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Mammalian cell culture

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https://www.readbyqxmd.com/read/28342833/a-high-density-cho-s-transient-transfection-system-comparison-of-expicho-and-expi293
#1
Nina K Jain, Susan Barkowski-Clark, Richard Altman, Krista Johnson, Fang Sun, Jonathan Zmuda, Chao Yan Liu, Adriana Kita, Ryan Schulz, Alyssa Neill, Robert Ballinger, Rekha Patel, Jian Liu, Alinafe Mpanda, Brian Huta, Henry Chiou, Walter Voegtli, Tadas Panavas
Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins...
March 22, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28339479/mouse-newborn-cells-allow-highly-productive-mouse-cytomegalovirus-replication-constituting-a-novel-convenient-primary-cell-culture-system
#2
Vu Thuy Khanh Le-Trilling, Mirko Trilling
Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation...
2017: PloS One
https://www.readbyqxmd.com/read/28339398/n-terminal-fragments-of-huntingtin-longer-than-residue-170-form-visible-aggregates-independently-to-polyglutamine-expansion
#3
Moore Z Chen, Sue-Ann Mok, Angelique R Ormsby, Paul J Muchowski, Danny M Hatters
BACKGROUND: A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. OBJECTIVE: It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation...
March 22, 2017: Journal of Huntington's Disease
https://www.readbyqxmd.com/read/28337817/the-cytotoxic-effects-of-camptothecin-and-mastoparan-on-the-unicellular-green-alga-chlamydomonas-reinhardtii
#4
Jürgen Voigt, Markus Morawski, Johannes Wöstemeyer
We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well-known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag-phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occured during the cell-division phase...
March 24, 2017: Journal of Eukaryotic Microbiology
https://www.readbyqxmd.com/read/28337294/hypoxic-preconditioning-of-human-cardiosphere-derived-cell-sheets-enhances-cellular-functions-via-activation-of-the-pi3k-akt-mtor-hif-1%C3%AE-pathway
#5
Yuya Tanaka, Tohru Hosoyama, Akihito Mikamo, Hiroshi Kurazumi, Arata Nishimoto, Koji Ueno, Bungo Shirasawa, Kimikazu Hamano
Cell sheet technology is a promising therapeutic strategy for the treatment of ischemic diseases such as myocardial infarction. We recently developed a novel protocol, termed "hypoxic preconditioning," capable of augmenting the therapeutic efficacy of cell sheets. Following this protocol, the pro-angiogenic and anti-fibrotic activity of cell sheets were enhanced by brief incubation of cell sheets under hypoxic culture conditions. However, the precise molecular mechanism underlying the hypoxic preconditioning of cell sheets is unclear...
2017: American Journal of Translational Research
https://www.readbyqxmd.com/read/28336600/complete-genome-sequences-before-and-after-mammalian-cell-culture-of-zika-virus-isolated-from-the-serum-of-a-symptomatic-male-patient-from-oaxaca-mexico
#6
Celia Boukadida, Jesús M Torres-Flores, Martha Yocupicio-Monroy, Elvira Piten-Isidro, Amaranta Y Rivero-Arrieta, Yara A Luna-Villalobos, Liliane Martínez-Vargas, Sofía L Alcaraz-Estrada, Klintsy J Torres, Rosalia Lira, Gustavo Reyes-Terán, Edgar E Sevilla-Reyes
Zika virus (ZIKV) is an emerging arthropod-borne flavivirus associated with severe congenital malformations and neurological complications. Although the ZIKV genome is well characterized, there is limited information regarding changes after cell isolation and culture adaptation. We isolated, and passaged in Vero cells, ZIKV from the serum of a symptomatic male patient and compared the viral genomes before and after culture. Single nucleotide polymorphisms were characteristic among serum-circulating genomes, while such diversity decreased after cell culture...
March 23, 2017: Genome Announcements
https://www.readbyqxmd.com/read/28333306/protein-aggregation-in-ehrlichia-chaffeensis-during-infection-of-mammalian-cells
#7
Dorota Kuczynska-Wisnik, Chuanmin Cheng, Roman R Ganta, Michal Zolkiewski
Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and other vertebrates. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. We also incubated the infected cells with guanidinium chloride (GuHCl), a known inhibitor of the protein-disaggregating molecular chaperone ClpB...
March 15, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/28333162/the-endoplasmic-reticulum-chaperone-grp78-bip-modulates-prion-propagation-in-vitro-and-in-vivo
#8
Kyung-Won Park, Gyoung Eun Kim, Rodrigo Morales, Fabio Moda, Ines Moreno-Gonzalez, Luis Concha-Marambio, Amy S Lee, Claudio Hetz, Claudio Soto
Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches...
