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https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#1
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28209207/recombinant-%C3%AE-actinin-subunit-antigens-of-trichomonas-vaginalis-as-potential-vaccine-candidates-in-protecting-against-trichomoniasis
#2
Yi-Ting Xie, Jiang-Mei Gao, Ya-Ping Wu, Petrus Tang, Geoff Hide, De-Hua Lai, Zhao-Rong Lun
BACKGROUND: Human trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T...
February 16, 2017: Parasites & Vectors
https://www.readbyqxmd.com/read/28209057/saturated-fatty-acid-analogues-of-cell-penetrating-peptide-pepfect14-role-of-fatty-acid-modification-in-complexation-and-delivery-of-splice-correcting-oligonucleotides
#3
Tõnis Lehto, Luis Vasconcelos, Helerin Margus, Ricardo Figueroa, Margus Pooga, Mattias Hällbrink, Ulo Langel
Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were non-covalently complexed with a splice-correcting oligonucleotide (SCO) and tested in the HeLa pLuc705 reporter cell line...
February 17, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28204840/inhibition-of-the-innate-immune-receptors-for-foreign-dna-sensing-improves-transfection-efficiency-of-gene-electrotransfer-in-melanoma-b16f10-cells
#4
Masa Bosnjak, Urska Kamensek, Gregor Sersa, Danijela Stolfa, Jaka Lavrencak, Maja Cemazar
Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype...
February 15, 2017: Journal of Membrane Biology
https://www.readbyqxmd.com/read/28188727/apoptin-gene-delivery-by-the-functionalized-polyamidoamine-pamam-dendrimer-derivatives-induces-cell-death-of-u87-mg-glioblastoma-cells
#5
Yoonhee Bae, Hyang-Shuk Rhim, Seulgi Lee, Kyung Soo Ko, Jin Han, Joon Sig Choi
Malignant glioma is the most common and aggressive form of primary brain tumor in adults. In this study, we describe the efficacy of nonviral gene delivery carriers, histidine and arginine or histidine and lysine grafted PAMAM dendrimers (PAMAM-H-R and PAMAM-H-K), in delivering a therapeutic, a tumor-selective killer gene, apoptin, using human glioma cells (U87-MG) and newborn human dermal fibroblast (NB-HDF) cells. We analyzed transfection efficiency using luciferase (Luc) and a pDNA encoding for enhanced fluorescent protein (EGFP), and assessed cell viability in both cells...
February 7, 2017: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/28178869/low-molecular-weight-polyethylenimine-conjugated-gold-nanospheres-a-platform-for-selective-gene-therapy-controlled-by-near-infrared-light
#6
Fei Liu, Fen-Fen Kong, Qing-Po Li, Hong Yuan, Yong-Zhong Du, Fu-Qiang Hu, Ji-Hong Sun, Jian You
AIM: Whether PEI2k-HAuNS could promote gene transfection efficiency controlled by near-infrared (NIR) light. MATERIALS & METHODS: This safe nonviral gene delivery system was obtained by conjugating low molecular weight (2 kDa) polyethylenimine (PEI) onto hollow gold nanospheres (PEI2k-HAuNS). Upon NIR laser irradiation, there was a conspicuous increase both in the in vitro and in vivo transfection achieved by the nanocomplexes. Furthermore, a plasmid encoding the tumor suppressor TP53 (pTP53) was applied to test antitumor activity...
February 9, 2017: Nanomedicine
https://www.readbyqxmd.com/read/28170185/magnetic-nanoparticle-based-upregulation-of-b-cell-lymphoma-2-enhances-bone-regeneration
#7
Elizabeth Brett, Elizabeth R Zielins, Anna Luan, Chin Chun Ooi, Siny Shailendra, David Atashroo, Siddarth Menon, Charles Blackshear, John Flacco, Natalina Quarto, Shan X Wang, Michael T Longaker, Derrick C Wan
Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an "off the shelf" prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester...
January 2017: Stem Cells Translational Medicine
https://www.readbyqxmd.com/read/28166958/in%C3%A2-vitro-production-of-functional-haploid-sperm-cells-from-male-germ-cells-of-saanen-dairy-goat
#8
Shoulong Deng, Xiuxia Wang, Zhipeng Wang, Suren Chen, Yuqian Wang, Xiaoxia Hao, Tiecheng Sun, Yi Zhang, Zhengxing Lian, Yixun Liu
The dairy goat is an important economic animal. Studies on in vitro differentiation of dairy goat spermatogonial stem cells (SSCs) could understand the molecular mechanisms for detecting mammalian sperm generation and innovative transgenetics. This study established an in vitro differentiation system for Saanen dairy goat SSCs. After 35 days incubation, single flagellum sperm-like cells with late round spermatid (Sa2) morphologically and the ability to express sperm-specific protein acrosin is detected. DNA ploid analysis showed that addition of testosterone to the culture system significantly improved the differentiation efficiency (P < 0...
