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Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.
Cadar AG, Feaster TK, Bersell KR, Wang L, Hong T, Balsamo JA, Zhang Z, Chun YW, Nam YJ, Gotthardt M, Knollmann BC, Roden DM, Lim CC, Hong CC
(2020) Am J Physiol Cell Physiol 318: C163-C173
MeSH Terms: Adult, Cell Differentiation, Cell Line, Connectin, Fluorescence Recovery After Photobleaching, Humans, Induced Pluripotent Stem Cells, Kinetics, Luminescent Proteins, Male, Microscopy, Fluorescence, Microscopy, Video, Myocytes, Cardiac, Recombinant Fusion Proteins, Reproducibility of Results, Sarcomeres
Show Abstract · Added March 24, 2020
Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.
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16 MeSH Terms
Targeted mobilization of Lrig1 gastric epithelial stem cell populations by a carcinogenic type IV secretion system.
Wroblewski LE, Choi E, Petersen C, Delgado AG, Piazuelo MB, Romero-Gallo J, Lantz TL, Zavros Y, Coffey RJ, Goldenring JR, Zemper AE, Peek RM
(2019) Proc Natl Acad Sci U S A 116: 19652-19658
MeSH Terms: Adenocarcinoma, Animals, Carcinogenesis, Disease Models, Animal, Epithelial Cells, Female, Gastric Mucosa, Gastritis, Helicobacter Infections, Helicobacter pylori, Humans, Male, Membrane Glycoproteins, Mice, Mice, Knockout, Nerve Tissue Proteins, Precancerous Conditions, Primary Cell Culture, Risk Factors, Stem Cells, Stomach, Stomach Neoplasms, Type IV Secretion Systems
Show Abstract · Added September 27, 2019
-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within -infected gastric mucosa. Lineage tracing was induced in (Lrig1/YFP) mice that were uninfected or subsequently infected with or an isogenic mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the mutant. WT -infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic infection stimulates Lrig1-expressing progenitor cells in a -dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.
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23 MeSH Terms
Heterogeneity of Neural Stem Cells in the Ventricular-Subventricular Zone.
Rushing GV, Bollig MK, Ihrie RA
(2019) Adv Exp Med Biol 1169: 1-30
MeSH Terms: Animals, Brain, Cell Lineage, Lateral Ventricles, Mice, Neural Stem Cells, Neurons, Stem Cell Niche
Show Abstract · Added March 9, 2020
In this chapter, heterogeneity is explored in the context of the ventricular-subventricular zone, the largest stem cell niche in the mammalian brain. This niche generates up to 10,000 new neurons daily in adult mice and extends over a large spatial area with dorso-ventral and medio-lateral subdivisions. The stem cells of the ventricular-subventricular zone can be subdivided by their anatomical position and transcriptional profile, and the stem cell lineage can also be further subdivided into stages of pre- and post-natal quiescence and activation. Beyond the stem cells proper, additional differences exist in their interactions with other cellular constituents of the niche, including neurons, vasculature, and cerebrospinal fluid. These variations in stem cell potential and local interactions are discussed, as well as unanswered questions within this system.
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MeSH Terms
Clonal Hematopoiesis: Crossroads of Aging, Cardiovascular Disease, and Cancer: JACC Review Topic of the Week.
Libby P, Sidlow R, Lin AE, Gupta D, Jones LW, Moslehi J, Zeiher A, Jaiswal S, Schulz C, Blankstein R, Bolton KL, Steensma D, Levine RL, Ebert BL
(2019) J Am Coll Cardiol 74: 567-577
MeSH Terms: Aging, Algorithms, Cardiovascular Diseases, Hematopoiesis, Hematopoietic Stem Cells, Humans, Mutation, Neoplasms, Risk Factors
Show Abstract · Added November 12, 2019
A novel, common, and potent cardiovascular risk factor has recently emerged: clonal hematopoiesis of indeterminate potential (CHIP). CHIP arises from somatic mutations in hematopoietic stem cells that yield clonal progeny of mutant leukocytes in blood. Individuals with CHIP have a doubled risk of coronary heart disease and ischemic stroke, and worsened heart failure outcomes independent of traditional cardiovascular risk factors. The recognition of CHIP as a nontraditional risk factor challenges specialists in hematology/oncology and cardiovascular medicine alike. Should we screen for CHIP? If so, in whom? How should we assess cardiovascular risk in people with CHIP? How should we manage the excess cardiovascular risk in the absence of an evidence base? This review explains CHIP, explores the clinical quandaries, strives to provide reasonable recommendations for the multidisciplinary management of cardiovascular risk in individuals with CHIP, and highlights current knowledge gaps.
Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
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9 MeSH Terms
Location-dependent maintenance of intrinsic susceptibility to mTORC1-driven tumorigenesis.
