Evaluation of Nesfatin-1 Expression in Lean, Overweight, and Diabetic Cats

Chapter Two Background: Rates of obesity and diabetes mellitus are increasing in domestic cats. Nesfatin-1 is a protein hormone implicated in controlling food intake and maintaining glycemic control. The role of nesfatin-1 in the pathophysiology of obesity and diabetes mellitus in cats has not been evaluated. This study aimed to characterize NUCB2/nesfatin-1 mRNA expression in feline gastric, pancreatic, abdominal adipose, and skeletal muscle tissue, and to evaluate how tissue nesfatin-1 expression differs between lean, overweight, and diabetic cats. Methods: Case control study. Four lean, three obese, and five diabetic cats were sampled. RNA was extracted from pancreas, stomach, abdominal fat, and skeletal muscle, and a one-step RT-PCR was performed. Relative NUCB2/nesfatin-1 expression was assessed by the comparative 2-Ct method. Results: NUCB2/nesfatin-1 mRNA expression was highest in the pancreas and there was a wide variance in tissue expression in all cats. Expression tended to be marginally decreased in pancreatic tissue of overweight cats in comparison to lean cats. NUCB2/nesfatin-1 expression in the gastric and pancreatic tissue of diabetic cats showed a biphasic pattern. Two diabetic cats had similar expression patterns to lean cats, while three diabetic cats had decreased expression compared to lean cats. This biphasic expression was unrelated to the body condition or treatment status of these diabetic cats. No patterns of expression were identified in adipose and muscle tissue. Conclusions: The pancreas had the highest levels of NUCB2/nesfatin-1 mRNA expression in cats. Changes in expression in diabetic cats were most pronounced in the stomach and pancreas. The biphasic pattern of expression in diabetic cats may reflect differences in pancreatic islet health and function in these cats. The clinicopathological implications of these changes need to be investigated further. Future studies may involve assessing changes in nesfatin-1 mRNA expression in conjunction with changes in nesfatin-1 tissue protein expression and circulating nesfatin-1 concentrations. Chapter Three Background: Obesity and diabetes mellitus are increasing issues in humans and domestic cats. Diabetes mellitus in cats shares similar clinicopathologic features to type 2 diabetes mellitus in humans. Nesfatin-1 is a recently discovered protein hormone that has been implicated in the development of obesity and diabetes, and alterations in plasma nesfatin-1 concentrations have been documented in type 2 diabetic humans. This study aimed to evaluate whether plasma nesfatin-1 concentrations in cats were associated with adiposity or insulin resistance, and to determine if nesfatin-1 concentrations were altered by diabetic treatment. Influences of sex and age on nesfatin-1 concentrations were also assessed. Methods: Case control study. Plasma was collected from 13 lean and 13 obese non-diabetic cats, and eight newly diagnosed and five long term diabetic cats. Newly diagnosed diabetic cats were sampled before and after four weeks of diabetic treatment. Plasma nesfatin-1 levels were measured by a rat nesfatin-1 ELISA, which was validated for use with feline plasma. Results: Median plasma nesfatin-1 concentrations in newly diagnosed and long-term treated diabetic cats showed a slight upward trend in comparison to lean and overweight non-diabetic cats, though the highest nesfatin-1 concentrations were seen in a subset of lean healthy cats. However, overall differences in plasma nesfatin-1 concentrations in lean, overweight, and diabetic cats were not statistically significant. Sex and age were also not significantly associated with plasma nesfatin-1 levels. Newly diagnosed diabetic cats showed a statistically significant decrease in nesfatin-1 concentrations following short-term treatment, which may suggest a link between decreased nesfatin-1 concentrations and diabetic treatment in cats. Conclusions: Our results suggest that changes in plasma nesfatin-1 concentrations may be more related to diabetic status and the presence of insulin resistance as opposed to differences in adiposity or body condition, though further research is required to determine the significance of this relationship. There may be additional, currently unknown factors that increase nesfatin-1 concentrations in cats. Future studies may include longitudinal studies to investigate if nesfatin-1 concentrations change as cats develop increasing adiposity, insulin resistance, and eventually become clinically diabetic

Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons.

Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation

Vascular changes in diabetic retinopathy-a longitudinal study in the Nile rat.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies

Novel components of the Toxoplasma inner membrane complex revealed by BioID.

