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Kalle Kipper, Ebba Gregorsson Lundius, Vladimir Curic, Ivana Nikic, Edward A Lemke, Manfred Wiessler, Johan Elf
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells...
October 24, 2016: ACS Synthetic Biology
Wenshu Luo, Hidenobu Mizuno, Ryohei Iwata, Shingo Nakazawa, Kosuke Yasuda, Shigeyoshi Itohara, Takuji Iwasato
Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood...
October 24, 2016: Scientific Reports
Min Chen, Yang Xu, Rongchen Huang, Yubin Huang, Shuchao Ge, Bing Hu
Stimulating neuronal activity increases myelin sheath formation by individual oligodendrocytes, but how myelination is regulated by neuronal activity in vivo is still not fully understood. While in vitro studies have revealed the important role of N-cadherin in myelination, our understanding in vivo remains quite limited. To obtain the role of N-cadherin during activity-dependent regulation of myelinating capacity of individual oligodendrocytes, we successfully built an in vivo dynamic imaging model of the Mauthner cell at the subcellular structure level in the zebrafish central nervous system...
October 22, 2016: Molecular Neurobiology
M J Aguado-Romeo, S Benot-López, A Romero-Tabares
Electrochemotherapy is a therapeutic option for the treatment of cutaneous and subcutaneous metastases from melanoma and other tumors. The procedure consists of the administration of anticancer drugs followed by locally applied electrical impulses to achieve an effect known as electroporation, which facilitates entry into the cytosol of drugs that cannot cross the cell membrane. The aim of this review is to evaluate the evidence that supports the use of electrochemotherapy as a therapeutic strategy in melanoma...
October 18, 2016: Actas Dermo-sifiliográficas
Margaret M Fleming, Anna I Holbrook, Mary S Newell
OBJECTIVE: The purpose of this article is to delineate the potential techniques for percutaneous ablation of breast cancer, discuss the advantages and disadvantages of each technique, and provide results from recent studies on these technologies. The techniques discussed are cryotherapy, laser irradiation, microwave irradiation, radiofrequency ablation, high-intensity focused ultrasound ablation, and irreversible electroporation. CONCLUSION: Although percutaneous ablation techniques have some promising potential for less-invasive treatment of breast cancer, larger multicenter trials are needed to confirm their efficacy, especially in comparison with the reference standard of lumpectomy...
October 20, 2016: AJR. American Journal of Roentgenology
Gaia Pigna, Taniya Dhillon, Elizabeth M Dlugosz, Joshua S Yuan, Connor Gorman, Piero Morandini, Scott C Lenaghan, C Neal Stewart
Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. We developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 μM 2,4-D and 4.4 μM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a 5-fold increase in tissue mass over 14 days...
October 20, 2016: Biotechnology Journal
H A Marsman, M G Besselink
- The incidence of pancreatic cancer is increasing due to the ageing population among other things, while 5-year survival has improved in the past two decades from 3 to 7%.- In case of biliary obstruction due to pancreatic cancer, biliary drainage before surgery or ablative therapy using a covered metal stent instead of plastic reduces the rate of complications.- In patients with metastasized pancreatic cancer a combination of folinic acid, fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX) results in improved survival...
2016: Nederlands Tijdschrift Voor Geneeskunde
George S Vidal, Maja Djurisic, Kiana Brown, Richard W Sapp, Carla J Shatz
Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood...
September 2016: ENeuro
Ying Li Gu, Guan Qun Gao, Ning Ma, Lin Lin Ye, Li Wei Zhang, Xu Gao, Zhuo Bo Zhang
The aim of the present study was to investigate whether ciliary neurotrophic factor (CNTF) plays its neuroprotective role following hypoxic injury through the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Firstly, to determine whether CNTF exerts its effects via STAT3 following hypoxic injury, cultured neurons from the cerebral cortex of mice were prepared and a neuronal model of hypoxia was then established. The neurons exposed to hypoxia were then pre-treated with CNTF and transfected with small interference RNA (siRNA) targeting STAT3 (STAT3 siRNA) using polybrene, or with STAT3Tyr705 mutant or STAT3Ser727 mutant using an electroporation system...
October 13, 2016: International Journal of Molecular Medicine
Thomas Broggini, Lisa Schnell, Ali Ghoochani, José María Mateos, Michael Buchfelder, Kurt Wiendieck, Michael K Schäfer, Ilker Y Eyupoglu, Nicolai E Savaskan
The Plasticity Related Gene family covers five, brain-specific, transmembrane proteins (PRG1-5, also termed LPPR1-5) that operate in neuronal plasticity during development, aging and brain trauma. Here we investigated the role of the PRG family on axonal and filopodia outgrowth. Comparative analysis revealed the strongest outgrowth induced by PRG3 (LPPR1). During development, PRG3 is ubiquitously located at the tip of neuronal processes and at the plasma membrane and declines with age. In utero electroporation of PRG3 induced dendritic protrusions and accelerated spine formations in cortical pyramidal neurons...
