Frequent transitions in self-assembly across the evolution of a central metabolic enzyme.

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy.

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

Characterization of the pathogenicity of strains of Pseudomonas syringae towards cherry and plum.

Bacterial canker is a major disease of Prunus avium (cherry), Prunus domestica (plum) and other stone fruits. It is caused by pathovars within the Pseudomonas syringae species complex including P. syringae pv. morsprunorum (Psm) race 1 (R1), Psm race 2 (R2) and P. syringae pv. syringae (Pss). Psm R1 and Psm R2 were originally designated as the same pathovar; however, phylogenetic analysis revealed them to be distantly related, falling into phylogroups 3 and 1, respectively. This study characterized the pathogenicity of 18 newly genome-sequenced P. syringae strains on cherry and plum, in the field and laboratory. The field experiment confirmed that the cherry cultivar Merton Glory exhibited a broad resistance to all clades. Psm R1 contained strains with differential specificity on cherry and plum. The ability of tractable laboratory-based assays to reproduce assessments on whole trees was examined. Good correlations were achieved with assays using cut shoots or leaves, although only the cut shoot assay was able to reliably discriminate cultivar differences seen in the field. Measuring bacterial multiplication in detached leaves differentiated pathogens from nonpathogens and was therefore suitable for routine testing. In cherry leaves, symptom appearance discriminated Psm races from nonpathogens, which triggered a hypersensitive reaction. Pathogenic strains of Pss rapidly induced disease lesions in all tissues and exhibited a more necrotrophic lifestyle than hemibiotrophic Psm. This in-depth study of pathogenic interactions, identification of host resistance and optimization of laboratory assays provides a framework for future genetic dissection of host-pathogen interactions in the canker disease.

Extracting drug mechanism and pharmacodynamic information from clinical electroencephalographic data using generalised semi-linear canonical correlation analysis.

Conventional analysis of clinical resting electroencephalography (EEG) recordings typically involves assessment of spectral power in pre-defined frequency bands at specific electrodes. EEG is a potentially useful technique in drug development for measuring the pharmacodynamic (PD) effects of a centrally acting compound and hence to assess the likelihood of success of a novel drug based on pharmacokinetic-pharmacodynamic (PK-PD) principles. However, the need to define the electrodes and spectral bands to be analysed a priori is limiting where the nature of the drug-induced EEG effects is initially not known. We describe the extension to human EEG data of a generalised semi-linear canonical correlation analysis (GSLCCA), developed for small animal data. GSLCCA uses data from the whole spectrum, the entire recording duration and multiple electrodes. It provides interpretable information on the mechanism of drug action and a PD measure suitable for use in PK-PD modelling. Data from a study with low (analgesic) doses of the µ-opioid agonist, remifentanil, in 12 healthy subjects were analysed using conventional spectral edge analysis and GSLCCA. At this low dose, the conventional analysis was unsuccessful but plausible results consistent with previous observations were obtained using GSLCCA, confirming that GSLCCA can be successfully applied to clinical EEG data.

Aspiration pneumonia in the Irish wolfhound: a possible breed predisposition.

BACKGROUND: Anecdotal reports suggest a recent high prevalence of aspiration pneumonia in Irish wolfhounds, prompting further investigation into the incidence of the disease in this breed. OBJECTIVES: To investigate the possibility that Irish wolfhounds have an increased incidence of aspiration pneumonia, and to consider possible predisposing causes in this breed. METHODS: Retrospective review of medical records from the Small Animal Specialist Hospital, Sydney, from January 2008 to December 2012 inclusive to determine the total hospital incidence and individual breed incidences of aspiration pneumonia. RESULTS: The total hospital incidence of aspiration pneumonia was 0 · 5%. The Irish wolfhound had the highest breed incidence, with 9 of 25 dogs (36%) diagnosed with aspiration pneumonia. Four of the Irish wolfhounds had a predisposing cause identified; one having a choking episode, one having gastric bloat, while two were diagnosed with laryngeal paralysis after it was specifically investigated because of clinical suspicion. Five had no underlying cause of their aspiration pneumonia determined. CLINICAL SIGNIFICANCE: On the basis of the hospital population studied, the Irish wolfhound has a high incidence of aspiration pneumonia. Further investigation into the possible predisposing cause(s) in this breed is warranted.