UNLABELLED:The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE:The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma

Localization of Canine Brachycephaly Using an Across Breed Mapping Approach

The domestic dog, Canis familiaris, exhibits profound phenotypic diversity and is an ideal model organism for the genetic dissection of simple and complex traits. However, some of the most interesting phenotypes are fixed in particular breeds and are therefore less tractable to genetic analysis using classical segregation-based mapping approaches. We implemented an across breed mapping approach using a moderately dense SNP array, a low number of animals and breeds carefully selected for the phenotypes of interest to identify genetic variants responsible for breed-defining characteristics. Using a modest number of affected (10–30) and control (20–60) samples from multiple breeds, the correct chromosomal assignment was identified in a proof of concept experiment using three previously defined loci; hyperuricosuria, white spotting and chondrodysplasia. Genome-wide association was performed in a similar manner for one of the most striking morphological traits in dogs: brachycephalic head type. Although candidate gene approaches based on comparable phenotypes in mice and humans have been utilized for this trait, the causative gene has remained elusive using this method. Samples from nine affected breeds and thirteen control breeds identified strong genome-wide associations for brachycephalic head type on Cfa 1. Two independent datasets identified the same genomic region. Levels of relative heterozygosity in the associated region indicate that it has been subjected to a selective sweep, consistent with it being a breed defining morphological characteristic. Genotyping additional dogs in the region confirmed the association. To date, the genetic structure of dog breeds has primarily been exploited for genome wide association for segregating traits. These results demonstrate that non-segregating traits under strong selection are equally tractable to genetic analysis using small sample numbers

Studies of Phase Turbulence in the One Dimensional Complex Ginzburg-Landau Equation

The phase-turbulent (PT) regime for the one dimensional complex Ginzburg-Landau equation (CGLE) is carefully studied, in the limit of large systems and long integration times, using an efficient new integration scheme. Particular attention is paid to solutions with a non-zero phase gradient. For fixed control parameters, solutions with conserved average phase gradient $\nu$ exist only for $|\nu|$ less than some upper limit. The transition from phase to defect-turbulence happens when this limit becomes zero. A Lyapunov analysis shows that the system becomes less and less chaotic for increasing values of the phase gradient. For high values of the phase gradient a family of non-chaotic solutions of the CGLE is found. These solutions consist of spatially periodic or aperiodic waves travelling with constant velocity. They typically have incommensurate velocities for phase and amplitude propagation, showing thereby a novel type of quasiperiodic behavior. The main features of these travelling wave solutions can be explained through a modified Kuramoto-Sivashinsky equation that rules the phase dynamics of the CGLE in the PT phase. The latter explains also the behavior of the maximal Lyapunov exponents of chaotic solutions.Comment: 16 pages, LaTeX (Version 2.09), 10 Postscript-figures included, submitted to Phys. Rev.

Assessment of New Hub-and-spoke and Point-to-point Airline Network Configurations

This paper aims to provide new measures of airline network configuration with a view to analyse effectively the complexity of modern carriers' network design. It studies network configurations in the airline sector by taking into account both spatial and temporal dimensions. The spatial dimension is measured by using both the Gini index and the Freeman index, which originate from social science research. The temporal dimension is measured by the connectivity ratio, i.e. the share of indirect connections over the total number of connections. According to these indicators, the configuration of the largest full-service carriers and the largest low-cost carriers in Europe is investigated. The results show that the temporal dimension provides a clear distinction between full-service carriers and low-cost carriers; while the spatial dimension appears useful when identifying the peculiarities within groups

Accuracy of Modified Checklist for Autism in Toddlers (M-CHAT) in Detecting Autism and Other Developmental Disorders in Community Clinics

This study determined the accuracy of Modified Checklist for Autism in Toddlers (M-CHAT) in detecting toddlers with autism spectrum disorder (ASD) and other developmental disorders (DD) in community mother and child health clinics. We analysed 19,297 eligible toddlers (15–36 months) who had M-CHAT performed in 2006–2011. Overall sensitivities for detecting ASD and all DD were poor but better in the 21 to 21 months) and a useful screening tool for all DD. © 2017, Springer Science+Business Media, LLC

Perinatal derivatives: How to best validate their immunomodulatory functions

Perinatal tissues, mainly the placenta and umbilical cord, contain a variety of different somatic stem and progenitor cell types, including those of the hematopoietic system, multipotent mesenchymal stromal cells (MSCs), epithelial cells and amnion epithelial cells. Several of these perinatal derivatives (PnDs), as well as their secreted products, have been reported to exert immunomodulatory therapeutic and regenerative functions in a variety of pre-clinical disease models. Following experience with MSCs and their extracellular vesicle (EV) products, successful clinical translation of PnDs will require robust functional assays that are predictive for the relevant therapeutic potency. Using the examples of T cell and monocyte/macrophage assays, we here discuss several assay relevant parameters for assessing the immunomodulatory activities of PnDs. Furthermore, we highlight the need to correlate the in vitro assay results with preclinical or clinical outcomes in order to ensure valid predictions about the in vivo potency of therapeutic PnD cells/products in individual disease settings