Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Developmental temperature stress and parental identity shape offspring burst swimming performance in sockeye salmon (Oncorhynchus nerka)

Abstract – The persistent effects of embryonic temperature stress and individual parentage on fry swimming performance were examined in a cross‐fertilisation experiment using sockeye salmon (Oncorhynchus nerka). A fixed‐velocity test of burst swimming was used to assess the endurance capacity and behavioural performance of individual fry from 10 offspring families incubated at 12, 14 or 16 °C to hatch and then reared through yolk absorption and exogenous feeding stages in a common posthatch environment (average 6.9 °C). Fry burst swim time (BST) was influenced by an interaction between incubation temperature and family identity. Average BST was longer for fry from the 12 °C prehatch treatment compared to 14 and 16 °C, although differences were largely attributable to temperature effects on average fry size. Behavioural observations revealed that fish incubated at 16 °C performed more poorly, having a larger proportion of individuals that required stimulation to swim, fatigued more frequently or were classified as ‘nonswimmers’. Within all three incubation temperature treatments, mean BST varied significantly among offspring families, independent of fry mass and length. An interesting relationship was observed within the 16 °C treatment, whereby families with higher survivorship were characterised with lower mean BSTs. Collectively, these findings demonstrate that exposure to high temperatures in early sockeye salmon development can result in persistent, parentally mediated effects on fry performance. As such, these results provide important insight into how elevated temperature events during egg incubation may affect early life history selection processes and survival in stages beyond when the stressor is experienced.     

Variability in in vitro macrophage activation by commercially diverse bulk echinacea plant material is predominantly due to bacterial lipoproteins and lipopolysaccharides

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends upon bacterial lipopolysaccharides and Braun-type bacterial lipoproteins. We determined the contribution made by these bacterial components to the overall immune-enhancing activity detected in E. purpurea and E. angustifolia bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze-dried, freshly harvested Echinacea plants to those harvested and dried using various commercially relevant conditions suggest that postharvesting procedures do not substantially contribute to the variation observed in the commercial material.     

Stocking density and artificial habitat influence stock structure and yield from intensive nursery systems for mud crabs Scylla serrata (Forsskål 1775)

Intensive nursery systems are designed to culture mud crab postlarvae through a critical phase in preparation for stocking into growout systems. This study investigated the influence of stocking density and provision of artificial habitat on the yield of a cage culture system. For each of three batches of postlarvae, survival, growth and claw loss were assessed after each of three nursery phases ending at crab instars C1/C2, C4/C5 and C7/C8. Survival through the first phase was highly variable among batches with a maximum survival of 80% from megalops to a mean crab instar of 1.5. Stocking density between 625 and 2300 m⁻² did not influence survival or growth in this first phase. Stocking densities tested in phases 2 and 3 were 62.5, 125 and 250 m⁻². At the end of phases 2 and 3, there were five instar stages present, representing a more than 20‐fold size disparity within the populations. Survival became increasingly density‐sensitive following the first phase, with higher densities resulting in significantly lower survival (phase 2: 63% vs. 79%; phase 3: 57% vs. 64%). The addition of artificial habitat in the form of pleated netting significantly improved survival at all densities. The mean instar attained by the end of phase 2 was significantly larger at a lower stocking density and without artificial habitat. No significant effect of density or habitat on harvest size was detected in phase 3. The highest incidence of claw loss was 36% but was reduced by lowering stocking densities and addition of habitat. For intensive commercial production, yield can be significantly increased by addition of a simple net structure but rapidly decreases the longer crablets remain in the nursery.     

