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Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissue.
Prentice BM, Hart NJ, Phillips N, Haliyur R, Judd A, Armandala R, Spraggins JM, Lowe CL, Boyd KL, Stein RW, Wright CV, Norris JL, Powers AC, Brissova M, Caprioli RM
(2019) Diabetologia 62: 1036-1047
MeSH Terms: Animals, Fluorescent Antibody Technique, Gangliosides, Humans, Immunohistochemistry, Islets of Langerhans, Mice, Pancreas, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added May 7, 2019
AIMS/HYPOTHESIS - The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases.
METHODS - We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 μm) of regions of interest and leads to reduced time requirements for data acquisition.
RESULTS - MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation.
CONCLUSIONS/INTERPRETATION - The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.
1 Communities
4 Members
0 Resources
9 MeSH Terms
Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
Bailey DM, Kovtun O, Rosenthal SJ
(2017) Methods Mol Biol 1570: 165-177
MeSH Terms: Algorithms, Animals, Fluorescent Antibody Technique, Immunoconjugates, Models, Theoretical, Molecular Imaging, Neurons, Neurotransmitter Transport Proteins, Primary Cell Culture, Quantum Dots, Rats, Single Molecule Imaging, Statistics as Topic
Show Abstract · Added April 3, 2018
Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.
0 Communities
2 Members
0 Resources
MeSH Terms
Larger core size has superior technical and analytical accuracy in bladder tissue microarray.
Eskaros AR, Egloff SA, Boyd KL, Richardson JE, Hyndman ME, Zijlstra A
(2017) Lab Invest 97: 335-342
MeSH Terms: Cell Proliferation, Fluorescent Antibody Technique, Humans, Ki-67 Antigen, Mitotic Index, Paraffin Embedding, Pilot Projects, Reproducibility of Results, Tissue Array Analysis, Urinary Bladder, Urinary Bladder Neoplasms
Show Abstract · Added April 6, 2017
The construction of tissue microarrays (TMAs) with cores from a large number of paraffin-embedded tissues (donors) into a single paraffin block (recipient) is an effective method of analyzing samples from many patient specimens simultaneously. For the TMA to be successful, the cores within it must capture the correct histologic areas from the donor blocks (technical accuracy) and maintain concordance with the tissue of origin (analytical accuracy). This can be particularly challenging for tissues with small histological features such as small islands of carcinoma in situ (CIS), thin layers of normal urothelial lining of the bladder, or cancers that exhibit intratumor heterogeneity. In an effort to create a comprehensive TMA of a bladder cancer patient cohort that accurately represents the tumor heterogeneity and captures the small features of normal and CIS, we determined how core size (0.6 vs 1.0 mm) impacted the technical and analytical accuracy of the TMA. The larger 1.0 mm core exhibited better technical accuracy for all tissue types at 80.9% (normal), 94.2% (tumor), and 71.4% (CIS) compared with 58.6%, 85.9%, and 63.8% for 0.6 mm cores. Although the 1.0 mm core provided better tissue capture, increasing the number of replicates from two to three allowed with the 0.6 mm core compensated for this reduced technical accuracy. However, quantitative image analysis of proliferation using both Ki67+ immunofluorescence counts and manual mitotic counts demonstrated that the 1.0 mm core size also exhibited significantly greater analytical accuracy (P=0.004 and 0.035, respectively, r=0.979 and 0.669, respectively). Ultimately, our findings demonstrate that capturing two or more 1.0 mm cores for TMA construction provides superior technical and analytical accuracy over the smaller 0.6 mm cores, especially for tissues harboring small histological features or substantial heterogeneity.
1 Communities
2 Members
0 Resources
11 MeSH Terms
Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
Chandrasekaran R, Kenworthy AK, Lacy DB
(2016) PLoS Pathog 12: e1006070
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Bacterial Toxins, Blotting, Western, Caco-2 Cells, Clathrin, Clostridium Infections, Clostridium difficile, Endocytosis, Enterotoxins, Fluorescent Antibody Technique, Gene Knockdown Techniques, HEK293 Cells, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Confocal, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Virulence Factors
Show Abstract · Added April 26, 2017
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
0 Communities
1 Members
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21 MeSH Terms
Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming.