March 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28333031/new-approaches-regarding-the-in-vitro-maturation-of-oocytes-manipulating-cyclic-nucleotides-and-their-partners-in-crime
#9
REVIEW
Ramon Cesar Botigelli, Eduardo Montanari Razza, Elisa Mariano Pioltine, Marcelo Fábio Gouveia Nogueira
Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production...
February 1, 2017: JBRA Assisted Reproduction
https://www.readbyqxmd.com/read/28324501/assessing-telomerase-activities-in-mammalian-cells-using-the-quantitative-pcr-based-telomeric-repeat-amplification-protocol-qtrap
#10
Shuai Jiang, Mengfan Tang, Huawei Xin, Junjiu Huang
Telomerase expression and activity appear elevated in >80% of human cancers. The activity of the telomerase may serve as a diagnostic marker for malignancy, and an indicator of the proliferative potential of somatic and stem cells. The telomeric repeat amplification protocol (TRAP) is a sensitive and accurate PCR-based assay for telomerase detection and measurement. Here, we describe a quantitative PCR-based TRAP assay (qTRAP) that is more convenient and amenable to high-throughput applications compared to traditional gel-based TRAP assays...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324494/analysis-of-average-telomere-length-in-human-telomeric-protein-knockout-cells-generated-by-crispr-cas9
#11
Jun Xu, Zhou Songyang, Dan Liu, Hyeung Kim
Telomeres play an important role in ensuring the integrity of the genome. Telomere shortening can lead to loss of genetic information and trigger DNA damage responses. Cultured mammalian cells have served as critical model systems for studying the function of telomere binding proteins and telomerase. Tremendous heterogeneity can be observed both between species and within a single cell population. Recent advances in genome editing (such as the development of the CRISPR/Cas9 platform) have further enabled researchers to carry out loss-of-function analysis of how disrupting key players in telomere maintenance affects telomere length regulation...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324489/monitoring-mitochondrial-changes-by-alteration-of-the-pink1-parkin-signaling-in-drosophila
#12
Tsuyoshi Inoshita, Kahori Shiba-Fukushima, Hongrui Meng, Nobutaka Hattori, Yuzuru Imai
Mitochondrial quality control is a key process in tissues with high energy demands, such as the brain and muscles. Recent studies using Drosophila have revealed that the genes responsible for familial forms of juvenile Parkinson's disease (PD), PINK1 and Parkin regulate mitochondrial function and motility. Cell biological analysis using mammalian cultured cells suggests that the dysregulation of mitophagy by PINK1 and Parkin leads to neurodegeneration in PD. In this chapter, we describe the methods to monitor mitochondrial morphology in the indirect flight muscles of adult Drosophila and Drosophila primary cultured neurons and the methods to analyze the motility of mitochondria in the axonal transport of living larval motor neurons...
March 22, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324217/rare-slc1a1-variants-in-hot-water-epilepsy
#13
Kalpita Rashimi Karan, P Satishchandra, Sanjib Sinha, Anuranjan Anand
Hot water epilepsy is sensory epilepsy, wherein seizures are triggered by an unusual stimulus: contact with hot water. Although genetic factors contribute to the etiology of hot water epilepsy, molecular underpinnings of the disorder remain largely unknown. We aimed to identify the molecular genetic basis of the disorder by studying families with two or more of their members affected with hot water epilepsy. Using a combination of genome-wide linkage mapping and whole exome sequencing, a missense variant was identified in SLC1A1 in a three-generation family...
March 21, 2017: Human Genetics
https://www.readbyqxmd.com/read/28321435/amyloid-precursor-protein-family-as-unconventional-go-coupled-receptors-and-the-control-of-neuronal-motility
#14
REVIEW
Jenna M Ramaker, Philip F Copenhaver
Cleavage of the Amyloid Precursor Protein (APP) generates amyloid peptides that accumulate in Alzheimer Disease (AD), but APP is also upregulated by developing and injured neurons, suggesting that it regulates neuronal motility. APP can also function as a G protein-coupled receptor that signals via the heterotrimeric G protein Gαo, but evidence for APP-Gαo signaling in vivo has been lacking. Using Manduca as a model system, we showed that insect APP (APPL) regulates neuronal migration in a Gαo-dependent manner...
