New method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside the host cell

oxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS. IMPORTANCE Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp

Epidemiological associations between brachycephaly and upper respiratory tract disorders in dogs attending veterinary practices in England

Background: Brachycephalic dog breeds are increasingly common. Canine brachycephaly has been associated with upper respiratory tract (URT) disorders but reliable prevalence data remain lacking. Using primary-care veterinary clinical data, this study aimed to report the prevalence and breed-type risk factors for URT disorders in dogs. Results: The sampling frame included 170,812 dogs attending 96 primary-care veterinary clinics participating within the VetCompass Programme. Two hundred dogs were randomly selected from each of three extreme brachycephalic breed types (Bulldog, French Bulldog and Pug) and three common small-to medium sized breed types (moderate brachycephalic: Yorkshire Terrier and non-brachycephalic: Border Terrier and West Highland White Terrier). Information on all URT disorders recorded was extracted from individual patient records. Disorder prevalence was compared between groups using the chi-squared test or Fisher’s test, as appropriate. Risk factor analysis used multivariable logistic regression modelling. During the study, 83 (6.9 %) study dogs died. Extreme brachycephalic dogs (median longevity: 8.6 years, IQR: 2.4-10.8) were significantly younger at death than the moderate and non-brachycephalic group of dogs (median 12.7 years, IQR 11.1-15.0) (P \u3c 0.001). A higher proportion of deaths in extreme brachycephalic breed types were associated with URT disorders (4/24 deaths, 16.7 %) compared with the moderate and non-brachycephalic group (0/59 deaths, 0.0 %) (P = 0.001). The prevalence of having at least one URT disorder in the extreme brachycephalic group was higher (22.0 %, 95 % confidence interval (CI): 18.0-26.0) than in the moderate and non-brachycephalic group (9.7 %, 95 % CI: 7.1-12.3, P \u3c 0.001). The prevalence of URT disorders varied significantly by breed type: Bulldogs 19.5 %, French Bulldogs 20.0 %, Pugs 26.5 %, Border Terriers 9.0 %, West Highland White Terriers 7.0 % and Yorkshire Terriers 13.0 % (P \u3c 0.001). After accounting for the effects of age, bodyweight, sex, neutering and insurance, extreme brachycephalic dogs had 3.5 times (95 % CI: 2.4-5.0, P \u3c 0.001) the odds of at least one URT disorder compared with the moderate and non-brachycephalic group. Conclusions: In summary, this study reports that URT disorders are commonly diagnosed in Bulldog, French Bulldog, Pug, Border Terrier, WHWT and Yorkshire Terrier dogs attending primary-care veterinary practices in England. The three extreme brachycephalic breed types (Bulldog, French Bulldog and Pug) were relatively short-lived and predisposed to URT disorders compared with three other small-to-medium size breed types that are commonly owned (moderate brachycephalic Yorkshire Terrier and non-brachycephalic: Border Terrier and WHWT). Conclusions: In summary, this study reports that URT disorders are commonly diagnosed in Bulldog, French Bulldog, Pug, Border Terrier, WHWT and Yorkshire Terrier dogs attending primary-care veterinary practices in England. The three extreme brachycephalic breed types (Bulldog, French Bulldog and Pug) were relatively short-lived and predisposed to URT disorders compared with three other small-to-medium size breed types that are commonly owned (moderate brachycephalic Yorkshire Terrier and non-brachycephalic: Border Terrier and WHWT)

Bosonic excitations of the AdS4 Reissner-Nordstrom black hole

We study the long-lived modes of the charge density and energy density correlators in the strongly-coupled, finite density field theory dual to the AdS4 Reissner-Nordstrom black hole. For small momenta q<<\mu, these correlators contain a pole due to sound propagation, as well as a pole due to a long-lived, purely imaginary mode analogous to the \mu=0 hydrodynamic charge diffusion mode. As the temperature is raised in the range T\lesssim\mu, the sound attenuation shows no significant temperature dependence. When T\gtrsim\mu, it quickly approaches the \mu=0 hydrodynamic result where it decreases like 1/T. It does not share any of the temperature-dependent properties of the 'zero sound' of Landau Fermi liquids observed in the strongly-coupled D3/D7 field theory. For such small momenta, the energy density spectral function is dominated by the sound mode at all temperatures, whereas the charge density spectral function undergoes a crossover from being dominated by the sound mode at low temperatures to being dominated by the diffusion mode when T \mu^2/q. This crossover occurs due to the changing residue at each pole. We also compute the momentum dependence of these spectral functions and their corresponding long-lived poles at fixed, low temperatures T<<\mu.Comment: 33 pages, 21 figures, 6 animation

