Optogenetic silencing of a corticotropin-releasing factor pathway from the central amygdala to the bed nucleus of the stria terminalis disrupts sustained fear.

The lateral central nucleus of the amygdala (CeAL) and the dorsolateral bed nucleus of the stria terminalis (BNSTDL) coordinate the expression of shorter- and longer-lasting fears, respectively. Less is known about how these structures communicate with each other during fear acquisition. One pathway, from the CeAL to the BNSTDL, is thought to communicate via corticotropin-releasing factor (CRF), but studies have yet to examine its function in fear learning and memory. Thus, we developed an adeno-associated viral-based strategy to selectively target CRF neurons with the optogenetic silencer archaerhodopsin tp009 (CRF-ArchT) to examine the role of CeAL CRF neurons and projections to the BNSTDL during the acquisition of contextual fear. Expression of our CRF-ArchT vector injected into the amygdala was restricted to CeAL CRF neurons. Furthermore, CRF axonal projections from the CeAL clustered around BNSTDL CRF cells. Optogenetic silencing of CeAL CRF neurons during contextual fear acquisition disrupted retention test freezing 24 h later, but only at later time points (>6 min) during testing. Silencing CeAL CRF projections in the BNSTDL during contextual fear acquisition produced a similar effect. Baseline contextual freezing, the rate of fear acquisition, freezing in an alternate context after conditioning and responsivity to foot shock were unaffected by optogenetic silencing. Our results highlight how CeAL CRF neurons and projections to the BNSTDL consolidate longer-lasting components of a fear memory. Our findings have implications for understanding how discrete amygdalar CRF pathways modulate longer-lasting fear in anxiety- and trauma-related disorders.

The Equine Movement Disorder "Shivers" Is Associated With Selective Cerebellar Purkinje Cell Axonal Degeneration.

"Shivers" is a progressive equine movement disorder of unknown etiology. Clinically, horses with shivers show difficulty walking backward, assume hyperflexed limb postures, and have hind limb tremors during backward movement that resembles shivering. At least initially, forward movements are normal. Given that neither the neurophysiologic nor the pathologic mechanisms of the disease is known, nor has a neuroanatomic locus been identified, we undertook a detailed neuroanatomic and neuropathologic analysis of the complete sensorimotor system in horses with shivers and clinically normal control horses. No abnormalities were identified in the examined hind limb and forelimb skeletal muscles nor the associated peripheral nerves. Eosinophilic segmented axonal spheroids were a common lesion. Calretinin-positive axonal spheroids were present in many regions of the central nervous system, particularly the nucleus cuneatus lateralis; however, their numbers did not differ significantly from those of control horses. When compared to controls, calretinin-negative, calbindin-positive, and glutamic acid decarboxylase-positive spheroids were increased 80-fold in Purkinje cell axons within the deep cerebellar nuclei of horses with shivers. Unusual lamellar or membranous structures resembling marked myelin decompaction were present between myelin sheaths of presumed Purkinje cell axons in the deep cerebellar nuclei of shivers but not control horses. The immunohistochemical and ultrastructural characteristics of the lesions combined with their functional neuroanatomic distribution indicate, for the first time, that shivers is characterized by end-terminal neuroaxonal degeneration in the deep cerebellar nuclei, which results in context-specific hypermetria and myoclonus.

Familial rare EGFR-mutant lung cancer syndrome: Review of literature and description of R776H family.

BACKGROUND: Interest in hereditary lung cancer is increasing, in particular germline mutations in the Epidermal Growth Factor Receptor (EGFR) gene. We review the current literature on this topic, discuss risk of developing lung cancer, treatment and screening options and describe a family of 3 sisters with lung cancer and their unaffected mother all with a rare EGFR germline mutation (EGFR p.R776H). METHODS: We searched PubMed, Medline, Embase, the Cochrane Library, Google Scholar and scanned reference lists of articles. Search terms included "EGFR germline" and "familial lung cancer" or "EGFR familial lung cancer". We also describe our experience of managing a family with rare germline EGFR mutant lung cancer. RESULTS: Although the numbers are small, the described cases in the literature show several similarities. The patients are younger and usually have no or light smoking history. 50% of the patients were treated with a tyrosine kinase inhibitor (TKIs) with OS over six months. CONCLUSION: Although rare, germline p.R776H EGFR lung cancer mutations are over-represented in light or never smoking female patients who often also possess an additional somatic EGFR mutation. Treatment with TKIs appears suitable but further research is needed into the appropriate screening regime for unaffected carriers or light/never smokers.

