Farm animal genomics and informatics: an update

Farm animal genomics is of interest to a wide audience of researchers because of the utility derived from understanding how genomics and proteomics function in various organisms. Applications such as xenotransplantation, increased livestock productivity, bioengineering new materials, products and even fabrics are several reasons for thriving farm animal genome activity. Currently mined in rapidly growing data warehouses, completed genomes of chicken, fish and cows are available but are largely stored in decentralized data repositories. In this paper, we provide an informatics primer on farm animal bioinformatics and genome project resources which drive attention to the most recent advances in the field. We hope to provide individuals in biotechnology and in the farming industry with information on resources and updates concerning farm animal genome projects

CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

PMC3744507Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.JH Libraries Open Access Fun

CD36−/− mice are protected from malaria-induced endothelial permeability.

<p><b>A</b>) Effect of <i>P. berghei</i> infection on filtration coefficient in the lungs from WT and CD36−/− mice. Control mice received 10⁶ uninfected RBCs whereas malaria mice received 10⁶<i>P. berghei</i>-iRBCs. Filtration coefficient was measured on day 6 post-injection as described in the Methods section. Data are means ± SE. n = 3–6; *<i>P</i><0.02 vs. WT control; #<i>P</i><0.05 vs. WT malaria by ANOVA interaction. <b>B</b>) Effect of <i>P. berghei</i> infection on the average fluid filtration in WT and CD36−/− mice. Average fluid filtration was determined from the change in perfusion circuit hematocrit and a time-weighted vascular pressure normalized to lung dry weight as described in the Methods section. Data are means ± SE. n = 3–6; *<i>P</i><0.006 vs. corresponding control; ^#<i>P</i><0.002 vs. WT malaria by ANOVA interaction. <b>C</b>) Effect of <i>P. berghei</i> infection on the albumin reflection coefficient (σ_alb) in the lungs from WT and CD36−/− mice. σ_alb was measured by a modified filtered volume method as described in Methods. Data are means ± SE. n = 3–5; *<i>P</i><0.005 vs. corresponding Control by ANOVA.</p

Altered Fyn protein level and activation in the lungs of malaria-infected CD36−/− mice.

<p><b>A)</b> Immunostained Western blots measuring total Fyn, P^Y417-Fyn (activated form) and P^Y528-Fyn (inactivated form) in lung homogenates from control and malaria-infected (day 6) WT and CD36−/− mice. <b>B–D</b>) Densitometry quantification of total Fyn (<b>B</b>), P^Y417-Fyn (<b>C</b>) and P^Y528-Fyn (<b>D</b>) normalized to actin expression. Data are expressed in arbitrary densitometry units (ADU) normalized to WT control values and represent the mean ± SE. n = 3–5; *<i>P</i><0.05 vs. corresponding control; #, <i>P</i><0.05 vs. WT malaria by ANOVA interaction; +,P<0.03 vs. WT values by ANOVA main factor effect.</p

CD36−/− lung microvascular endothelial cells are protected from increases in H₂O₂-induced endothelial barrier permeability.

<p>Time course of trans-endothelial electrical resistance (TER) of mouse lung microvascular endothelial cell (MLMVEC) monolayers from WT (<b>A</b>) or CD36−/− (<b>B</b>) mice following exposure to increasing concentrations of H₂O₂ (0.25, 0.50 and 1.0 mM). All TER values were normalized to the TER measured 0.5 h before the addition of H₂O₂. Baseline TER for CD36−/− MLMVEC monolayers (1098±25 Ω; n = 63) was significantly greater (<i>P</i><0.0001) than WT TER (902±25 Ω; n = 119). Data are means ± SE. n = 16–26; all symbols indicate <i>P</i><0.00001 vs. diluent by 2-factor (H₂O₂ dose, time) ANOVA interaction; * refers to 0.25 mM H₂O₂, + refers to 0.5 mM H₂O₂ and # refers to 1 mM H₂O₂). The result of a 3-factor (genotype, H₂O₂ dose, time) ANOVA interaction F ratio was P<0.00001 for TER differing as a function of CD36 expression.</p

Fyn cellular compartmentalization and the impact of Fyn knockdown on H₂O₂-mediated endothelial barrier function.

<p><b>A</b>) Effect of H₂O₂ exposure on WT (left panels) and CD36−/− (right panels) MLMVEC Fyn protein localization. MLMVECs stained for total Fyn (anti-Fyn, green), actin (Texas red-conjugated phalloidin) and nuclei (DAPI 4′, 6-diamidino-2-phenylindole, blue) without (top row) or with (bottom row) 60 min of exposure to 500 μm H₂O₂ (60×). Inserts show magnified images (100×). <b>B</b>) Efficiency of Fyn knockdown by sticky small interfering (ssi) RNA. Fyn expression in WT MLMVECs 48 hours after transfection with lipofectamine only (Control), scrambled (ssiScr) or Fyn (ssiFyn) ssiRNA was assessed by Western blot with quantification by densitometry. The data are expressed in arbitrary absorption units before normalization to GAPDH levels. Data are means ± SE. n = 3; *<i>P</i><0.05 vs. ssScr. <b>C</b>, <b>D</b>) Time series of normalized trans-endothelial electrical resistance (TER) in WT MLMVEC monolayers following exposure to (<b>C</b>) 0.25 mM or (<b>D</b>) 0.50 mM H₂O₂. Open symbols include Lipofectamine only, ssiScr and ssiFyn all in the absence of H₂O₂. Closed symbols include ssiScr-transfected or ssiFyn-transfected MLMVEC monolayers exposed to H₂O₂. Data are means ± SE. n = 3–5; *<i>P</i><0.002 vs. ssScr by ANOVA interaction.</p

CD36−/− mice exhibit minimal malaria-induced lung pathology.

