Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

BACKGROUND: The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. RESULTS: Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. CONCLUSION: Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution

Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse

BACKGROUND: cDNA microarrays have the potential to identify the genes involved in invasion and metastasis. However, when used with whole tumor tissue, the results average the expression patterns of different cell types. We have combined chemotaxis-based cell collection of the invasive subpopulation of cells within the primary tumor with array-based gene expression analysis to identify the genes necessary for the process of carcinoma cell invasion. RESULTS: Invasive cells were collected from live primary tumors using microneedles containing chemotactic growth factors to mimic chemotactic signals thought to be present in the primary tumor. When used with mammary tumors of rats and mice, carcinoma cells and macrophages constitute the invasive cell population. Microbeads conjugated with monoclonal anti-CD11b (Mac-1α) antibodies were used to separate macrophages from carcinoma cells. We utilized PCR-based cDNA amplification from small number of cells and compared it to the quality and complexity of conventionally generated cDNA to determine if amplified cDNA could be used with fidelity for array analysis of this cell population. These techniques showed a very high level of correlation indicating that the PCR based amplification technique yields a cDNA population that resembles, with high fidelity, the original template population present in the small number of cells used to prepare the cDNA for use with the chip. CONCLUSIONS: The specific collection of invasive cells from a primary tumor and the analysis of gene expression in these cells are is now possible. By further comparing the gene expression patterns of cells collected by invasion into microneedles with that of carcinoma cells obtained from the whole primary tumor, the blood, and whole metastatic tumors, genes that contribute to the invasive process in carcinoma cells may be identified

Transferable Pair Potentials for CdS and ZnS Crystals

A set of interatomic pair potentials is developed for CdS and ZnS crystals. We show that a simple energy function, which has been used to describe the properties of CdSe [J. Chem. Phys. 116, 258 (2002)], can be parametrized to accurately describe the lattice and elastic constants, and phonon dispersion relations of bulk CdS and ZnS in the wurtzite and rocksalt crystal structures. The predicted coexistence pressure of the wurtzite and rocksalt structures, as well as the equation of state are in good agreement with experimental observations. These new pair potentials enable the study of a wide range of processes in bulk and nanocrystalline II-VI semiconductor materials

If You Don't Look, You Won't See: Intravital Multiphoton Imaging of Primary and Metastatic Breast Cancer

A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interaction

The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors

Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine–threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119–1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868–36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247–7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349–359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

BACKGROUND: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. RESULTS: We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism. CONCLUSION: The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at . Data and source code are available by request at [email protected]

Formulation development and characterization of cellulose acetate nitrate based propellants for improved insensitive munitions properties

AbstractCellulose acetate nitrate (CAN) was used as an insensitive energetic binder to improve the insensitive munitions (IM) properties of gun propellants to replace the M1 propellant used in 105 mm artillery charges. CAN contains the energetic nitro groups found in nitrocellulose (NC), but also acetyl functionalities, which lowered the polymer's sensitivity to heat and shock, and therefore improved its IM properties relative to NC. The formulation, development and small-scale characterization testing of several CAN-based propellants were done. The formulations, using insensitive energetic solid fillers and high-nitrogen modifiers in place of nitramine were completed. The small scale characterization testing, such as closed bomb testing, small scale sensitivity, thermal stability, and chemical compatibility were done. The mechanical response of the propellants under high-rate uni-axial compression at, hot, cold, and ambient temperatures were also completed. Critical diameter testing, hot fragment conductive ignition (HFCI) tests were done to evaluate the propellants' responses to thermal and shock stimuli. Utilizing the propellant chemical composition, theoretical predictions of erosivity were completed. All the small scale test results were utilized to down-select the promising CAN based formulations for large scale demonstration testing such as the ballistic performance and fragment impact testing in the 105 mm M67 artillery charge configurations. The test results completed in the small and large scale testing are discussed

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors

Introduction The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. Methods To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. Results Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. Conclusions Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.National Cancer Institute (U.S.). Integrative Cancer Biology Program (grant U54 CA112967)Virginia and D.K. Ludwig Fund for Cancer Researc

A One Health overview, facilitating advances in comparative medicine and translational research.

Table of contentsA1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17 Polycystic kidney disease: cellular mechanisms to emerging therapiesJames P. CalvetA18 The domestic cat as a large animal model for polycystic kidney diseaseLeslie A. Lyons, Barbara GandolfiA19 The support of basic and clinical research by the Polycystic Kidney Disease FoundationDavid A. BaronA20 Using naturally occurring large animal models of human disease to enable clinical translation: treatment of arthritis using autologous stromal vascular fraction in dogsMark L. WeissA21 Regulatory requirements regarding clinical use of human cells, tissues, and tissue-based productsDebra A. WebsterA22 Regenerative medicine approaches to Type 1 diabetes treatmentFrancis N. KaranuA23 The zoobiquity of canine diabetes mellitus, man's best friend is a friend indeed-islet transplantationEdward J. RobbA24 One Medicine: a development model for cellular therapy of diabetesRobert J. Harman