Inhibition of circRNA NGFR promotes ferroptosis in gallbladder carcinoma cells.

Background: Gallbladder carcinoma (GBC) is a formidably aggressive malignancy. Circular RNAs (circRNAs) play crucial regulatory roles in cancer. NGFR is a novel circRNA implicated in various types of cancers. The primary goal of this study was to elucidate the role of NGFR in GBC. Methods: NGFR variants exhibiting discernible discrepancies were identified using RNA sequencing and validated using real-time PCR. Cell proliferation was assessed using 5-ethynyl-2'-deoxyuridine and Cell Counting Kit-8 assays. The ferroptotic phenotype was characterized by assessing the reactive oxygen species and Fe2+ levels. Western blotting was used to analyze ferroptosis-associated proteins. Superoxide dismutase, malondialdehyde, and glutathione levels were measured using commercially available reagent kits. The severity of mitochondrial damage was evaluated by assessing JC-1, MitoSOX, and ATP activities. Results: NGFR was upregulated, and its suppression inhibited cell proliferation and increased Fe2+ levels in GBC cells. Furthermore, NGFR downregulation disrupted mitochondrial function. Conclusion: Circular RNA NGFR can impede the advancement of GBC by modulating the ferroptotic phenotype, thereby potentially offering a novel avenue for the clinical diagnosis and treatment strategies of GBC.

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway.

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial-mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A-NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.

A Smartphone-Based Sensing for Portable and Sensitive Visual Detection of Hg (II) via Nitrogen Doped Carbon Quantum Dots Modified Paper Strip.

The development of portable and cost-effective sensing system for Hg2+ quantitation is highly demanded for environmental monitoring. Herein, an on-site, rapid and portable smartphone readout device based Hg2+ sensing system integrating nitrogen-doped carbon quantum dots (NCDs) modified paper strip was proposed, and the physicochemical properties of NCDs were characterized by high resolution TEM, FTIR, UV-vis absorption spectrum and fluorescence spectral analysis. The modified paper strip was prepared via "ink-jet" printing technology and exhibits sensitive fluorescence response to Hg2+ with fluorescence color of bright blue (at the excitation/emission wavelength of 365/440 nm). This portable smartphone-based sensing platform is highly selective and sensitive to Hg2+ with the limit of detection (LOD) of 10.6 nM and the concentration range of 0-130 nM. In addition, the recoveries of tap water and local lake water were in the range of 89.4% to 109%. The cost-effective sensing system based on smartphone shows a great potential for trace amounts of Hg2+ monitoring in environmental water samples.

Risk Factors and Nomogram for Postoperative Pulmonary Infection in Patients with Cervical Spinal Cord Injury.

OBJECTIVE: To identify the risk factors for developing postoperative pulmonary infection in patients with acute cervical spinal cord injury (CSCI), and to develop a nomogram prediction model. METHODS: Patients with CSCI who were admitted to 3 different medical centers between July 2011 and July 2021 were included in this study. All patients underwent cervical spine surgery. Data for patients admitted to the first 2 centers were included in a training set to establish the nomogram prediction model, and data for patients admitted to the third center were included in a validation set to externally verify the efficacy of the prediction model. For the training set, patients were divided into an infected group and a noninfected group (control group). Independent risk factors for postoperative pulmonary infection in patients with CSCI were identified by univariate and multivariate logistic regression analyses. Additionally, a nomogram prediction model was developed and validated based on the risk factors. RESULTS: A total of 689 patients were enrolled, including 574 for the training set and 115 for the validation set. Of the patients included for the training set, 144 developed pulmonary infection, with an incidence of 25.09%; 40 patients included for the validation set developed pulmonary infection (34.78%). Multivariate logistic regression analysis showed that age, American Spinal Injury Association grade, steroid pulse, high-level injury, smoking, multistage surgery, and operation duration were risk factors for the development of postoperative pulmonary infection in patients with CSCI. The area under the curve of the receiver operating characteristic curve of the model built by the training set was 0.905, and that of the receiver operating characteristic curve of the verification set was 0.917. The decision curve indicated that the model was in the range 1%-100%, and the predicted net benefit value of the model was high. CONCLUSIONS: Age, American Spinal Injury Association grade, steroid pulse, CSCI site, smoking history, number of surgical levels, and surgical duration are correlated with the development of postoperative pulmonary infection in patients with CSCI. The risk prediction model of postoperative pulmonary infection has a good prediction efficiency and accuracy.