Prevalence of Disorders Recorded in Dogs Attending Primary-Care Veterinary Practices in England

Purebred dog health is thought to be compromised by an increasing occurence of inherited diseases but inadequate prevalence data on common disorders have hampered efforts to prioritise health reforms. Analysis of primary veterinary practice clinical data has been proposed for reliable estimation of disorder prevalence in dogs. Electronic patient record (EPR) data were collected on 148,741 dogs attending 93 clinics across central and south-eastern England. Analysis in detail of a random sample of EPRs relating to 3,884 dogs from 89 clinics identified the most frequently recorded disorders as otitis externa (prevalence 10.2%, 95% CI: 9.1-11.3), periodontal disease (9.3%, 95% CI: 8.3-10.3) and anal sac impaction (7.1%, 95% CI: 6.1-8.1). Using syndromic classification, the most prevalent body location affected was the head-and-neck (32.8%, 95% CI: 30.7-34.9), the most prevalent organ system affected was the integument (36.3%, 95% CI: 33.9-38.6) and the most prevalent pathophysiologic process diagnosed was inflammation (32.1%, 95% CI: 29.8-34.3). Among the twenty most-frequently recorded disorders, purebred dogs had a significantly higher prevalence compared with crossbreds for three: otitis externa (P = 0.001), obesity (P = 0.006) and skin mass lesion (P = 0.033), and popular breeds differed significantly from each other in their prevalence for five: periodontal disease (P = 0.002), overgrown nails (P = 0.004), degenerative joint disease (P = 0.005), obesity (P = 0.001) and lipoma (P = 0.003). These results fill a crucial data gap in disorder prevalence information and assist with disorder prioritisation. The results suggest that, for maximal impact, breeding reforms should target commonly-diagnosed complex disorders that are amenable to genetic improvement and should place special focus on at-risk breeds. Future studies evaluating disorder severity and duration will augment the usefulness of the disorder prevalence information reported herein

Are mice good models for human neuromuscular disease? Comparing muscle excursions in walking between mice and humans

The mouse is one of the most widely used animal models to study neuromuscular diseases and test new therapeutic strategies. However, findings from successful pre-clinical studies using mouse models frequently fail to translate to humans due to various factors. Differences in muscle function between the two species could be crucial but often have been overlooked. The purpose of this study was to evaluate and compare muscle excursions in walking between mice and humans

Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog

Border Terriers under primary veterinary care in England: demography and disorders

The Border Terrier is a working terrier type that is generally considered to be a relatively healthy and hardy breed. This study aimed to characterise the demography and common disorders of Border Terriers receiving veterinary care in England using de-identified electronic patient record data within the VetCompass™ Programme

Miniature Schnauzers under primary veterinary care in the UK in 2013: demography, mortality and disorders

Individual dog breeds are often reported as predisposed to specific breed-related disorders but reliable epidemiological data on disease prevalence are sparse. The Miniature Schnauzer in the UK is a popular small breed dog that is often considered as relatively healthy and long-lived, but is this really true? This study aimed to use data from the VetCompass™ Programme at the Royal Veterinary College to characterise the demography, mortality and common disorders of the general population of Miniature Schnauzers under veterinary care in the UK

The effect of weight loss on lameness in obese dogs with osteoarthritis

This paper describes the effect of weight loss on lameness in obese dogs with osteoarthritis (OA). Fourteen obese client-owned dogs with clinical and radiographic signs of OA participated in an open prospective clinical trial. After a screening visit and a visit for collection of baseline data, the dogs were fed a restricted-calorie diet over a study period of 16 weeks that incorporated six follow-up visits. At each visit, body weight and pelvic circumference were measured and severity of lameness was assessed using a numeric rating scale (NRS), a visual analogue scale (VAS) and kinetic gait analysis. This is the first study to assess both subjectively and objectively, the effect of weight loss alone on lameness in obese dogs with OA. The results indicate that body weight reduction causes a significant decrease in lameness from a weight loss of 6.10% onwards. Kinetic gait analysis supported the results from a body weight reduction of 8.85% onwards. These results confirm that weight loss should be presented as an important treatment modality to owners of obese dogs with OA and that noticeable improvement may be seen after modest weight loss in the region of 6.10 – 8.85% body weight