March 1, 2017: Theriogenology
https://www.readbyqxmd.com/read/28166401/maximizing-synergistic-activity-when-combining-rnai-and-platinum-based-anticancer-agents
#9
Haihua Xiao, Ruogu Qi, Ting Li, Samuel G Awuah, Yaorong Zheng, Wei Wei, Xiang Kang, Haiqin Song, Yongheng Wang, Yingjie Yu, Molly A Bird, Xiabin Jing, Michael B Yaffe, Michael J Birrer, P Peter Ghoroghchian
RNAi approaches have been widely combined with platinum-based anticancer agents to elucidate cellular responses and to target gene products that mediate acquired resistance. Recent work has demonstrated that platination of siRNA prior to transfection may negatively influence RNAi efficiency based on the position and sequence of its guanosine nucleosides. Here, we used detailed spectroscopic characterization to demonstrate rapid formation of Pt-guanosine adducts within 30 min after coincubation of oxaliplatin [OxaPt(II)] or cisplatin [CisPt(II)] with either guanosine monophosphate or B-cell lymphoma 2 (BCL-2) siRNA...
February 16, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28165726/synthetic-protein-mimics-for-functional-protein-delivery
#10
Arife Ozgul Tezgel, Paejonette Jacobs, Coralie M Backlund, Janice C Telfer, Gregory N Tew
The use of proteins as biological tools and therapeutic agents is limited due to the fact that proteins do not effectively cross the plasma membrane of cells. Here, we report a novel class of protein transporter molecules based on protein transduction domain mimics (PTDMs) synthesized via ring opening metathesis polymerization (ROMP). The PTDMs reported here were specifically inspired by amphiphilic peptides known to deliver functional proteins into cells via non-covalent interactions between the peptide and cargo...
February 6, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28163178/over-expression-of-jagged1-ameliorates-aged-rat-derived-endothelial-progenitor-cell-functions-and-improves-its-transfusion-efficiency-for-rat-balloon-induced-arterial-injury
#11
Guangxu Zhu, Jinxiang Wang, Mingbao Song, Fang Zhou, Dagan Fu, Guangping Ruan, Yingying Bai, Zhengping Yu, Leilei Zhang, Xiangqing Zhu, Lan Huang, Rongqing Pang, Xinghua Pan
BACKGROUND: Endothelial progenitor cell (EPC) has significant age-dependent alterations in properties, but the role of Jagged1 in ageing-induced decline of EPC functions remains unclear. METHODS: 2 and 20 months old healthy male Sprague-Dawley rats were used in present study. Jagged1 gene transfection was performed in EPC isolated from aged (AEPC) and young rats (YEPC) respectively. Experiments were divided into four groups: a) pIRES2-EGFP (PE) group, b) PE combined N-[N-(3, 5-difluoro-phenacetyl)-1- alany1]-S-phenyglycine t-butyl ester (DAPT) (PE + D) group, c) pIRES2 EGFP- Jagged1 (PEJ) group and d) PEJ combined DAPT (PEJ+D) group...
February 2, 2017: Annals of Vascular Surgery
https://www.readbyqxmd.com/read/28159719/improvement-of-osteogenesis-in-dental-pulp-pluripotent-like-stem-cells-by-oligopeptide-modified-poly-%C3%A3-amino-ester-s
#12
Raquel Núñez-Toldrà, Pere Dosta, Sheyla Montori, Víctor Ramos, Maher Atari, Salvador Borrós
: Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. Recently, a new family of poly (β-amino ester)s (pBAEs) with oligopeptide-modified termini showing high transfection efficiency of both siRNA and DNA plasmid has been developed. In this study, oligopeptide-modified pBAEs were used to simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid to cells from the dental pulp with pluripotent-like characteristics (DPPSC) in order to promote their osteogenic differentiation...
January 31, 2017: Acta Biomaterialia
https://www.readbyqxmd.com/read/28159514/self-assembling-complexes-between-binary-mixtures-of-lipids-with-different-linkers-and-nucleic-acids-promote-universal-mrna-dna-and-sirna-delivery
#13
Thibault Colombani, Pauline Peuziat, Laurence Dallet, Thomas Haudebourg, Mathieu Mével, Mathieu Berchel, Olivier Lambert, Damien Habrant, Bruno Pitard
Protein expression and RNA interference require efficient delivery of DNA or mRNA and small double stranded RNA into cells, respectively. Although cationic lipids are the most commonly used synthetic delivery vectors, a clear need still exists for a better delivery of various types of nucleic acids molecules to improve their biological activity. To optimize the transfection efficiency, a molecular approach consisting in modifying the chemical structure of a given cationic lipid is usually performed, but an alternative strategy could rely on modulating the supramolecular assembly of lipidic lamellar phases sandwiching the nucleic acids molecules...