Outcomes of different zero-profile spacers in the treatment of two-level cervical degenerative disk disease.

PURPOSE: The purpose of this study was to analyze the clinical and radiological outcomes of two different zero-profile spacers (ROI-C and anchor-C) in contiguous two-level ACDF for CDDD patients. METHODS: We retrospectively analyzed patients who underwent contiguous two-level ACDF due to CDDD between January 2015 and December 2020 in our hospital. Patients who received ROI-C and anchor-C were included as the study groups, and those who underwent plate-cage construct (PCC) were included as the control group. The primary outcome measures were radiographical parameters, and the secondary outcome measures were dysphagia, JOA scores and VAS scores for these patients. RESULTS: A total of 91 patients were enrolled in the study; there were 31, 21 and 39 patients in the ROI-C, anchor-C and PCC groups, respectively. The mean follow-up duration was 24.52 months (range, 18-48 months) in the ROI-C group, 24.38 months (range, 16-52 months) in the anchor-C group and 25.18 months (range, 15-54 months) in the PCC group. The loss of the intervertebral space height and cage subsidence rate in the ROI-C group were significantly higher than those in the anchor-C group and PCC group at the final follow-up (P < 0.05). The ROI-C group showed a lower incidence of adjacent segment degeneration than the anchor-C group and PCC group, but the difference was not significant. The fusion rates were not different among these three groups. The early dysphagia rate was significantly lower in the patients with zero-profile spacers than in the PCC group (P < 0.05), but the difference was not significant at the last follow-up. No relevant differences were found in the JOA scores and VAS scores. CONCLUSIONS: Zero-profile spacers showed promising clinical outcomes in CDDD patients having contiguous two-level ACDF. However, ROI-C resulted in a higher intervertebral space height loss and a higher cage subsidence rate than anchor-C during the follow-up.

A novel classification of osteotomized debridement based on the range of focus in treating active thoracolumbar tuberculosis: a multicenter study.

INTRODUCTION: Osteotomized debridement (OD) is increasingly used in the treatment of active thoracolumbar tuberculosis (TB). So far, no nomenclature has been established to describe the patterns of OD, and thus the surgical outcomes cannot be directly analyzed and compared among the patients treated with different extents of OD. The purpose of this study was to establish a reliable classification of OD for further study of spinal TB. MATERIALS AND METHODS: This was a multicenter retrospective study. The proposed classification included 6 grades of OD based on sagittal range of vertebral body destruction: grade 0 involves single-level intervertebral disc and adjacent superficial endplates; grade 1 involves adjacent endplates and vertebral bodies, but no pedicle is involved; grade 2 involves adjacent endplates, vertebral bodies, and a lower or upper pedicle; grade 3 involves adjacent endplates, vertebral bodies, and both of lower and upper pedicles; grade 4 involves an entire vertebral body and an adjacent lower or upper pedicle; grade 5 involves two continuous entire vertebral bodies. Two hundred and five patients with active thoracolumbar TB who underwent OD surgery were included, and all ODs were classified. The reliability of this classification was evaluated twice by 10 readers, and Fleiss kappa coefficients were calculated. RESULTS: In the 205 patients, 208 ODs were performed. Grade 2 OD was the commonest type (98/208, 47.1%), followed by grade 1 (50/208, 24.0%), grade 3 (26/208, 12.5%), grade 0 (20/208, 9.6%), grade 4 (8/208, 3.8%), and grade 5 (6/208, 2.9%). The average accuracy of the two readings was 86.2% and 90.1%, respectively. The intra-rater reliability for the classification was "almost perfect agreement" with a Fleiss kappa coefficient average of 0.92. The inter-rater reliability was "almost perfect agreement" with a coefficient average of 0.89 for two readings. CONCLUSIONS: This classification proved to be intuitive and reliable. The graded OD provides a platform for preoperative evaluation and allows comparative analysis of clinical outcomes in different extents of OD.

