CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

2-Hydroxy-3-methylanthraquinone inhibits homologous recombination repair in osteosarcoma through the MYC-CHK1-RAD51 axis.

BACKGROUND: Osteosarcoma is a malignant bone tumor that usually affects adolescents aged 15-19 y. The DNA damage response (DDR) is significantly enhanced in osteosarcoma, impairing the effect of systemic chemotherapy. Targeting the DDR process was considered a feasible strategy benefitting osteosarcoma patients. However, the clinical application of DDR inhibitors is not impressive because of their side effects. Chinese herbal medicines with high anti-tumor effects and low toxicity in the human body have gradually gained attention. 2-Hydroxy-3-methylanthraquinone (HMA), a Chinese medicine monomer found in the extract of Oldenlandia diffusa, exerts significant inhibitory effects on various tumors. However, its anti-osteosarcoma effects and defined molecular mechanisms have not been reported. METHODS: After HMA treatment, the proliferation and metastasis capacity of osteosarcoma cells was detected by CCK-8, colony formation, transwell assays and Annexin V-fluorescein isothiocyanate/propidium iodide staining. RNA-sequence, plasmid infection, RNA interference, Western blotting and immunofluorescence assay were used to investigate the molecular mechanism and effects of HMA inhibiting osteosarcoma. Rescue assay and CHIP assay was used to further verified the relationship between MYC, CHK1 and RAD51. RESULTS: HMA regulate MYC to inhibit osteosarcoma proliferation and DNA damage repair through PI3K/AKT signaling pathway. The results of RNA-seq, IHC, Western boltting etc. showed relationship between MYC, CHK1 and RAD51. Rescue assay and CHIP assay further verified HMA can impair homologous recombination repair through the MYC-CHK1-RAD51 pathway. CONCLUSION: HMA significantly inhibits osteosarcoma proliferation and homologous recombination repair through the MYC-CHK1-RAD51 pathway, which is mediated by the PI3K-AKT signaling pathway. This study investigated the exact mechanism of the anti-osteosarcoma effect of HMA and provided a potential feasible strategy for the clinical treatment of human osteosarcoma.

Pan-sarcoma characterization of lncRNAs in the crosstalk of EMT and tumour immunity identifies distinct clinical outcomes and potential implications for immunotherapy.

The epithelial-to-mesenchymal transition (EMT) is a reversible process that may interact with tumour immunity through multiple approaches. There is increasing evidence demonstrating the interconnections among EMT-related processes, the tumour microenvironment, and immune activity, as well as its potential influence on the immunotherapy response. Long non-coding RNAs (lncRNAs) are emerging as critical modulators of gene expression. They play fundamental roles in tumour immunity and act as promising biomarkers of immunotherapy response. However, the potential roles of lncRNA in the crosstalk of EMT and tumour immunity are still unclear in sarcoma. We obtained multi-omics profiling of 1440 pan-sarcoma patients from 19 datasets. Through an unsupervised consensus clustering approach, we categorised EMT molecular subtypes. We subsequently identified 26 EMT molecular subtype and tumour immune-related lncRNAs (EILncRNA) across pan-sarcoma types and developed an EILncRNA signature-based weighted scoring model (EILncSig). The EILncSig exhibited favourable performance in predicting the prognosis of sarcoma, and a high-EILncSig was associated with exclusive tumour microenvironment (TME) characteristics with desert-like infiltration of immune cells. Multiple altered pathways, somatically-mutated genes and recurrent CNV regions associated with EILncSig were identified. Notably, the EILncSig was associated with the efficacy of immune checkpoint inhibition (ICI) therapy. Using a computational drug-genomic approach, we identified compounds, such as Irinotecan that may have the potential to convert the EILncSig phenotype. By integrative analysis on multi-omics profiling, our findings provide a comprehensive resource for understanding the functional role of lncRNA-mediated immune regulation in sarcomas, which may advance the understanding of tumour immune response and the development of lncRNA-based immunotherapeutic strategies for sarcoma.

Quercetin encapsulated in folic acid-modified liposomes is therapeutic against osteosarcoma by non-covalent binding to the JH2 domain of JAK2 Via the JAK2-STAT3-PDL1.