Fitting Emax models to clinical trial dose-response data when the high dose asymptote is ill defined.

We consider fitting the so-called Emax model to continuous response data from clinical trials designed to investigate the dose-response relationship for an experimental compound. When there is insufficient information in the data to estimate all of the parameters because of the high dose asymptote being ill defined, maximum likelihood estimation fails to converge. We explore the use of either bootstrap resampling or the profile likelihood to make inferences about effects and doses required to give a particular effect, using limits on the parameter values to obtain the value of the maximum likelihood when the high dose asymptote is ill defined. The results obtained show these approaches to be comparable with or better than some others that have been used when maximum likelihood estimation fails to converge and that the profile likelihood method outperforms the method of bootstrap resampling used.

Metaphyseal osteopathy in three Australian Kelpie siblings.

CASE REPORT: Metaphyseal osteopathy (MO) was diagnosed in three Australian Kelpie puppies that were presented for veterinary assessment of lameness. The three puppies were siblings. Each was from a different litter by the same breeding pair. The puppy in case one was seen by the authors, and the puppies in cases two and three were patients at other veterinary hospitals. However, the medical records and radiographs were examined and reviewed for this report. Radiographic investigation of the lameness revealed pathognomonic appearance of MO affecting the metaphyseal region of the long bones in all three puppies. The diagnosis was confirmed on histopathology in one patient. CONCLUSION: MO is considered a disease of large and giant-breed dogs, being rarely reported in non-large-breed dogs, and has not been reported in the Australian Kelpie, which is considered a medium-breed dog. This case series suggests a previously unreported breed predisposition to MO in the Australian Kelpie.

Influence of needle gauge used for venipuncture on automated platelet count and coagulation profile in dogs.

OBJECTIVE: To determine if differing gauge (G) needles used for venipuncture altered the automated platelet count and coagulation profile (prothrombin time (PT) and activated partial thromboplastin time (aPTT)) in clinically healthy dogs. DESIGN: Prospective, observational, randomised, clinical study. METHODS: We enrolled 20 clinically healthy dogs. Blood was collected via direct venipuncture of the jugular veins with 21G, 23G and 25G needles in a random order. Automated haematology and automated coagulation times were performed on the blood samples. Values were analysed for differences among the needle gauges and also the order of sample collection. RESULTS: No difference was found in the automated platelet count or automated coagulation times for the three needle gauges used or the order in which the samples were collected. CONCLUSION: Venipuncture can be performed with a 21G, 23G or 25G needle to obtain blood from dogs for automated platelet count and PT/aPTT measurement without affecting the results.

Vaginal discharge in a spayed dog with multiple distinct malignancies.

BACKGROUND: An 11-year-old female spayed Labrador Retriever was presented with clinical signs of oestrus and vaginal bleeding. Historically, the dog had not cycled and had been spayed at less than 6 months of age. RESULTS: Extensive investigation culminated in the diagnosis of an ovarian granulosa cell tumour. The patient had a history of localised grade III mast cell tumour 5 years prior and hepatic haemangiosarcoma 8 months prior to diagnosis. CONCLUSIONS: Both conditions were successfully treated with a combination of surgery and chemotherapy and there was no evidence of metastasis or recurrence at the time of evaluation for signs of oestrus.

Development and evaluation of a pyriproxyfen-treated device to control the dengue vector, Aedes aegypti (L.) (Diptera:Culicidae).