Role of magnetic resonance imaging features in diagnosing and localization of disc rupture related to cervical spinal cord injury without radiographic abnormalities.

STUDY DESIGN: A retrospective study. OBJECTIVE: Traumatic cervical spinal cord injury (TSCI) is often associated with disc rupture. It was reported that high signal of disc and anterior longitudinal ligament (ALL) rupture on magnetic resonance imaging (MRI) were the typical signs of ruptured disc. However, for TSCI with no fracture or dislocation, there is still difficult to diagnose disc rupture. The purpose of this study was to investigate the diagnostic efficiency and localization method of different MRI features for cervical disc rupture in patient with TSCI but no any signs of fracture or dislocation. SETTING: Affiliated hospital of University in Nanchang, China. METHODS: Patients who had TSCI and underwent anterior cervical surgery between June 2016 and December 2021 in our hospital were included. All patients received X-ray, CT scan, and MRI examinations before surgery. MRI findings such as prevertebral hematoma, high-signal SCI, high-signal posterior ligamentous complex (PLC), were recorded. The correlation between preoperative MRI features and intraoperative findings was analyzed. Also, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these MRI features in diagnosing the disc rupture were calculated. RESULTS: A total of 140 consecutive patients, 120 males and 20 females with an average age of 53 years were included in this study. Of these patients, 98 (134 cervical discs) were intraoperatively confirmed with cervical disc rupture, but 59.1% (58 patients) of them had no definite evidence of an injured disc on preoperative MRI (high-signal disc or ALL rupture signal). For these patients, the high-signal PLC on preoperative MRI had the highest diagnostic rate for disc rupture based on intraoperative findings, with a sensitivity of 97%, specificity of 72%, PPV of 84% and NPV of 93%. Combined high-signal SCI with high-signal PLC had higher specificity (97%) and PPV (98%), and a lower FPR (3%) and FNR (9%) for the diagnosis of disc rupture. And combination of three MRI features (prevertebral hematoma, high-signal SCI and PLC) had the highest accuracy in diagnosing traumatic disc rupture. For the localization of the ruptured disc, the level of the high-signal SCI had the highest consistency with the segment of the ruptured disc. CONCLUSION: MRI features, such as prevertebral hematoma, high-signal SCI and PLC, demonstrated high sensitivities for diagnosing cervical disc rupture. High-signal SCI on preoperative MRI could be used to locate the segment of ruptured disc.

Global realignment after posterior vertebral column resection in severe thoracolumbar post-tubercular kyphosis: correlation with patient-reported outcomes.