Osteosarcoma (OS) is a malignant solid tumor prone to lung metastasis that occurs in adolescents aged 15-19 years. Neoadjuvant chemotherapy and surgical treatment aimed at curing OS have gained limited progress over the last 30 years. Exploring new effective second-line therapies for OS patients is a serious challenge for researchers. Quercetin, a multiple biologically active polyphenolic flavonoid, has been used in tumor therapy. However, the exact mechanism of quercetin is still unknown, which limits the application of quercetin. In the current study, we found that quercetin could inhibit JAK2 through the JH2 domain in a non-covalent manner, resulting in the inhibition of OS proliferation and immune escape via the JAK2-STAT3-PD-L1 signaling axis. More importantly, to overcome the shortcomings of quercetin, including low water solubility and low oral availability, we encapsulated it with folic acid-modified liposomes. The transportation of quercetin by folic acid-modified liposomes may provide a feasible strategy to cure OS.

Prognostic role of c-Jun activation domain-binding protein-1 in cancer: A systematic review and meta-analysis.

c-Jun activation domain-binding protein-1 (Jab1) is aberrantly overexpressed in multiple cancers and plays an oncogenic role in cancer progression. We examined the association between Jab1 expression and prognosis in patients with cancer by conducting a meta-analysis. A comprehensive search strategy was performed using the PubMed, Web of Science, Ovid and EMBASE in July 2020. Eligible studies were enrolled according to definite criteria. Twenty-seven studies involving 2609 patients were enrolled in this meta-analysis. A significant association between high Jab1 expression and poor overall survival (pooled hazard ratio [HR] 2.344, 95% confidence interval [CI]: 2.037-2.696) was observed. Subgroup analyses of the type of cancer, sample size, follow-up period, Jab1 detection method and preoperative treatment did not alter the significance. On pooling data from Cox multivariate analyses, high Jab1 expression was found to be an independent prognostic indicator for overall survival. In addition, high Jab1 expression was found to be associated with advanced clinicopathological features such as clinical stage, lymphatic metastasis, histological grade and distant metastasis in cancers. Our meta-analysis is the first to demonstrate that high Jab1 expression may be a promising indicator of poor prognosis and has an independent prognostic value for overall survival in patients with cancer.

A 3D Hologram With Mixed Reality Techniques to Improve Understanding of Pulmonary Lesions Caused by COVID-19: Randomized Controlled Trial.

BACKGROUND: The COVID-19 outbreak has now become a pandemic and has had a serious adverse impact on global public health. The effect of COVID-19 on the lungs can be determined through 2D computed tomography (CT) imaging, which requires a high level of spatial imagination on the part of the medical provider. OBJECTIVE: The purpose of this study is to determine whether viewing a 3D hologram with mixed reality techniques can improve medical professionals' understanding of the pulmonary lesions caused by COVID-19. METHODS: The study involved 60 participants, including 20 radiologists, 20 surgeons, and 20 medical students. Each of the three groups was randomly divided into two groups, either the 2D CT group (n=30; mean age 29 years [range 19-38 years]; males=20) or the 3D holographic group (n=30; mean age 30 years [range 20=38 years]; males=20). The two groups completed the same task, which involved identifying lung lesions caused by COVID-19 for 6 cases using a 2D CT or 3D hologram. Finally, an independent radiology professor rated the participants' performance (out of 100). All participants in two groups completed a Likert scale questionnaire regarding the educational utility and efficiency of 3D holograms. The National Aeronautics and Space Administration Task Load Index (NASA-TLX) was completed by all participants. RESULTS: The mean task score of the 3D hologram group (mean 91.98, SD 2.45) was significantly higher than that of the 2D CT group (mean 74.09, SD 7.59; P<.001). With the help of 3D holograms, surgeons and medical students achieved the same score as radiologists and made obvious progress in identifying pulmonary lesions caused by COVID-19. The Likert scale questionnaire results showed that the 3D hologram group had superior results compared to the 2D CT group (teaching: 2D CT group median 2, IQR 1-2 versus 3D group median 5, IQR 5-5; P<.001; understanding and communicating: 2D CT group median 1, IQR 1-1 versus 3D group median 5, IQR 5-5; P<.001; increasing interest: 2D CT group median 2, IQR 2-2 versus 3D group median 5, IQR 5-5; P<.001; lowering the learning curve: 2D CT group median 2, IQR 1-2 versus 3D group median 4, IQR 4-5; P<.001; spatial awareness: 2D CT group median 2, IQR 1-2 versus 3D group median 5, IQR 5-5; P<.001; learning: 2D CT group median 3, IQR 2-3 versus 3D group median 5, IQR 5-5; P<.001). The 3D group scored significantly lower than the 2D CT group for the "mental," "temporal," "performance," and "frustration" subscales on the NASA-TLX. CONCLUSIONS: A 3D hologram with mixed reality techniques can be used to help medical professionals, especially medical students and newly hired doctors, better identify pulmonary lesions caused by COVID-19. It can be used in medical education to improve spatial awareness, increase interest, improve understandability, and lower the learning curve. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100045845; http://www.chictr.org.cn/showprojen.aspx?proj=125761.

