Development of a SCAR Marker-Based Diagnostic Method for the Detection of the Citrus Target Spot Pathogen Pseudofabraea citricarpa

Target spot, a recently observed citrus disease that is caused by Pseudofabraea citricarpa, can cause substantial economic losses in citrus production. In this study, a 797 bp marker specific to Ps. citricarpa was identified via random amplified polymorphic DNA (RAPD) technique. The primer pair Pc-SFP/Pc-SRP, which was designed from RAPD amplicons, was utilized as a sequence-characterized amplified region (SCAR) marker. This marker identified Ps. citricarpa with a single and distinct band of 389 bp but did not amplify DNA from other tested fungal species. The PCR assay was highly sensitive to the target DNA at picogram levels and could reliably amplify Ps. citricarpa sequences with the Pc-SFP/Pc-SRP primer pair. The SCAR marker that was identified in the present study can facilitate rapid decision-making and precise disease forecasting and management

Regional arterial infusion with lipoxin A4 attenuates experimental severe acute pancreatitis.

Investigate the therapeutic effect of regional arterial infusion (RAI) with Aspirin-Triggered Lipoxin A4 (ATL) in experimental severe acute pancreatitis (SAP) in rats.SAP was induced by injection of 5% sodium taurocholate into the pancreatic duct. Rats with SAP were treated with ATL (the ATL group) or physiological saline (the SAP group) infused via the left gastric artery 30 min after injection of sodium taurocholate. The sham group was subjected to the same surgical procedure, though without induction of SAP. Serum levels of amylase, phospholipase A2 (PLA2), interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured at 12 and 24 h after induction of SAP. Ascitic fluid, the pancreatic index (wet weight ratio) and myeloperoxidase (MPO) levels in the pancreas were determined and histopathological findings were evaluated. The expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), NF-κB p65, and heme oxygenase-1 (HO-1) in the pancreas were estimated by immunofluorescence and western blot, respectively.ATL rats had lower serum levels of TNF-α, IL-1β, and IL-6 (P<0.01), PLA2 (P<0.05), and amylase levels (P<0.05) studied as compared with the SAP group. The pancreatic index in the ATL group decreased only at 24 h as compared with the SAP group (P<0.05). The histopathological findings and MPO levels in the pancreas significantly decreased in the ATL group as compared to the SAP group (P<0.05 and P<0.01, respectively). Immunofluorescence and western blot showed that ATL attenuated the expression of NF-κB p65, ICAM-1 and PECAM-1 in the pancreas, and increased the expression of HO-1 in SAP animals.We demonstrated that RAI with ATL attenuated the severity of experimental SAP, maybe achieved by improving the expression of HO-1, and down-regulating the NF-κB signaling pathway, with decreased expression of ICAM-1 and PECAM-1 and reduced generation of pro-inflammatory cytokines

The weight of ascetic, serum levels of amylase and PLA₂, and MPO activity in pancreas.

<p><b>A)</b> The weight of ascitic fluid increased after induction of severe acute pancreatitis (SAP), though without significant difference after regional arterial infusion (RAI) with Asprin-Triggered Lipoxin A₄ (ATL). <b>B)</b> The amylase activity increased rapidly in the SAP group both at 12 and 24 h compared to the sham group and decreased significantly after RAI with ATL (<i>P</i><0.05). <b>C)</b> Serum levels of PLA₂ (ELISA) increased in the SAP group vs. the sham group, higher at 24 h than at 12 h both in the SAP and ATL groups. PLA₂ serum levels in the ATL group decreased (<i>P</i><0.05 vs. SAP). <b>D)</b> MPO levels in the pancreas increased after SAP induction, significantly attenuated after RAI with ATL both at 12 and 24 h (<i>P</i><0.05 and <i>P</i><0.01, respectively). Seven rats were studied in each experimental group at each time point. The results are expressed as means and SD. * <i>P</i><0.05 and ** <i>P</i><0.01, ATL group vs. the SAP group.</p

Representative photographs of HE stained pancreas.

