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Mammalian cell culture

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https://www.readbyqxmd.com/read/27934094/fixation-and-permeabilization-approaches-for-scanning-electrochemical-microscopy-of-living-cells
#1
Alexandra Bondarenko, Tzu-En Lin, Petar Stupar, Andreas Lesch, Fernando Cortés-Salazar, Hubert H Girault, Horst Pick
Scanning electrochemical microscopy (SECM) has been widely used for the electrochemical imaging of dynamic topographical and metabolic changes in alive adherent mammalian cells. However, extracting intracellular information by SECM is challenging, since it requires redox species to travel in and out the lipid cell membrane. Herein, we present cell fixation and permeabilization approaches as an alternative tool for visualizing cell properties by SECM. With this aim, adherent cells were analyzed in the SECM feedback mode in three different conditions: (i) alive; (ii) fixed, and (iii) fixed and permeabilized...
December 6, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27932950/amyloid-precursor-proteins-are-dynamically-trafficked-and-processed-during-neuronal-development
#2
Jenna M Ramaker, Robert S Cargill, Tracy L Swanson, Hanil Quirindongo, Marlène Cassar, Doris Kretzschmar, Philip F Copenhaver
Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aβ) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2) that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP...
2016: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/27932756/-inhibitory-effect-of-jianpi-jiedu-prescription-contained-serum-on-colorectal-cancer-sw48-cell-proliferation-by-mtor-p53-p21-signalling-pathway
#3
Fengxia Lin, Sanlin Lei, Jin'an Ma, Li Shi, Dan Mao, Shaofan Zhang, Jianhua Huang, Xinyi Liu, Dengfeng Ding, Yingjin Zhang, Sifang Zhang
To investigate the effect of jianpi-jiedu (JPJD) prescription-contained serum on colorectal cancer SW48 cell proliferation and the underlying mechanisms.
 Methods: Crude extract from JPJD was made by water extract method and the main components of crude extract from JPJD were analyzed by ultra-performance liquid phase high resolution time of flight mass spectrometry (UPLC-Q-TOF/MS). The low, medium, and high-concentration of JPJD-contained serum were prepared by the serum pharmacological method. The effect of serum containing JPJD on SW48 cell proliferation was determined by MTT assay...
November 28, 2016: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/27932422/significance-of-low-mtorc1-activity-in-defining-the-characteristics-of-brain-tumor-stem-cells
#4
Yi-Peng Han, Atsushi Enomoto, Yukihiro Shiraki, Shen-Qi Wang, Xiaoze Wang, Shinya Toyokuni, Naoya Asai, Kaori Ushida, Hosne Ara, Fumiharu Ohka, Toshihiko Wakabayashi, Jie Ma, Atsushi Natsume, Masahide Takahashi
BACKGROUND: The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs). METHODS: Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs)...
December 8, 2016: Neuro-oncology
https://www.readbyqxmd.com/read/27930338/pegylated-ifn-%C3%AE-suppresses-hepatitis-c-virus-by-promoting-the-dapk-mtor-pathway
#5
Wei-Liang Liu, Hung-Chih Yang, Ching-Sheng Hsu, Chih-Chiang Wang, Tzu-San Wang, Jia-Horng Kao, Ding-Shinn Chen
Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)-induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting...
December 6, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27930325/development-of-high-yield-influenza-b-virus-vaccine-viruses
#6
Jihui Ping, Tiago J S Lopes, Gabriele Neumann, Yoshihiro Kawaoka
The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates...
December 5, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27929735/development-of-adsorptive-hybrid-filters-to-enable-two-step-purification-of-biologics
#7
Nripen Singh, Abhiram Arunkumar, Michael Peck, Alexei M Voloshin, Angela M Moreno, Zhijun Tan, Jonathan Hester, Michael C Borys, Zheng Jian Li
Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process- and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze™ AEX) and salt-tolerant biomimetic (Emphaze™ ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus...