En bloc resection and intercalary prosthesis implantation for the treatment of humeral diaphyseal bone metastases.

PURPOSE: To evaluate the short-term clinical efficacy and complications of en bloc resection and intercalary prosthesis implantation for the treatment of humeral diaphyseal bone metastases. METHODS: A total of 21 patients with humeral diaphyseal bone metastases treated with en bloc resection and intercalary prosthesis implantation from August 2014 to August 2019 were retrospectively analysed. The visual analogue scale (VAS), Musculoskeletal Tumour Society (MSTS) scale, International Society of Limb Salvage (ISOLS) scoring system, Karnofsky Performance Status (KPS) scale, and Nottingham Health Profile (NHP) scale were used to assess pain, limb function, and quality of life. Survival of the patients was analysed using the Kaplan-Meier method. RESULTS: The patients were followed up for 12-57 months (mean: 22 months); the operative time was 68-114 minutes (mean: 76.24 min); the osteotomy length was 6.5-10 cm (mean: 8.02 cm); and the intra-operative blood loss was 95-125 ml (mean: 104.71 ml). At three, six and 12 months after surgery, the VAS and NHP scores were lower, whereas the MSTS, ISOLS, and KPS scores were higher than those before surgery, and the differences were statistically significant (P < 0.05). The survival time was four to 24 months (mean: 19.46 months). Thesix month and one year survival rates were 80.95% and 52.38%, respectively. During the follow-up period, no complications occurred except for aseptic prosthesis loosening in one patient. CONCLUSIONS: En bloc resection and intercalary prosthesis implantation can reduce pain, improve limb function, prolong survival time, and improve quality of life in patients with humeral diaphyseal bone metastases.

Total sacrectomy with a combined antero-posterior surgical approach for malignant sacral tumours.

PURPOSE: To investigate the indications, approaches, resection methods, and complications of total sacrectomy with a combined antero-posterior approach for malignant sacral tumours. METHODS: Fourteen cases of primary malignant sacral tumours treated with total sacrectomy between January 2012 and 2018 were retrospectively analysed. All patients presented with pre-operative lumbosacral pain or constipation. A combined antero-posterior approach was used for tumour resection, and the spinal pedicle screw rod system was used to achieve ilio-lumbar stability. The visual analogue scale (VAS) and Musculoskeletal Tumor Society (MSTS) scores were used to assess pain and lower limb function, respectively. The mean operative time and intra-operative blood loss were 6.54 hours and 2935 mL, respectively. The mean follow-up period was 62 months. RESULTS: None of the patients died peri-operatively. At the last follow-up, ten patients were continuously disease-free, three were alive with disease, and one died of disease from lung metastasis. Tumour recurrence occurred in three patients. The MSTS scores ranged from 6 to 28 (20.00-93.33%, 6/30-28/30) with an average of 20 (66.67%, 20/30). Seven patients could walk independently in public, five could only walk at home using a walking aid, and two could only lie down and stand for a short time. Thirteen patients developed post-operative complications such as skin necrosis, screw loosening, connecting rod fracture, neuropathic pain, sciatic nerve injury, dysuria, and urinary incontinence. CONCLUSION: Total sacrectomy can effectively treat malignant sacral tumours with good resection boundaries and prognosis. However, the high incidence of post-operative complications may impact post-operative neurological function.

PTBP1 modulates osteosarcoma chemoresistance to cisplatin by regulating the expression of the copper transporter SLC31A1.