The resurgence of dengue fever and the chikungunya epidemic make the control of Aedes aegypti mosquitoes, the vectors of these diseases, critically important. We developed and evaluated an Ae. aegypti control device that is visually-attractive to mosquitoes. This pyriproxyfen-treated device was evaluated for its impact on Ae. aegypti egg production and population dynamics in dengue-endemic areas in Thailand. The device consists of a "high rise" shaped ovitrap/ resting station covered with black cotton cloth. The device is easily collapsible and transportable. Ae. aegypti are generally drawn towards darker, shadier areas making this device physically attractive as a resting station to mosquitoes of all physiological stages. The results show this device suppressed Ae. aegypti populations after it was introduced into a village. The observed effect was primarily the result of the Ae. aegypti exposure to pyriproxyfen shortly after adult emergence or after taking a blood meal resulting in decreased egg production. We believe the device may be further improved physically and the formulation should be replaced to provide even better efficacy for controlling Ae. aegypti mosquito, populations.

Resolution of life-threatening dysphagia caused by caudal occipital malformation syndrome following foramen magnum decompressive surgery.

A Cavalier King Charles Spaniel was presented with acute onset, life-threatening dysphagia suspected to be secondary to medulla oblongata compression caused by caudal occipital malformation syndrome. The patient required urgent tracheostomy tube placement to remain stable and was subsequently cured of the presenting neurological deficits by foramen magnum decompressive surgery. Neurogenic dysphagia is a relatively common presenting sign in human Chiari malformation syndromes, but has not been described as a major clinical sign in veterinary patients. Caudal occipital malformation syndrome should be included in the differential diagnosis list for susceptible breeds presenting with dysphagia. Early recognition favours expeditious surgical intervention and a positive outcome in human patients, and this may also be the case in veterinary patients.

Omentalisation of a caudal mediastinal abscess in a dog.

A caudal mediastinal abscess was diagnosed in a 2-year-old German Short-haired Pointer with acute onset of lethargy, pyrexia and tachypnoea. The abscess was managed surgically by median sternotomy, abscess content evacuation and omentalisation of the abscess cavity via the diaphragm. Although surgical management of mediastinal abscesses has been reported previously, this is the first report to describe successful management of the condition in a dog by omentalisation.

Mucinous nonneoplastic cyst of the pancreas: apomucin phenotype distinguishes this entity from intraductal papillary mucinous neoplasm.

Mucinous nonneoplastic cyst of the pancreas is a newly described and rare cystic lesion with unknown histogenesis. It is defined as a cystic lesion lined with mucinous epithelium, supported by hypocellular stroma and not communicating with the pancreatic ducts. It is very challenging to differentiate this lesion from other cystic mucinous neoplasms of the pancreas such as branch-duct intraductal papillary mucinous neoplasm by morphology. In this study, a total of 436 pancreatic specimens resected between 2002 and 2007 in our institution were reviewed. Fifteen (3.4%, 15/436) mucinous nonneoplastic cysts were identified. They included 3 males and 12 females, with a median age of 60 years. Forty-six percent of cases (7/15) occurred in pancreatic head, 27% (4/15) in neck, 7% (1/15) in body, and 20% (3/15) in tail. The size of lesions ranged from 0.5 to 3.5 cm in greatest dimension. In most cases (12/15, 80%), mucinous nonneoplastic cyst was associated or adjacent to acinar-ductal mucinous metaplasia. These morphologic data indicate that mucinous nonneoplastic cyst is not really a rare disease and may originate from acinar-duct mucinous metaplasia histogenestically. Furthermore, apomucin immunostains of mucinous nonneoplastic cyst showed MUC1 expressed in 27% (4/15) cases, MUC5AC in 67% (10/15 cases), and MUC2 was were negative in all cases, whereas intraductal papillary mucinous neoplasm (n = 17; 5 main duct type, 12 branch-duct type) showed focal and weak MUC1 positivity in 18% (3/17) cases, MUC2 positivity in 71% (12/17) cases, and all intraductal papillary mucinous neoplasm (17/17) were MUC5AC positive. The clonality assay with the HUMARA gene revealed that the mucinous nonneoplastic cysts were of polyclonal origin. For the first time, using HUMARA assay, we demonstrate the nonneoplastic nature of these cysts and further characterize morphologic and immunophenotypic properties that allow differentiation from intraductal papillary mucinous neooplasm.