Transmission of canine transmissible venereal tumour between two dogs in the UK.

Canine transmissible venereal tumour (CTVT) is a contagious cancer spread by transfer of living cancer cells. Occasional cases are observed in the UK in dogs imported from endemic regions. Here, we report a case of imported canine transmissible venereal tumour that was transmitted to a second dog within the UK. Transmission of genital canine transmissible venereal tumour occurred despite neutered status of the second dog. The aggressive course of disease in both cases, which included metastasis, resistance to therapeutic interventions and ultimate euthanasia of both dogs, is described. The diagnosis of canine transmissible venereal tumour was made using a combination of cytology, histology, immunohistochemistry and PCR to detect the LINE-MYC rearrangement. Practitioners unfamiliar with canine transmissible venereal tumour are reminded of this disease of concern, particularly when imported dogs are placed in multi-dog households, irrespective of neuter status.

Testing spatial working memory in pigs using an automated T-maze.

Pigs are an important large animal model for translational clinical research but underutilized in behavioral neuroscience. This is due, in part, to a lack of rigorous neurocognitive assessments for pigs. Here, we developed a new automated T-maze for pigs that takes advantage of their natural tendency to alternate. The T-maze has obvious cross-species value having served as a foundation for cognitive theories across species. The maze (17' × 13') was constructed typically and automated with flanking corridors, guillotine doors, cameras, and reward dispensers. We ran nine pigs in (1) a simple alternation task and (2) a delayed spatial alternation task. Our assessment focused on the delayed spatial alternation task which forced pigs to wait for random delays (5, 60, 120, and 240 s) and burdened spatial working memory. We also looked at self-paced trial latencies, error types, and coordinate-based video tracking. We found pigs naturally alternated but performance declined steeply across delays (R2 = 0.84). Self-paced delays had no effect on performance suggestive of an active interference model of working memory. Positional and head direction data could differentiate subsequent turns on short but not long delays. Performance levels were stable over weeks in diverse strains and sexes, and thus provide a benchmark for future neurocognitive assessments in pigs.

Qualitative research in nutrition and dietetics: data collection issues.

The wide scope of qualitative enquiry presents the researcher with a number of choices regarding data collection and sampling. Selecting data collection and sampling techniques can therefore be somewhat daunting, particularly because, often, there is no single, universally accepted 'correct' option. Appropriate research methods are, however, crucial to ensure high-quality research. This review, the second in the series, provides an overview of the principal techniques of data collection and sampling that may be used for qualitative research in nutrition and dietetics. In addition, it describes a process for choosing appropriate data collection and sampling methods that considers the extent to which they provide data that answers the research question(s) and are compatible with the philosophical assumptions about ontology, epistemology and methodology that underpin the overall design of a study. Finally, it discusses the central role that the researcher plays in qualitative data collection and encourages researchers to acknowledge and reflect upon their impact on the data. Other reviews in this series provide a model for embarking on a qualitative research project in nutrition and dietetics, an overview of the principal techniques of data analysis and quality assessment of this kind of research, and some practical advice relevant to nutrition and dietetics, along with glossaries of key terms.

Association of the melioidosis agent Burkholderia pseudomallei with water parameters in rural water supplies in Northern Australia.

We analyzed water parameters and the occurrence of the melioidosis agent Burkholderia pseudomallei in 47 water bores in Northern Australia. B. pseudomallei was associated with soft, acidic bore water of low salinity but high iron levels. This finding aids in identifying water supplies at risk of contamination with this pathogenic bacterium.

Dual sensitization to rat and mouse urinary allergens reflects cross-reactive molecules rather than atopy.

BACKGROUND: Sensitization to rats and mice can develop in laboratory animal workers exposed to only one species. Reasons for this dual sensitization are unclear but may reflect a genetic predisposition to developing allergy (atopy) or alternatively cross-reactivity between rat and mouse urinary allergens. We examined cross-reactivity between rat and mouse urine and the effect atopy has on dual sensitization in laboratory animal workers. METHODS: In a cross-sectional study the frequency of sensitization to rat and/or mouse was analysed in 498 employees exposed to both rat and mouse at work and 220 to rat only. RAST inhibitions, western blots and blot inhibitions were carried out on a subset of five individuals to assess cross-reactivity. RESULTS: Fourteen per cent of workers were sensitized to rats and 9% to mouse. Over half (62%) of rat sensitized individuals were also mouse sensitized and the majority (91%) of mouse sensitized individuals were also rat sensitized. IgE cross-reactivity was demonstrated between rat and mouse urine using RAST inhibitions. Rates of atopy did not differ between rat only sensitized individuals compared with those sensitized to both species. Sensitization to cats and rabbits was more common amongst those with dual sensitization. CONCLUSIONS: Dual sensitization to rat and mouse reflects IgE cross-reactivity rather than atopy. Individuals with dual sensitization are more likely to be sensitized to other animal allergens. These findings will have implications for individuals working with only one rodent species who develop sensitization and symptoms to be aware of the potential for allergy to other species.

Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids.

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

The nutritional vulnerability of older Guyanese in residential homes.

OBJECTIVE: To assess the nutritional status, functional ability and food intake of older Guyanese in residential care. METHODS: Eighty-four residents of one public and two private homes underwent an anthropometric and functional ability assessment including height, weight, armspan, arm and calf circumferences and handgrip strength. Food intake in two private homes was measured over seven days by direct weighing and the use of consumption units. RESULTS: The overall prevalence of underweight was 26.2% and of overweight was 17.8% but the prevalence of underweight was higher in the public home (29.3% underweight and 17.2% overweight in the public home, and 19.2% underweight and 19.2% overweight in the private home). Mean handgrip strength was 26 kg in males and 17.7 kg in females. The nutritional adequacy of the diet provided by one of the homes was poor with the food providing less than 50% of the required amount of zinc and vitamins A, D and C. Neither home met the requirement for energy. CONCLUSION: A high prevalence of malnutrition exists in a public home for the elderly and, to a lesser degree, in two private homes. In the context of a rapidly ageing population and tight financial constraints, the challenge of providing an adequate diet must be given priority.

Epidemiology of shivering (shivers) in horses.

REASONS FOR PERFOMING STUDY: Investigating the epidemiology of shivering in horses. OBJECTIVES: The purpose of this study was to characterise the signalment, clinical signs and management factors associated with shivering (also known as shivers), a relatively rare, poorly defined movement disorder in horses. STUDY DESIGN: Web-based case series survey and case-control study. METHODS: A Web-based survey was used to obtain information from owners, worldwide, who suspected that their horse had shivering. Survey respondents were asked to answer standardised questions and to provide a video of the horse. Authors reviewed the surveys and videos, and horses were diagnosed with shivering if they displayed normal forward walking, with difficulty during manual lifting of the hoof and backward walking due to hyperflexion or hyperextension of the pelvic limbs. Cases confirmed by video were designated 'confirmed shivering', while those with compatible clinical signs but lacking video confirmation were designated 'suspected shivering'. Owners of confirmed shivering horses were asked to provide information on 2 horses without signs of shivering (control group). RESULTS: Three hundred and five surveys and 70 videos were received; 27 horses were confirmed shivering (50 controls), 67 were suspected shivering and the rest had a variety of other movement disorders. Suspected shivering horses resembled confirmed shivering cases, except that the suspected shivering group contained fewer draught breeds and fewer horses with exercise intolerance. Confirmed shivering signs often began at <5 years of age and progressed in 74% of cases. Owner-reported additional clinical signs in confirmed cases included muscle twitching (85%), muscle atrophy (44%), reduced strength (33%) and exercise intolerance (33%). Shivering horses were significantly taller (confirmed shivering, mean ∼173 cm; control horses, ∼163 cm) with a higher male:female ratio (confirmed shivering, 3.2:1 vs. control, 1.7:1). No potential triggering factors or effective treatments were reported. CONCLUSIONS: Shivering is a chronic, often gradually progressive movement disorder that usually begins before 7 years of age and has a higher prevalence in tall male horses.

Posture and movement characteristics of forward and backward walking in horses with shivering and acquired bilateral stringhalt.

