Clinical features of muscle stiffness in 37 dogs with concurrent naturally occurring hypercortisolism.

BACKGROUND: Severe muscle stiffness (SMS) in dogs with hypercortisolism (HC) is uncommon. OBJECTIVES: To evaluate signalment, presentation, treatments, and long-term outcomes of dogs with concurrent HC and SMS. ANIMALS: Thirty-seven dogs. METHODS: Medical records of dogs with HC and concurrent SMS were recruited from 10 institutions. Clinical information, test results, therapeutic responses, and survival times were reviewed. RESULTS: All 37 dogs with HC and SMS had pituitary-dependent hypercortisolism (PDH); 36/37 weighed <20 kg. Signs and test results were typical of PDH aside from SMS, initially diagnosed in all 4 limbs in 9, pelvic limbs of 22, and thoracic limbs of 6 dogs. Hypercortisolism and SMS were diagnosed together in 3 dogs; HC 1-36 months before SMS in 23; SMS 1-12 months before HC in 11. Mitotane or trilostane, given to control HC in 36/37 dogs, improved or resolved HC signs in 28; SMS did not resolve, remaining static or worsening in 31/36 dogs, mildly improving in 5/19 dogs given additional therapies. Progression of SMS included additional limbs in 10 dogs and the masticatory muscles of 2. The median survival time from diagnosis of SMS was 965 days (range, 8-1188). CONCLUSIONS AND CLINICAL IMPORTANCE: Concurrent SMS and HC is uncommon, possibly affecting only dogs with PDH. Development of SMS might occur before or after diagnosis of HC. Apart from SMS, the clinical picture and survival time of these dogs seem indistinguishable from those of dogs with HC in general. However, while muscle weakness usually resolves with HC treatment SMS does not.

Discovery and characterization of Hv1-type proton channels in reef-building corals.

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH-regulation processes and possible consequences of ocean acidification related to the function of these channels.

'We should classify pet obesity as a disease'.

Alexander German, Ian Ramsey and Philip Lhermette address some of the concerns raised over calling pet obesity a disease and argue that this classification can bring many management opportunities.

Clinical and Clinicopathological Features in Dogs with Uncomplicated Spontaneous Hyperadrenocorticism Diagnosed in Primary Care Practice (2013-2014).

The objective of this study was to report clinical and clinicopathological abnormalities in canine spontaneous hyperadrenocorticism (HAC) and assess the relationship between patient size and those findings. A questionnaire was made available to primary care veterinarians to gather information on cases diagnosed with canine spontaneous HAC. Inclusion criteria were an adrenocorticotropic stimulation test and/or low-dose dexamethasone suppression test consistent with HAC. Exclusion criteria included concurrent systemic diseases, any clinical sign (CS) not typically associated with HAC, a urinary corticoid:creatinine ratio within reference interval, administration of steroids during the 3 mo before diagnosis, treatment with any drug causing CSs of HAC, and ongoing treatment for canine spontaneous HAC. Sixty-two cases were identified. The prevalences of various CSs were similar to those historically reported. No association between the patient weight and CSs was identified. The platelet count was negatively correlated to the weight (P = .005, r2 = 0.3). Alanine aminotransferase (P = .016, r2 = 0.17) and alkaline phosphatase (P = .05, r2 = 0.0014) activities were positively correlated to the CS ratio. In this group of dogs, CSs were not significantly different between dogs ≤20 kg and dogs >20 kg. The prevalences of various clinical findings appeared to be similar to those historically reported. Dogs with more CSs tended to have higher alanine aminotransferase and alkaline phosphatase activities.

Gating Currents in the Hv1 Proton Channel.

The Hv1 proton channel shares striking structural homology with fourth transmembrane helical segment-type voltage-sensor (VS) domains but manifests distinctive functional properties, including a proton-selective "aqueous" conductance and allosteric control of voltage-dependent gating by changes in the transmembrane pH gradient. The mechanisms responsible for Hv1's functional properties remain poorly understood, in part because methods for measuring gating currents that directly report VS activation have not yet been described. Here, we describe an approach that allows robust and reproducible measurement of gating-associated charge movements in Hv1. Gating currents reveal that VS activation and proton-selective aqueous conductance opening are thermodynamically distinct steps in the Hv1 activation pathway and show that pH changes directly alter VS activation. The availability of an assay for gating currents in Hv1 may aid future efforts to elucidate the molecular mechanisms of gating cooperativity, pH-dependent modulation, and H+ selectivity in a model VS domain protein.

Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation.

The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H+-selective "aqueous" conductance (GAQ) that is potently modulated by extracellular Zn2+ Although two conserved His residues are required for Zn2+ effects on GAQ gating, the atomic structure of the Zn2+ coordination site and mechanism by which extracellular Zn2+ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn2+ potency and are therefore likely to participate in first solvation shell interactions with Zn2+ Experimental Zn2+-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn2+ coordination spheres in Zn2+-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn2+-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg "gating charges" in S4 as well as acidic "counter-charges" in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn2+ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting GAQ activation to more negative voltages, E153A also decreases Zn2+ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

Pyrexia in cats: Retrospective analysis of signalment, clinical investigations, diagnosis and influence of prior treatment in 106 referred cases.

