Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A) PPI network was constructed basing on the top 300 DEGs. The red icons represent up-regulated genes and the blue ones represent down-regulated genes. Furthermore, the connecting lines between the icons indicate potential interaction between different DEGs. (B) Module analysis of the DEGs enrolled in PPI network using MCODE method. The top five ranking clusters are shown according to MCODE score. (C) Functional enrichment analysis of genes in cluster 1. Likewise, the y axis and x axis represent enrichment terms and scores, respectively, and the color means p- value. (D) The top 10 hub genes are identified from PPI network using MCC method. The color represents MCC score. A darker red icon means higher MCC score and a lighter yellow one represents lower MCC score. (E) mRNA expression levels of CCL13, CCR2, FPR3 and PLAU in alveolar macrophages isolated from better prognosis subjects (VFDs > 12) and from poor prognosis individuals (VFDs <12).

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Construct, Functional Assay, Expressing, Isolation

Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A) Relative mRNA expressions of CCL13, CCR2, CX3CR1, CXCL16, FPR3 and PLAU on THP-1-derived macrophages in response to LPS were assessed using qRT-PCR. (B) The protein levels of CCR2 and FPR3 under a varying time of LPS treatment were evaluated by Western blot analysis. (C) CCR2 expression on surface of THP-1-derived macrophages after LPS treatment was detected by flow cytometry analysis. (D) Relative mRNA expressions of CCR2 and FPR3 on primary AMs collected from patients and volunteers were evaluated using qRT-PCR. (E) The protein levels of CCR2 and FPR3 in primary AMs were examined using Western blot assay. (F) CCR2 expression on surface of primary AMs was detected by flow cytometry analysis. Data are presented as mean ± SD and are representative of three independent experiments. ∗ P < 0.05, by unpaired, 2-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A)Diagrammatic illustration for the chemotaxis assay procedure: Upper:1 ×10 4 of wild-type or siRNA-transfected macrophages were cultured in serum-free medium with or without LPS. Lower: Culture medium with or without CCL2/fMLP. (B) LPS-induced macrophage chemotaxis toward chemokine CCL2 was assessed using a transwell assay as described in the Methods. (C) Likewise, LPS-triggered macrophage migration toward fMLP was evaluated as above. (D) The correlation between CCR2 and FPR3 expression was analyzed using pearson correlation analysis with expression data of GSE89953 and GSE116560 . (E) Elevated protein expression of CCR2 induced by LPS stimulation was significantly attenuated by siRNA-FPR3 transfection. Data are presented as mean ± SD and are representative of 3 independent experiments. ∗ P < 0.05, by unpaired, two-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Transwell Assay, Migration, Expressing, Two Tailed Test

Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: MPO activity level in pancreas ( A ) and lungs ( B ) of healthy mice, mice with evoked acute pancreatitis (AP), and mice with AP after administration of 100% GBBC and FFAR1 or FFAR4 antagonist. Data presented as raw data (circles) and means (horizontal bolded lines) with SEM (boxes). Significance estimated with the use of one-way ANOVA followed by Dunnett’s post hoc test. # (hash) denotes comparison to control. * (asterisk) indicates comparison to AP. n = 4–8; * denotes p < 0.05; ** denotes p < 0.01; ###/*** denotes p < 0.001.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activity Assay

Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: Microphotographs of the pancreas. H&E staining: control group—normal architecture of the exocrine pancreas with functional acinar cells rich in zymogen granules ( A ); acute pancreatitis (AP) group—diffuse inflammatory infiltrates in periacinar area with the predominance of neutrophils ( B ); AP + 100% ghee butter from bovine colostrum (GBBC) group—acinar cell vacuolization, minimal atrophy of acinar cells with no evidence of inflammatory cell infiltrates ( C ); AP + FFAR1 antagonist + 100% GBBC group—dissociation and moderate atrophy of acinar cells ( D ); AP + FFAR4 antagonist + 100% GBBC group—dissociation of acinar cells, strong remodeling of the stroma, and marked atrophy of acinar cells ( E ). Masson’s trichrome staining: control group—minimal collagen deposition as a delicate framework for acinar cells ( F ); AP group—minimal collagen deposition in inflamed and edematous stroma ( G ); AP + 100% GBBC group—minimal collagen deposition between well-compacted acinar cells ( H ); AP + FFAR1 antagonist + 100% GBBC group—moderate collagen deposition in periacinar area and between individual acinar cells ( I ); AP + FFAR4 antagonist + 100% GBBC group—marked deposition of collagen between atrophied acinar cells ( J ). Immunoreactivity of α-SMA protein: control group—negative reaction ( K ); AP group—positive reaction in individual myofibroblast-like cells ( L ); AP + 100% GBBC group—moderate positive cytoplasmic reaction in migrating myofibroblast-like spindle-shaped cells penetrating periacinar spaces ( M ); AP + FFAR1 antagonist + 100% GBBC group—strong immunohistochemical reaction in myofibroblast-like cells ( N ); AP + FFAR4 antagonist + 100% GBBC group—strong immunohistochemical reaction in numerous myofibroblast-like cells ( O ). Nikon’s Eclipse E600 light microscope (Nikon Instruments Inc., Tokyo, Japan) was used to examine tissue samples. Microphotographs were taken with a digital imaging system that included a microscopy digital camera (Nikon DS-Fil, Nikon Instruments Inc., Tokyo, Japan) and image analysis software (NIS-Elements BR-2.20, Laboratory Imaging, Praha, Czech Republic); 400× magnification.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Staining, Functional Assay, Immunohistochemical staining, Light Microscopy, Imaging, Microscopy, Software