Effects of oil pollution at Kuwait's Greater Al-Burgan oil field on the timing of morning emergence, basking and foraging behaviors by the sand lizard Acanthodactylus scutellatus.

An attempt was made to study the effects of oil pollution in a desert location (the Greater Al-Burgan oil fields, an area damaged in the second Gulf War) in Kuwait on the behaviour of the Sand lizard A. scutellatus. Polluted sites with apparently different degrees of contamination (namely tar mat, soot and clear sites) were compared with control areas outside this region. Between 2002 and 2003, ten lizards (5 of each sex) on each polluted and each control site were observed in the field at a time of the year when they were highly active. Air, substrate and burrow temperatures were recorded and lizards were monitored for their morning emergence times, as well as their basking and foraging activities. The present study confirmed that the morning emergence times and the basking behavior varied in sand lizards among the different pollution site categories. Physical changes in the tar mat sites caused the substrate temperatures in these locations to rise more quickly in the morning in response to solar gain than was the case in the other sites. This gives lizards in these locations the opportunity to emerge earlier and to start eating more quickly, giving them an energetic advantage (perhaps, in turn, influencing their rates of growth and fecundity). The clear sites had the next earliest emergence and were the next hottest but it is difficult to account for this in terms of the physical characteristics of this site. The basking times were clearly shorter on the dark soot and tar mat sites that appeared to have higher solar gain than control or clear sites. There did not appear to be any obvious differences in foraging activity of lizards in the different locations. It appears that some aspects of simple behaviour in these lizards provides a reliable, noninvasive indices for assessing oil pollution in desert locations. The precise impact of these changes in these reptiles on their long-term viability needs to be evaluated.

High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening.

To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12+/-22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 microM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.

Time-dependent behavioral, neurochemical, and immune consequences of repeated experiences of social defeat stress in male mice and the ameliorative effects of fluoxetine.

This study attempted to determine whether differing numbers of days of repeated defeat experience altered behavior, immune measures, and neuroendocrine mediators in mice. OF1 male mice were socially stressed by repeated experiences of defeat in a sensorial contact model. Subjects exposed to nine defeats showed more stretch-attend postures and fewer active defense elements than counterparts exposed to 23 defeats. Submissive subjects with nine experiences of defeat also had a lower splenocyte proliferative response than unmanipulated controls. The proliferation index progressively increased but at a higher rate in manipulated controls than in socially stressed subjects, resulting in a significant immunosuppressive effect after 23 days of exposure to social stressors. Nine days of such exposure resulted in higher hypothalamic ratios of serotonin and dopamine to their major metabolites than in unmanipulated or manipulated controls and subjects socially stressed for 23 days. The data generally indicate that the acute social stressors (such as nine defeats) produce a profile of behavioral and physiological variables characteristic of a state of anxiety. The proliferation index was also lower after 52 days of social stress than in manipulated controls. Fluoxetine treatment appeared to have an anxiolytic effect, reducing immobility, and even seemed to protect subjects from the immune impairment and endocrine alteration caused by social stressors. The results generally provide clues that improve our knowledge of the consequences of social stressors and their possible treatment.

Time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration.

