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The precise calculation of GRACE and GRACE-FO satellite orbits is reliant on knowledge of accurate non-gravitational accelerations acting on the spacecraft. These are measured by the on-board accelerometers that require a thermal environment stabilised to ∼ ± 0.1 °C per revolution. However, during periods of the GRACE mission with reduced thermal control, internal temperature variations reached up to 10 °C within a revolution, causing low-frequency and non-linear drifts in the accelerometer observations. Additionally, accelerometer bias drifts occurred throughout the GRACE mission as changes in the orientation of the orbital plane with respect to the Earth-to-Sun vector caused the satellites to absorb more or less solar energy. These temperature-induced drifts degrade the quality of mass change estimates, particularly during the latter half of the GRACE mission after thermal control of the satellites was terminated. We filter (in the frequency domain) the accelerometer observations to remove these low-frequency components ( f 0.045 mHz). The bias drift removed from the cross-track is then scaled to derive a thermally-based correction for the highly sensitive along-track observations. We then estimate temporal gravity fields using the ANU GRACE software, our filtered accelerometer observations and the range acceleration as the inter-satellite observation. The use of our thermally-corrected accelerometer measurements significantly improves the accuracy of both orbit modelling and gravity field estimation.

We present updated mascon solutions of the temporal gravity field, derived from the analysis of Level-1B GRACE and GRACE-FO data using the range acceleration observable. Our previous solutions contained significant noise in particular regions of high mass loss (e.g. West Antarctica), which was caused by inappropriate levels of regularisation. Here we show a new approach of both iteration and regularisation of the solutions which generates more reliable estimates of the mass variations across the whole Earth, with both temporal and spatial consistency despite not applying any temporal or spatial inter-mascon constraints. We compare mascon solutions at spatial resolutions from 300 km to 100 km, show estimates of ocean mass increase and mass loss in ice-covered regions as well as assess the C20 time series estimated directly from GRACE data alone. Our new mascon estimates, at ~200 km x 200 km spatial resolution, are now publicly available.

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. To test the efficacy of the autoclave cycle on larger biomasses, 1, 2, or 4 ferret carcasses were placed in a biohazard bag. A total of 4 carcasses were placed in each pan. An autoclave cycle was created to begin the study. After initial tests, minor modifications to the initial test cycle parameters were made, and a new cycle was validated for ferret carcasses up to 2 kg each. Parameters for the validated cycle were as follows: sterilization time 240 minutes, temperature 125°C, 5 prevacuum pulses, and chamber pressure 15 psi.Results:The results of this study indicate that an extended sterilization time is required to successfully decontaminate animal carcasses compared with regular, solid, and biohazardous waste.Conclusions:This study demonstrates that it is possible to sterilize multiple intact ferret carcasses per load under validated autoclave cycle conditions.

We present an updated oceanic crustal age grid and a set of complementary grids including spreading rate, asymmetry, direction, and obliquity. Our data set is based on a selected set of magnetic anomaly identifications and the plate tectonic model of Muller et al. (2019, 10.1029/2018TC005462 ). We find the mean age of oceanic crust is 64.2 Myr, slightly older than previous estimates, mainly due to the inclusion of pockets of Mesozoic aged crust in the Atlantic and Mediterranean and improvements to the Jurassic Pacific triangle. This older crust is partly compensated by additional Cenozoic‐aged back‐arc basin crust not included in previous models. The distribution of spreading modes based on area of preserved crust is relatively equal between slow (20–55 mm/yr) and fast (75–180 mm/yr) spreading systems at 33% and 39%, respectively. Crust transitional between fast and slow, or intermediate systems (55–75 mm/yr), cover 20% of the preserved ocean floor with much smaller proportions of crust formed at ultraslow (5%) and super‐fast (3%) spreading systems. Slow and intermediate spreading systems exhibit the most stable behavior in terms of spreading asymmetry and obliquity, with the widest distribution of obliquities occurring at ultraslow spreading systems, consistent with present‐day observations. Our confidence grid provides a complementary resource for nonexperts to identify those parts of the age grid that are least well constrained. Our grids in 6, 2, and 1 arc min resolution as well as our python workflow, isopolate, used to compute our data sets are freely available in online repositories and on the GPlates data portal.

GitHub repository link to plate motion model (GPlates) and slab-tracker Python code, plate reconstruction methodology, and supplementary figures.