Cryptic genetic diversity in “widespread” Southeast Asian bird species suggests that Philippine avian endemism is gravely underestimated

Mistakenly classifying morphologically cryptic endemic species as populations of widespread species potentially interferes with the conservation of biodiversity because undetected endemics that are imperilled may lack appropriate protection. It also impedes the reconstruction of the evolutionary history of a taxon by obscuring the number and distributional limits of species. Here, we present genetic and phylogenetic evidence corroborated by morphology that Philippine populations of seven widespread, non-migratory passerine birds might represent unrecognized, distinct species. An extrapolation based on this finding suggests that the proportion of endemic bird species in the Philippines could be much higher than currently estimated. This high degree of cryptic diversity in a well-studied, volant taxon implies that large numbers of unrecognized species can be expected in less thoroughly studied groups. We predict that genetic investigations of insular populations of widespread species will frequently reveal unrecognized island endemics, and because of the vulnerability of island habitats and their biota, these taxa may be particularly susceptible to extinction.     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

The roles of subsurface carbon and hydrogen in palladium-catalyzed alkyne hydrogenation

Alkynes can be selectively hydrogenated into alkenes on solid palladium catalysts. This process requires a strong modification of the near-surface region of palladium, in which carbon (from fragmented feed molecules) occupies interstitial lattice sites. In situ x-ray photoelectron spectroscopic measurements under reaction conditions indicated that much less carbon was dissolved in palladium during unselective, total hydrogenation. Additional studies of hydrogen content using in situ prompt gamma activation analysis, which allowed us to follow the hydrogen content of palladium during catalysis, indicated that unselective hydrogenation proceeds on hydrogen-saturated beta-hydride, whereas selective hydrogenation was only possible after decoupling bulk properties from the surface events. Thus, the population of subsurface sites of palladium, by either hydrogen or carbon, governs the hydrogenation events on the surface.     

Body weight of newborn and suckling piglets affects their intestinal gene expression

Modern hyperprolific sows must deal with large litters (16–20 piglets) which reduce piglet birthweight with a concomitant increase in the proportion of small and intrauterine growth retarded piglets. However, larger litters do not only have a greater variation of piglet weights, but also a greater variation in colostrum and milk consumption within the litter. To further understand the impact that body weight has on piglets, the present study aimed to evaluate the degree of physiological weakness of the smallest piglets at birth and during the suckling period (20 d) compared to their middle-weight littermates through their jejunal gene expression. At birth, light piglets showed a downregulation of genes related to immune response (FAXDC2, HSPB1, PPARGC1α), antioxidant enzymes (SOD2m), digestive enzymes (ANPEP, IDO1, SI), and nutrient transporter (SLC39A4) (P < 0.05) but also a tendency for a higher mRNA expression of GBP1 (inflammatory regulator) and HSD11β1 (stress hormone) genes compared to their heavier littermates (P < 0.10). Excluding HSD11β1 gene, all these intestinal gene expression differences initially observed at birth between light and middle-weight piglets were stabilized at the end of the suckling period, when others appeared. Genes involved in barrier function (CLDN1), pro-inflammatory response (CXCL2, IL6, IDO1), and stress hormone signaling (HSD11β1) over-expressed compared to their middle-weight littermates (P < 0.05). In conclusion, at birth and at the end of suckling period, light body weight piglets seem to have a compromised gene expression and therefore impaired nutrient absorption, immune and stress responses compared to their heavier littermates.     

日光温室保温被卷放位置对温度环境的影响

温室保温被卷放位置影响着室内的太阳能得热及通过前屋面的散热,从而影响着温室温度。该研究以室外气象因子为模拟输入条件,分别模拟晴天、阴天不同保温被卷放位置对12 m跨度日光温室温度环境的影响。保温被卷放位置分别为保温被卷至前屋面一半与至顶2种方案。模拟结果显示：白天保温被卷至前屋面一半的室内空气温度高于保温被卷至屋顶的温度,而夜间2种方案室内空气温度差异不明显;晴天保温被卷至屋顶与卷至前屋面一半相比,可明显提高夜间墙体表面温度,但对后坡的夜间表面温度影响较小。该研究结果对温室生产中晴天、阴天保温被卷的合理放