<p>Pathological section of pancreatic tissue under light microscope (HE staining × 100). The sham group (A and D) was normal. Tissue edema, inflammatory cell infiltration, hemorrhage, and necrosis in the pancreas were observed in the severe acute pancreatitis (SAP) group (B and E). Changes were substantially ameliorated by regional arterial infusion (RAI) with Asprin-Triggered Lipoxin A₄ (ATL) (Fig. C and F).</p

The serum levels of pro-inflammatory cytokines.

<p>Increases in interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) generation at 12 and 24 h after induction of severe acute pancreatitis (SAP). These increases in cytokine levels were significantly ameliorated by regional arterial infusion (RAI) with Asprin-Triggered Lipoxin A₄ (ATL) (<i>P</i><0.01). Seven rats were studied in each experimental group at each time point. The results are expressed as means and SD. * <i>P</i><0.05 and ** <i>P</i><0.01, ATL vs. the SAP group.</p

The scores of pathological grading of pancreatic injury and pancreatic index.

<p>SAP = severe acute pancreatitis.</p><p>ATL = SAP+regional arterial infusion ATL.</p><p><i>P</i> value = the statistical value of the SAP vs. the ATL group.</p><p>The scores of pathological grading of pancreatic injury and pancreatic index.</p

Immunofluorescence staining for ICAM-1 and PECAM-1 in the pancreas.

<p><b>A)</b> The expression of ICAM-1 in vascular endothelial cells in the pancreas increased after induction of severe acute pancreatitis (SAP) compared to the sham group; findings significantly attenuated by regional arterial infusion (RAI) with Asprin-Triggered Lipoxin A₄ (ATL). <b>C)</b> Fluorescence intensity for ICAM-1. The results are expressed as means and SD. The mean fluorescence intensity of ICAM-1 decreased significantly in the ATL group both at 12 and 24 h (<i>P</i><0.05 vs. SAP). <b>B)</b> The expression of PECAM-1 in vascular endothelial cells in the pancreas increased significantly after induction of SAP compared to the sham group, attenuated by RAI with ATL. <b>D)</b> Bar graph demonstrating fluorescence intensity for PECAM-1. The results are expressed as means and SD. The mean fluorescence intensity of PECAM-1 decreased significantly in the ATL group both at 12 and 24 h (<i>P</i><0.05). * <i>P</i><0.05 and ** <i>P</i><0.01, ATL vs. the SAP group.</p

Western blot analysis of heme oxygenase-1 (HO-1), NF-κB p65, ICAM-1 and PECAM-1 in the pancreas.

<p><b>A)</b> Representative photographs of HO-1 protein levels in the pancreas in the three groups. <b>E)</b> Bar graph quantifying the HO-1 protein level in the pancreas. HO-1 protein levels in the pancreas increased after induction of severe acute pancreatitis (SAP) compared to the sham group, significantly increased following regional arterial infusion with Asprin-Triggered Lipoxin A₄ (ATL) (<i>P</i><0.01 vs. SAP). <b>B)</b> Representative photographs of nuclear NF-κB protein level in the pancreas in the three groups. <b>F)</b> Bar graph quantifying nuclear NF-κB protein levels in the pancreas. Protein levels of nuclear NF-κB in the pancreas increased after induction of SAP compared to the sham group, and decreased significantly in the ATL group both at 12 and 24 h (<i>P</i><0.05 or <i>P</i><0.01, respectively, vs. SAP). <b>C)</b> Representative photographs of ICAM-1 protein levels in the pancreas in the three groups. <b>G)</b> Bar graph quantifying ICAM-1 protein levels in the pancreas, increased after induction of SAP as compared to the sham group, and significantly decreased in the ATL group (<i>P</i><0.05 vs. SAP). <b>D)</b> Representative photographs of PECAM-1 protein levels in the pancreas in the three groups. <b>H)</b> Bar graph quantifying PECAM-1 protein levels in the pancreas, increased after induction of SAP (vs. sham) and significantly decreased in the ATL group both at 12 and 24 h (<i>P</i><0.05 vs. SAP). The results are expressed as means and SD. * <i>P</i><0.05 and ** <i>P</i><0.01, ATL vs. the SAP group.</p