Passage of SARS-CoV-2 in cells expressing human and mouse ACE2 selects for mouse-adapted and ACE2-independent viruses

Human ACE2 (hACE2) is required for cell attachment and entry of SARS-CoV-2. Mouse ACE2 (mACE2) does not support infection of early SARS-CoV-2 isolates. Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with a series of spike protein amino acid changes, all of which have been reported in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. Remarkably, one mouse-adapted virus was able to replicate efficiently in ACE2-negative cell lines, a characteristic not described for any SARS-CoV-2 variants. ACE2-independent entry by SARS-CoV-2 represents a new biology for SARS-CoV-2 with potential widespread implications for disease and intervention development.

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Precision cancer therapy through nanoparticle delivery of siRNA against KRAS.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.260 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 260-260

Author(s):

Matthew S. Strand ◽

Hua Pan ◽

Julie G. Grossman ◽

Peter S. Goedegebuure ◽

Timothy Fleming ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Colorectal Cancers ◽

Mouse Pancreas ◽

Colorectal Cancer Cell Lines ◽

Human And Mouse

260 Background: Small interfering RNA (siRNA) has potential for highly specific gene manipulation, making it attractive for delivering precision therapy to cancer patients. However, efforts to employ siRNA therapeutically have been limited by its short half-life in circulation, low target tissue specificity, and cellular entrapment within endosomes. We utilized serum-stable, cell-penetrating, and endosomolytic peptide-based nanoparticles (NPs) to overcome these obstacles and deliver siRNA against KRAS to KRAS-mutant human and mouse pancreas and colorectal cancers. Methods: Human and mouse pancreas and colorectal cancer cell lines were tested for NP uptake in vitro utilizing fluorescent siRNAs. Uptake was assessed via fluorescent microscopy and flow cytometry (FC). Mice bearing tumors from these cells were injected IV with the same NP, and uptake was assessed with an in vivo imaging system (IVIS), and FC. Cell lines were treated with KRAS-siRNA NP and KRAS knockdown was assessed by real-time PCR. Results: Mouse and human pancreas and colorectal cancer cell lines took up NP in vitro, with signal detected within > 93% of cells at 24 hours. Tumors from these cells grown in mice were strongly fluorescent after IV injection of fluorescent NP within 2 hours, and until at least 30 hours. FC of a tumor treated with fluorescent NP showed that 86% of tumor cells expressed fluorescent signal 24 hours post-injection. IVIS revealed signal in mouse liver and kidneys, but when assessed by FC, only 17.8% and 13.5% of cells from these tissues were fluorescent, respectively. The brain, heart, lungs, spleen, and pancreas of mice receiving injections were negative. Cancer cell lines exposed to KRAS-siRNA NP for 48 hours express KRAS at levels that are 4.5 to 15.1% of untreated cells. Conclusions: Human and mouse pancreas and colorectal cancers efficiently and specifically take up NP in vitro and in vivo. Selected limitations of siRNA are overcome with this NP delivery system, and NP-packaged siRNA effectively inhibits KRAS. This platform represents a highly specific approach to targeting tumor genes of interest, which may ultimately enable selective knockdown of putative drivers of tumor progression.

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Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2015.05.011 ◽

2015 ◽

Vol 789-790 ◽

pp. 7-27 ◽

Cited By ~ 26

Author(s):

James Whitwell ◽

Robert Smith ◽

Karen Jenner ◽

Heather Lyon ◽

Deborah Wood ◽

...

Keyword(s):

Cell Lines ◽

Mouse Cell ◽

P53 Status ◽

Human And Mouse

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NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

Journal of Clinical Investigation ◽

10.1172/jci36022 ◽

2009 ◽

Vol 119 (5) ◽

pp. 1251-1263 ◽

Cited By ~ 217

Author(s):

Tadepally Lakshmikanth ◽

Shannon Burke ◽

Talib Hassan Ali ◽

Silvia Kimpfler ◽

Francesco Ursini ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Nk Cell ◽

Cell Recognition ◽

Mouse Melanoma ◽

Mouse Melanoma Cell ◽

Human And Mouse ◽

Melanoma Cell Lines

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DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

Blood ◽

10.1182/blood-2001-11-0032 ◽

2002 ◽

Vol 100 (12) ◽

pp. 3968-3974 ◽

Cited By ~ 55

Author(s):

Hiroshi Nishihara ◽

Masae Maeda ◽

Atsushi Oda ◽

Masumi Tsuda ◽

Hirofumi Sawa ◽

...

Keyword(s):

Cell Lines ◽

Cell Attachment ◽

Leukemia Cell ◽

Jurkat Cells ◽

Human Leukemia ◽

Lymphocyte Migration ◽

Leukemia Cell Lines ◽

Src Homology

The CDM (ced-5 of Caenorhabditis elegans,DOCK180 [downstream of Crkwith molecular weight of 180 kDa] of humans, andmyoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor moleculec-CT10–related kinase II (c–Crk-II) in adherent cells. Recently, hematopoietic cell–specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c–Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.

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Efficient lentiviral transduction of different human and mouse cells

10.1101/253732 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gang Zhang ◽

Taihua Wang

Keyword(s):

Cell Lines ◽

Human Cell ◽

Efficient Method ◽

Genetic Research ◽

Human Cell Lines ◽

Primary Cells ◽

Lentiviral Transduction ◽

Mouse Cells ◽

Human And Mouse

AbstractBackgroundLentiviral vectors (LVs) allowing efficient establishment of stable transgene overexpression mammalian and human cell lines are invaluable tools for genetic research. Currently, although LV transductions are broadly adopted, they are often limited due to their low titers for efficient transduction.ResultsHere, we described a set of optimized, efficient techniques, which could produce sufficiently high LV titers, and, provide efficient transduction of cells. According to these optimizations, most of the mammalian and human cells, both primary cells and cell lines, could be transduced successfully with high levels of transgene stable expression, including both constitutive and induced expressions.ConclusionsOur data demonstrated the highly usefulness of our optimized methods. Therefore, this study provided an efficient method for most of LV transduction experiments in vitro.

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T-Cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and Requires p21CIP1/WAF1 Expression

Molecular and Cellular Biology ◽

10.1128/mcb.23.2.566-578.2003 ◽

2003 ◽

Vol 23 (2) ◽

pp. 566-578 ◽

Cited By ~ 57

Author(s):

Zhi-yong Huang ◽

YanLi Wu ◽

Nicolé Hedrick ◽

David H. Gutmann

Keyword(s):

Cell Lines ◽

Growth Regulation ◽

Cell Attachment ◽

Growth Arrest ◽

Cell Aggregation ◽

Growth Suppression ◽

Serum Starvation ◽

Multiple Tumor ◽

Phase Arrest

ABSTRACT Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53 −/− mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21CIP1/WAF1, but not p27Kip1, expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21CIP1/WAF1 expression and was eliminated in p21CIP1/WAF1-deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21CIP1/WAF1 expression and G2 arrest.

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Some Structural Features of Metaphase Chromosomes of Mice and Men

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060969 ◽

1967 ◽

Vol 25 ◽

pp. 190-191

Author(s):

Godfrey C. Hoskins ◽

Betty B. Hoskins

Keyword(s):

Electron Microscopy ◽

Electron Micrographs ◽

Structural Features ◽

Metaphase Chromosomes ◽

The Future ◽

Of Mice And Men ◽

Mouse Cells ◽

Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

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Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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