<p><b>A</b>) Hematoxylin and eosin staining of histological lung sections from control and P. <i>berghei</i>-infected (day 6) WT and CD36−/− mice (10×). Inserts are representative higher-magnification fields (40×) of the septal thickening and edema seen in WT lungs compared to the lack of pathology in observed in the lungs from CD36−/− animals. <b>B</b>) Total number of cells isolated from the bronchoalveolar lavage fluid from uninfected and <i>P. berghei</i>-infected WT and CD36−/− mice. Data are means ± SE; n = 3–6. *<i>P</i><0.05 vs. Control; #<i>P</i><0.05 vs. WT malaria by ANOVA interaction and Kruskal Wallace testing. <b>C</b>) <i>P. berghei</i> within lung conduit vessel (arrows) and alveolar capillaries (arrowheads) of WT and CD36−/− mice (60×). <b>D</b>) The number of <i>P. berghei</i>/high power field (HPF) in the alveolar capillaries or in all vessels (Total) in the lungs of WT and CD36−/− mice. Data are means ± SE. n = 3 mice per group and 10 HPFs per lung section.*<i>P</i><0.05 vs. WT by ANOVA.</p

iRBCs generate reactive oxygen species (ROS) in the peripheral blood and pulmonary vasculature.

<p><b>A</b>) Flow cytometric analysis of peripheral blood isolated from WT mice 6 days after infection with <i>P. berghei</i> ANKA (left panel) or the red fluorescent strain of <i>P. berghei</i> ANKA tdTomato (PbtdT) (right panel). The gating strategy used to identify iRBCs based on tdTomato fluorescence forward scatter (FSC) is defined in the right panel. <b>B</b>) ROS production in the peripheral blood of control or PbAtdT-infected mice. ROS production was measured by flow cytometry of cells isolated from control and PbAtdT-infected (day 6) animals and labeled with dihydrorhodamine (DHR) 123. Uninfected RBCs and iRBCs were separated based on the presence of the erythroid marker, ter119, and PbAtdT fluorescence. The data are expressed as mean fluorescence intensity (MFI) of the DHR 123. Data are means ± SE. n = 3–6; *<i>P</i><0.001 vs. RBCs. Inset plot: histograms of DHR fluorescence of peripheral unstained control RBCs (white histogram), DHR-stained control RBCs (gray histogram), and DHR-stained iRBCs (black histogram). <b>C</b>) ROS production of control or PbAtdT iRBCs sequestered in the pulmonary vasculature. Lung-sequestered RBCs were isolated via dispersion of lung tissue after vascular perfusion with 10 ml of PBS to remove non-adherent cells. Lung-associated RBCs and iRBCs were stained and sorted for the presence of the erythroid marker, ter119, and PbAtdT fluorescence. Sorted cells were stained with DHR 123 for assessment of ROS production. The data are expressed as mean fluorescence intensity (MFI) of the DHR 123. Data are means ± SE. n = 3–6; *<i>P</i><0.001 vs RBCs. Inset plot: histograms of DHR fluorescence of lung sequestered unstained control RBCs (white histogram), DHR-stained control RBCs (gray histogram), and DHR-stained iRBCs (black histogram).</p

Fyn−/− mice are protected from malaria-induced increases in pulmonary endothelial barrier permeability.

<p><b>A</b>) Effect of <i>P. berghei</i> infection on filtration coefficient in the lungs from WT and Fyn−/− mice. Filtration coefficient was measured via isolated lung perfusion on day 6 post-infection as described in the Methods. Data are means ± SE. n = 3–6; *<i>P</i><0.02 vs. corresponding control; #<i>P</i><0.05 vs. WT malaria by ANOVA interaction. <b>B</b>) Effect of <i>P. berghei</i> infection on the average fluid filtration in WT and Fyn−/− mice. Average fluid filtration was determined from the change in perfusion circuit hematocrit and a time-weighted vascular pressure normalized to lung dry weight as described in the Methods. Data are means ± SE. n = 3–6; *<i>P</i><0.006 vs. corresponding control; ^#<i>P</i><0.002 vs. WT malaria by ANOVA interaction. <b>C</b>) Effect of <i>P. berghei</i> infection on the albumin reflection coefficient (σ_alb) in the lungs from WT and Fyn−/− mice. σ_alb was measured by a modified filtered volume method as described in Methods. Data are means ± SE. n = 3–5; *<i>P</i><0.01 vs. corresponding control by ANOVA main factor (malaria infection) effect. <b>D</b>) Hematoxylin and eosin staining of histological lung sections from P. <i>berghei</i>-infected (day 6) Fyn−/− mice (60×). Circles identify <i>P. berghei</i> ANKA within alveolar capillaries. <b>E</b>) The number of <i>P. berghei</i>/high power field (HPF) in the alveolar capillaries in the lungs of WT and Fyn−/− mice. Data are means ± SE. n = 3 mice per group and 10 HPFs per lung section.</p