Validating Autoclave Cycles for Carcass Disposal in Animal Biosafety Level 2/3 Containment Laboratories.

Introduction: Animal carcasses differ in composition from other types of solid waste, and through prior testing it was determined that cycle parameters applied to general, solid biohazardous waste did not ensure proper sterilization of ferret carcasses.

Objectives: The goals of this study were to develop and validate an autoclave cycle that would ensure the decontamination of infectious animal carcasses before removal from an animal biosafety level 2/3 containment suite for downstream disposal and to test different ways to prepare and package animal carcasses for autoclaving.

Methods: Intact ferret carcasses were implanted with biological indicators, and the carcasses were placed in biohazard bags, then into metal pans.

MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation.

Changes in the Cardiac GHSR1a-Ghrelin System Correlate With Myocardial Dysfunction in Diabetic Cardiomyopathy in Mice.

Ghrelin and its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), are present in cardiac tissue. Activation of GHSR1a by ghrelin promotes cardiomyocyte contractility and survival, and changes in myocardial GHSR1a and circulating ghrelin track with end-stage heart failure, leading to the hypothesis that GHSR1a is a biomarker for heart failure. We hypothesized that GHSR1a could also be a biomarker for diabetic cardiomyopathy (DCM).

Characterization of 5-(2- F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas.

: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2- F-fluoroethoxy)-L-tryptophan ( F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes.

Synthesis and evaluation of optical and PET GLP-1 peptide analogues for GLP-1R imaging.

A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68Ga-labeled GLP-1(7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies.

Application of 3-d echocardiography and gated micro-computed tomography to assess cardiomyopathy in a mouse model of duchenne muscular dystrophy.

The purpose of this study was to measure changes in cardiac function as cardiomyopathy progresses in a mouse model of Duchenne muscular dystrophy using 3-D ECG-gated echocardiography. This study is the first to correlate cardiac volumes acquired using 3-D echocardiography with those acquired using retrospectively gated micro-computed tomography (CT). Both were further compared with standard M-mode echocardiography and histologic analyses.

Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway.

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides.

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes.

Molecular imaging to target transplanted muscle progenitor cells.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD.

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals.

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia.

Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a.

Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl)(3), Lys(fluorescein)(19)]ghrelin(1-19)).

Towards PET imaging of intact pancreatic beta cell mass: a transgenic strategy.

Purpose: We have generated transgenic mouse lines expressing the positron emission tomography (PET) reporter gene, sr39tk, under the control of the mouse insulin I promoter (MIP-sr39tk) to image endogenous islets using PET.

Procedures: The MIP-sr39tk transgene was constructed using the 8.3 kb fragment of the mouse insulin I promoter and the sr39tk coding sequence from the mrfp-hrl-ttk trifusion construct.

Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging.

Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA, a putative iron transporter. We have examined expression of MagA in mouse neuroblastoma N2A cells and characterized their response to iron loading and cellular imaging by MRI.

Imaging of gene expression in live pancreatic islet cell lines using dual-isotope SPECT.

Unlabelled: We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule.

Glucose dependence of the regulated secretory pathway in alphaTC1-6 cells.

We have investigated the effects of chronically elevated glucose concentrations on the pancreatic alpha-cell line alphaTC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2.