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Jianxun Feng, Chang Lu, Qin Dai, Junqin Sheng, Min Xu
BACKGROUND/AIMS: Amniotic fluid stem cells (AFSCs) transplantation is a promising therapeutic strategy for diabetic nephropathy. Sirtuin3 (SIRT3) is a novel mitochondrial protective factor. In the present study, we aimed to investigate whether SIRT3 protects against hyperglycemia-induced AFSCs damage and enhances the therapeutic efficiency of AFSCs in diabetic nephropathy. METHODS: To establish the diabetic nephropathy model, db/ db mice were used. AFSCs were obtained and transplanted into the kidney tissue of db/ db mice...
April 19, 2018: Cellular Physiology and Biochemistry
Daisuke Morita, Nobuhiro Nishio, Shoji Saito, Miyuki Tanaka, Nozomu Kawashima, Yusuke Okuno, Satoshi Suzuki, Kazuyuki Matsuda, Yasuhiro Maeda, Matthew H Wilson, Gianpietro Dotti, Cliona M Rooney, Yoshiyuki Takahashi, Yozo Nakazawa
Adoptive T cell therapy using chimeric antigen receptor (CAR)-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs) as feeder cells and virus-specific T cell receptor (TCR) stimulation, we achieved 51...
March 16, 2018: Molecular Therapy. Methods & Clinical Development
Yang Yu, Shun-Guang Wei, Robert M Weiss, Robert B Felder
Inflammation in the hypothalamic paraventricular nucleus (PVN) contributes to neurohumoral excitation and its adverse consequences in systolic heart failure (HF). The stimuli that trigger inflammation in the PVN in HF are not well understood. Angiotensin II (AngII) has pro-inflammatory effects, and circulating levels of AngII increase in HF. The subfornical organ (SFO), a circumventricular structure that lacks an effective blood-brain barrier and senses circulating AngII, contains PVN-projecting neurons. We hypothesized that activation of AngII type 1a receptors (AT1a R) in the SFO induces neuroinflammation downstream in the PVN...
April 20, 2018: Neuroscience
Xi Chen, Song-Man Li, Yan-Wei Li, Zi-Hao Han, Hao Liang
AIM: To study the effect of senescence marker protein 30 (SMP30) on the proliferation and apoptosis of human lens epithelial cell (HLEC) SRA01/04. METHODS: SMP30 overexpression (OE) and knock down (KD) type cell lines were cultivated by using two groups regucalcin (RGN; SMP30) lentiviral vectors (LV-RGN, LV-RGN-RNAi) and the respective negative control virus infect SRA01/04 cells. Western blot and real-time quantitative polymerase chain reaction (q-PCR) analysis were used to determine RGN overexpression and knock down efficiency...
2018: International Journal of Ophthalmology
Patricia Dranchak, John J Moran, Ryan MacArthur, Camila Lopez-Anido, James Inglese, John Svaren
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter...
2018: Methods in Molecular Biology
Satoshi Komoto, Saori Fukuda, Tomihiko Ide, Naoto Ito, Makoto Sugiyama, Tetsushi Yoshikawa, Takayuki Murata, Koki Taniguchi
An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (EGFP and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages...
April 18, 2018: Journal of Virology
Mengyu Zhang, Mingxing Zhang, Jing Wang, Qingqing Cai, Ran Zhao, Yi Yu, Haiyan Tai, Xiaoyan Zhang, Congjian Xu
BACKGROUND: The development of nanoparticle drug delivery systems with targeted ligands has the potential to increase treatment efficiency in ovarian cancer. METHODS: We developed a 21-amino acid peptide, YTRDLVYGDPARPGIQGTGTF (L-FP21) conjugated to polyethylenimine (PEI) and methoxy polyethylene glycol (mPEG) to prepare a nanoparticle drug vehicle to target follicle-stimulating hormone receptor (FSHR) in ovarian cancer. At the same time, we optimized the ligand of the nanoparticle vehicle using D-peptides, which consist of D-amino acids (D-FP21)...
November 2018: Drug Delivery
Benjamin L Weis, Nadine Guth, Simon Fischer, Silke Wissing, Simon Fradin, Karl-Heinz Holzmann, René Handrick, Kerstin Otte
Chinese hamster ovary (CHO) cells still represent the major production host for therapeutic proteins. However, multiple limitations have been acknowledged leading to the search for alternative expression systems. CEVEC's amniocyte production (CAP) cells are human production cells demonstrated to enable efficient overexpression of recombinant proteins with human glycosylation pattern. However, CAP cells have not yet undergone any engineering approaches to optimize process parameters for a cheaper and more sustainable production of biopharmaceuticals...
April 17, 2018: Biotechnology and Bioengineering
Zhi-Qiang Yin, Wan-Hong Xing
Chronic heart failure (CHF) is still the leading cause of morbidity and mortality worldwide, and carries with it large economic and social burdens. Although steady and substantial progress has been made in reducing mortality from heart failure using conventional treatments, novel pharmacologic and surgical interventions have not been effective in extending five year survival rates. Therefore, it is necessary to explore new therapies. Gene therapy was introduced in 1970s with the development of recombinant DNA technology...