Chemoresistance is the main obstacle of treatment in patients with osteosarcoma. RNA-binding protein PTBP1 has been identified as an oncogene in various cancers. However, the role of PTBP1 in osteosarcoma, especially in chemoresistant osteosarcoma, and the underlying mechanism remain unclear. In this study, we aimed to explore the functions of PTBP1 in chemoresistance of osteosarcoma. We found that PTBP1 was significantly increased in chemotherapeutically insensitive osteosarcoma tissues and cisplatin-resistant osteosarcoma cell lines (MG-63CISR and U-2OSCISR ) as compared to chemotherapy-sensitive osteosarcoma tissues and cell lines. Knock-down of PTBP1 can enhance the anti-proliferation and apoptosis-induced effects of cisplatin in MG-63CISR and U-2OSCISR cells. Moreover, PTBP1 knock-down significantly up-regulated the expression of the copper transporter SLC31A1, as indicated by transcriptome sequencing. Through RNA immunoprecipitation, dual-luciferase reporter assay and RNA stability detection, we confirmed that PTBP1 binds to SLC31A1 mRNA and regulates the expression level of SLC31A1 by affecting mRNA stability. Additionally, SLC31A1 silencing abrogated the chemosensitizing effect of PTBP1 knock-down in MG-63CISR and U-2OSCISR cells. Using a nude mouse xenograft model, we further confirmed that PTBP1 knock-down enhanced chemoresistant osteosarcoma responsiveness to cisplatin treatment in vivo. Collectively, the present study suggests that PTBP1 is a crucial determinant of chemoresistance in osteosarcoma.

Hsa_circ_0000285 functions as a competitive endogenous RNA to promote osteosarcoma progression by sponging hsa-miRNA-599.

Circular RNA (circRNA) is important in the pathogenesis of many diseases. By analyzing the GSE96964 microarray, hsa_circ_0000285 (circ-0000285) was found to be highly expressed in osteosarcoma. Recent studies have shown that circ-0000285 is capable of regulating proliferative and migratory potentials. Here, we investigated the potential functions in regulating osteosarcoma cells to proliferate and migrate. First of all, qRT-PCR data revealed a higher level of circ-0000285 in osteosarcoma cell lines relative to normal osteoblasts. Through dual-luciferase reporter gene assay and RIP assay, we confirmed that both circ-0000285 and TGFB2 could directly bind to miRNA-599. Regulatory effects of circ-0000285 and miRNA-599 on proliferative and migratory potentials were evaluated by EdU assay and transwell migration assay. It is indicated that circ-0000285 overexpression enhanced the proliferative and migratory potentials of osteosarcoma, which could be reversed by miRNA-599 overexpression. This study revealed a vital role of circ-0000285/miRNA-599/TGFB2 axis in regulating the progression of osteosarcoma, providing a novel perspective for clarifying its pathogenesis.

Dysregulated circRNAs serve as prognostic and diagnostic markers in osteosarcoma by sponging microRNA to regulate the downstream signaling pathway.

Circular RNAs (circRNAs) have been extensively studied in many tumors. The aim of this study was to demonstrate the relationship between circRNAs and clinical features, prognosis, and diagnosis of osteosarcoma patients. We mainly included studies about circRNAs expression and osteosarcoma. The odds ratio (ORs) and 95% confidence intervals (CIs) were used for clinical features, sensitivity, and specificity, while the hazard ratios (HRs) and 95% CIs were used to assess overall survival (OS). A number of 13 articles were included in this study, including 9 about clinical features, 11 about prognosis, and 5 about diagnosis. The results showed that increased circRNAs expression was significantly correlated with adverse clinical characteristics. In terms of prognosis, oncogenic circRNAs had adverse effects on overall survival (OS: HR = 2.54; 95%Cl: 2.05-3.03), and increased expression of cancer-suppressor circRNAs prolonged survival (OS: HR = 0.42; 95%Cl: 0.210.64). Our study further showed an AUC of 0.85, with an 80% sensitivity and 77% specificity to distinguish osteosarcoma patients from healthy controls. In conclusion, circRNAs may be new promising indicators for prognostic evaluation and early diagnosis of osteosarcoma patients.

Reconstruction with constrained scapular prosthesis after total scapulectomy for scapular malignant tumor.