PURPOSE: To investigate spinal realignment in patients with severe post-tubercular kyphosis (PTK) who underwent posterior vertebral column resection (PVCR) and its correlation with patient-reported outcomes (PROs). METHODS: Eighty-two patients were included in this study. Spinopelvic parameters (focal scoliosis (FS), coronal balance (CB), sagittal vertical axis (SVA), focal kyphosis (FK), C2-7 lordosis (CL), thoracic kyphosis (TK), lumbar lordosis (LL), sacral slope (SS), pelvic tilt (PT), pelvic incidence (PI), and pelvic incidence minus lumbar lordosis (PI-LL)) and PROs (Visual Analog Scale (VAS) and Oswestry Disability Index (ODI)) were analyzed. The correlation between spinopelvic parameters and PROs was evaluated. RESULTS: FK, FS, CL, TK, LL, and PI-LL significantly changed after surgery. FK decreased from pre-operative 108.5 ± 16.4° to 31.8 ± 4.5° at three months after surgery and increased to 38.7 ± 6.6° at final follow-up (P < 0.001). FS decreased from pre-operative 20.9 ± 2.2° to 5.1 ± 2.2° at final follow-up (P < 0.001). CL decreased from pre-operative 7.2 ± 7.3° to 3.3 ± 8.3° at final follow-up (P = 0.002). TK improved from pre-operative - 5.6 ± 7.1° to 12.9 ± 8.2° at final follow-up (P < 0.001). LL decreased from pre-operative 75.5 ± 12.6° to 45.5 ± 7.9° at final follow-up (P < 0.001). PI-LL improved from pre-operative - 24.8 ± 13.4° to 4.8 ± 9.9° at final follow-up (P < 0.001). The improvement of PROs was found to be significantly correlated with the variations of FK, CL, TK, LL, and PI-LL. The multiple regression analysis revealed that FK was an independent predictor for the improvement of VAS and ODI. CONCLUSIONS: PVCR is effective in treating severe PTK, which can significantly improve patients' clinical and radiographic outcomes. Spine surgeons should pay more attention to reducing the residual kyphosis.

Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

The three-dimensional printed template guided technique for S2 alar iliac screw placement and a comparison with freehand technique.

PURPOSE: Sacropelvic fixation continues to present challenges when involved in the adult spinal deformity correction. The S2 alar iliac (S2AI) fixation is commonly used in sacropelvic fixation. Several techniques, including intraoperative navigation and freehand technique, were used for S2AI screws placement. The aim of this study is to analyze the anatomic parameters for S2AI screw trajectory in Asian population and introduce a novel technique described as a three-dimensional printed template guided technique (TGT). Meanwhile, the accuracy and safety of this technique were compared with the conventional freehand technique. METHODS: The S2AI trajectory parameters were measured in 100 Asian adult volunteers. Parameters were compared between different genders. Forty-eight adult patients who underwent S2AI screw placement were reviewed: 28 patients received freehand technique and 20 patients received TGT technique. Postoperative computed tomography was used to assess the accuracy of screw trajectory and cortex violation-related complications were recorded. RESULTS: The cephalocaudal angles (CAs), maximal length of screw pathway, narrowest width of pathway within the iliar teardrop, distance from the center of teardrop to sciatic notch, and distance of the start point distal to S1 dorsal foramen showed significant gender-related difference (p < 0.05). All 48 patients were placed S2AI screws bilaterally (40 screws in TGT vs. 56 screws in freehand). One screw penetrated iliac cortex in the TGT group but 10 screws penetrated iliac cortex in the freehand group (3% vs. 17.9%) (p < 0.05). CONCLUSION: Approximately 30-35° of CA and 39° mediolateral angle are appropriate for S2AI screw placement in Asian patients. Either freehand or TGT technique is safe for S2AI screw placement. TGT technique is more accurate compared with the conventional freehand technique. TRIAL REGISTRATION: This is a retrospective study.