REASONS FOR PERFORMING STUDY: To investigate and further characterise posture and movement characteristics during forward and backward walking in horses with shivering and acquired, bilateral stringhalt. OBJECTIVES: To characterise the movement of horses with shivering (also known as shivers) in comparison with control horses and horses with acquired bilateral stringhalt. STUDY DESIGN: Qualitative video analysis of gait in horses. METHODS: Owners' and authors' videos of horses with shivering or stringhalt and control horses walking forwards and backwards and manually lifting their limbs were examined subjectively to characterise hyperflexion, hyperextension and postural abnormalities of the hindlimbs. The pattern and timing of vertical displacement of a hindlimb over one stride unit was evaluated among control, shivering and stringhalt cases. RESULTS: Gait patterns of shivering cases were characterised as follows: shivering-hyperextension (-HE, n = 13), in which horses subjectively showed hyperextension when backing and lifting the limb; shivering-hyperflexion (-HF, n = 27), in which horses showed hindlimb hyperflexion and abduction during backward walking; and shivering-forward hyperflexion (-FHF, n = 4), which resembled shivering-HF but included intermittent hyperflexion and abduction with forward walking. Horses with shivering-HF, shivering-FHF and stringhalt (n = 7) had a prolonged swing phase duration compared with control horses and horses with shivering-HE during backward walking. With the swing phase of forward walking, horses with stringhalt had a rapid ascent to adducted hyperflexion of the hindlimb, compared with a rapid descent of the hindlimb after abducted hyperflexion in horses with shivering-FHF. CONCLUSIONS: Shivering affects backward walking, with either HE or HF of hindlimbs, and can gradually progress to involve intermittent abducted hyperflexion during forward walking. Shivering-HF and shivering-FHF can look remarkably similar to acquired bilateral stringhalt during backward walking; however, stringhalt can be distinguished from shivering-HF by hyperflexion during forward walking and from shivering-FHF by an acute onset of a more consistent, rapidly ascending, hyperflexed, adducted hindlimb gait at a walk.

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.

Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.

Seven unique food consumption patterns identified among women in the UK Women's Cohort Study.

OBJECTIVE: [corrected] To identify groups of subjects with similar food consumption patterns so that complex disease-diet relationships can be investigated at the level of the whole diet, rather than just in terms of nutrient intake. SUBJECTS: 33,971 women in the UK Women's Cohort Study. 60,000 women on the World Cancer Research Fund mailing list were initially invited to take part. Subjects were selected to include a high proportion of vegetarians. DESIGN: The cohort completed a 217 item food frequency questionnaire. Cluster analysis was used to identify groups of women with similar food consumption patterns. Clusters were compared on socio-demographic characteristics, indicators of health and diet, and nutrient intakes. RESULTS: Seven clusters were identified including two vegetarian clusters. Groups appeared to be differentiated by differences in food types and in diversity of diet. Socio-demographic, health and diet characteristics and nutrient intakes all differed significantly between groups. CONCLUSION: Classifying diets in more pragmatic terms than just nutrient intake should provide valuable insight into understanding complex diet-disease relationships. Dietary advice, whilst based on nutrient content of meals, needs to take account of the combinations of different food types that people naturally choose to use together. SPONSORSHIP: World Cancer Research Fund.

Consumer focus can spur group practice turnaround.

Many healthcare organizations have lost money on their employed group practices. The solution to this dilemma is not necessarily divestment of the group practices. Instead, some healthcare organizations should view their physicians as an asset. Healthcare organizations and physicians need to develop a new framework for their relationship to optimize their competitive advantage. Three guiding principles that will help accomplish this objective are to recast the healthcare organization-physician relationship to focus on the consumer, reconfigure the economic model to exceed consumer demands, and restructure the group practice to encourage fiscal and service excellence. In developing a new relationship framework, the stakeholders need to define the group practice's mission, strategic direction, composition, infrastructure, compensation model, and structure.

Strategic planning: going beyond the basics.

Boards are discovering that many of the strategies of the '90s that took their organizations far afield from their core mission did not pan out as intended. Now they need to take a new strategic approach--combining the basics with a view toward a dynamic health care environment.

Pituitary null cell adenoma in a domestic llama (Lama glama).

Pituitary gland neoplasia has been reported rarely in camelids. A 12-year-old neutered male llama (Lama glama) presented with lethargy, inappetence and neurological signs. On physical examination, the llama was mentally dull and exhibited compulsive pacing and circling to the left. Complete blood count and serum biochemistry revealed haemoconcentration, mild hypophosphataemia, hyperglycaemia, hypercreatininaemia and hyperalbuminaemia. Humane destruction was elected due to rapid clinical deterioration and poor prognosis. Post-mortem examination revealed a pituitary macroadenoma and bilateral internal hydrocephalus. Microscopically, the pituitary tumour was composed of neoplastic chromophobic pituitary cells. Ultrastructural studies revealed similar neoplastic cells to those previously described in human null cell adenomas. Immunohistochemically, the neoplastic cells were strongly immunoreactive for neuroendocrine markers (synaptophysin and chromogranin A), but did not exhibit immunoreactivity for epithelial, mesenchymal, neuronal and all major pituitary hormone markers (adrenocorticotropic hormone, follicle stimulating hormone, growth hormone, luteinizing hormone, melanocyte-stimulating hormone, prolactin and thyroid stimulating hormone), consistent with the diagnosis of a pituitary null cell adenoma. This is the first report of pituitary neoplasia in a llama.