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.     

Anaemia, iron status and vitamin A deficiency among adolescent refugees in Kenya and Nepal

OBJECTIVE: To investigate the prevalence of anaemia (haemoglobin<11.0 to 13.0 g dl(-1) depending on age and sex group), iron deficiency (transferrin receptor concentration>8.3 microg ml(-1)) and vitamin A deficiency (serum retinol <0.7 micromol l(-1)) in adolescent refugees. DESIGN: Cross-sectional surveys. SETTING: Kakuma refugee camp in Kenya and seven refugee camps in Nepal. SUBJECTS: Adolescent refugee residents in these camps. RESULTS: Anaemia was present in 46% (95% confidence interval (CI): 42-51) of adolescents in Kenya and in 24% (95% CI: 20-28) of adolescents in Nepal. The sensitivity of palmar pallor in detecting anaemia was 21%. In addition, 43% (95% CI: 36-50) and 53% (95% CI: 46-61) of adolescents in Kenya and Nepal, respectively, had iron deficiency. In both surveys, anaemia occurred more commonly among adolescents with iron deficiency. Vitamin A deficiency was found in 15% (95% CI: 10-20) of adolescents in Kenya and 30% (95% CI: 24-37) of adolescents in Nepal. Night blindness was not more common in adolescents with vitamin A deficiency than in those without vitamin A deficiency. In Kenya, one of the seven adolescents with Bitot's spots had vitamin A deficiency. CONCLUSIONS: Anaemia, iron deficiency and vitamin A deficiency are common among adolescents in refugee populations. Such adolescents need to increase intakes of these nutrients; however, the lack of routine access makes programmes targeting adolescents difficult. Adolescent refugees should be considered for assessment along with other at-risk groups in displaced populations.     

Organics captured from comet 81P/Wild 2 by the Stardust spacecraft

Organics found in comet 81P/Wild 2 samples show a heterogeneous and unequilibrated distribution in abundance and composition. Some organics are similar, but not identical, to those in interplanetary dust particles and carbonaceous meteorites. A class of aromatic-poor organic material is also present. The organics are rich in oxygen and nitrogen compared with meteoritic organics. Aromatic compounds are present, but the samples tend to be relatively poorer in aromatics than are meteorites and interplanetary dust particles. The presence of deuterium and nitrogen-15 excesses suggest that some organics have an interstellar/protostellar heritage. Although the variable extent of modification of these materials by impact capture is not yet fully constrained, a diverse suite of organic compounds is present and identifiable within the returned samples.     

Evaluation of ilial screw loosening after triple pelvic osteotomy in dogs: 227 cases (1991-1999)

OBJECTIVE: To investigate factors influencing screw loosening after triple pelvic osteotomy (TPO) and ischial wire stabilization of the acetabular segment. DESIGN: Retrospective case series. Animals-227 dogs with congenital hip dysplasia or subluxated hip joints. PROCEDURES: Medical records and radiographs of 227 dogs that underwent 332 TPO procedures were evaluated, and data pertaining to screw type, plate position, sacral screw engagement, use of ischial interfragmentary wires, and pelvic alignment were assessed for associations with screw loosening. RESULTS: Complications developed in 96 of the 332 (29%) procedures. Cancellous screws without sacral engagement were associated with the lowest frequency (6%) of loosening, compared with cancellous and cortical screws engaging the sacrum and cortical screws that did not engage the sacrum. Frequency of screw loosening increased when cortical or cancellous screws engaged the sacrum and when cortical screws were used. In dogs that had surgery bilaterally, the first limb on which TPO was performed had a higher frequency of screw loosening than the second limb. Pelvic alignment loss was greatest (5.4 degrees ) when the 3 most cranial screws were loosened. Loss of pelvic alignment was significantly different between dogs that underwent surgery and had complications and those that underwent surgery and did not have complications in association with loosening of 1, 2, and 3 screws. CONCLUSIONS AND CLINICAL RELEVANCE: TPO screw loosening was multifactorial and related to stability of the affected ilium, screw type, and screw position. Placing cancellous screws that do not engage the sacrum in pelvic osteotomy plate positions 1 through 3 may decrease the number of screws that loosen.     

A gain-of-function polymorphism controlling complex traits and fitness in nature

Identification of the causal genes that control complex trait variation remains challenging, limiting our appreciation of the evolutionary processes that influence polymorphisms in nature. We cloned a quantitative trait locus that controls plant defensive chemistry, damage by insect herbivores, survival, and reproduction in the natural environments where this polymorphism evolved. These ecological effects are driven by duplications in the BCMA (branched-chain methionine allocation) loci controlling this variation and by two selectively favored amino acid changes in the glucosinolate-biosynthetic cytochrome P450 proteins that they encode. These changes cause a gain of novel enzyme function, modulated by allelic differences in catalytic rate and gene copy number. Ecological interactions in diverse environments likely contribute to the widespread polymorphism of this biochemical function.     

Biogenic potassium salt particles as seeds for secondary organic aerosol in the Amazon

The fine particles serving as cloud condensation nuclei in pristine Amazonian rainforest air consist mostly of secondary organic aerosol. Their origin is enigmatic, however, because new particle formation in the atmosphere is not observed. Here, we show that the growth of organic aerosol particles can be initiated by potassium-salt-rich particles emitted by biota in the rainforest. These particles act as seeds for the condensation of low- or semi-volatile organic compounds from the atmospheric gas phase or multiphase oxidation of isoprene and terpenes. Our findings suggest that the primary emission of biogenic salt particles directly influences the number concentration of cloud condensation nuclei and affects the microphysics of cloud formation and precipitation over the rainforest.     

Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE

Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [-3-xylose-α1,3-glucuronic acid-β1-]. This modification allowed α-DG to bind laminin-G domain-containing ECM ligands.