The Amount of Relative Curve Correction Is More Important Than Upper Instrumented Vertebra Selection for Ensuring Postoperative Shoulder Balance in Lenke Type 1 and Type 2 Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Retrospective review of a prospectively collected multicenter database. OBJECTIVE: To assess how "overcorrection" of the main thoracic curve without control of the proximal curve increases the risk for shoulder imbalance in Lenke type 1 Adolescent Idiopathic Scoliosis (AIS). SUMMARY OF BACKGROUND DATA: Postop shoulder imbalance is a common complication following AIS surgery. It is thought that a more cephalad upper-instrumented vertebra (UIV) decreases the risk of shoulder imbalance in Lenke type 1 and 2 curves; however, this has not been proven. METHODS: Thirteen surgeons reviewed preop and 5-year postop clinical photos and PA radiographs of patients from a large multicenter database with Lenke type 1 and 2 AIS curves who were corrected with pedicle screw/rod constructs. Predictors of postop shoulder imbalance were identified by univariate analysis; multivariate analysis was done using the classification and regression tree method to identify independent drivers of shoulder imbalance. RESULTS: One hundred forty-five patients were reviewed. The UIV was T3-T5 in 87% of patients, with 8.9% instrumented up to T1 or T2. Fifty-two (36%) had shoulder imbalance at 5 years. On classification and regression tree analysis when the proximal thoracic (PT) Cobb angle was corrected more than 52%, 80% of the patients had balanced shoulders. Similarly, when the PT curve was corrected less than 52% and the main thoracic (MT) curve was corrected less than 54%, 87% were balanced. However, when the PT curve was corrected less than 52%, and the MT curve was corrected more than 54%, only 41% of patients had balanced shoulders (P = 0.05). This relationship was maintained regardless of the UIV level. CONCLUSION: In Lenke type 1 and 2 AIS curves, significant correction of the main thoracic curve (>54%) with simultaneous "under-correction" (<52%) of the upper thoracic curve resulted in shoulder height imbalance in 59% of patients, regardless of the UIV. This suggests the PT curve must be carefully scrutinized in order to optimize shoulder balance, especially when larger correction of the MT curve is performed. LEVEL OF EVIDENCE: 2.

miR­494­BAG­1 axis is involved in cinobufacini­induced cell proliferation and apoptosis in gastric cancer.

Cinobufacini is widely used in the treatment of advanced cancers. It has been previously reported that microRNA (miR)­494 was upregulated in cinobufacini­treated gastric cancer cells; however, the detailed role of miR­494 in the anti­tumor activity of cinobufacini is unclear. The present study aimed to clarify the function of miR­494 in cinobufacini­induced cell behavior changes. Cell viability and proliferation ability were investigated using a Cell Counting Kit­8 assay. Flow cytometry was performed to investigate the apoptosis rate of gastric cancer (GC) cells. The mRNA expression levels of microRNA (miR)­494 and BCL2 associated athanogene 1 (BAG­1) were investigated using reverse transcription­quantitative polymerase chain reaction, and the protein expression level of BAG­1 was investigated using western blot assays. The results demonstrated that treatment with cinobufacini suppressed proliferation and promoted apoptosis of gastric cancer cells. miR­494 acts as a tumor suppressor gene in gastric cancer. In cinobufacini­treated cells, miR­494 and BAG­1 exhibited opposing expression trends. Furthermore, knockdown of miR­494 in cinobufacini­treated cells upregulated the protein expression level of BAG­1, promoted cell proliferation and inhibited cell apoptosis. In addition, inhibition of BAG­1 using small interfering RNA in cinobufacini­treated cells partially abrogated the effects of miR­494 inhibitor on cell proliferation and apoptosis. Thus, these results suggest that cinobufacini suppresses GC cells proliferation and promotes apoptosis partially through the regulation of miR­494­BAG­1 axis, which may provide a novel insight into the functional mechanism of cinobufacini.

Overexpression of miR-15b-5p promotes gastric cancer metastasis by regulating PAQR3.

Gastric cancer (GC) is the fifth most common cancer in the world, with 952,000 new cases diagnosed in 2012. Tumor metastasis is the major cause of cancer recurrence and death. miR-15b-5p has been reported to be dysregulated in numerous types of cancers. However, the role of miR-15b-5p in GC metastasis remains unclear. An miRNA microarray was adopted to analyze the miRNA expression profile. By employing quantitative real-time polymerase chain reaction (qRT-PCR), miR-15b-5p expression levels were detected in GC cell lines, tissues and plasma samples. In addition, the effects of miR-15b-5p on cell proliferation, migration and invasion were studied by applying gain-of-function approaches. Moreover, the target of miR-15b-5p was assessed by dual-luciferase assay, and the mechanism underlying the regulation of GC metastasis by miR-15b-5p was assessed by rescue experiments. The results revealed that miR-15b-5p was upregulated in GC cell lines, tissues and plasma samples. A high plasma level of miR-15b-5p was correlated with distant tumor metastasis. In addition, overexpression of miR­15b-5p in GC cells promoted cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT). Moreover, progestin and adipoQ receptor family member 3 (PAQR3) was found to be a direct target of miR-15b-5p and re-expression of PAQR3 in miR-15b-5p-overexpressing GC cells partly attenuated the proliferation, migration and invasion. These findings revealed that miR-15b-5p promotes the metastasis of GC cells through PAQR3 and may represent a potential biomarker of GC.