March 5, 2018: Heart Surgery Forum
Bita Javan, Fatemeh Atyabi, Majid Shahbazi
AIMS: This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. MAIN METHODS: To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression...
April 12, 2018: Life Sciences
Li-Ming Li, Ling-Ling Huang, Xin-Chi Jiang, Jia-Chen Chen, Hong-Wei OuYang, Jian-Qing Gao
Mesenchymal stem cells (MSCs) are promising candidates for nerve tissue engineering. Brain derived neurotrophic factor (BDNF) secreted by MSCs can function to increase neural differentiation and relieve inflammation response. Gene transfection technology is an efficient strategy to increase the secretion levels of cytokines and enhance cellular functions. However, transfection and in vivo gene expression of environmentally sensitive stem cells have been one of the most challenging subjects due to the requirement in both safety and transfection efficiency...
April 13, 2018: Current Gene Therapy
Ce Dou, Ning Ding, Fei Luo, Tianyong Hou, Zhen Cao, Yun Bai, Chuan Liu, Jianzhong Xu, Shiwu Dong
The objective of this study is to design a graphene-based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR-7b overexpression plasmid. GO-PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR-7b after GO-PEI-miR-7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell-specific transmembrane protein...
February 2018: Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
Leonid Gaidukov, Liliana Wroblewska, Brian Teague, Tom Nelson, Xin Zhang, Yan Liu, Kalpana Jagtap, Selamawit Mamo, Wen Allen Tseng, Alexis Lowe, Jishnu Das, Kalpanie Bandara, Swetha Baijuraj, Nevin M Summers, Timothy K Lu, Lin Zhang, Ron Weiss
Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection...
April 2, 2018: Nucleic Acids Research
Jingbo Zhai, Qiubo Wang, Yunfeng Gao, Ran Zhang, Shengjun Li, Bing Wei, Yong You, Xun Sun, Changlong Lu
Low immunogenicity is one of the major problems limiting the clinical use for DNA vaccines, which makes it impossible to obtain a strong protective immune response after vaccination. In order to explore whether Ag85A DNA vaccine could mount more efficiently protective immune response through new RNA sensor and its signal transduction pathway of antigen presentation we designed and synthesized Ag85A gene fragment containing multiple points mutations and transfected the gene fragment into the dendritic cell line (DC2...
March 28, 2018: International Immunopharmacology
Ming Liu, Yongqing Wang, Huifen Lu, Hao Wang, Xiaoming Shi, Xuan Shao, Yu-Xia Li, Yangyu Zhao, Yan-Ling Wang
Preeclampsia is a pregnancy-specific complication defined as newly onset gestational hypertension and proteinuria. Deficiency in placental development is considered as the predominant cause of preeclampsia. Our previous study found that the expression of miR-518b increased significantly in the preeclamptic placentas, indicating the potential participation of this small RNA in the occurrence of preeclampsia. In this study, data analysis using multiple databases predicted Rap1b as a candidate target of miR-518b...
2018: Frontiers in Endocrinology
Xiaohui Li, Xingtao Han, Jinhui Yang, Jiantao Sun, Pengtao Wei
Objective To observe the effect of microRNA-519d-3p (miR-519d-3p) on the proliferation of prostate cancer cells and explore the possible molecular mechanism. Methods The expression level of miR-519d-3p in PC-3, DU-145, 22RV1, PC-3M, LNCaP human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time quantitative PCR. miR-519d-3p mimics or negative control microRNAs (miR-NC) was transfected into the prostate cancer cells with the lowest level of miR-519d-3p expression...
January 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Yvon Woappi, Maria Hosseinipour, Kim E Creek, Lucia Pirisi
Human papillomavirus (HPV) infection of the genital tract is common, however only about 10-15% of infections persist, and approximately 10-15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. Yet, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA...
March 28, 2018: Journal of Virology
David P Walsh, Robert D Murphy, Angela Panarella, Rosanne Marie Raftery, Brenton Cavanagh, Jeremy Charles Simpson, Fergal J O'Brien, Andreas Heise, Sally-Ann Cryan
The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(L-lysine) polypeptide (star-PLL) with varying number and length of poly(L-Lysine) arms as potential non-viral gene delivery vectors for MSCs...
March 28, 2018: Molecular Pharmaceutics
H A Tomás, A F Rodrigues, M J T Carrondo, A S Coroadinha
Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already demonstrated in gene therapy clinical trials for the treatment of diverse disorders. For large scale LV production, a stable producer system is desirable since it allows scalable and cost-effective viral productions, with increased reproducibility and safety. However, the development of stable systems has been challenging and time-consuming, being the selection of cells presenting high expression levels of Gag-Pro-Pol polyprotein and the cytotoxicity associated with some viral components, the main limitations...
March 27, 2018: Scientific Reports
Julia N Khantakova, Alexander N Silkov, Valeriy P Tereshchenko, Elena V Gavrilova, Rinat A Maksyutov, Sergey V Sennikov
In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death...
March 23, 2018: Cytokine
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