Development of 'gastrostomy tube - is it for me?', a web-based patient decision aid for people living with motor neurone disease considering having a gastrostomy tube placed.

Objective: To develop and pilot a web-based patient decision aid (PDA) to support people living with motor neurone disease (plwMND) considering having a gastrostomy tube placed. Methods: In Phase 1, content and design were informed by semi-structured interviews, literature reviews and a prioritization survey. In Phase 2, the prototype PDA was tested with users and developed iteratively with feedback from surveys and 'think-aloud' interviews. Phase 1 and 2 participants were plwMND, carers and healthcare professionals (HCPs). In Phase 3, the PDA was evaluated by plwMND using validated questionnaires and HCPs provided feedback in focus groups. Results: Sixteen plwMND, 16 carers and 25 HCPs took part in Phases 1 and 2. Interviews and the literature review informed a prioritization survey with 82 content items. Seventy-seven per cent (63/82) of the content of the PDA was retained. A prototype PDA, which conforms to international standards, was produced and improved during Phase 2. In Phase 3, 17 plwMND completed questionnaires after using the PDA. Most plwMND (94%) found the PDA completely acceptable and would recommend it to others in their position, 88% had no decisional conflict, 82% were well prepared and 100% were satisfied with their decision-making. Seventeen HCPs provided positive feedback and suggestions for use in clinical practice. Conclusion: Gastrostomy Tube: Is it for me? was co-produced with stakeholders and found to be acceptable, practical and useful. Freely available from the MND Association website, the PDA is a valuable tool to support the shared decision-making process for gastrostomy tube placement.

From safety to frustration: The neural substrates of inhibitory learning in aversive and appetitive conditioning procedures.

Inhibitory associative learning counters the effects of excitatory learning, whether appetitively or aversively motivated. Moreover, the affective responses accompanying the inhibitory associations are of opponent valence to the excitatory conditioned responses. Inhibitors for negative aversive outcomes (e.g. shock) signal safety, while inhibitors for appetitive outcomes (e.g. food reward) elicit frustration and/or disappointment. This raises the question as to whether studies using appetitive and aversive conditioning procedures should demonstrate the same neural substrates for inhibitory learning. We review the neural substrates of appetitive and aversive inhibitory learning as measured in different procedural variants and in the context of the underpinning excitatory conditioning on which it depends. The mesocorticolimbic dopamine pathways, retrosplenial cortex and hippocampus are consistently implicated in inhibitory learning. Further neural substrates identified in some procedural variants may be related to the specific motivation of the learning task and modalities of the learning cues. Finally, we consider the translational implications of our understanding of the neural substrates of inhibitory learning, for obesity and addictions as well as for anxiety disorders.

Immunohistochemical differentiation of reactive from malignant mesothelium as a diagnostic aid in canine pericardial disease.

OBJECTIVES: To develop a provisional immunohistochemistry panel for distinguishing reactive pericardium, atypical mesothelial proliferation and mesothelioma in dogs. MATERIALS AND METHODS: Archived pericardial biopsies were subject to haematoxylin and eosin staining, immunohistochemistry for cytokeratin, vimentin, insulin-like growth factor II mRNA-binding protein 3, glucose transporter 1 and desmin. Samples were scored for intensity and number of cells stained. RESULTS: Ten biopsies of reactive mesothelium, 17 of atypical mesothelial proliferation, 26 of mesothelioma and five of normal pericardium were identified on the basis of haematoxylin and eosin staining. Cytokeratin and vimentin were expressed in all biopsies, confirming mesothelial origin. Normal pericardial samples had the lowest scores for insulin-like growth factor II mRNA-binding protein 3, glucose transporter 1 and desmin. Mesothelioma and atypical proliferative samples were similar to each other, with higher scores for insulin-like growth factor II mRNA-binding protein 3 and glucose transporter 1 than the reactive samples. Desmin staining was variable. Insulin-like growth factor II mRNA-binding protein 3 was the best to distinguish between disease groups. CLINICAL SIGNIFICANCE: An immunohistochemistry panel of cytokeratin, vimentin, insulin-like growth factor II mRNA-binding protein 3 and glucose transporter 1 could provide superior information compared with haematoxylin and eosin staining alone in the diagnosis of cases of mesothelial proliferation in canine pericardium, but further validation is warranted.