February 1, 2017: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/28157292/structurally-programmed-assembly-of-translation-initiation-nanoplex-for-superior-mrna-delivery
#14
Jiahe Li, Wade Wang, Yanpu He, Yingzhong Li, Emily Z Yan, Ketian Zhang, Darrell J Irvine, Paula T Hammond
Messenger RNA (mRNA) represents a promising class of nucleic-acid-based therapeutics. While numerous nanocarriers have been developed for mRNA delivery, the inherent labile nature of mRNA results in a very low transfection efficiency and poor expression of desired protein. Here we preassemble the mRNA translation initiation structure through an inherent molecular recognition between 7-methylguanosine (m(7)G)-capped mRNA and eukaryotic initiation factor 4E (eIF4E) protein to form ribonucleoproteins (RNPs), thereby mimicking the first step of protein synthesis inside cells...
February 14, 2017: ACS Nano
https://www.readbyqxmd.com/read/28155604/a-vector-based-on-the-chicken-hypersensitive-site-4-insulator-element-replicates-episomally-in-mammalian-cell
#15
Xi Zhang, Xiao-Yin Wang, Yan-Long Jia, Xiao Guo, Yan-Fang Wang, Tian-Yun Wang
Gene therapy in mammalian cells requires vectors exhibiting long-term stability and high expression. Episomal gene expression vectors offer a safe and attractive alternative to those that integrate into the host cell genome. In the present study, we developed a new episomal vector based on the insulator, chicken hypersensitive site 4 (cHS4). The cHS4 element was artificially synthesized, cloned into the pEGFP-C1 vector, and used to transfect Chinese hamster ovary (CHO) and human Chang liver cells. The stably transfected cell colonies were further cultured in either the presence or absence of G418 selection...
February 2, 2017: Current Gene Therapy
https://www.readbyqxmd.com/read/28150318/determine-exogenous-human-ddah2-gene-function-in-rabbit-bone-marrow-derived-endothelial-progenitor-cells-in-vitro
#16
Sara Shoeibi, Shabnam Mohammadi, Hamid Reza Sadeghnia, Elahe Mahdipour, Majid Ghayour-Mobarhan
The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery...
February 1, 2017: Cell Biochemistry and Function
https://www.readbyqxmd.com/read/28137669/effective-gene-delivery-to-trypanosoma-cruzi-epimastigotes-through-nucleofection
#17
Lisandro Pacheco-Lugo, Yirys Díaz-Olmos, José Sáenz-García, Christian Macagnan Probst, Wanderson Duarte DaRocha
New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T...
January 27, 2017: Parasitology International
https://www.readbyqxmd.com/read/28125685/lack-of-casein-kinase-1-delta-promotes-genomic-instability-the-accumulation-of-dna-damage-and-down-regulation-of-checkpoint-kinase-1
#18
Yoshimi Endo Greer, Bo Gao, Yingzi Yang, Andre Nussenzweig, Jeffrey S Rubin
Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells...
2017: PloS One
https://www.readbyqxmd.com/read/28124909/enhanced-spacer-length-between-mannose-mimicking-shikimoyl-and-quinoyl-head-groups-and-hydrophobic-region-of-cationic-amphiphile-increases-efficiency-of-dendritic-cell-based-dna-vac-cination-a-structure-activity-investigation
#19
Chandrashekhar Voshavar, Rakeshchandra Reddy Meka, Sanjoy Samanta, Srujan Kumar Marepally, Arabinda Chaudhuri
In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl head-group are promising DNA vaccine carriers for DC transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking head groups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57/BL6J mice under prophylactic settings...
January 26, 2017: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/28120892/antimicrobial-nanoplexes-meet-model-bacterial-membranes-the-key-role-of-cardiolipin
#20
Alejandro Marín-Menéndez, Costanza Montis, Teresa Díaz-Calvo, Davide Carta, Kostas Hatzixanthis, Christopher J Morris, Michael McArthur, Debora Berti
Antimicrobial resistance to traditional antibiotics is a crucial challenge of medical research. Oligonucleotide therapeutics, such as antisense or Transcription Factor Decoys (TFDs), have the potential to circumvent current resistance mechanisms by acting on novel targets. However, their full translation into clinical application requires efficient delivery strategies and fundamental comprehension of their interaction with target bacterial cells. To address these points, we employed a novel cationic bolaamphiphile that binds TFDs with high affinity to form self-assembled complexes (nanoplexes)...
January 25, 2017: Scientific Reports
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