December 8, 2016: MAbs
https://www.readbyqxmd.com/read/27929471/isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors
#8
Thomas Duncan, Aileen Lowe, Marshall A Dalton, Michael Valenzuela
The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature...
November 29, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27928028/mitochondrial-pyruvate-carrier-regulates-autophagy-inflammation-and-neurodegeneration-in-experimental-models-of-parkinson-s-disease
#9
Anamitra Ghosh, Trevor Tyson, Sonia George, Erin N Hildebrandt, Jennifer A Steiner, Zachary Madaj, Emily Schulz, Emily Machiela, William G McDonald, Martha L Escobar Galvis, Jeffrey H Kordower, Jeremy M Van Raamsdonk, Jerry R Colca, Patrik Brundin
Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of Parkinson's disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neurodegeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP(+)) insult in murine and cultured human midbrain dopamine neurons and in an α-synuclein-based Caenorhabditis elegans model...
December 7, 2016: Science Translational Medicine
https://www.readbyqxmd.com/read/27924556/in-vitro-enzyme-kinetics-analysis-of-egfr
#10
Zhihong Wang, Christine Candelora
The epidermal growth factor receptor (EGFR) is an essential receptor tyrosine kinase (RTK) that regulates cell proliferation and differentiation, and its abnormal activation contributes to a variety of human cancers. EGFR is activated by the binding of growth factors, such as epidermal growth factor (EGF), resulting in the formation of an asymmetric dimer in which one EGFR molecule allosterically activates the other molecule. This chapter provides a detailed protocol to purify and characterize full-length EGFR bound with EGF...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924271/amino-acid-residues-of-bitter-taste-receptor-tas2r16-that-determine-sensitivity-in-primates-to-%C3%AE-glycosides
#11
Hiroo Imai, Nami Suzuki-Hashido, Yoshiro Ishimaru, Takanobu Sakurai, Lijie Yin, Wenshi Pan, Masaji Ishiguro, Katsuyoshi Masuda, Keiko Abe, Takumi Misaka, Hirohisa Hirai
In mammals, bitter taste is mediated by TAS2Rs, which belong to the family of seven transmembrane G protein-coupled receptors. Since TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we analyzed the amino acids responsible for the difference in sensitivities of TAS2R16s of various primates using a cultured cell expression system. We found that the sensitivity of TAS2R16 varied due to several amino acid residues...
2016: Biophysics and Physicobiology
https://www.readbyqxmd.com/read/27922179/a-carbon-dioxide-stripping-model-for-mammalian-cell-culture-in-manufacturing-scale-bioreactors
#12
Zizhuo Xing, Amanda M Lewis, Michael C Borys, Zheng Jian Li
Control of carbon dioxide within the optimum range is important in mammalian bioprocesses at the manufacturing scale in order to ensure robust cell growth, high protein yields, and consistent quality attributes. The majority of bioprocess development work is done in laboratory bioreactors, in which carbon dioxide levels are more easily controlled. Some challenges in carbon dioxide control can present themselves when cell culture processes are scaled up, because carbon dioxide accumulation is a common feature due to longer gas-residence time of mammalian cell culture in large scale bioreactors...
December 6, 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/27919266/arrest-at-the-diplotene-stage-of-meiotic-prophase-i-is-delayed-by-progesterone-but-is-not-required-for-primordial-follicle-formation-in-mice
#13
Sudipta Dutta, Deion M Burks, Melissa E Pepling
BACKGROUND: In mammalian females, reproductive capacity is determined by the size of the primordial follicle pool. During embryogenesis, oogonia divide mitotically but cytokinesis is incomplete so oogonia remain connected in germ cell cysts. Oogonia begin to enter meiosis at 13.5 days postcoitum in the mouse and over several days, oocytes progress through the stages of meiotic prophase I arresting in the diplotene stage. Concurrently, germ cell cysts break apart and individual oocytes become surrounded by granulosa cells forming primordial follicles...