Canine NAPEPLD-associated models of human myelin disorders.

Canine leukoencephalomyelopathy (LEMP) is a juvenile-onset neurodegenerative disorder of the CNS white matter currently described in Rottweiler and Leonberger dogs. Genome-wide association study (GWAS) allowed us to map LEMP in a Leonberger cohort to dog chromosome 18. Subsequent whole genome re-sequencing of a Leonberger case enabled the identification of a single private homozygous non-synonymous missense variant located in the highly conserved metallo-beta-lactamase domain of the N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) gene, encoding an enzyme of the endocannabinoid system. We then sequenced this gene in LEMP-affected Rottweilers and identified a different frameshift variant, which is predicted to replace the C-terminal metallo-beta-lactamase domain of the wild type protein. Haplotype analysis of SNP array genotypes revealed that the frameshift variant was present in diverse haplotypes in Rottweilers, and also in Great Danes, indicating an old origin of this second NAPEPLD variant. The identification of different NAPEPLD variants in dog breeds affected by leukoencephalopathies with heterogeneous pathological features, implicates the NAPEPLD enzyme as important in myelin homeostasis, and suggests a novel candidate gene for myelination disorders in people.

Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

The membrane bound matrix metalloproteinase MT1-MMP plays roles in modulating cell movement, independent of its abilities to remodel the extracellular matrix. Unlike many MMPs, MT1-MMP is activated in the Golgi prior to secretion by a pro-protein convertase, primarily furin. Regulation of the activation of pro-MT1-MMP has been methodically investigated, as altering the level of the active protein has broad implications in both activating other pro-MMPs, including pro-MMP-2, and many subsequent remodelling events. Our previous work in MCF-7 cells has demonstrated that modest, and not extremely high, levels of active MT1-MMP manifests into altered cell morphology and movement. At this low but optimal amount of MT1-MMP protein, changes to MT1-MMP levels are always mirrored by MMP-9 and pERK levels, and always opposite to MMP-2 levels. In this study, stable expression of the furin inhibitor α1-antitrypsin Portland (α1-PDX) in MDA-MB-231 cells increased overall MT1-MMP levels, but cells maintained a 21% proportion of pro-MT1-MMP. The increase in MT1-MMP was mirrored by increases in MMP-9 and pERK, but a decrease in MMP-2. These changes were associated with increased NF-κB transcription. In vitro analysis showed that α1-PDX decreased cell protrusions and migration, and this manifested as decreased tumourigenesis when examined in vivo using a chick CAM assay.

Grow with the flow: a latitudinal cline in physiology is associated with more variable precipitation in Erythranthe cardinalis.

Local adaptation is commonly observed in nature: organisms perform well in their natal environment, but poorly outside it. Correlations between traits and latitude, or latitudinal clines, are among the most common pieces of evidence for local adaptation, but identifying the traits under selection and the selective agents is challenging. Here, we investigated a latitudinal cline in growth and photosynthesis across 16 populations of the perennial herb Erythranthe cardinalis (Phrymaceae). Using machine learning methods, we identify interannual variation in precipitation as a likely selective agent: southern populations from more variable environments had higher photosynthetic rates and grew faster. We hypothesize that selection may favour a more annualized life history - grow now rather than save for next year - in environments where severe droughts occur more often. Thus, our study provides insight into how species may adapt if Mediterranean climates become more variable due to climate change.

Prevalence of FoxP3+ Cells Does Not Correlate With Ki67 Expression in Canine Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of canine lymphoma and survival times are currently <1 year. Manipulation of the tumour microenvironment, of which the regulatory T cell (Treg) is a principal player, represents a potentially exciting way to curb the rapid proliferation of neoplastic cells. Tregs, characterized by the stable expression of the transcription factor FoxP3, suppress innate and adaptive arms of the immune response and represent a potential therapeutic target within neoplastic lymph nodes. This retrospective study explored the hypothesis that Tregs promote the proliferation of neoplastic large B cells, employing immunohistochemistry to assess both FoxP3 and Ki67 expression within canine lymph nodes. Fifty-seven biopsy samples of canine nodal DLBCL were examined. There were significantly fewer FoxP3+ cells in lymph nodes effaced by DLBCL than in reactive lymph nodes (27 versus 369 cells/mm2; Mann-Whitney U = 16, P = 0.011). There was no relationship between the number of intratumoural FoxP3+ cells and neoplastic cell proliferation (Spearman's rank r = 0.058, P = 0.670, 95% confidence interval). The results of this study show that FoxP3+ cells are reduced in lymph nodes effaced by DLBCL and that this change is unrelated to the expression of Ki67. This study also describes a robust digital method to standardize cell counts and facilitate future comparative studies.

