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Lipid Droplet Accumulation in Human Pancreatic Islets Is Dependent On Both Donor Age and Health.
Tong X, Dai C, Walker JT, Nair GG, Kennedy A, Carr RM, Hebrok M, Powers AC, Stein R
(2020) Diabetes 69: 342-354
MeSH Terms: Acinar Cells, Adolescent, Adult, Age Factors, Aged, Animals, Child, Child, Preschool, Diabetes Mellitus, Experimental, Diabetes Mellitus, Type 2, Embryonic Stem Cells, Female, Glucagon-Secreting Cells, Humans, Infant, Insulin-Secreting Cells, Islets of Langerhans, Islets of Langerhans Transplantation, Lipid Droplets, Male, Mice, Microscopy, Electron, Microscopy, Fluorescence, Middle Aged, Rats, Tissue Donors, Young Adult
Show Abstract · Added March 29, 2020
Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet β-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and β-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet β-like cells produced from human embryonic cell-derived β-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.
© 2019 by the American Diabetes Association.
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27 MeSH Terms
Ligand-conjugated quantum dots for fast sub-diffraction protein tracking in acute brain slices.
Thal LB, Mann VR, Sprinzen D, McBride JR, Reid KR, Tomlinson ID, McMahon DG, Cohen BE, Rosenthal SJ
(2020) Biomater Sci 8: 837-845
MeSH Terms: Animals, Brain, Brain Chemistry, Ligands, Mice, Microscopy, Fluorescence, Microtomy, Proteins, Quantum Dots, Selenium Compounds, Staining and Labeling, Zinc Compounds
Show Abstract · Added March 18, 2020
Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.
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12 MeSH Terms
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
neurons have functional dendritic spines.
Cuentas-Condori A, Mulcahy B, He S, Palumbos S, Zhen M, Miller DM
(2019) Elife 8:
MeSH Terms: Animals, Caenorhabditis elegans, Dendritic Spines, Intravital Microscopy, Microscopy, Electron, Microscopy, Fluorescence, Motor Neurons, Organelles
Show Abstract · Added March 3, 2020
Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.
© 2019, Cuentas-Condori et al.
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8 MeSH Terms
MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies.
Ryan DJ, Patterson NH, Putnam NE, Wilde AD, Weiss A, Perry WJ, Cassat JE, Skaar EP, Caprioli RM, Spraggins JM
(2019) Anal Chem 91: 7578-7585
MeSH Terms: Animals, Fluorescent Dyes, Kidney, Liquid-Liquid Extraction, Mice, Microscopy, Fluorescence, Peptides, Proteins, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Staphylococcus aureus, Trypsin
Show Abstract · Added January 22, 2020
The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of ∼110 μm in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (∼250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.
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12 MeSH Terms
Myocardial differentiation is dependent upon endocardial signaling during early cardiogenesis .
Saint-Jean L, Barkas N, Harmelink C, Tompkins KL, Oakey RJ, Baldwin HS
(2019) Development 146:
MeSH Terms: Animals, Cell Differentiation, Endocardium, Female, Flow Cytometry, Male, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Myocardium, NFATC Transcription Factors, Organogenesis, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction
Show Abstract · Added November 25, 2019
The endocardium interacts with the myocardium to promote proliferation and morphogenesis during the later stages of heart development. However, the role of the endocardium in early cardiac ontogeny remains under-explored. Given the shared origin, subsequent juxtaposition, and essential cell-cell interactions of endocardial and myocardial cells throughout heart development, we hypothesized that paracrine signaling from the endocardium to the myocardium is crucial for initiating early differentiation of myocardial cells. To test this, we generated an , endocardial-specific ablation model using the diphtheria toxin receptor under the regulatory elements of the genomic locus (). Early treatment of mouse embryoid bodies with diphtheria toxin efficiently ablated endocardial cells, which significantly attenuated the percentage of beating EBs in culture and expression of early and late myocardial differentiation markers. The addition of Bmp2 during endocardial ablation partially rescued myocyte differentiation, maturation and function. Therefore, we conclude that early stages of myocardial differentiation rely on endocardial paracrine signaling mediated in part by Bmp2. Our findings provide novel insight into early endocardial-myocardial interactions that can be explored to promote early myocardial development and growth.
© 2019. Published by The Company of Biologists Ltd.
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14 MeSH Terms
Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures.