Jones-Paris CR, Paria S, Berg T, Saus J, Bhave G, Paria BC, Hudson BG
(2017) Matrix Biol 57-58: 347-365
MeSH Terms: Animals, Basement Membrane, Collagen Type IV, Embryo Implantation, Extracellular Matrix Proteins, Female, Fluorescent Antibody Technique, Gene Expression Regulation, Injections, Laminin, Mice, Peptide Fragments, Peroxidase, Pregnancy, Protein-Serine-Threonine Kinases, RNA, Messenger, Sesame Oil, Uterus
Show Abstract · Added October 30, 2016
Basement membranes (BMs) are specialized extracellular scaffolds that influence behaviors of cells in epithelial, endothelial, muscle, nervous, and fat tissues. Throughout development and in response to injury or disease, BMs are fine-tuned with specific protein compositions, ultrastructure, and localization. These features are modulated through implements of the BM toolkit that is comprised of collagen IV, laminin, perlecan, and nidogen. Two additional proteins, peroxidasin and Goodpasture antigen-binding protein (GPBP), have recently emerged as potential members of the toolkit. In the present study, we sought to determine whether peroxidasin and GPBP undergo dynamic regulation in the assembly of uterine tissue BMs in early pregnancy as a tractable model for dynamic adult BMs. We explored these proteins in the context of collagen IV and laminin that are known to extensively change for decidualization. Electron microscopic analyses revealed: 1) a smooth continuous layer of BM in between the epithelial and stromal layers of the preimplantation endometrium; and 2) interrupted, uneven, and progressively thickened BM within the pericellular space of the postimplantation decidua. Quantification of mRNA levels by qPCR showed changes in expression levels that were complemented by immunofluorescence localization of peroxidasin, GPBP, collagen IV, and laminin. Novel BM-associated and subcellular spatiotemporal localization patterns of the four components suggest both collective pericellular functions and distinct functions in the uterus during reprogramming for embryo implantation.
Copyright © 2016 Elsevier B.V. All rights reserved.
1 Communities
2 Members
3 Resources
18 MeSH Terms
Quantitative [F]FMISO PET Imaging Shows Reduction of Hypoxia Following Trastuzumab in a Murine Model of HER2+ Breast Cancer.
Sorace AG, Syed AK, Barnes SL, Quarles CC, Sanchez V, Kang H, Yankeelov TE
(2017) Mol Imaging Biol 19: 130-137
MeSH Terms: Animals, Biomarkers, Tumor, Cell Line, Tumor, Disease Models, Animal, Female, Fluorescent Antibody Technique, Mammary Neoplasms, Animal, Misonidazole, Nitroimidazoles, Positron-Emission Tomography, Receptor, ErbB-2, Trastuzumab, Tumor Burden, Tumor Hypoxia
Show Abstract · Added April 6, 2017
PURPOSE - Evaluation of [F]fluoromisonidazole ([F]FMISO)-positron emission tomography (PET) imaging as a metric for evaluating early response to trastuzumab therapy with histological validation in a murine model of HER2+ breast cancer.
PROCEDURES - Mice with BT474, HER2+ tumors, were imaged with [F]FMISO-PET during trastuzumab therapy. Pimonidazole staining was used to confirm hypoxia from imaging.
RESULTS - [F]FMISO-PET indicated significant decreases in hypoxia beginning on day 3 (P < 0.01) prior to changes in tumor size. These results were confirmed with pimonidazole staining on day 7 (P < 0.01); additionally, there was a significant positive linear correlation between histology and PET imaging (r  = 0.85).
CONCLUSIONS - [F]FMISO-PET is a clinically relevant modality which provides the opportunity to (1) predict response to HER2+ therapy before changes in tumor size and (2) identify decreases in hypoxia which has the potential to guide subsequent therapy.
0 Communities
1 Members
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14 MeSH Terms
Mammalian retinal Müller cells have circadian clock function.
Xu L, Ruan G, Dai H, Liu AC, Penn J, McMahon DG
(2016) Mol Vis 22: 275-83
MeSH Terms: Animals, CLOCK Proteins, Cells, Cultured, Circadian Clocks, Circadian Rhythm, Ependymoglial Cells, Female, Fluorescent Antibody Technique, Indirect, Genetic Vectors, Humans, Lentivirus, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA, Small Interfering, Transfection
Show Abstract · Added March 18, 2020
PURPOSE - To test whether Müller glia of the mammalian retina have circadian rhythms.
METHODS - We used Müller glia cultures isolated from mouse lines or from humans and bioluminescent reporters of circadian clock genes to monitor molecular circadian rhythms. The clock gene dependence of the Müller cell rhythms was tested using clock gene knockout mouse lines or with siRNA for specific clock genes.
RESULTS - We demonstrated that retinal Müller glia express canonical circadian clock genes, are capable of sustained circadian oscillations in isolation from other cell types, and exhibit unique features of their molecular circadian clock compared to the retina as a whole. Mouse and human Müller cells demonstrated circadian clock function; however, they exhibited species-specific differences in the gene dependence of their clocks.