Clinical Features, Imaging Characteristics, and Long-term Outcome of Dogs with Cranial Meningocele or Meningoencephalocele.

BACKGROUND: The term meningoencephalocele (MEC) describes a herniation of cerebral tissue and meninges through a defect in the cranium, whereas a meningocele (MC) is a herniation of the meninges alone. HYPOTHESIS/OBJECTIVES: To describe the clinical features, magnetic resonance imaging (MRI) characteristics, and outcomes of dogs with cranial MC and MEC. ANIMALS: Twenty-two client-owned dogs diagnosed with cranial MC or MEC. METHODS: Multicentric retrospective descriptive study. Clinical records of 13 institutions were reviewed. Signalment, clinical history, neurologic findings and MRI characteristics as well as treatment and outcome were recorded and evaluated. RESULTS: Most affected dogs were presented at a young age (median, 6.5 months; range, 1 month - 8 years). The most common presenting complaints were seizures and behavioral abnormalities. Intranasal MEC was more common than parietal MC. Magnetic resonance imaging identified meningeal enhancement of the protruded tissue in 77% of the cases. Porencephaly was seen in all cases with parietal MC. Cerebrospinal fluid (CSF) analysis identified mild abnormalities in 4 of 11 cases. Surgery was not performed in any affected dog. Seventeen patients were treated medically, and seizures were adequately controlled with anti-epileptic drugs in 10 dogs. Dogs with intranasal MEC and mild neurologic signs had a fair prognosis with medical treatment. CONCLUSION AND CLINICAL IMPORTANCE: Although uncommon, MC and MEC should be considered as a differential diagnosis in young dogs presenting with seizures or alterations in behavior. Medical treatment is a valid option with a fair prognosis when the neurologic signs are mild.

A pulse-front-tilt-compensated streaked optical spectrometer with high throughput and picosecond time resolution.

A high-throughput, broadband optical spectrometer coupled to the Rochester optical streak system equipped with a Photonis P820 streak tube was designed to record time-resolved spectra with 1-ps time resolution. Spectral resolution of 0.8 nm is achieved over a wavelength coverage range of 480 to 580 nm, using a 300-groove/mm diffraction grating in conjunction with a pair of 225-mm-focal-length doublets operating at an f/2.9 aperture. Overall pulse-front tilt across the beam diameter generated by the diffraction grating is reduced by preferentially delaying discrete segments of the collimated input beam using a 34-element reflective echelon optic. The introduced delay temporally aligns the beam segments and the net pulse-front tilt is limited to the accumulation across an individual sub-element. The resulting spectrometer design balances resolving power and pulse-front tilt while maintaining high throughput.

Markers of biological stress and mucosal immunity during a week leading to competition in adolescent swimmers.

In this study we examined changes in the salivary concentrations of immunoglobulin A (sIgA), cortisol (sC), testosterone (sT), and testosterone-to-cortisol ratio (T/C) in 21 competitive swimmers, 11-15 years old, during a week leading to competition as compared to a control (noncompetition) week. No day-to-day changes or significant differences between weeks were observed for sIgA (47.9 ± 4.4 versus 54.9 ± 5.2 µg/mL for control versus competition week, resp.), sC (2.7 ± 0.2 versus 2.5 ± 0.2 ng/mL for control versus competition week, resp.), and T/C ratio (83.4 ± 7.0 versus 77.9 ± 7.7 for control versus competition week, resp.). In contrast, sT was significantly lower during the week of competition (154.5 ± 11.3 pg/mL) as compared to the control week (181.3 ± 11.5 pg/mL) suggesting that the swimmers were in a catabolic state, although this did not have a negative effect on their performance. In conclusion, salivary cortisol did not change between the two weeks, and thus competition stress was relatively low, and mucosal immunity was unaffected in these young athletes prior to competition.

