Generation of Buffalo Mammary Epithelial Cells with Targeted Knockout of 18s rDNA by a CRISPR/Cas9 Adenovirus System

Peng Zhu,An-Qin Duan,Ting-Xian Deng,Xing-Rong Lu,Xiao-Ya Ma,Sha-Sha Liang,Chun-Ying Pang,Xian-Wei Liang 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.5

In recent years, CRISPR/Cas9 has rapidly become one of the most promising genome editing tools because it is simple and easy to use and cost effective. However, the large size of Cas9 sequences limits its application in clinically promising vectors and it also impacts non-viral transfection. In this study, CRISPR/Cas9 adenovirus vectors that target the buffalo 18s rDNA gene were constructed, transfected into 293 cells for adenovirus packaging, and the adenovirus was used to knockout the 18s rDNA gene in buffalo mammary epithelial cells. The results demonstrated that the CRISPR/Cas9 adenovirus vectors for the buffalo 18s rDNA gene could efficiently target the sites as revealed by the fluorescence reporter system. After amplification, the adenovirus titer of Sn458- 18s1 and Sn458-18s2 reached 1.03 × 109PFU/mL and 1.05 × 109 PFU/mL, respectively. For buffalo mammary epithelial cell infection, the efficiency was 100% when the multiplicity of infection (MOI) 100 PFU/mL. There were 9 mutational clones found in the 20 clones, and the gene mutagenesis rate reached 45%. Of these, 2 clones were 35-bp deleted and 7 clones were 12-bp deleted. These results suggested that the adenovirus system overcame the low transfection efficiency of the buffalo mammary epithelial cells associated with using lipid-based methods or electroporation. Moreover, we preliminary developed an efficient technique for multiple-locus gene targeting at repeated sequences of the buffalo genome.

A Novel Molecular Grading Model: Combination of Ki67 and VEGF in Predicting Tumor Recurrence and Progression in Non-invasive Urothelial Bladder Cancer

Chen, Jun-Xing,Deng, Nan,Chen, Xu,Chen, Ling-Wu,Qiu, Shao-Peng,Li, Xiao-Fei,Li, Jia-Ping Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.5

Purpose: To assess efficacy of Ki67 combined with VEGF as a molecular grading model to predict outcomes with non-muscle invasive bladder cancer (NMIBC). Materials: 72 NMIBC patients who underwent transurethral resection (TUR) followed by routine intravesical instillations were retrospectively analyzed in this study. Univariate and multivariate analyses were performed to confirm the prognostic values of the Ki67 labeling index (LI) and VEGF scoring for tumor recurrence and progression. Results: The novel molecular grading model for NMIBC contained three molecular grades including mG1 (Ki67 $LI{\leq}25%$, VEGF $scoring{\leq}8$), mG2 (Ki67 LI>25%, VEGF $scoring{\leq}8$; or Ki67 $LI{\leq}25%$, VEGF scoring > 8), and mG3 (Ki67 LI > 25%, VEGF scoring > 8), which can indicate favorable, intermediate and poor prognosis, respectively. Conclusions: The described novel molecular grading model utilizing Ki67 LI and VEGF scoring is helpful to effectively and accurately predict outcomes and optimize personal therapy.

Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

Xiang Wei,Peng Yongbo,Zeng Hongliang,Yu Chunping,Zhang Qun,Liu Biao,Liu Jiahao,Hu Xing,Wei Wensu,Deng Minhua,Wang Ning,Liu Xuewen,Xie Jianfei,Hou Weibin,Tang Jin,Long Zhi,Wang Long,Liu Jianye 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

Gemcitabine (GEM) is one of the first-line chemotherapies for bladder cancer (BC), but the GEMs cannot recognize cancer cells and have a low long-term response rate and high recurrence rate with side effects during the treatment of BC. Targeted transport of GEMs to mediate cytotoxicity to tumor and avoid the systemic side effects remains a challenge in the treatment of BC.Based on a firstly confirmed biomarker in BC-protein tyrosine kinase 7 (PTK7), which is overexpressed on the cell membrane surface in BC cells, a novel targeting system protein tyrosine kinase 7 aptamer-Gemcitabine conjugate (PTK7-GEMs) was designed and synthesized using a specific PTK7 aptamer and GEM through auto-synthesis method to deliver GEM against BC. In addition, the antitumor effects and safety evaluation of PTK7-GEMs was assessed with a series of in vitro and in vivo assays.PTK7-GEMs can specifically bind and enter to BC cells dependent on the expression levels of PTK7 and via the macropinocytosis pathway, which induced cytotoxicity after GEM cleavage from PTK7-GEMs respond to the intracellular phosphatase. Moreover, PTK7-GEMs showed stronger anti-tumor efficacy and excellent biosafety in three types of tumor xenograft mice models.These results demonstrated that PTK7-GEMs is a successful targeted aptamer-drug conjugates strategy (APDCs) to treat BC, which will provide new directions for the precision treatment of BC in the field of biomarker-oriented tumor targeted therapy.

