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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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Rhodol-based thallium sensors for cellular imaging of potassium channel activity.
Dutter BF, Ender A, Sulikowski GA, Weaver CD
(2018) Org Biomol Chem 16: 5575-5579
MeSH Terms: Fluorescent Dyes, HEK293 Cells, Humans, Methylation, Microscopy, Confocal, Optical Imaging, Potassium Channels, Spectrometry, Fluorescence, Thallium, Xanthones
Show Abstract · Added April 10, 2019
Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.
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Automated quantification of microvascular perfusion.
McClatchey PM, Mignemi NA, Xu Z, Williams IM, Reusch JEB, McGuinness OP, Wasserman DH
(2018) Microcirculation 25: e12482
MeSH Terms: Animals, Automation, Blood Flow Velocity, Hematocrit, Mice, Microcirculation, Microscopy, Fluorescence, Microscopy, Video, Microvessels, Perfusion, Phenylephrine, Reproducibility of Results, Saline Solution, Software
Show Abstract · Added March 26, 2019
OBJECTIVE - Changes in microvascular perfusion have been reported in many diseases, yet the functional significance of altered perfusion is often difficult to determine. This is partly because commonly used techniques for perfusion measurement often rely on either indirect or by-hand approaches.
METHODS - We developed and validated a fully automated software technique to measure microvascular perfusion in videos acquired by fluorescence microscopy in the mouse gastrocnemius. Acute perfusion responses were recorded following intravenous injections with phenylephrine, SNP, or saline.
RESULTS - Software-measured capillary flow velocity closely correlated with by-hand measured flow velocity (R = 0.91, P < 0.0001). Software estimates of capillary hematocrit also generally agreed with by-hand measurements (R = 0.64, P < 0.0001). Detection limits range from 0 to 2000 μm/s, as compared to an average flow velocity of 326 ± 102 μm/s (mean ± SD) at rest. SNP injection transiently increased capillary flow velocity and hematocrit and made capillary perfusion more steady and homogenous. Phenylephrine injection had the opposite effect in all metrics. Saline injection transiently decreased capillary flow velocity and hematocrit without influencing flow distribution or stability. All perfusion metrics were temporally stable without intervention.
CONCLUSIONS - These results demonstrate a novel and sensitive technique for reproducible, user-independent quantification of microvascular perfusion.
© 2018 John Wiley & Sons Ltd.
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14 MeSH Terms
Interplay between ER Ca Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca Entry.
Nelson HA, Leech CA, Kopp RF, Roe MW
(2018) Int J Mol Sci 19:
MeSH Terms: 3T3 Cells, Animals, Calcium, Calcium Signaling, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Humans, Membrane Microdomains, Mice, Neoplasm Proteins, ORAI1 Protein, Protein Binding, Stromal Interaction Molecule 1, Stromal Interaction Molecule 2
Show Abstract · Added July 6, 2018
Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca-binding protein, with Ca-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.
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Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).
Abney KK, Ramos-Hunter SJ, Romaine IM, Goodwin JS, Sulikowski GA, Weaver CD
(2018) Chemistry 24: 8985-8988
MeSH Terms: Animals, Esterases, HEK293 Cells, Humans, Liver, Microscopy, Confocal, Rhodamines, Spectrometry, Fluorescence, Swine
Show Abstract · Added April 10, 2019
This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Nanoscale architecture of the contractile ring.
McDonald NA, Lind AL, Smith SE, Li R, Gould KL
(2017) Elife 6:
MeSH Terms: Cell Cycle Proteins, Cell Division, Cell Membrane, Cytoplasm, Fluorescence Resonance Energy Transfer, Macromolecular Substances, Microscopy, Fluorescence, Schizosaccharomyces, Schizosaccharomyces pombe Proteins
Show Abstract · Added March 14, 2018
The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.
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9 MeSH Terms
Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles.
Uddin MI, Jayagopal A, Wong A, McCollum GW, Wright DW, Penn JS
(2018) Nanomedicine 14: 63-71
MeSH Terms: Animals, Cell Survival, Cells, Cultured, DNA, Antisense, Endothelium, Vascular, Fluorescence, Gold, Metal Nanoparticles, Mice, Molecular Imaging, RNA, Messenger, Retinal Vessels, Tumor Necrosis Factor-alpha, Vascular Cell Adhesion Molecule-1
Show Abstract · Added December 21, 2017
Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.
Copyright © 2017 Elsevier Inc. All rights reserved.
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14 MeSH Terms
The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage.
Wani TH, Chakrabarty A, Shibata N, Yamazaki H, Guengerich FP, Chowdhury G
(2017) Chem Res Toxicol 30: 1622-1628
MeSH Terms: Catalase, DNA Cleavage, DNA Damage, Free Radical Scavengers, HEK293 Cells, Hep G2 Cells, Human Umbilical Vein Endothelial Cells, Humans, Microscopy, Fluorescence, Plasmids, Poly(ADP-ribose) Polymerases, Reactive Oxygen Species, Teratogens, Thalidomide
Show Abstract · Added March 14, 2018
Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10 000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS and inhibition of angiogenesis and cereblon. All of the theories have their merits and limitations. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low-dose requirement reported by a number of groups. Recently, we have provided convincing structural evidence in support of the presence of arene oxide and the quinone-reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide, is responsible for generating ROS and causing DNA damage. We show, using cell lines, the formation of comet (DNA damage) and ROS. Using DNA-cleavage assays, we also show that catalase, radical scavengers, and desferal are capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA-damaging property but also the metabolism, low concentration, and species-specificity requirements of thalidomide.
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14 MeSH Terms
A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo.
Yang YP, Ma H, Starchenko A, Huh WJ, Li W, Hickman FE, Zhang Q, Franklin JL, Mortlock DP, Fuhrmann S, Carter BD, Ihrie RA, Coffey RJ
(2017) Cell Rep 19: 1257-1267
MeSH Terms: Adult Stem Cells, Amphiregulin, Animals, Embryo, Mammalian, ErbB Receptors, Genes, Reporter, Green Fluorescent Proteins, Hepatocytes, Intestinal Mucosa, Mice, Microscopy, Fluorescence, Protein Transport, Recombinant Proteins, Transgenes
Show Abstract · Added June 21, 2017
EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
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14 MeSH Terms
Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.
Nakase H, Sakuma S, Fukuchi T, Yoshino T, Mohri K, Miyata K, Kumagai H, Hiwatari KI, Tsubaki K, Ikejima T, Tobita E, Zhu M, Wilson KJ, Washington K, Gore JC, Pham W
(2017) Int J Nanomedicine 12: 1747-1755
MeSH Terms: Adenocarcinoma, Adenoma, Antigens, Tumor-Associated, Carbohydrate, Colorectal Neoplasms, Fluorescent Dyes, Humans, Microscopy, Fluorescence, Molecular Probes, Nanoparticles, Optical Imaging, Peanut Agglutinin
Show Abstract · Added April 6, 2017
The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.
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11 MeSH Terms