Effects of lasalocid and intermittent feeding of chlortetracycline on the growth of prepubertal dairy heifers.

Forty Holstein heifers entered the 12-wk study at approximately 12 wk of age. At enrollment, heifers were blocked by birth date and assigned to 1 of 4 treatments: (1) carrier (30 g; control); (2) lasalocid + carrier (1 mg/kg of body weight; L); (3) chlortetracycline + carrier (22 mg/kg of body weight; CTC); (4) L + CTC + carrier (CTCL). Heifers on CTC and CTCL were provided treatment Monday through Friday and carrier only on Saturday and Sunday. These heifers were provided their respective treatment during wk 1 to 4, 6, and 10; wk 5, 7 to 9, and 11 to 12 heifers were provided the nonmedicated carrier. Heifers were individually fed a total mixed ration with treatments top-dressed at 1200 h daily. Dry matter intake was monitored for each heifer and feed provided was adjusted according to individual intakes. Skeletal measurements were taken weekly and blood samples were obtained every Monday, Wednesday, and Friday. Blood samples were analyzed for thyroxine concentration via radial immunoassay. Heifers supplemented with L had lower average daily gain , overall body weight gain, and trends for lower daily body length gain and overall girth gain compared with CTC heifers, but similar to control and CTCL heifers. Heifers fed L had lower hip height gain and overall hip height gain compared with CTCL heifers, but similar to control and CTC heifers. Heifers fed L had lower overall withers height gain compared with control heifers, but similar to CTC and CTCL heifers. No treatment effect on thyroxine concentrations was observed. These data indicate that L did not increase growth. Results from this experiment indicate that supplementing heifers with L was not beneficial and no benefits to supplementing heifers with CTC or the combination of CTC and L were evident compared with control heifers. Heifers in this study experienced minimal health problems and were regarded to be under low stress levels. Supplementing CTC and L may be beneficial to growing heifers under conditions where disease exposure and stressors are greater.

A qualitative analysis of the nutritional requirements of palliative care patients.

BACKGROUND: The National Health Servive (NHS) Quality Improvement Scotland developed nutritional Clinical Standards to address the problem of malnutrition in hospitals. NHS palliative care units are obliged to incorporate these standards into nutritional aspects of care. The nutritional needs of this patient population are under-researched. The present study aimed to explore patients' views of nutrition, to begin to understand their concerns and to determine whether such standards meet the needs of patients in the palliative care setting. METHODS: A qualitative study was conducted in 2009 in an NHS Palliative Care Unit. Six inpatients were involved in one-to-one interviews, which were audiotaped and transcribed verbatim. The transcripts were subject to qualitative data analysis in accordance with a previous framework. RESULTS: A recurring theme that emerged was that of change and uncertainty. Four main areas subject to change were: disease state, symptoms, oral dietary intake and weight. Each change could exert control over, or be controlled by, the patient. When patients were eventually unable to exert control, they accepted the change, either willingly or enforced, thereby unintentionally setting their own targets. CONCLUSIONS: The present study enables a deeper understanding of the concerns that palliative care patients have regarding their oral dietary intake and weight. Their 'malnutrition' not only refers to physical malnutrition alone, but also incorporates psychological and social 'malnutrition'. When applying standards or protocols regarding nutritional care, these wider issues must be taken into account to meet patients' nutritional needs.

Genetic population structure of an anchialine shrimp, Metabetaeus lohena (Crustacea: Alpheidae), in the Hawaiian Islands.