BACKGROUND: Surgical reconstruction after total scapulectomy presents a great challenge. Currently, the constrained scapular prosthesis reconstruction is considered as the most effective and promising method. However, the mid- to long-term functional outcomes and complications of this procedure are unclear. METHODS: We retrospectively analyzed eight patients with scapular malignant tumors treated by total scapulectomy and constrained scapular prosthesis reconstruction between 2011 and 2013. The results of functional improvement were evaluated using MSTS-93 score at the final follow-up. We also analyzed tumor recurrence, metastases, and complications associated with the reconstruction procedure. RESULTS: Three patients died of pulmonary metastasis, and five patients were alive at the final follow-up. For the five surviving patients, the mean follow-up period was 61.8 months (range, 51-72 months). Each patient recovered satisfactory contour of the shoulder. The mean MSTS-93 functional score of the upper extremity was 23.5 (range, 20-27). Rib fractures developed two patients, while prosthesis exposure occurred in one patient. No infection, skin flap necrosis, dislocation, and aseptic loosening developed in this small series. Pulmonary metastasis was observed in two patients (2/5). CONCLUSIONS: Although a few of prosthesis-related complications not reported in previous studies are observed during the mid- to long-term follow-up, reconstruction with a constrained scapular prosthesis can provide a stable shoulder joint, and obtained a satisfactory shoulder contour and an acceptable mid-to long-term functional outcomes. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Anticancer Action and Mechanism of Ergosterol Peroxide from Paecilomyces cicadae Fermentation Broth.

Isaria cicadae, a medicinal food fungus, is a fruit from Paecilomyces cicadae. In this study, we purified ergosterol peroxide (EP) from the fermentation broth of P. cicadae and investigated its effects on renal cell carcinoma (RCC) cells, in vitro. EP was purified from P. cicadae fermentation broth. The human RCC cell line 786-0 was used to analyze the anticancer mechanism of EP and inhibit its effect on cancer cell proliferation, in vitro. EP with a validated structure showed a yield rate of 20.1 mg/L and a purity of 96%. EP significantly inhibited RCC cell growth and clone formation in vitro. In addition, EP suppressed the migration and invasion, triggered the apoptosis, and modulated the cell cycle of RCC cells, in a dose-dependent manner. It also downregulated ß-catenin expression. EP could be routinely produced through P. cicadae. It fights RCC cells in vitro through multiple mechanisms, including suppressing cell growth, colonization, migration, and invasion, arresting the cell cycle, attenuating ß-catenin pathways, and triggering apoptosis.

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients.

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.

Cx43- and Smad-Mediated TGF-ß/ BMP Signaling Pathway Promotes Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells and Inhibits Osteoblast Differentiation.

BACKGROUND/AIMS: The aim of this study was to investigate the influence of Cx43- and Smad-mediated TGF-ß/BMP signaling pathway on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cartilage and inhibition of ossification. METHODS: BMSCs of Wistar rats were cultured and assigned into 5 groups for transfection with adenoviruses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect mRNA and protein expressions of target genes. The condition of cartilage and ossification were measured by a series of staining methods. Subcutaneous injection of mesenchymal stem cells (MSCs) into nude rats was performed. RESULTS: After transfection, compared to the AdGFP group, the corresponding target mRNAs were overexpressed in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups, and overexpression of BMP2 at the mRNA and protein expression was observed in the AdSmad1 and AdCx43 + AdSmad1 groups. The mRNA expressions of aggrecan (ACAN) and collagen type II alpha 1 (Col2a1), the glycosaminoglycan content of the extracellular matrix and the expression of type II collagen, Col2a1, osteopontin (OPN) and osteocalcin (OC) were higher in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups than in the AdGFP group; alkaline phosphatase (ALP) activity and mRNA and protein expressions of Runx2 were also higher in these groups than in the AdGFP group. Heterotopic osteogenesis tests demonstrated evident cartilage differentiation ability in the AdCx43 + AdSmad1 + AdBMP2 groups. In comparison, the AdCx43 + AdSmad1 and AdSmad1 groups exhibited weaker cartilage differentiation abilities. CONCLUSION: Cx43 and Smad1 promote BMP-induced cartilage differentiation of BMSCs and inhibit osteoblast differentiation, which provide a new strategy for cartilage tissue engineering using exogenous Cx43 and Smad1.

Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses.

BACKGROUND/AIMS: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. METHODS: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. RESULTS: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. CONCLUSION: The RAPA cells are highly efficient for adenovirus production and amplification.

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs).

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. METHODS: Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. RESULTS: BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. CONCLUSION: Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering.

What are the Functional Results, Complications, and Outcomes of Using a Custom Unipolar Wrist Hemiarthroplasty for Treatment of Grade III Giant Cell Tumors of the Distal Radius?