Chondrocyte-Specific Knockout of TSC-1 Leads to Congenital Spinal Deformity in Mice.

Congenital spinal deformity is the most severe clinical orthopedic issue worldwide. Among all the pathological processes of congenital spinal deformity, the imbalance of endochondral ossification is considered to be the most important developmental cause of spinal dysplasia. We established chondrocyte-specific TSC-1 knockout (KO) mice to overactivate the energy metabolic component, mammalian target of rapamycin complex 1 (mTORC1), and measured the spinal development by general, imaging, histological, and Western-blot assessments. In addition to skeletal dysplasia, the KO mice displayed severe congenital spinal deformity and significant intervertebral disc changes. This study suggests that, in the process of endochondral ossification, excessive activation of mTORC1 signaling in chondrocytes induces obvious spinal deformity, and the chondrocytes may be the cell type responsible for congenital spinal deformity.

[Effect of ursolic acid on proliferation and apoptosis of human osteosarcoma cell line U2-OS].

Objective: To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods: Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 µmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results: CCK-8 tests showed that the absorbance ( A) value of each group was not significant at 0 and 24 hours ( P>0.05); but the differences between groups were significant at 48 and 72 hours ( P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G 1 phase of U2-OS cells increased, the S phase and G 2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups ( P<0.05). Compared with the 0 µmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 µmol/L groups significantly decreased ( P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups ( P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups ( P<0.05). Conclusion: Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G 0/G 1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.

[Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: A meta-analysis of 41 studies.

Mutation of oncogene KRAS is common in non-small cell lung cancer (NSCLC), however, its clinical significance is still controversial. Independent studies evaluating its prognostic and predictive value usually drew inconsistent conclusions. Hence, We performed a meta-analysis with 41 relative publications, retrieved from multi-databases, to reconcile these controversial results and to give an overall impression of KRAS mutation in NSCLC. According to our findings, KRAS mutation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in early stage resected NSCLC (hazard ratio or HR=1.56 and 1.57, 95% CI 1.39-1.76 and 1.17-2.09 respectively), and with inferior outcomes of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) treatment and chemotherapy (relative risk or RR=0.21 and 0.66 for objective response rate or ORR, 95% CI 0.12-0.39 and 0.54-0.81 respectively; HR=1.46 and 1.30 for progression-free survival or PFS, 95%CI 1.23-1.74 and 1.14-1.50 respectively) in advanced NSCLC. When EGFR mutant patients were excluded, KRAS mutation was still significantly associated with worse OS and PFS of EGFR-TKIs (HR=1.40 and 1.35, 95 % CI 1.21-1.61 and 1.11-1.64). Although KRAS mutant patients presented worse DFS and PFS of chemotherapy (HR=1.33 and 1.11, 95% CI 0.97-1.84 and 0.95-1.30), and lower response rate to EGFR-TKIs or chemotherapy (RR=0.55 and 0.88, 95 % CI 0.27-1.11 and 0.76-1.02), statistical differences were not met. In conclusion, KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC. There's a need for developing target therapies for KRAS mutant lung cancer and other tumors.

Influence of marital status on the survival of patients with gastric cancer.