Anchialine habitats in the Hawaiian Islands, characterized as coastal bodies of land-locked salt or brackish water that fluctuate with the tides due to subterranean connections, are the only ecosystems of this type found within the United States. These habitats are currently subject to anthropogenic impacts that threaten their future existence. Previous research has shown strong genetic population structure of an endemic atyid shrimp, Halocaridina rubra, in these habitats. The native alpheid shrimp, Metabetaeus lohena, whose known range entirely overlaps that of H. rubra, has feeding and reproductive behaviors that are biologically distinct from H. rubra. Its historic scarcity and status as a candidate for the US Fish and Wildlife Department's Endangered Species List, make M. lohena an ideal species to compare against the known genetic structure of H. rubra. We investigated the population structure of this native anchialine shrimp to test the hypothesis that genetic population structure differs between the two shrimp species and that M. lohena is genetically unstructured across its range. A survey of 605 bp of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 127 individuals collected at 7 sites spanning the islands of O'ahu, Maui and Hawaii revealed 43 haplotypes. The most common haplotype was represented in similar proportions from all sites sampled, accounting for 44% of the surveyed sequences. Analyses of molecular variation (AMOVA), pairwise PhiST values, Bayesian estimates of migration (M), Mantel tests and Nested Clade Analyses (NCAs) all failed to reveal evidence of major barriers to gene flow among most populations separated by inter-island channels. This lack of genetic structure in M. lohena is found to be in stark contrast with the highly structured population of H. rubra, and may be attributed to oceanic dispersal strategies and/or a recent introduction to the Hawaiian Islands.

Genetic population structure of an anchialine shrimp, Metabetaeus lohena (Crustacea: Alpheidae), in the Hawaiian Islands

Anchialine habitats in the Hawaiian Islands, characterized as coastal bodies of land-locked salt or brackish water that fluctuate with the tides due to subterranean connections, are the only ecosystems of this type found within the United States. These habitats are currently subject to anthropogenic impacts tha t threaten their future existence. Previous research has shown strong genetic population structure of an endemic atyid shrimp, Halocaridina rubra, in these habitats. The native alpheid shrimp, Metabetaeus lohena, whose known range entirely overlaps that of H. rubra, has feeding and reproductive behaviors that are biologically distinct from H. rubra. Its historic scarcity and status as a candidate for the US Fish and Wildlife Department’s Endangered Species List, make M. lohena an ideal species to compare against the known genetic structure of H. rubra. We investigated the population structure of this native anchialine shrimp to test the hypothesis that genetic population structure differs between the two shrimp species and that M. lohena is genetically unstructured across its range. A survey of 605 bp of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 127 individuals collected at 7 sites spanning the islands of O’ahu, Maui and Hawaii revealed 43 haplotypes. The most common haplotype was represented in similar proportions from all sites sampled, accounting for 44% of the surveyed sequences. Analyses of molecular variation (AMOVA), pairwise ΦST values, Bayesian estimates of migration (M), Mantel tests and Nested Clade Analyses (NCAs) all failed to reveal evidence of major barriers to gene flow among most populations separated by inter-island channels. This lack of genetic structure in M. lohena is found to be in stark contrast with the highly structured population of H. rubra, and may be attributed to oceanic dispersal strategies and/or a recent introduction to the Hawaiian Islands. Rev. Biol. Trop. 58 (1): 159-170. Epub 2010 March 01.

Los hábitats de los alfeidos de las islas de Hawaii, se caracterizan por ser zonas cerradas de aguas saladas o salobres, que fluctúan con las mareas, debido a las conexiones subterráneas, son los únicos ecosistemas de este tipo que se encuentran en Estados Unidos. Estos hábitats actualmente están sujetos a impactos antropogénicos que amenazan su existencia futura. La investigación anterior ha demostrado una fuerte estructura genética de una población de camarones atíidos endémicos, Halocaridina rubra, en estos hábitats. El camarón alfeido nativo, Metabetaeus lohena, cuya área de distribución conocida se superpone totalmente con la de H. rubra, tiene comportamientos alimenticios y reproductivos que son biológicamente diferentes a los de H. rubra. Su escasez histórica y su condición de candidato para aparecer en la Lista de Especies en Peligro del Departamento de Pesca y Vida Silvestre de Estados Unidos, hace de M. lohena una especie ideal para comparar su estructura genética con la de H. rubra. Se investigó la estructura de la población de este camarón alfeido nativo para probar la hipótesis que la estructura genética de la población difiere entre las dos especies y que la de M. lohena está genéticamente no estructurada en todo su ámbito. El análisis de 605 pb de la oxidasa mitocondrial citocromo c subunidad I (COI) de genes de 127 individuos recolectados en 7 sitios que abarcan las islas de Oahu, Maui y Hawaii reveló 43 haplotipos. El haplotipo más común fue representado en proporciones similares en todos los sitios incluidos en la muestra, de acuerdo al 44% de las secuencias estudiadas. El análisis de variación molecular (AMOVA), los valores de ΦST pareados, la estimación bayesiana de la migración (M), las pruebas de Mantel y los Análisis Cladísticos no pudieron revelar la existencia de importantes barreras al flujo genético entre las poblaciones más separadas por los canales entre las islas. La falta de estructura genética en M. lohena contrasta con la muy estructurada población de H. rubra, y puede ser atribuida a las estrategias de dispersión oceánica y/o una introducción reciente en las islas hawaianas.