Celastrol, produced by Tripterygium wilfordii Hook F. enhances defense response in cucumber seedlings against diverse environmental stresses

Li-jun Zhu,Xing-guang Deng,Li-juan Zou,Peng-xu Li,Jun-qiang Wu,Da-wei Zhang,Honghui Lin 한국식물학회 2017 Journal of Plant Biology Vol.60 No.1

Celastrol is an active triterpenoid compound isolated from Tripterygium wilfordii Hook F.. Many reports have highlighted that celastrol is an effective, safe and desirable approach to the treatment of cancers. However, their biological function during environmental stresses in plants is rarely reported. In the present study, the effects of celastrol on the tolerance against high light (HL), polyethylene glycol (PEG) and cold stress in cucumber (Cucumis sativus L.) were investigated. Celastrol pretreatment could enhance cucumber seedlings stress tolerance at a concentration of 1 μg ml–1. The results showed that pretreatment with 1 μg ml–1 celastrol clearly induced the activities of antioxidative enzymes, which subsequently alleviated stress-induced oxidative damage in plant cells. We also provided evidence that celastrol upregulated ABA biosynthetic gene NCED2 expression and ABA accumulation in cucumber seedlings, which resulted to the enhanced tolerance in response to environmental stresses. Furthermore, the celastrol-pretreated seedlings showed less photosystem damaged caused by the stress conditions, when compared with the control. Therefore, our findings provide a novel role of celastrol in plant against environmental stresses and indicate that the celastrol-induced activities of antioxidative enzymes and ABA content might contribute to the stress tolerance.

An Active Radial Compliance Method with Anisotropic Stiffness Learning for Precision Assembly

Fang-Bo Qin,De Xu,Deng-Peng Xing,Zheng-Tao Zhang 한국정밀공학회 2017 International Journal of Precision Engineering and Vol.18 No.4

Compliance is essential for precision assembly, which provides motion guidance and damage avoidance. Force control can offer flexible implementation of active compliance for manipulators. In this paper, an active radial compliance method is developed for the insertion task of thin walled millimeter-sized cylinders. A radial force controller is designed to satisfy the contact force constraint in the component's radial directions, which integrates the explicit force control with the high precision attitude measurement based on microscopic vision. An anisotropic stiffness learning method is proposed based on clustering and support vector machines. It can obtain the hidden anisotropic stiffness characteristics of the mechanical system from experience data. Experimental results verify the effectiveness of the proposed methods.

Long-term Survivals, Toxicities and the Role of Chemotherapy in Early-Stage Nasopharyngeal Carcinoma Patients Treated with Intensity-modulated Radiation Therapy: A Retrospective Study with 15-year Follow-up

Lin Wang,Jingjing Miao,Huageng Huang,Boyu Chen,Xiao Xiao,Manyi Zhu,Yingshan Liang,Weiwei Xiao,Shaomin Huang,Yinglin Peng,Xiaowu Deng,Xing Lv,Weixiong Xia,Yanqun Xiang,Xiang Guo,Fei Han,Chong Zhao 대한암학회 2022 Cancer Research and Treatment Vol.54 No.1

Purpose This study was aimed to investigate long-term survivals and toxicities of early-stage nasopharyngeal carcinoma (NPC) in endemic area, evaluating the role of chemotherapy in stage II patients. Materials and Methods Totally 187 patients with newly diagnosed NPC and restaged American Joint Committee on Cancer/ International Union Against Cancer 8th T1-2N0-1M0 were retrospectively recruited. All received intensity-modulated radiotherapy (IMRT)±chemotherapy (CT) from 2001 to 2010. Results With 15.7-year median follow-up, 10-year locoregional recurrence-free survival, distant metastasis-free survival (DMFS), disease-specific survival (DSS), and overall survival (OS) were 93.3%, 93.5%, 92.9% and 88.2%, respectively. Multivariable analyses showed cervical lymph nodes positive and pre-treatment prognostic nutritional index ≥ 52.0 could independently predict DMFS (p=0.036 and p=0.011), DSS (p=0.014 and p=0.026), and OS (p=0.002 and p < 0.001); Charlson comorbidity index < 3 points could predict DSS (p=0.011); age > 45 years (p=0.002) and pre-treatment lactate dehydrogenase ≥ 240 U/L (p < 0.001) predicted OS. No grade 4 late toxicity happened; grade 3 late toxicities included subcutaneous fibrosis (4.3%), deafness or otitis (4.8%), skin dystrophy (2.1%), and xerostomia (1.1%). No differences on survivals were shown between IMRT+CT vs. IMRT alone in stage II patients, even in T2N1M0 (p > 0.05). Unsurprising, patients in IMRT+CT had more acute gastrointestinal reaction, myelosuppression, mucositis, late ear toxicity, and cranial nerve injury (all p < 0.05) than IMRT alone group. Conclusion Superior tumor control and satisfying long-term outcomes could be achieved with IMRT in early-stage NPC with mild late toxicities. As CT would bring more toxicities, it should be carefully performed to stage II patients.