Journal: Emerging Infectious Diseases

Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

doi: 10.3201/eid2302.161239

Figure Lengend Snippet: Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

Article Snippet: In brief, we performed DPP4 IHC staining by using 5 μg/mL of polyclonal goat IgG anti-human DPP4 antibody (R&D Systems, Abingdon, UK) and peroxidase-labeled rabbit anti-goat IgG (1:200; DAKO; Agilent Technologies Company, Santa Clara, CA, USA) as a secondary antibody.

Techniques: Membrane, Staining, Immunohistochemistry

Journal: Cells

Article Title: Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion

doi: 10.3390/cells10020259

Figure Lengend Snippet: ARV p17-mediated release of Dipeptidyl Peptidase 4 (DPP4) expression. ( A ) Wound healing assay performed after HUVECs’ overnight stimulation with conditioned medium from Mock- or ARV p17-nucleofected HUVEC cells. Confluent cell monolayers were scratched using a 200 μL pipette tip and cell migration was recorded by light microscopy 10 h after wound scratch (original magnification, 4×). The wound width was measured, and the relative wound area was calculated as the ratio of the remaining area at the 10 h time point to the 0 h starting point. Pictures are representative of one out of two independent experiments with similar results. Values are the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( B ) Tube formation assay performed with HUVECs co-cultivated for 48 h with Mock- or ARV p17-nucleofected HUVEC cells. The pictures were taken 6 h after cell seeding (original magnification, 4×). Closed rings were counted as a parameter for quantification of tube formation. Pictures are representative of one out of two independent experiments with similar results. Values are the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( C ) Angiogenesis array performed with the supernatants of Mock- or ARV p17-nucleofected cells recovered 24 h post-nucleofection. Relative pixel intensity was calculated using ImageJ software and expressed as the mean of duplicate dots. Values are representative of one experiment out of two with similar results. ( D ) Analysis of DPP4 gene expression performed using quantitative real-time PCR in Mock- and ARV p17-nucleofected cells. Analysis of real-time PCR data were performed with the 2 -DDCt method using relative quantitation study software. Quantification of DPP4 mRNA was normalized according to the internal β-actin control. Values represent the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( E ) soluble (s) DPP4 levels in supernatants of HUVECs stimulated with GST-ARV p17 for 48 h were quantified with human sDPP4 ELISA. Supernatant from GST stimulated cells was used as a negative control. Bar graph displays the concentration of DPP4 determined by absorbance quantification. Values are the mean ± SD of one representative experiment out of three independent experiments with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001.

Article Snippet: The release of DPP4 in the concentrated conditioned medium was evaluated using a human CD26 ELISA Kit (R&D systems) according to manufacturer’s instructions.

Techniques: Expressing, Wound Healing Assay, Transferring, Migration, Light Microscopy, Tube Formation Assay, Software, Gene Expression, Real-time Polymerase Chain Reaction, Quantitation Assay, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay