervertebral Disc Degeneration by Ameliorating Endoplasmic Reticulum Stress.

OBJECTIVE: This study is aimed at determining the effects of human urine-derived stem cell-derived exosomes (USCs-exos) on pressure-induced nucleus pulposus cell (NPC) apoptosis and intervertebral disc degeneration (IDD) and on the ERK and AKT signaling pathways. METHODS: The NPCs were obtained from patients with herniated lumbar discs. Western blot analysis (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine endoplasmic reticulum (ER) stress levels of NPCs under stress. Human USCs were identified using an inverted microscope, three-line differentiation experiments, and flow cytometry. A transmission microscope, nanoparticle size analysis, and WB procedures were used to identify the extracted exosomes and observe NPC uptake. A control group, a 48 h group, and a USCs-exos group were established. The control group was untreated, and the 48 h group was pressure-trained for 48 h, while the USCs-exos group was pressure-trained for 48 h and treated with USCs-exos. WB, qRT-PCR, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis were used to determine the ER stress levels in stress conditions and after exosomal treatment. The AKT and ERK pathways were partially detected. Magnetic Resonance Imaging (MRI) and computed tomography (CT) were used to evaluate cell degeneration while exosomal effects on the intervertebral disc (IVD) tissue were determined by hematoxylin and eosin (HE) staining, Safranin O-fast green staining, immunohistochemical staining (IHC), nuclear magnetic resonance (NMR), spectrometric detection, and total correlation spectroscopy (TOCSY). IVD metabolites were also identified and quantified. RESULTS: After pressure culture, ER stress markers (GRP78 and C/EBP homologous protein (CHOP)) in the NPCs were significantly elevated with time (p < 0.05). Human USCs are short and spindle-shaped. They can successfully undergo osteogenic, adipogenic, and chondrogenic differentiation. In this study, these stem cells were found to be positive for CD29, CD44, and CD73. The exosomes were centrally located with a diameter of 50-100 nm. CD63 and Tsg101 were highly expressed while the expression of Calnexin was suppressed. The exosomes can be ingested by NPCs. USCs-exos significantly improved ER stress responses and inhibited excessive activation of the unfolded protein response (UPR) as well as cell apoptosis and disc degeneration through the AKT and ERK signaling pathways (p < 0.05). CONCLUSION: Through the AKT and ERK signaling pathways, USCs-exos significantly inhibit ER stress-induced cell apoptosis and IDD under pressure conditions. It is, therefore, a viable therapeutic strategy.

Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice.

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1ß in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.