Using 3D animations to teach intracellular signal transduction mechanisms: taking the arrows out of cells

Traditional methods of teaching intracellular biological processes and pathways use figures or flowcharts with the names of molecules linked with arrows. Many veterinary students, presented with such material, simply memorize the names or chemical structures of the molecules and are then likely to forget the material once the examination is completed. To address this problem, the authors designed, created, and field-tested new teaching media that incorporate realistic three-dimensional (3D) animations depicting the dynamic changes that occur in intracellular molecules during cellular activation. Testing found that veterinary students taught using traditional teaching media (e.g., lectures, handouts, textbooks) are proficient in memorizing the names and order of intracellular molecules but unable to appreciate the interactions between these elements or their spatial relationships within cells. In contrast, more than 90% of veterinary students taught using 3D animations not only recall the facts about the intracellular elements but also develop accurate mental images of the interactions among these molecules and their spatial relationships. These findings strongly suggest that the comprehension of complex biological processes by veterinary students can be enhanced by the use of dynamic 3D depictions of these processes in the classroom.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

<Emphasis Type="Italic">Streptococcus uberis</Emphasis>-specific T cells are present in mammary gland secretions of cows and can be activated to kill <Emphasis Type="Italic">S. uberis</Emphasis>

The presence, phenotype and function of Streptococcus uberis-specific T cells in the mammary gland secretion (MGS) and blood of cows exposed to S. uberis were assessed. MGS T cells in the udder were purified and incubated with autologous blood monocytes as antigen-presenting cells (APC). Most cows, irrespective of prior S. uberis infection status and lactation status, were shown to have S. uberis-specific T cells both in MGS and in the blood. When cells from a subgroup of cows were studied, it was found that the S. uberis-specific T cells produced high levels of interferon-gamma (IFN-γ), but low levels of interleukin-10 (IL-10). A high percentage of responding T cells were of the CD8 ⁺ memory (CD45RO) subset. T cells from the MGS specific for S. uberis were propagated from animals during the drying off period and expanded in vitro using interleukin-2 (IL-2) and S. uberis antigens. This led to the accumulation of T cells of the CD8 ⁺ subset bearing memory cell markers (CD45A ⁻ , CD45RO ⁺ ), which released high levels of IFN-γ. Four of the five T cell lines derived from the MGS of three animals had substantial direct killing activity towards S. uberis in vitro. It is concluded that there is an emergence of S. uberis-specific bactericidal T cells in the MGS of cows after infection or environmental exposure to S. uberis. Vaccines aimed at activating and expanding this T cell population in the mammary glands of cattle may offer an avenue for the prevention of mastitis caused by S. uberis.     

Prevalence and risk factors for Salmonella spp. contamination in French broiler-chicken flocks at the end of the rearing period

A nation-wide survey was carried out in 370 randomly chosen French commercial broiler chicken flocks from October 2005 to September 2006 to determine Salmonella spp. prevalence and to identify risk factors for contamination, at the end of the rearing period. The Salmonella status of the flocks was assessed from five faecal samples (litter swabs) analysed by classical bacteriological methods. A flock with at least one contaminated sample was classified as a Salmonella-positive flock. The apparent prevalence of Salmonella was 8.6% (95% CI: 5.7, 11.5%). The most prevalent serovar was S. hadar followed by S. anatum and S. mbandaka. Logistic regression methods were used to analyse the associations between husbandry practices, farm characteristics, general hygiene and the Salmonella status of the sample. The risk for Salmonella contamination of the flock at the end of the rearing period increased when neighbours helped in the placement of day-old chicks. On the contrary, the risk decreased when mobile equipment was dismantled before cleaning and disinfection, when the farm had a specific container for dead-bird disposal and when acetic acid was added to the drinking water.     

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.     

Agroenvironmental determinants associated with the presence of antimicrobial-resistant Escherichia coli in beach waters in Quebec, Canada

Exposure to microorganisms resistant to antimicrobials may constitute a health risk to human populations. It is believed that one route of exposure occurs when people engage in recreational activities in water contaminated with these microorganisms. The main objective of this study was to explore population-level and environmental determinants specifically associated with the presence of antimicrobial resistant (AMR) generic Escherichia coli isolated from recreational waters sampled from beaches located in southern Quebec, Canada. Water samples originated from the Quebec provincial beach surveillance program for the summers of 2004 and 2005. This study focused on three classes of determinants, namely: agricultural, population-level and beach characteristics for a total of 19 specific factors. The study was designed as a retrospective observational analysis and factors were assessed using logistic regression methods. From the multivariable analysis, the data suggested that the percentage of land used for spreading liquid manure was a significant factor associated with the presence of AMR E. coli (OR=27.73). Conceptually, broad factors potentially influencing the presence of AMR bacteria in water must be assessed specifically in addition to factors associated with general microbial contamination. Presence of AMR E. coli in recreational waters from beaches in southern Quebec may represent a risk for people engaging in water activities and this study provides preliminary evidence that agricultural practices, specifically spreading liquid manure in agricultural lands nearby beaches, may be linked to the contamination of these waters by AMR E. coli.     

Prediction of parturition date in the bitch and queen

Assessment of gestational age and parturition date prediction is a challenging, yet common task in clinical management of the pregnant bitch or queen. Knowing the approximate parturition date is essential for a thorough pregnancy monitoring. Radiographic and ultrasonographic methods are suitable in bitches and queens. In the bitch, moreover, ovulation timing by means of LH or progesterone assay, determination of onset of vaginal dioestrus by means of examination of vaginal cytology, and recognition of impending parturition by monitoring prepartal progesterone levels and body temperature variation are practical methods. A combination of different methods increases the accuracy of parturition date prediction.     

Ripening influences banana and plantain peels composition and energy content

Musa sp. peels are widely used by smallholders as complementary feeds for cattle in the tropics. A study of the influence of the variety and the maturation stage of the fruit on fermentability and metabolisable energy (ME) content of the peels was performed using banana (Yangambi Km5) and plantain (Big Ebanga) peels at three stages of maturation in an in vitro model of the rumen. Peel samples were analysed for starch, free sugars and fibre composition. Samples were incubated in the presence of rumen fluid. Kinetics of gas production were modelled, ME content was calculated using prediction equation and short-chain fatty acids production and molar ratio were measured after 72 h of fermentation. Final gas production was higher in plantain (269–339 ml g⁻¹) compared to banana (237–328 ml g⁻¹) and plantain exhibited higher ME contents (8.9–9.7 MJ/kg of dry matter, DM) compared to banana (7.7–8.8 MJ/kg of DM). Butyrate molar ratio decreased with maturity of the peels. The main influence of the variety and the stage of maturation on all fermentation parameters as well as ME contents of the peels was correlated to changes in the carbohydrate fraction of the peels, including starch and fibre.