Journal: BMC Microbiology

Article Title: Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells

doi: 10.1186/1471-2180-13-251

Figure Lengend Snippet: Binding of purified recombinant PIII protein to epithelial cells. A . Ectocervical cells were incubated for 1 h at 37°C with increasing concentrations of the purified PIII protein (range 2 nM-4.2 μM). The binding was analysed by FACS using mouse anti-PIII antibodies and an R-Phycoerythrin-conjugated secondary antibody. The values are reported as net mean fluorescence intensity (MFI). B . Saturation curve of PIII binding to ectocervical cells. Analysis was performed on data reported in A. The K d value was calculated as the PIII concentration that determines the saturation of 50% of the receptors present on the cells. C . Representative flow cytometry profiles of the binding of 1 μM PIII to ectocervical, endocervical and urethral cells. Grey line profiles represent the cells incubated with the primary and secondary antibodies in absence of the PIII protein.

Article Snippet: Ectocervical and Endocervical cells (Ect1/E6E7 and End1/E6E7 from ATCC) were maintained in keratinocyte serum-free medium (KSFM, Gibco) supplemented with 50 μg/mL bovine pituitary extract, 0.1 ng/mL epidermal growth factor, 0.4 mM CaCl 2 and antibiotics at 37°C in 5% CO 2 .

Techniques: Binding Assay, Purification, Recombinant, Incubation, Fluorescence, Concentration Assay, Flow Cytometry

Journal: BMC Microbiology

Article Title: Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells

doi: 10.1186/1471-2180-13-251

Figure Lengend Snippet: Adhesion (A) and invasion (B) of F62 wild-type (black columns) and F62Δ pIII (white columns) strains to ectocervical, endocervical and urethral cells. Cells were infected for 3 hours at an MOI of 100:1. Total cell-associated bacteria and intracellular gentamicin-resistant bacteria were quantified after lysis of cell membranes with 1% saponin followed by dilution and plating. In competition experiments, ectocervical cells were pre-incubated with 25 μg/mL of pIII protein before infection (grey column). Results are means ± SEM from three independent experiments, each performed in triplicate. The high variability in the values shown in the Figure B was due to the very low number of the intracellular bacteria. ** p < 0.01. C . Ectocervical cells were infected for 3 hours with F62 wild-type (left panel) and F62Δ pIII (right panel) strains and, after washing, were fixed and stained for confocal immunoflurescent microscopy. Bacteria were labeled by an anti-OM serum and a secondary fluorescent antibody (green). DNA and cellular actin were stained with DAPI (blue) and Phalloidin-Alexa Fluor 568 (red), respectively.

Article Snippet: Ectocervical and Endocervical cells (Ect1/E6E7 and End1/E6E7 from ATCC) were maintained in keratinocyte serum-free medium (KSFM, Gibco) supplemented with 50 μg/mL bovine pituitary extract, 0.1 ng/mL epidermal growth factor, 0.4 mM CaCl 2 and antibiotics at 37°C in 5% CO 2 .

Techniques: Infection, Bacteria, Lysis, Incubation, Staining, Microscopy, Labeling

Journal: PLoS ONE

Article Title: Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy

doi: 10.1371/journal.pone.0048418

Figure Lengend Snippet: ( A ) A dose of 2 U/ml efficiently digested purified TVV1 dsRNA: 1 = No Rnase control, 2 = Company buffer provided by manufacturer with the RNase kit, 3 = KSFM culture medium, 4 = modified Diamond medium. ( B ) Comparison of enzymatic effects on intact TVV1 (lanes 1–4) versus pre-heated TVV1 (lanes 5–8): 1, 5 = no RNase, 2, 6 = 200 U/mL, 3,7 = 20 U/mL, 4,8 = 2 U/mL of RNase III, all efficiently digesting the dsRNA but only if released from pre-heated virions. ( C ) IFNβ measurement in endocervical epithelial cells exposed to medium (med.), supernatants (sup.) from B7RC2 and UH9 TV isolates treated with metronidazole or DMSO control, intact virions (UH9 TVV) and poly(I:C). Bars represent means and S.E.M. of biological triplicates. ***p<0.001, RNase different from medium control (two-way ANOVA, Bonferroni).

Article Snippet: Human immortalized endocervical (End1/E6E7), ectocervical (Ect1/E6E7) and vaginal (Vk2/E6E7) epithelial cell lines, were grown in KSFM (Invitrogen, Carlsbad, CA) modified as described .

Techniques: Purification, Modification