Rushing GV, Brockman AA, Bollig MK, Leelatian N, Mobley BC, Irish JM, Ess KC, Fu C, Ihrie RA
(2019) Life Sci Alliance 2:
MeSH Terms: Adolescent, Animals, Astrocytoma, Carcinogenesis, Cells, Cultured, Child, Child, Preschool, Disease Susceptibility, Female, Humans, Lateral Ventricles, Male, Mechanistic Target of Rapamycin Complex 1, Mice, Mice, Inbred C57BL, Neural Stem Cells, Signal Transduction, Thyroid Nuclear Factor 1, Tuberous Sclerosis
Show Abstract · Added March 27, 2019
Neural stem/progenitor cells (NSPCs) of the ventricular-subventricular zone (V-SVZ) are candidate cells of origin for many brain tumors. However, whether NSPCs in different locations within the V-SVZ differ in susceptibility to tumorigenic mutations is unknown. Here, single-cell measurements of signal transduction intermediates in the mechanistic target of rapamycin complex 1 (mTORC1) pathway reveal that ventral NSPCs have higher levels of signaling than dorsal NSPCs These features are linked with differences in mTORC1-driven disease severity: introduction of a pathognomonic mutation only results in formation of tumor-like masses from the ventral V-SVZ. We propose a direct link between location-dependent intrinsic growth properties imbued by mTORC1 and predisposition to tumor development.
© 2019 Rushing et al.
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19 MeSH Terms
p120ctn-Mediated Organ Patterning Precedes and Determines Pancreatic Progenitor Fate.
Nyeng P, Heilmann S, Löf-Öhlin ZM, Pettersson NF, Hermann FM, Reynolds AB, Semb H
(2019) Dev Cell 49: 31-47.e9
MeSH Terms: Animals, Body Patterning, Cadherins, Catenins, Cell Differentiation, Cell Lineage, Cell Movement, Embryonic Development, Flow Cytometry, Gene Expression Regulation, Developmental, Humans, Islets of Langerhans, Mice, Pancreas, Pancreatic Ducts, Receptors, Notch, Signal Transduction, Stem Cells
Show Abstract · Added March 29, 2019
The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.
Copyright © 2019 Elsevier Inc. All rights reserved.
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18 MeSH Terms
Spatiotemporal control and modeling of morphogen delivery to induce gradient patterning of stem cell differentiation using fluidic channels.
O'Grady B, Balikov DA, Wang JX, Neal EK, Ou YC, Bardhan R, Lippmann ES, Bellan LM
(2019) Biomater Sci 7: 1358-1371
MeSH Terms: Cell Differentiation, Cells, Cultured, Humans, Microfluidic Analytical Techniques, Models, Biological, Morphogenesis, Stem Cells
Show Abstract · Added March 18, 2020
The process of cell differentiation in a developing embryo is influenced by numerous factors, including various biological molecules whose presentation varies dramatically over space and time. These morphogens regulate cell fate based on concentration profiles, thus creating discrete populations of cells and ultimately generating large, complex tissues and organs. Recently, several in vitro platforms have attempted to recapitulate the complex presentation of extrinsic signals found in nature. However, it has been a challenge to design versatile platforms that can dynamically control morphogen gradients over extended periods of time. To address some of these issues, we introduce a platform using channels patterned in hydrogels to deliver multiple morphogens to cells in a 3D scaffold, thus creating a spectrum of cell phenotypes based on the resultant morphogen gradients. The diffusion coefficient of a common small molecule morphogen, retinoic acid (RA), was measured within our hydrogel platform using Raman spectroscopy and its diffusion in our platform's geometry was modeled using finite element analysis. The predictive model of spatial gradients was validated in a cell-free hydrogel, and temporal control of morphogen gradients was then demonstrated using a reporter cell line that expresses green fluorescent protein in the presence of RA. Finally, the utility of this approach for regulating cell phenotype was demonstrated by generating opposing morphogen gradients to create a spectrum of mesenchymal stem cell differentiation states.
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MeSH Terms
iPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.
Faley SL, Neal EH, Wang JX, Bosworth AM, Weber CM, Balotin KM, Lippmann ES, Bellan LM
(2019) Stem Cell Reports 12: 474-487
MeSH Terms: Albumins, Blood-Brain Barrier, Brain, Cells, Cultured, Dextrans, Endothelial Cells, Endothelium, Fluorescein, Humans, Hydrogels, Induced Pluripotent Stem Cells, Microvessels
Show Abstract · Added March 18, 2020
There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10-100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
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Active Kras Expression in Gastric Isthmal Progenitor Cells Induces Foveolar Hyperplasia but Not Metaplasia.
Choi E, Means AL, Coffey RJ, Goldenring JR
(2019) Cell Mol Gastroenterol Hepatol 7: 251-253.e1
MeSH Terms: Animals, Biomarkers, Humans, Hyperplasia, Metaplasia, Mice, Proto-Oncogene Proteins p21(ras), Stem Cells, Stomach
Added February 7, 2019
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9 MeSH Terms
p73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton.
Fuertes-Alvarez S, Maeso-Alonso L, Villoch-Fernandez J, Wildung M, Martin-Lopez M, Marshall C, Villena-Cortes AJ, Diez-Prieto I, Pietenpol JA, Tissir F, Lizé M, Marques MM, Marin MC
(2018) Cell Death Dis 9: 1183
MeSH Terms: Animals, Carrier Proteins, Cell Polarity, Cilia, Cytoskeleton, Ependyma, Female, Frizzled Receptors, Gene Expression Regulation, Gene Ontology, HCT116 Cells, Humans, Male, Membrane Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Microtubules, Molecular Sequence Annotation, Nerve Tissue Proteins, Nonmuscle Myosin Type IIA, Pluripotent Stem Cells, Signal Transduction, Tumor Protein p73
Show Abstract · Added March 27, 2019
Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.
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24 MeSH Terms