CONCLUSIONS - Müller cells are the first mammalian retinal cell type in which sustained circadian rhythms have been demonstrated in isolation from other retinal cells.
0 Communities
1 Members
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MeSH Terms
Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia.
Grabowska MM, Kelly SM, Reese AL, Cates JM, Case TC, Zhang J, DeGraff DJ, Strand DW, Miller NL, Clark PE, Hayward SW, Gronostajski RM, Anderson PD, Matusik RJ
(2016) Endocrinology 157: 1094-109
MeSH Terms: Animals, Cell Line, Tumor, Cell Proliferation, Chromatin Immunoprecipitation, Fluorescent Antibody Technique, Gene Expression Regulation, Gene Expression Regulation, Neoplastic, Gene Knockdown Techniques, Gene Regulatory Networks, Hepatocyte Nuclear Factor 3-alpha, Humans, Immunohistochemistry, Male, Mice, Mice, Knockout, NFI Transcription Factors, Prostate, Prostatic Hyperplasia, Prostatic Neoplasms, Receptors, Androgen, Sequence Analysis, DNA, Sequence Analysis, RNA
Show Abstract · Added February 15, 2016
A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.
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2 Members
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22 MeSH Terms
Urinary oncofetal ED-A fibronectin correlates with poor prognosis in patients with bladder cancer.
Arnold SA, Loomans HA, Ketova T, Andl CD, Clark PE, Zijlstra A
(2016) Clin Exp Metastasis 33: 29-44
MeSH Terms: Adult, Aged, Aged, 80 and over, Alternative Splicing, Area Under Curve, Biomarkers, Tumor, Blotting, Western, Carcinoma, Transitional Cell, Enzyme-Linked Immunosorbent Assay, Female, Fibronectins, Fluorescent Antibody Technique, Humans, Kaplan-Meier Estimate, Male, Middle Aged, Prognosis, Protein Isoforms, ROC Curve, Sensitivity and Specificity, Urinary Bladder Neoplasms
Show Abstract · Added October 13, 2015
The extracellular matrix protein fibronectin (FN) contributes to the structural integrity of tissues as well as the adhesive and migratory functions of cells. While FN is abundantly expressed in adult tissues, the expression of several alternatively spliced FN isoforms is restricted to embryonic development, tissue remodeling and cancer. These FN isoforms, designated ED-A and ED-B, are frequently expressed by cancer cells, tumor-associated fibroblasts and newly forming blood vessels. Using a highly sensitive collagen-based indirect ELISA, we evaluated the correlation of urinary ED-A and ED-B at time of cystectomy with overall survival in patients with high-grade bladder cancer (BCa). Detectable levels of total FN as well as ED-A and ED-B were found in urine from 85, 73 and 51 % of BCa patients, respectively. The presence of urinary ED-A was a significant independent predictor of 2-year overall survival (OS) after adjusting for age, tumor stage, lymph node stage, and urinary creatinine by multivariable Logistic Regression (p = 0.029, OR = 4.26, 95 % CI 1.16-15.71) and improved accuracy by 3.6 %. Furthermore, detection of ED-A in the urine was a significant discriminator of survival specifically in BCa patients with negative lymph node status (Log-Rank, p = 0.006; HR = 5.78, 95 % CI 1.39-24.13). Lastly, multivariable Cox proportional hazards analysis revealed that urinary ED-A was an independent prognostic indicator of 5-year OS rate for patients with BCa (p = 0.04, HR = 2.20, 95 % CI 1.04-4.69). Together, these data suggest that cancer-derived, alternatively spliced FN isoforms can act as prognostic indicators and that additional studies are warranted to assess the clinical utility of ED-A in BCa.
1 Communities
2 Members
0 Resources
21 MeSH Terms
Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma.
Taylor C, Loomans HA, Le Bras GF, Koumangoye RB, Romero-Morales AI, Quast LL, Zaika AI, El-Rifai W, Andl T, Andl CD
(2015) Oncotarget 6: 34228-44
MeSH Terms: Activins, Adenocarcinoma, Barrett Esophagus, Blotting, Western, Cell Line, Tumor, Cell Proliferation, Cell Transformation, Neoplastic, Enzyme-Linked Immunosorbent Assay, Esophageal Neoplasms, Fluorescent Antibody Technique, Humans, Neoplasm Invasiveness
Show Abstract · Added October 13, 2015
TGFβ signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells.In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis.
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3 Members
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12 MeSH Terms