BACKGROUND AND AIMS: The influence of marital status on gastric cancer (GC) survival is controversial. The aim of our study was to investigate the relationship between marital status and the survival of GC patients. METHODS: The data of current study was from the Surveillance, Epidemiology, and End-Results database. We identified 18,815 GC patients between 2004 and 2009, who were categorized into married, never married, widowed, and divorced/separated groups. Kaplan-Meier analysis was performed to compare gastric cancer specific survival (GCSS), and Cox regression analysis was used to estimate the risk factors for different survival outcomes in four groups with diverse marital status. RESULTS: Married patients had better 5-year GCSS compared with patients who were never married, separated/widowed, or divorced. In stratified analyses by histological type and pathological differentiation, unmarried patients with gastric adenocarcinoma, signet ring cell carcinoma, moderately differentiated caicinoma, and poorly differentiated/undifferentiated carcinoma had an increased risk of cancer mortality. Widowed patients had the highest percentage of women and elderly, and were more susceptible to well and moderately differentiated gastric carcinomars and gastric adenocarcinoma with earlier TNM stage and lowest rate of surgery and radiation therapy. In addition, they also had the worst 5-year GCSS. CONCLUSIONS: Our study suggests that unmarried GC patients, especially widowed patients, are at a high risk of GCSS. Additionally, the survival benefit is more significant among married GC patients in higher malignancy status.

DNA methyltransferase 3A promotes cell proliferation by silencing CDK inhibitor p18INK4C in gastric carcinogenesis.

Little is known about the roles of DNA methyltransferase 3A (DNMT3A) in gastric carcinogenesis. Here, we reported that the exogenous expression of DNMT3A promoted gastric cancer (GC) cell proliferation by accelerating the G1/S transition. Subsequently, p18INK4C was identified as a downstream target of DNMT3A. The elevated expression of DNMT3A suppressed p18INK4C at least at the transcriptional level. Depletion of p18INK4C expression in GC cells induced cell cycle progression, whereas its re-expression alleviated the effect of DNMT3A overexpression on G1/S transition. Furthermore, we found that DNMT3A modulated p18INK4C by directly binding to and silencing the p18INK4C gene via promoter hypermethylation. In clinical GC tissue specimens analyzed, the level of methylation of p18INK4C detected in tumor tissues was significantly higher than that in paired non-tumor tissues. Moreover, elevated level of DNMT3A expression was associated with the differentiation of GC tissues and was negatively correlated with the p18INK4C expression level. Taken together, our results found that DNMT3A contributes to the dysregulation of the cell cycle by repressing p18INK4C in a DNA methylation-dependent manner, suggesting that DNMT3A-p18INK4C axis involved in GC. These findings provide new insights into gastric carcinogenesis and a potential therapeutic target for GC that may be further investigated in the future.

Reduced miR-29a-3p expression is linked to the cell proliferation and cell migration in gastric cancer.

BACKGROUND: MicroRNAs (miRNAs) play an important role in a tumor-suppressive or oncogenic manner in carcinogenesis. Alteration expression patterns of miRNAs in gastric cancer (GC) are associated with cancer initiation and progression. In the present study, we evaluated miR-29a-3p expression pattern and its function in gastric carcinogenesis. METHODS: The expression of miR-29a-3p in GC tissue samples and cell lines was detected by quantitative real-time PCR (qRT-PCR). After transfected with miR-29a-3p mimics or inhibitor, the cell proliferation, cell migration, and invasion ability were assessed by CCK-8 assay, wound healing assay, and Trans-well assay, respectively. The level of CDK2, CDK4, CDK6, and CyclinD1 were determined by qRT-PCR and Western blot. RESULTS: Compared with the corresponding non-tumor tissues, miR-29a-3p showed a significant down-regulated expression in tumor tissues. In vitro functional assays demonstrated that enforced miR-29a-3p expression inhibited cell proliferation by reducing the expression of CDK2, CDK4, and CDK6. Wound healing and Transwell assays revealed that miR-29a-3p suppressed tumor metastasis in GC. CONCLUSIONS: Our preliminary results suggest that altered expression of miR-29a-3p is involved in gastric cancer process. The present study provides the first insight into the specific role of miR-29a-3p in gastric carcinogenesis.