Enzyme immunoassay of testosterone, 17beta-estradiol, and progesterone in perspiration and urine of preadolescents and young adults: exceptional levels in men's axillary perspiration.

Enzyme immunoassays for testosterone, 17beta-estradiol, and progesterone were validated for human facial and axillary perspiration and compared to levels in urine. In study 1, these assays were applied to samples from preadolescent girls and boys and young women and men. Men's axillary perspiration contained substantially higher levels of steroids than seen in other substrates from men or in any sample from women, boys, and girls. Male axillary steroid levels were very variable across individuals, and on average they exceeded levels in facial perspiration by 90-fold for testosterone and 45-fold for estradiol. Men's urinary testosterone also exceeded urinary levels of the other subjects. In study 2, axillary perspiration, urine, and saliva were collected from young men. Substantial axillary levels of testosterone and estradiol were again observed. Correlations of the same hormone among the different substrates were generally very low, except for a small correlation between estradiol levels measured in axillary perspiration and urine in study 2. High unconjugated steroid content in men's axillary excretions could, if absorbed by women during intimacy, be implicated in pheromonal activity.

Conservation implications of hybridization in Hawaiian picture-winged Drosophila.

In this review, we discuss the importance of hybridization among species for the conservation of Hawaiian picture-winged Drosophila. Hybridization can be a positive evolutionary process that creates new species and increases the adaptation of populations and species through the spread of adaptive alleles and traits. Conversely, hybridization can disrupt the genetic integrity of species or populations and this may be most detrimental among taxa that are recently hybridizing due to recent ecological changes. The loss of biodiversity in Hawaiian Drosophila through hybridization may be facilitated by habitat alteration and introduced species that reduce population sizes and alter geographic distributions of native species. We briefly review the evidence for hybridization in the genus Drosophila and then focus on hybridization in the Hawaiian picture-winged Drosophila. We examine three general approaches for identifying hybrids and for assessing the factors that appear to contribute to hybridization and the potential ecological and evolutionary outcomes of hybrids in natural populations. Overall, the potential for hybridization among species will likely increase the risk of extinction for Hawaiian picture-winged Drosophila species. Thus, it is important to consider the potential for hybridization among species when developing plans for the conservation of Hawaiian Drosophila.

Involvement of noradrenergic and corticoid receptors in the consolidation of the lasting anxiogenic effects of predator stress.