December 5, 2016: Reproductive Biology and Endocrinology: RB&E
https://www.readbyqxmd.com/read/27918591/transcriptomic-analysis-of-mouse-cochlear-supporting-cell-maturation-reveals-large-scale-changes-in-notch-responsiveness-prior-to-the-onset-of-hearing
#14
Juan C Maass, Rende Gu, Tiantian Cai, Ying-Wooi Wan, Silvia C Cantellano, Joanna S T Asprer, Hongyuan Zhang, Hsin-I Jen, Renée K Edlund, Zhandong Liu, Andrew K Groves
Neonatal mouse cochlear supporting cells have a limited ability to divide and trans-differentiate into hair cells, but this ability declines rapidly in the two weeks after birth. This decline is concomitant with the morphological and functional maturation of the organ of Corti prior to the onset of hearing. However, despite this association between maturation and loss of regenerative potential, little is known of the molecular changes that underlie these events. To identify these changes, we used RNA-seq to generate transcriptional profiles of purified cochlear supporting cells from 1- and 6-day-old mice...
2016: PloS One
https://www.readbyqxmd.com/read/27915978/cell-compatibility-of-an-eposimal-vector-mediated-by-the-characteristic-motifs-of-matrix-attachment-regions
#15
Tian-Yun Wang, Li Wang, Yu-Xin Yang, Chun-Peng Zhao, Yan-Long Jia, Qin Li, Jun-He Zhang, Yi-You Peng, Miao Wang, Hong-Yan Xu, Xiao-Yin Wang
The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening...
December 2, 2016: Current Gene Therapy
https://www.readbyqxmd.com/read/27914912/identification-of-foxj1-effectors-during-ciliogenesis-in-the-foetal-respiratory-epithelium-and-embryonic-left-right-organiser-of-the-mouse
#16
Michael Stauber, Marina Weidemann, Oliver Dittrich-Breiholz, Katharina Lobschat, Leonie Alten, Michaela Mai, Anja Beckers, Michael Kracht, Achim Gossler
Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary...
November 30, 2016: Developmental Biology
https://www.readbyqxmd.com/read/27913309/sequence-determinants-of-the-caenhorhabditis-elegansdopamine-transporter-dictating-in-vivoaxonal-export-and-synaptic-localization
#17
Sarah B Robinson, J Andrew Hardaway, Shannon L Hardie, Jane Wright, Ryan M Glynn, Daniel P Bermingham, Qiao Han, Sarah M Sturgeon, Phyllis Freeman, Randy D Blakely
The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies...
November 29, 2016: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/27908581/sonodynamic-inactivation-of-gram-positive-and-gram-negative-bacteria-using-a-rose-bengal-antimicrobial-peptide-conjugate
#18
David Costley, Heather Nesbitt, Nigel Ternan, James Dooley, Ying-Ying Huang, Michael R Hamblin, Anthony P McHale, John F Callan
Combating antimicrobial resistance is one of the most serious public health challenges facing society today. The development of new antibiotics or alternative techniques that can help combat antimicrobial resistance is being prioritised by many governments and stakeholders across the globe. Antimicrobial photodynamic therapy is one such technique that has received considerable attention but is limited by the inability of light to penetrate through human tissue, reducing its effectiveness when used to treat deep-seated infections...
November 17, 2016: International Journal of Antimicrobial Agents
https://www.readbyqxmd.com/read/27907214/derivation-of-induced-trophoblast-cell-lines-in-cattle-by-doxycycline-inducible-piggybac-vectors
#19
Takamasa Kawaguchi, Dooseon Cho, Masafumi Hayashi, Tomoyuki Tsukiyama, Koji Kimura, Shuichi Matsuyama, Naojiro Minami, Masayasu Yamada, Hiroshi Imai
Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique...
2016: PloS One
https://www.readbyqxmd.com/read/27906496/aerobic-expression-of-vitreoscilla-hemoglobin-improves-the-growth-performance-of-cho-k1-cells
#20
Mariana Juárez, Claudia H González-De la Rosa, Elisa Memún, Juan-Carlos Sigala, Alvaro R Lara
Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated...
December 1, 2016: Biotechnology Journal
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