2017: Neurogenesis (Austin, Tex.)
https://www.readbyqxmd.com/read/28321016/4%C3%AE-8c-inhibits-insulin-secretion-independent-of-ire1%C3%AE-rnase-activity
#15
Hitomi Sato, Yoko Shiba, Yuichi Tsuchiya, Michiko Saito, Kenji Kohno
IRE1α plays an important role in the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. 4μ8C, a well-known inhibitor of IRE1α RNase activity, is commonly used to analyze IRE1α function during ER stress in cultured mammalian cells. However, the off-target effects of 4μ8C remain elusive. Pancreatic β-cells synthesize a large amount of insulin in response to high glucose stimulation, and IRE1α plays an important role in insulin secretion from pancreatic β-cells...
March 18, 2017: Cell Structure and Function
https://www.readbyqxmd.com/read/28321005/the-concentration-dependent-effect-of-progesterone-on-follicle-growth-in-the-mouse-ovary
#16
Kouji Komatsu, Satoru Masubuchi
Follicle growth in the mammalian ovary is coordinately controlled by multiple factors to sustain periodic ovulation. In this study, we investigated the role of progesterone on follicle growth in the mouse ovary. As the concentration of progesterone changes during the estrus cycle, we cultured the sliced mouse ovary in a medium containing 10 ng/ml, 100 ng/ml, and 1 μg/ml progesterone. Progesterone promoted the growth of primordial to primary follicles at 100 ng/ml, while it suppressed the growth of secondary follicles at 1 μg/ml...
March 17, 2017: Journal of Reproduction and Development
https://www.readbyqxmd.com/read/28319334/biofunctionalized-plants-as-diverse-biomaterials-for-human-cell-culture
#17
Gianluca Fontana, Joshua Gershlak, Michal Adamski, Jae-Sung Lee, Shion Matsumoto, Hau D Le, Bernard Binder, John Wirth, Glenn Gaudette, William L Murphy
The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells...
March 20, 2017: Advanced Healthcare Materials
https://www.readbyqxmd.com/read/28315598/n-piperonyl-substitution-on-aminoquinoline-pyrimidine-hybrids-effect-on-the-antiplasmodial-potency
#18
Rohit Kholiya, Shabana I Khan, Aparna Bahuguna, Mohit Tripathi, Diwan S Rawat
A series of 4-aminoquinoline-piperonyl-pyrimidine hybrids were synthesized with the aim of identifying compounds with enhanced antimalarial activity. All the synthesized molecules were evaluated in vitro against cultured Plasmodium falciparum W2 and D6 strains and exhibited potent antiplasmodial activities with IC50 values in the range of 0.02-5.16 μM. Out of the 22 synthesised hybrids, 12 were found to be better (up to eight-fold more active) than chloroquine (CQ), particularly against the CQ-resistant W2 strain of P...
March 11, 2017: European Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/28314612/a-simple-imagej-macro-tool-for-analyzing-mitochondrial-network-morphology-in-mammalian-cell-culture
#19
Andrew J Valente, Lucas A Maddalena, Ellen L Robb, Fereshteh Moradi, Jeffrey A Stuart
Mitochondria exist in a dynamic cycle of fusion and fission whose balance directly influences the morphology of the 'mitochondrial network', a term that encompasses the branched, reticular structure of fused mitochondria as well as the separate, punctate individual organelles within a eukaryotic cell. Over the past decade, the significance of the mitochondrial network has been increasingly appreciated, motivating the development of various approaches to analyze it. Here, we describe the Mitochondrial Network Analysis (MiNA) toolset, a relatively simple pair of macros making use of existing ImageJ plug-ins, allowing for semi-automated analysis of mitochondrial networks in cultured mammalian cells...
March 14, 2017: Acta Histochemica
https://www.readbyqxmd.com/read/28314188/pharmaceutical-analysis-of-a-novel-propargyl-linked-antifolate-antibiotic-in-the-mouse
#20
John Hoody, Jeremy B Alverson, Santosh Keshipeddy, Patrick A Barney, Larissa Walker, Amy C Anderson, Dennis L Wright, Nigel D Priestley
Antimicrobial resistance to current antibiotics is a significant public health problem and the need for new antibiotics is a compelling one. We have been developing a new series of antibiotics, propargyl-linked diaminopyrimidines, based on the structure of trimethoprim. To date we have discovered compounds that are effective inhibitors of dihydrofolate reductase (the target of trimethoprim), that are potent antibiotics in vitro against a range of Gram-positive pathogens including methicillin-resistant S. aureus, and that are non-toxic in mammalian cell culture...
March 6, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
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