The roles of beta-NER (beta-noradrenergic receptor), GR (glucocorticoid) and mineral corticoid receptors (MR) in the consolidation of anxiogenic effects of predator stress were studied. One minute after predator stress, different groups of rats were injected (ip) with vehicle, propranolol (beta-NER blocker, 5 and 10 mg/kg), mifepristone (RU486, GR blocker, 20 mg/kg), spironolactone (MR blocker, 50 mg/kg), propranolol (5 mg/kg) plus RU486 (20 mg/kg) or the anxiolytic, chloradiazepoxide (CPZ, 10 mg/kg). One week later, rodent anxiety was assessed in elevated plus maze, hole board, light/dark box, social interaction and acoustic startle. Considering all tests except startle, propranolol dose dependently blocked consolidation of lasting anxiogenic effects of predator stress in all tests. GR receptor block alone was ineffective. However, GR block in combination with an ineffective dose of propranolol did blocked consolidation of predator stress effects in all tests, suggesting a synergism between beta-NER and GR. Surprisingly, MR block prevented consolidation of anxiogenic effects in all tests except the light/dark box. CPZ post stress was ineffective against the anxiogenic impact of predator stress. Study of startle was complicated by the fact that anxiogenic effects of stress on startle amplitude manifested as both an increase and a decrease in startle amplitude. Suppression of startle occurred in stressed plus vehicle injected groups handled three times prior to predator stress. In contrast, stressed plus vehicle rats handled five times prior to predator stress showed increases in startle, as did all predator stressed only groups. Mechanisms of consolidation of the different startle responses appear to differ. CPZ post stress blocked startle suppression but not enhancement of startle. Propranolol post stress had no effect on either suppression or enhancement of startle. GR block alone post stress prevented suppression of startle, but not enhancement. In contrast blocking GR and beta-NER together prevented startle enhancement. MR block also prevented startle enhancement. Effects of MR block on startle suppression were not tested. Delay of habituation to startle was found in all stressed rats. Consolidation of delay of habituation was blocked or attenuated by post stress MR block, GR plus beta-NER block and CPZ but not by post stress GR or beta-NER block alone. Taken together, present findings suggest consolidation of lasting anxiogenic effects of predator stress may share some of the same neurochemical mechanisms implicated in some forms of fear memory consolidation. Implications of these findings for the study of stress-induced changes in affect including posttraumatic stress disorder (PTSD) are discussed.

Nitric oxide attenuates resistance to doxorubicin in three-dimensional aggregates of human breast carcinoma cells.

Compared with monolayer culture, tumour cells cultured as multicellular aggregates (spheroids) exhibit much higher levels of resistance to chemotherapeutic agents, a phenomenon known as multicellular resistance (MCR). Associated with multicellular aggregates is a heterogeneous microenvironment characterised by gradients in oxygen, pH, and nutrients. We previously showed that nitric oxide (NO) signalling plays an important role in the regulation of chemosensitivity in cancer cells cultured as monolayer, and that hypoxia increases resistance to anti-cancer agents largely through a mechanism involving the inhibition of NO signalling. The goal of the present study was to determine whether NO mimetics chemosensitize breast cancer cells in spheroid cultures. Survival of MDA-MB-231 breast carcinoma cells was determined by clonogenic assay following spheroid culture, doxorubicin exposure, and NO mimetic administration. When spheroids were incubated for 24 h with the NO mimetics diethylenetriamine/nitric oxide adduct (DETA/NO) and glyceryl trinitrate (GTN), cell survival after doxorubicin (200 microM) exposure was decreased by 33% (p<0.006) and by up to 47% (p<0.02), respectively. Nitric oxide-mediated signalling involves the generation of the second messenger cyclic guanosine monophosphate (cGMP). Administration of a non-hydrolysable cGMP analogue, 8-Bromo-cGMP, significantly decreased MCR (p<0.04). The effect of NO mimetic exposure on tumour cell chemosensitivity was not due to increased penetration of doxorubicin into spheroids, nor was it associated with an increase in cell proliferation. These results suggest that NO mimetics attenuate MCR to doxorubicin through a mechanism involving cGMP-dependent signalling. Therefore, NO-mimetics may potentially be used as chemosensitizers in cancer therapy.

Enzymeimmunoassay of oestradiol, testosterone and progesterone in urine samples from female mice before and after insemination.

ELISA measurements of 17beta-oestradiol, testosterone and progesterone were determined for urine samples collected non-invasively from female mice. Initial samples were collected during 5 successive days while mature female mice were isolated and cyclic. Subsequently, female mice were inseminated and additional urine samples were collected during days 2-6 after observation of copulatory plugs. Measurements of oestradiol and testosterone showed variance over days within individuals and did not significantly differ in measurements taken before or after insemination. Progesterone concentrations were significantly higher after insemination compared with before mating. In a second sample of inseminated females, urinary progesterone was measured during days 2-18 of pregnancy. Most females showed high urinary progesterone up to day 10 of pregnancy and lower concentrations during the remainder of gestation. These results indicate that urinary progesterone reflects established systemic increases of this hormone during pregnancy.