© 2019 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. Hem-agglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R20 (R2* – R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

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). We used two models of streptozotocin (STZ)-induced DCM: group 1, adult mice treated with 35 mg/kg STZ for 3 days; and group 2, neonatal mice treated with 70 mg/kg STZ at days 2 and 5 after birth. In group 1, mild fasting hyperglycemia (11 mM) was first detected 8 weeks after the last injection, and in group 2, severe fasting hyperglycemia (20 mM) was first detected 1 to 3 weeks after the last injection. In group 1, left ventricular function was slightly impaired as measured by echocardiography, and Western blot analysis showed a significant decrease in myocardial GHSR1a. In group 2, GHSR1a levels were also decreased as assessed by Cy5-ghrelin(1–19) fluorescence microscopy, and there was a significant negative correlation between GHSR1a levels and glucose tolerance. There were significant positive correlations between GHSR1a and ghrelin and between GHSR1a and sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), a marker for contractility, but not between GHSR1a and B-type natriuretic peptide, a marker for heart failure. We conclude that the subclinical stage of DCM is accompanied by alterations in the myocardial ghrelin-GHSR1a system, suggesting the possibility of a biomarker for DCM., Induction of diabetes in mice was associated with a decrease in myocardial GHSR1a that correlated with impairments in left ventricular function, glucose tolerance, and the sarcoplasmic reticulum protein SERCA2a.

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. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."

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. We also generated the des-acyl analog, des-acyl [Lys 19 (Cy5)]ghrelin (1–19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.

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. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown ofCPEdisrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

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)). Receptor binding affinity of this novel fluorescein-ghrelin(1–18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1–18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1–18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1–18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.

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.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. Expression of sr39TK in beta cells was confirmed by fluorescence immunohistochemistry of pancreatic sections. Histological sections were used to determine beta cell mass, islet area and islet number. Beta cell function was determined using intraperitoneal glucose tolerance tests. For ex vivo biodistrubution, mice were injected i.v. with 9.25 MBq [(18)F]fluorohydroxymethyl-butyl-guanine (FHBG), euthanized 1 h later and pancreata and other organs were collected and counted. For PET scans, mice were injected i.v. with 9.25 MBq [(18)F]FHBG, and dynamic scans were conducted for 1 h, followed by a 30 min static acquisition. To induce type 1 diabetes-like symptoms, MIP-sr39tk mice were injected i.p. with 40 mg/kg streptozotocin (STZ) once per day for five consecutive days, and biodistribution and PET scans were conducted thereafter.Ex vivo quantification of [(18)F]FHBG uptake in the pancreas showed a 4.5-fold difference in transgenic vs. non-transgenics, confirming that expression of sr39TK results in the retention of the PET tracer. In STZ-treated MIP-sr39tk mice, impairments in glucose tolerance and decreases in beta cell mass correlated significantly with a diminishment in [(18)F]FHBG uptake before fasting hyperglycemia became apparent.The MIP-sr39tk mouse demonstrates that PET imaging can detect changes in beta cell mass that precede the onset of diabetes.

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 68 Ga-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. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.

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. We found that although each modality measures a decrease in cardiac function as disease progresses in mdx/utrn(-/-) mice (n = 5) compared with healthy C57BL/6 mice (n = 8), 3-D echocardiography has higher agreement with gold-standard measurements acquired by gated micro-CT, with little standard deviation between measurements. M-Mode echocardiography measurements, in comparison, exhibit considerably greater variability and user bias. Given the radiation dose associated with micro-CT and the geometric assumptions made in M-mode echocardiography to calculate ventricular volume, we suggest that use of 3-D echocardiography has important advantages that may allow for the measurement of early disease changes that occur before overt cardiomyopathy.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration(1, 2). In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient(4). Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities(9, 10). A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations(11). Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse) (12-14). Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans(10,11), PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

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. MagA expression augmented both Prussian blue staining and the elemental iron content of N2A cells, without altering cell proliferation, in cultures grown in the presence of iron supplements. Despite evidence for iron incorporation in both MagA and a variant, MagAE137V, only MagA expression produced intracellular contrast detectable by MRI at 11 Tesla. We used this stable expression system to model a new sequence for cellular imaging with MRI, using the difference between gradient and spin echo images to distinguish cells from artifacts in the field of view. Our results show that MagA activity in mammalian cells responds to iron supplementation and functions as a contrast agent that can be deactivated by a single point mutation. We conclude that MagA is a candidate MRI reporter gene that can exploit more fully the superior resolution of MRI in noninvasive medical imaging.

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.INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously.alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation.Dual-isotope SPECT is a promising method to detect gene expression in and location of transplanted pancreatic cells in vivo.