Adverse drug reactions are a major clinical problem. Drug-induced hepatotoxicity constitutes a large percentage of these reactions. A thorough understanding of the genetic events, specifically, the early "decision-making" processes underlying biological changes caused by drugs and metabolites, is required. To assist in the understanding of these events, we have employed the model hepatotoxin, paracetamol (APAP), and GeneChip technology to investigate global genetic events seen after nontoxic and toxic doses in the mouse. Mice were dosed [vehicle, nontoxic APAP (1 mmol/kg), and toxic APAP (3.5 mmol/kg)], and individual hepatic RNA samples were hybridized to separate chips to determine interanimal variation. Statistical analysis detected 175 CD-1 mouse genes that were significantly regulated (P < 4.1 x 10(-6)), and nonsignificant genes were discarded. For clarity, the significantly regulated genes were then binned into categories according to their major function-antioxidant, glutathione, metabolism, transcription, immune, and apoptosis. There was no hepatic stress observed after dosing 1 mmol/kg APAP, when measured by serum alanine aminotransferase levels. Hepatic toxicity was observed at both 4 and 24 h after a 3.5 mmol/kg dose of APAP. Time course expression profiles for selected genes have been created. These results demonstrate that most active gene expression occurs around 4 h after a toxic dose of APAP. Down-regulation of these genes is observed over 24 h, coinciding with the development of overt toxicity. These data provide a deeper understanding of the in vivo time course of physiological responses of the liver to chemical stress and provide a logical step forward for the investigation of new chemical entities demonstrated positive in chemically reactive metabolite screens. The complete data set can be viewed at http://www.ebi.ac.uk/arrayexpress/. The accession number is E-MEXP-82.

Effects of paternal heat stress on the in vivo development of preimplantation embryos in the mouse.

The objective of this study was to examine the effect of paternal heat stress on the in vivo development of preimplantation embryos in the mouse. Synchronised B6CBF1 female mice were mated either to a control male mouse or to one that had been exposed at 7, 21 or 35 days previously, for 24 h to an ambient temperature of 36+/-0.3 degrees C and 66+/-5.6% relative humidity. Embryos were collected from the oviducts of mice at 14-16 h, 34-39 h or 61-65 h after mating or from the uterus at 85-90 h after mating and their developmental status was evaluated morphologically. The number of cells within blastocysts was also determined using bisbenzimide-propidium iodide staining. Paternal heat stress 7 days before mating reduced the proportion of embryos developing from 4-cell (4-C) to morulae (M), hatched blastocysts, total blastocysts and the number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocyst. Paternal heat stress 21 days prior to mating reduced the proportion of 2-C and 4-C to M embryos with no embryos developing to blastocysts. There were also increases in the number of 1-C and abnormal embryos recorded at this time. Paternal heat stress 35 days before mating decreased the proportion of 2-C embryos, expanded blastocysts and ICM and TE cells in the blastocyst. These results support previous work demonstrating that both the sperm in the epididymis and germ cells in the testis are susceptible to damage by environmental heat stress, with spermatocytes being the most vulnerable. This study also demonstrates that subtle effects on the male such as a short exposure to elevated environmental temperatures can translate to quite profound paternal impacts on early embryo development.

First-order differential equation models with estimable parameters as functions of environmental variables and their application to a study of vascular development in young hybrid aspen stems.

A novel method is described for solving systems of differential equations pertaining to organism development, where this development is assumed to be directly influenced by fluctuation in measurable environmental variables. The system parameters are written as functions of these variables and, because these functions involve the accumulation of "environment time" (e.g., "day-degrees"), the system is therefore regulated by the prevailing environmental conditions. This method contrasts with the more usual descriptions of development along a time-line. The parameters of the differential equations involved are estimated by modelling data, which show evidence of changes in the dependent variable(s), i.e. the components of the system. They are expressed in terms of their response to continuous fluctuations in one or more independent, environmental variables. Accumulated thermal time (including day-degrees) or more complex units may be derived by using either linear or nonlinear functions. Critical environmental parameters such as the basal thresholds of a given developmental process or parameters describing a nonlinear relationship with the environment may then be estimated. This paper develops the methodology of this environmentally driven approach to describing organism development in general terms, and gives a specific example of its application with reference to the cellular development within the secondary vascular tissues in the stems of young hybrid aspen trees.