PURPOSE: A giant cell tumor (GCT) of bone presenting in the distal radius is rare, however, when they occur, Campanacci Grade III tumors can present formidable reconstructive challenges. They are associated with a high local recurrence rate with intralesional treatment, therefore approaches to reconstruct the wrist after en bloc resection warrant study. QUESTIONS: We asked: (1) What are the functional outcomes after en bloc resection and reconstruction of the wrist with a unipolar prosthesis in patients with Grade III GCT of the distal radius? (2) What complications occur with use of a unipolar prosthesis in these patients? (3) What are the oncologic outcomes with using en bloc resection and reconstruction with a custom unipolar wrist hemiarthroplasty for Grade III GCTs of the distal radius? METHODS: We retrospectively analyzed 10 patients with Campanacci Grade III GCTs of the distal radius treated by a unipolar prosthesis after wide resection of the tumor between January 2008 and October 2013. During that period, all patients at our medical group who presented with a Grade III GCT of the distal radius were treated with wide resection and reconstruction using a custom unipolar implant. Pre- and postoperative pain at rest were assessed according to a 10-cm VAS score. The functional outcomes of the wrist were assessed using the modified Mayo wrist score, and the degenerative changes were evaluated radiographically by a new rating system based on the Knirk and Jupiter scale. We also analyzed tumor recurrence, metastases, and complications associated with the reconstruction procedure. All patients were available for followup at a mean of 52 months (range, 24-90 months). RESULTS: Although the complication rate associated with prosthetic arthroplasty was relatively high (six of 10), none of our patients experienced severe complications. Two patients reported having occasional pain of the involved wrist at the time of final followup (VAS, preoperative versus postoperative: 0 versus 3; 5 versus 2, respectively). The mean modified Mayo wrist score was 68 (range, 45-90). Degenerative changes were found in three wrists (Grade 1, two patients; Grade 2, one patient). Aseptic loosening occurred in one patient and wrist subluxation occurred in two patients. Lung metastases or local tumor recurrence were not observed. CONCLUSIONS: Because of the proportion of patients who had complications and progressive degeneration with this approach, we recommend first exploring alternatives to reconstruction with custom unipolar wrist hemiarthroplasty after resection of Grade III GCTs of the distal radius, such as fibular autografting. However, this technique provides an alternative for patients with concerns regarding possible morbidity associated with autografting, and for situations when allograft is not available. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Transformation of multi-component ginkgolide into ginkgolide B by Coprinus comatus.

BACKGROUND: As the strongest antagonist of the platelet activating factor, ginkgolide B (GB) possesses anti-ischemic, anti-oxidant and anti-convulsant properties, and it is used for the treatment of thrombosis in clinical practice. Till now, GB is usually obtained from extraction of Ginkgo biloba leaves through column chromatography with an extremely low yield and high cost, which can not meet clinical requirement. Therefore, it is urgent to find a new method to prepare GB. RESULTS: In the current study, we studied the ability and mechanism to transform multi-component ginkgolide into GB by Coprinus comatus in order to enhance the GB yield. Except for ginkgolide A (GA) and GB, all the other ginkgolides in the extract were transformed by the strain. In the case of culture medium containing 20 g/L glucose, the transformation product was identified as 12% GA and 88% GB by high performance liquid chromatography-Mass spectrometry (HPLC-MS), two stage mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR). Partial GA was also transformed into GB according to the yield (76%) and the content of GA in the raw ginkgolide (28.5%). Glucose was the key factor to transform ginkgolides. When glucose concentration in medium was higher than 40 g/L, all ginkgolides were transformed into the GB. Proteomic analysis showed that C. comatus transformed ginkgolide into GB by producing 5 aldo/keto reductases and catalases, and enhancing the metabolism of glucose, including Embden-Meyerhof pathway (EMP), hexose monophophate pathway (HMP) and tricarboxylic acid (TCA). CONCLUSIONS: C. comatus could transform ginkgolides into GB when the medium contained 40 g/L glucose. When the strain transformed ginkgolides, the glucose metabolism was enhanced and the strain synthesized more aldo/keto reductases and catalases. Our current study laid the groundwork for industrial production of GB.

Mechanistic insight into transition metal-catalyzed reaction of enynal/enynone with alkenes: metal-dependent reaction pathway.

A systematic study of the transition metal-catalyzed reaction of enynal/enynone with alkenes has been reported. It was found that the reaction has two metal-dependent reaction pathways. One led to the formation of 1,2-DHN, while another led to cyclic-o-QDM.