Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs

An Age-Associated Decline in Thymic Output Differs in Dog Breeds According to Their Longevity

The age associated decline in immune function is preceded in mammals by a reduction in thymic output. Furthermore, there is increasing evidence of a link between immune competence and lifespan. One approach to determining thymic output is to quantify signal joint T cell receptor excision circles (sj-TRECs), a method which has been developed and used in several mammalian species. Life expectancy and the rate of aging vary in dogs depending upon their breed. In this study, we quantified sj-TRECs in blood samples from dogs of selected breeds to determine whether there was a relationship between longevity and thymic output. In Labrador retrievers, a breed with a median expected lifespan of 11 years, there was an age-associated decline in sj-TREC values, with the greatest decline occurring before 5 years of age, but with sj-TREC still detectable in some geriatric animals, over 13 years of age. In large short-lived breeds (Burnese mountain dogs, Great Danes and Dogue de Bordeaux), the decline in sj-TREC values began earlier in life, compared with small long-lived breeds (Jack Russell terriers and Yorkshire terriers), and the presence of animals with undetectable sj-TRECs occurred at a younger age in the short-lived breeds. The study findings suggest that age-associated changes in canine sj-TRECs are related to breed differences in longevity, and this research highlights the use of dogs as a potential model of immunosenescence

Yellow and Red Supergiants in the Large Magellanic Cloud

Due to their transitionary nature, yellow supergiants provide a critical challenge for evolutionary modeling. Previous studies within M31 and the SMC show that the Geneva evolutionary models do a poor job at predicting the lifetimes of these short-lived stars. Here we extend this study to the LMC while also investigating the galaxy's red supergiant content. This task is complicated by contamination by Galactic foreground stars that color and magnitude criteria alone cannot weed out. Therefore, we use proper motions and the LMC's large systemic radial velocity (\sim278 km/s) to separate out these foreground dwarfs. After observing nearly 2,000 stars, we identified 317 probable yellow supergiants, 6 possible yellow supergiants and 505 probable red supergiants. Foreground contamination of our yellow supergiant sample was \sim80%, while that of the the red supergiant sample was only 3%. By placing the yellow supergiants on the H-R diagram and comparing them against the evolutionary tracks, we find that new Geneva evolutionary models do an exemplary job at predicting both the locations and the lifetimes of these transitory objects.Comment: Accepted for publication in the Ap

Residue 39 of Kir6.2 drives a difference in ATP sensitivity in human and canine beta-cell K ATP channels

ATP-sensitive potassium (KATP) channels link beta-cell metabolism to electrical activity. By modulating the beta-cell membrane potential, they finely regulate glucose-stimulated insulin secretion. KATP channels are hetero-octameric complexes composed of four pore-forming subunits (Kir6.2, encoded by KCNJ11) and four regulatory subunits (SUR1, encoded by ABCC8). A multi-species alignment of the KCNJ11 gene revealed that, although the sequence is highly conserved, residue 39 varies among different mammals. Previous studies have shown that this residue plays a critical role in regulating KATP channel activity and its mutation results in neonatal diabetes in humans. We therefore explored whether the canine and human KATP channel show different ATP sensitivities as a result of their sequence variation. We used patch-clamp electrophysiology to investigate species variation in the ATP sensitivity of the KATP channel. Functional studies showed that canine KATP channels exhibit reduced ATP sensitivity compared to human channels. However, stimulation by MgADP was unaffected. We next compared the ATP sensitivity of hybrid channels (human Kir6.2 with canine SUR1, and vice versa), as well as KATP channels in which residue 39 was swapped between human and canine Kir6.2. In each case, ATP sensitivity was mainly determined by the identity of the residue at position 39. Our study suggests that the ATP sensitivity of the pancreatic KATP channel differs between human and dog. This suggests that the beta-cell membrane potential and potentially insulin release may be fine-tuned differently across species

Syndromic surveillance: two decades experience of sustainable systems – its people not just data!

