Journal: Journal of Clinical Medicine

Article Title: Urinary Proteomics Reveals Promising Biomarkers in Menstrually Related and Post-Menopause Migraine

doi: 10.3390/jcm10091854

Figure Lengend Snippet: Representative 2DE urinary maps. The differentially expressed protein spots among ( A ) control group, ( B ) menstrually related migraine group, and ( C ) post-menopause migraine group are evidenced by ellipses; protein entry names refer to those reported in . Two-dimensional separation was performed by 17 cm IPG strip, pH range 3–10 (first-dimension), and 8–16% polyacrylamide gradient gel (second-dimension). Molecular weight marker (PrecisionPlus All Blue, Bio-Rad) is expressed in kilodalton (kDa).

Article Snippet: Large size 8–16% polyacrylamide gradient gels (29:1 acrylamide/bis solution, 1.5 M Tris, pH 8.8, 10% SDS, 1% TEMED, 10% ammonium persulfate, from Bio-Rad Laboratories) and TGS 1X running buffer (Bio-Rad Laboratories) were employed for the electrophoretic run.

Techniques: Control, Stripping Membranes, Molecular Weight, Marker

Journal: PLoS ONE

Article Title: Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

doi: 10.1371/journal.pone.0126650

Figure Lengend Snippet: (A) Construct of M2e-displaying or control peptide-displaying f88 plasmid. (B) Schematic diagram of f88 and f88M2e2-16 bacteriophages. (C, D) Characterization of CsCl-gradient-centrifuge purified f88M2e2-16 and f88ctr phages using coomassie blue staining and western blot. Two μg of f88M2e2-16 and f88ctr bacteriophages were loaded on SDS-PAGE and the corresponding Western blot was visualized using anti-pVIII mAb, anti-pIII mAb, anti-M2e mAb 37 and anti-M2e mAb 148, as indicated. MAb 37 is an anti-M2e IgG1 specifically recognizing amino acids 4–14 of human consensus M2e; mAb 148 is an anti-M2e IgG1 that specifically binds the conserved first nine amino acids of M2e. (E) Recombinant pVIII-M2e2-16 and f88 phages were separated using Tricine-SDS-PAGE with 15% acrylamide gel. Mouse anti-pVIII antibody was then used to probe protein samples in Western Blot. The upper faint protein band in the left lane corresponds to pVIII-M2e2-16 and is absent in f88 phages. The faster migrating and more intense band corresponds to wild type pVIII. (F) phage preparation titration using TG1 Escherichia coli strain containing F pilus. 100 μl bacteriophage containing 10 10 tetracycline transducing units were diluted with 5x10 6 , 5x10 7 and 5x10 8 times and were plated on tetracycline-containing LB agar plates. Each colony forming unit represents one viable bacteriophage particle.

Article Snippet: The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP) (GE Healthcare UK Ltd.), or HRP-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotech, U.S.) for 45 min at room temperature.

Techniques: Construct, Control, Plasmid Preparation, Gradient Centrifugation, Purification, Staining, Western Blot, SDS Page, Recombinant, Acrylamide Gel Assay, Phage Preparation, Titration

Journal: PLoS ONE

Article Title: Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

doi: 10.1371/journal.pone.0126650

Figure Lengend Snippet: (A-D) Groups of female BALB/c mice (n = 10 per group) were intraperitoneally vaccinated with antigens in the presence of incomplete Freund’s adjuvant via the intraperitoneal route. Purified phages were administered at 10 10 tetracycline transducing units (f88M2e2-16 and f88ctr groups). As a positive control, one group was immunized with 10 μg M2eHBc per vaccination. A PBS plus adjuvant was included as a negative control. Blood was collected 10 days after priming and each boost injection and serum was prepared and tested in ELISA. (A) Anti-f88 total IgG titration following priming and boosting. (B) Anti-human consensus M2e total IgG following priming and boosting. (C) Anti-human consensus M2e IgG1 and (D) IgG2a endpoint titration following priming and boosting. Endpoint serum IgG titers after the third boost were determined from BALB/c mice that had been immunized with 10 10 tetracycline transducing units of f88ctr, 10 10 tetracycline transducing units f88M2e2-16 or 10 μg of avian M2e-HBc using ELISA plates coated with M2e peptides from different influenza A virus strains: (E) M2e from A/swine/Ontario/42729A/01 (sOnM2e), (F) M2e from A/chicken/HongKong/258/1997 (cHKM2e), (G) M2e from A/chicken/Vietnam/36/2004 (cVNM2e). In A-D error bars represent standard deviations.

Article Snippet: The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP) (GE Healthcare UK Ltd.), or HRP-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotech, U.S.) for 45 min at room temperature.

Techniques: Adjuvant, Purification, Positive Control, Negative Control, Injection, Enzyme-linked Immunosorbent Assay, Titration, Virus

Journal: PLoS ONE

Article Title: Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

doi: 10.1371/journal.pone.0126650

Figure Lengend Snippet: Preimmune and immune sera collected after the third immunization with avian M2e-HBc, f88M2e2-16 or f88ctr were used to immuno-stain Madin-Darby canine kidney (MDCK) cells that had been infected with A/Memphis/106/76 (H3N2), A/Belgium/145/2009 (pandemic H1N1), A/chicken/Nanchang/3-120/2001 (H3N2), PR8 (H1N1) or mock infected. Alexa Fluor 555 Donkey anti-Mouse secondary antibody (red fluorescence) was used to reveal mouse IgG binding. An Alexa Fluor 488 labeled polyclonal goat anti-RNP staining (green fluorescence) was used to trace influenza A virus infected cells. All cells were stained with DAPI (blue fluorescence).

Article Snippet: The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP) (GE Healthcare UK Ltd.), or HRP-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotech, U.S.) for 45 min at room temperature.

Techniques: Immunostaining, Infection, Fluorescence, Binding Assay, Labeling, Staining, Virus

Journal: PLoS ONE

Article Title: Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

doi: 10.1371/journal.pone.0126650

Figure Lengend Snippet: BALB/c mice (n = 10) were vaccinated with f88M2e2-16, f88-ctr, human M2eHBc or PBS three times in the presence of incomplete Freund’s adjuvant. Three weeks after the last boost, mice were challenged with 0.2 LD 50 of mouse-adapted pandemic H1N1 2009 virus. Serum was collected 10 day after the last booster immunization (-) and 15 days after infection (+). The anti-M2e serum IgG response was determined by ELISA coated with (A) human consensus M2e (M2e2-23), M2e from (B) A/swine/Ontario/42729A/01 (sOnM2e), (C) M2e from A/chicken/Hong Kong/258/1997 (cHKM2e), (D) M2e from A/swine/Belgium/1/1998 (sBelM2e) and (E) M2e from A/duck/Vietnam/NCVD-9/2007 (dVNM2e). (F) Hemagglutination inhibition titers against pandemic H1N1 2009 virus before and after infection of mice. Error bars represent standard deviations.

Article Snippet: The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP) (GE Healthcare UK Ltd.), or HRP-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotech, U.S.) for 45 min at room temperature.

Techniques: Adjuvant, Virus, Infection, Enzyme-linked Immunosorbent Assay, HI Assay