OBJECTIVES: The main aim of the study was to describe the features and diagnoses of a population of cats referred with pyrexia. Other aims were to report and evaluate the utility of clinical investigations performed, and describe any effect of treatment before referral on temperature at presentation and ability to make a diagnosis. METHODS: Clinical records of cats with pyrexia (⩾39.2°C) documented at least twice were retrospectively reviewed. Cases were assigned to disease categories (infectious, inflammatory, immune-mediated, neoplastic, miscellaneous and no diagnosis [pyrexia of unknown origin, PUO]) based on diagnosis. The overall value of clinical investigations was assessed by classifying them as 'enabling', 'assisting' or 'no assistance' in achieving each diagnosis. The effect of treatment before referral was assessed for any association with temperature at presentation and ability to make a diagnosis (PUO vs other disease categories). RESULTS: One hundred and six cases were identified. The most common cause of pyrexia was feline infectious peritonitis (22 cats, 20.8%) and the largest disease category was infectious (41/106, 38.7%). Inflammatory conditions were found in 19 (17.9%) cats, neoplasia in 13 (12.3%), miscellaneous causes in 11 (10.4%) and immune-mediated disease in six (5.7%). No diagnosis was reached in 16 (15.0%) cats, often despite extensive diagnostic investigations. Cytology and histopathology most often 'enabled' or 'assisted' in obtaining a diagnosis. Most cats (91, 85.8%) received treatment before referral, with antimicrobial treatment given to 87 (82.1%). Prior treatment before referral was not associated with temperature at presentation nor with success in establishing a diagnosis. CONCLUSIONS AND RELEVANCE: This is the first study investigating causes of pyrexia in cats. Infectious diseases were most common and immune-mediated diseases were comparatively rare.

Validation and determination of a reference interval for canine HbA1c using an immunoturbidimetric assay.

BACKGROUND: Hemoglobin A1c (HbA1c) provides a reliable measure of glycemic control over 2-3 months in human diabetes mellitus. In dogs, presence of HbA1c has been demonstrated, but there are no validated commercial assays. OBJECTIVE: The purpose of the study was to validate a commercially available automated immunoturbidimetric assay for canine HbA1c and determine an RI in a hospital population. METHODS: The specificity of the assay was assessed by inducing glycosylation in vitro using isolated canine hemoglobin, repeatability by measuring canine samples 5 times in succession, long term inter-assay imprecision by measuring supplied control materials, stability using samples stored at 4°C over 5 days and -20°C over 8 weeks, linearity by mixing samples of known HbA1c in differing proportions, and the effect of anticoagulants with paired samples. An RI was determined using EDTA-anticoagulated blood samples from 60 nondiabetic hospitalized animals of various ages and breeds. Hemoglobin A1c was also measured in 10 diabetic dogs. RESULTS: The concentration of HbA1c increased proportionally with glucose concentration in vitro. For repeat measurements, the CV was 4.08% (range 1.16-6.10%). Samples were stable for 5 days at 4°C. The assay was linear within the assessed range. Heparin- and EDTA-anticoagulated blood provided comparable results. The RI for HbA1c was 9-18.5 mmol/mol. There was no apparent effect of age or breed on HbA1c. In diabetic dogs, HbA1c ranged from 14 to 48 mmol/mol. CONCLUSIONS: The assay provides a reliable method for canine HbA1c measurement with good analytic performance.

Proton currents constrain structural models of voltage sensor activation.

The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic 'aqueous' H+ conductance (GAQ). Mutation of a highly conserved 'gating charge' residue in the S4 helix (R1H) confers a resting-state H+ 'shuttle' conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1.

Clinical factors associated with death before discharge and overall survival time in dogs with generalized megaesophagus.

OBJECTIVE: To investigate the association of 6 clinical features with outcome of dogs with generalized megaesophagus. DESIGN: Retrospective cohort study. ANIMALS: 71 client-owned dogs with radiographic evidence of generalized esophageal dilation. PROCEDURES: Medical records were reviewed for data on signalment, age at onset of clinical signs, body weight, evidence of undernutrition, and the administration of drugs to treat or prevent esophagitis. Radiographs were reviewed for evidence of aspiration pneumonia (AP) and to calculate the relative esophageal diameter. Details of outcome were collected from the medical records and by contacting owners and referring veterinarians. The association of 6 factors with death before discharge and overall survival time was assessed. RESULTS: Overall median survival time was 90 days. Nineteen (26.7%) patients died before discharge from the hospital. Radiographic evidence of AP was both positively associated with death before discharge and negatively associated with overall survival time. An age at onset of clinical signs of >13 months was negatively associated with overall survival time. No evidence of an association of the degree of esophageal dilation or the use of drugs to prevent or treat esophagitis with death before discharge or overall survival time was found. CONCLUSIONS AND CLINICAL RELEVANCE: Radiographic evidence of AP and the age at onset of clinical signs were the only variables found to be significantly associated with survival time in this study, and this should be considered when advising on prognosis in dogs with megaesophagus.