Syndromic surveillance is a form of surveillance that generates information for public health action by collecting, analysing and interpreting routine health-related data on symptoms and clinical signs reported by patients and clinicians rather than being based on microbiologically or clinically confirmed cases. In England, a suite of national real-time syndromic surveillance systems (SSS) have been developed over the last 20 years, utilising data from a variety of health care settings (a telehealth triage system, general practice and emergency departments). The real-time systems in England have been used for early detection (e.g. seasonal influenza), for situational awareness (e.g. describing the size and demographics of the impact of a heatwave) and for reassurance of lack of impact on population health of mass gatherings (e.g. the London 2012 Olympic and Paralympic Games).We highlight the lessons learnt from running SSS, for nearly two decades, and propose questions and issues still to be addressed. We feel that syndromic surveillance is an example of the use of ‘big data’, but contend that the focus for sustainable and useful systems should be on the added value of such systems and the importance of people working together to maximise the value for the public health of syndromic surveillance services

Polymorphisms in the CTLA4 promoter sequence are associated with canine hypoadrenocorticism

Canine hypoadrenocorticism is an immune-mediated endocrinopathy that shares both clinical and pathophysiological similarities with Addison’s disease in humans. Several dog breeds are overrepresented in the disease population, suggesting that a genetic component is involved, although this is likely to be polygenic. Previous research has implicated CTLA4 as a potential susceptibility gene. CTLA4 is an important regulator of T cell function and polymorphisms/mutations in CTLA4 have been associated with a number of autoimmune phenotypes in both humans and rodent models of autoimmunity. The aim of the current study was to undertake a case:control association study of CTLA4 promotor polymorphisms in three dog breeds, cocker spaniels, springer spaniels and West Highland white terriers (WHWT)

Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes

Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n = 942) compared with non-diabetic dogs (n = 100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic dogs

The Star-Forming Galaxy Contribution to the Cosmic MeV and GeV Gamma-Ray Background

While star-forming galaxies could be major contributors to the cosmic GeV $\gamma$-ray background, they are expected to be MeV-dim because of the "pion bump" falling off below ~100 MeV. However, there are very few observations of galaxies in the MeV range, and other emission processes could be present. We investigate the MeV background from star-forming galaxies by running one-zone models of cosmic ray populations, including Inverse Compton and bremsstrahlung, as well as nuclear lines (including $^{26}$Al), emission from core-collapse supernovae, and positron annihilation emission, in addition to the pionic emission. We use the Milky Way and M82 as templates of normal and starburst galaxies, and compare our models to radio and GeV--TeV $\gamma$-ray data. We find that (1) higher gas densities in high-z normal galaxies lead to a strong pion bump, (2) starbursts may have significant MeV emission if their magnetic field strengths are low, and (3) cascades can contribute to the MeV emission of starbursts if they emit mainly hadronic $\gamma$-rays. Our fiducial model predicts that most of the unresolved GeV background is from star-forming galaxies, but this prediction is uncertain by an order of magnitude. About ~2% of the claimed 1 MeV background is diffuse emission from star-forming galaxies; we place a firm upper limit of <~10% based on the spectral shape of the background. The star-formation contribution is constrained to be small, because its spectrum is peaked, while the observed background is steeply falling with energy through the MeV-GeV range.Comment: Published in ApJ, 27 pages, emulateapj format. Readers may be interested in the concurrent paper by Chakraborty and Fields (arXiv:1206.0770), a calculation of the Inverse Compton background from star-forming galaxie

A genome-wide association study identifies novel candidate genes for susceptibility to diabetes mellitus in non-obese cats

Diabetes mellitus (DM) is a common feline endocrinopathy, which is similar to human type 2 diabetes (T2DM) in terms of its pathophysiology. T2DM occurs due to peripheral insulin resistance and/or β-cell dysfunction. Several studies have identified genetic and environmental factors that contribute to susceptibility to human T2DM. In cats, environmental factors such as obesity and physical inactivity have been linked with DM, although to date, the only genetic association that has been demonstrated is with a polymorphism in the feline MC4R gene. The aim of this study was to perform a genome-wide association study (GWAS) to identify polymorphisms associated with feline DM.Illumina Infinium 63k iSelect DNA arrays were used to analyse genomic DNA samples from 192 diabetic domestic shorthair cats and 389 non-diabetic control cats. Data was analysed using PLINK whole genome data analysis toolset. Significance was established at