October 15, 2016: Aging
Ziyang Sheng, Na Gao, Xiaoyun Cui, Dongying Fan, Hui Chen, Na Wu, Jianchun Wei, Jing An
Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV...
October 12, 2016: Vaccine
Zhaogang Yang, Jing Xie, Jing Zhu, Chen Kang, Chiling Chiang, Xinmei Wang, Xiaobing Wang, Tairong Kuang, Feng Chen, Zhou Chen, Aili Zhang, Bo Yu, Robert J Lee, Lesheng Teng, L James Lee
Exosomes, the smallest subgroup of extracellular vesicles, have been recognized as extracellular organelles that contain genetic and proteomic information for long distance intercellular communication. Exosome-based drug delivery is currently a subject of intensive research. Here, we report a novel strategy to produce nanoscale exosome-mimics (EMs) in sufficient quantity for gene delivery in cancer both in vitro and in vivo. Size-controllable EMs were generated at a high yield by serial extrusion of non-tumorigenic epithelial MCF-10A cells through filters with different pore sizes...
October 11, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
Matthew R Collinson-Pautz, Kevin M Slawin, Jonathan M Levitt, David M Spencer
Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways...
2016: PloS One
Ying Zhu, Yan Yang, Pingping Den, Yong Huang, Mengxiang Ni, Hongqing Fang
We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts: cma (which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region...
October 2016: Science China. Life Sciences
L Bond, B Schulz, T VanMeter, R C G Martin
INTRODUCTION: Irreversible electroporation (IRE) uses multiple needles and a series of electrical pulses to create pores in cell membranes and cause cell apoptosis. One of the demands of IRE is the precise needle spacing required. Two-dimensional intraoperative ultrasound (2-D iUS) is currently used to measure inter-needle distances but requires significant expertise. This study evaluates the potential of three-dimensional (3-D) image guidance for placing IRE needles and calculating needle spacing...
September 29, 2016: European Journal of Surgical Oncology
Emma R Cold, Gerardo R Vasta, Josà A Fernà Ndez-Robledo
Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite". This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant...
October 10, 2016: Journal of Parasitology
Suguru Shigemori, Masaki Ihara, Takashi Sato, Yoshinari Yamamoto, Shireen Nigar, Tasuku Ogita, Takeshi Shimosato
Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L...
October 8, 2016: Applied Microbiology and Biotechnology
Sho Ohta, Baolin Wang, Suzanne L Mansour, Gary C Schoenwolf
During development of the inner ear, secreted morphogens act coordinately to establish otocyst dorsoventral polarity. Among these, Sonic hedgehog (SHH) plays a critical role in determining ventral polarity. However, how this extracellular signal is transduced intracellularly to establish ventral polarity is unknown. In this study, we show that cAMP dependent protein kinase A (PKA) is a key intracellular factor mediating SHH signaling through regulation of GLI3 processing. Gain-of-function experiments using targeted gene transfection by sonoporation or electroporation revealed that SHH signaling inactivates PKA, maintaining a basal level of PKA activity in the ventral otocyst...
October 5, 2016: Developmental Biology
Haruhiko Fujiwara, Hideki Nishikawa
In addition to the genome editing technology, novel functional analyses using electroporation are powerful tools to reveal the gene function in the color pattern formation. Using these methods, several genes involved in various larval color pattern formation are clarified in the silkworm Bombyx mori and some Papilio species. Furthermore, the coloration pattern mechanism underlying the longtime mystery of female-limited Batesian mimicry of Papilio polytes has been recently revealed. This review presents the recent progress on the molecular mechanisms and evolutionary process of coloration patterns contributing to various mimicry in Lepidoptera, especially focusing on the gene function in the silkworm and Papilio species...
October 2016: Current Opinion in Insect Science
Lingqian Chang, Lei Li, Junfeng Shi, Yan Sheng, Wu Lu, Daniel Gallego-Perez, Ly James Lee
Electroporation has been one of the most popular non-viral technologies for cell transfection. However, conventional bulk electroporation (BEP) shows significant limitations in efficiency, cell viability and transfection uniformity. Recent advances in microscale-electroporation (MEP) resulted in improved cell viability. Further miniaturization of the electroporation system (i.e., nanoscale) has brought up many unique advantages, including negligible cell damage and dosage control capabilities with single-cell resolution, which has enabled more translational applications...
October 18, 2016: Lab on a Chip
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