Tomlinson ID, Kovtun O, Crescentini TM, Rosenthal SJ
(2019) Bioorg Med Chem Lett 29: 959-964
MeSH Terms: Biotin, Dopamine D2 Receptor Antagonists, HEK293 Cells, Humans, Ligands, Microscopy, Fluorescence, Quantum Dots, Receptors, Dopamine D2, Spiperone, Streptavidin
Show Abstract · Added March 26, 2019
We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.
Copyright © 2019. Published by Elsevier Ltd.
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10 MeSH Terms
Beyond the H&E: Advanced Technologies for in situ Tissue Biomarker Imaging.
Himmel LE, Hackett TA, Moore JL, Adams WR, Thomas G, Novitskaya T, Caprioli RM, Zijlstra A, Mahadevan-Jansen A, Boyd KL
(2018) ILAR J 59: 51-65
MeSH Terms: Animals, Biomarkers, Humans, Immunohistochemistry, In Situ Hybridization, Microscopy, Fluorescence, Quality Control, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added April 10, 2019
For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.
© The Author(s) 2018. Published by Oxford University Press on behalf of the National Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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8 MeSH Terms
Next Generation Histology-Directed Imaging Mass Spectrometry Driven by Autofluorescence Microscopy.
Patterson NH, Tuck M, Lewis A, Kaushansky A, Norris JL, Van de Plas R, Caprioli RM
(2018) Anal Chem 90: 12404-12413
MeSH Terms: Animals, Female, Humans, Kidney Diseases, Malaria, Mice, Mice, Inbred BALB C, Microscopy, Fluorescence, Optical Imaging, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added October 15, 2019
Histology-directed imaging mass spectrometry (IMS) is a spatially targeted IMS acquisition method informed by expert annotation that provides rapid molecular characterization of select tissue structures. The expert annotations are usually determined on digital whole slide images of histological stains where the staining preparation is incompatible with optimal IMS preparation, necessitating serial sections: one for annotation, one for IMS. Registration is then used to align staining annotations onto the IMS tissue section. Herein, we report a next-generation histology-directed platform implementing IMS-compatible autofluorescence (AF) microscopy taken prior to any staining or IMS. The platform enables two histology-directed workflows, one that improves the registration process between two separate tissue sections using automated, computational monomodal AF-to-AF microscopy image registration, and a registration-free approach that utilizes AF directly to identify ROIs and acquire IMS on the same section. The registration approach is fully automated and delivers state of the art accuracy in histology-directed workflows for transfer of annotations (∼3-10 μm based on 4 organs from 2 species) while the direct AF approach is registration-free, allowing targeting of the finest structures visible by AF microscopy. We demonstrate the platform in biologically relevant case studies of liver stage malaria and human kidney disease with spatially targeted acquisition of sparsely distributed (composing less than one tenth of 1% of the tissue section area) malaria infected mouse hepatocytes and glomeruli in the human kidney case study.
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MeSH Terms
Advanced Registration and Analysis of MALDI Imaging Mass Spectrometry Measurements through Autofluorescence Microscopy.
Patterson NH, Tuck M, Van de Plas R, Caprioli RM
(2018) Anal Chem 90: 12395-12403
MeSH Terms: Animals, Brain, Kidney, Mice, Microscopy, Fluorescence, Optical Imaging, Rats, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added March 3, 2020
The correlation of imaging mass spectrometry (IMS) with histopathology can help relate novel molecular findings obtained through IMS to the well-characterized and validated histopathology knowledge base. The quality of correlation between these two modalities is limited by the quality of the spatial mapping that is obtained by registration of the two image types. In this work, we develop novel workflows for MALDI IMS-to-microscopy data registration and analysis using nondestructive IMS-compatible wide field autofluorescence (AF) microscopy combined with computational image registration. First, a substantially automated procedure for high-accuracy registration between IMS and microscopy data of the same section is described that explicitly links the MALDI laser ablation pattern imaged by microscopy to its corresponding IMS pixel. Subsequent examination of the registered data allows for high-confidence colocalization of image features between the two modalities, down to single-cell scales within tissue. Building on this IMS-microscopy spatial mapping, we furthermore demonstrate the automated spatial correlation between IMS measurements from serial sections. This AF-registration-driven inter-section analysis, using a combination of nonlinear AF-to-AF and IMS-to-AF image registrations, can be applied to tissue sections that are prepared and imaged with different sample preparations (e.g., lipids vs proteins) and/or that are measured using different spatial resolutions. Importantly, all registrations, whether within a single section or across serial sections, are entirely independent of the IMS intensity signal content and thus unbiased by it.
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MeSH Terms