Journal: PLoS Pathogens

Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing

doi: 10.1371/journal.ppat.1006582

Figure Lengend Snippet: (A) In vitro antimicrobial tolerance assays for survival of paired blood-isolated pneumococci (BIP) and heart-isolated pneumococci (HIP) from the same mouse (n = 6). Tolerance to penicillin and erythromycin killing at the designated concentration was tested. Statistical analysis was performed using Mann-Whitney test. (B) Representative high magnification immunofluorescent microscopy images of cardiac microlesions showing presence of biofilm extracellular matrix components: nucleic acids (stained with DAPI [DAPI], blue ), capsule (stained with anti-serotype 4 capsule antibody [CPS], red ), and mouse extracellular DNA (stained with anti-mH2A histone antibody [mH2A], green ). A minimum of 5 stained heart sections were examined. (C) Representative tile-stitched image of whole heart sections from TIGR4 infected mice (n = 5). The cardiac sections (stained with DAPI, blue ) were probed for TIGR4 ( red ), using serotype 4 capsule polysaccharide antisera, and for exposed galactose residues ( green ), using fluorescein labeled Erythrina crystagalli lectin, within the heart. Inset, Representative tile-stitched image of an uninfected control heart (n = 3). (D) Static biofilm-forming ability of TIGR4 (T4) and T4 ΔspxB was assessed in 48-hour 6-well plate model (n = 3 biological replicates, each with 2 technical replicates). Biofilm biomass was measured using crystal violet staining. Statistical analysis was performed using Student’s t -test. (E) Average number of cardiac microlesions detected per cardiac section in T4 and T4 Δ spxB infected mice post infection. For each time point, cardiac sections from at least 5 mice were examined. Averages were calculated by enumerating the number of microlesions in 3 non-adjacent cardiac sections. Statistical analysis was performed using Mann-Whitney test. P value: * ≤ 0.05, ** ≤ 0.01, **** ≤ 0.0001; data are represented as mean ± SEM.

Article Snippet: Exposed galactose was stained for with fluorescein labeled Erythrina crystagalli lectin (Vector Laboratories, FL-1141) after blocking with carbo-free blocking solution for 1 hour (Vector Laboratories, SP-5040).

Techniques: In Vitro, Isolation, Concentration Assay, MANN-WHITNEY, Microscopy, Staining, Infection, Labeling

Journal: PLoS Neglected Tropical Diseases

Article Title: Serological Screening of the Schistosoma mansoni Adult Worm Proteome

doi: 10.1371/journal.pntd.0002745

Figure Lengend Snippet: A) S. mansoni major egg antigen (MjE) and hemoglobinase precursor (Hem) proteins, lanes 1 and 2 respectively, were expressed using TNT SP6 High-Yield Wheat Germ Protein Expression System and the FluoroTect Green Lys labeled proteins were analyzed in SDS-PAGE 4–20%. The gel image was obtained with a laser-based fluorescent gel scanner. The negative control using no DNA template was indicated in lane 3 and using no DNA template and no FluoroTect Green Lys , in lane 4. Lane 5 contains the Precision Plus Protein Kaleidoscope molecular weight marker (BioRad). B) Western blotting analysis of the in vitro recombinant proteins probed with INF (2, 3, 4), NE (5, 6, 7) and NI (8, 9, 10) serum pools. The TNT wheat germ extract using MjE flexi-vector as DNA template was loaded in lanes 2, 5 and 8, and using Hem flexi-vector in lanes 3, 6 and 9. TNT wheat germ extract with no DNA template was loaded in lanes 4, 7 and 10. Rabbit anti-human IgG and anti-rabbit IgG HRP conjugated were used as secondary and tertiary antibodies. The membranes were developed using ECL Plus-Western Blotting Detection System and the proteins were visualized by chemiluminescence detection using a Fujifilm LAS-4000 Imaging System. Pre-stained Precision Plus Protein Kaleidoscope molecular weight marker was loaded in lane 1.

Article Snippet: After 2×30 min washes in TBS-T/1% nonfat dry milk, membranes were incubated with rabbit anti-human IgG antibody (Sigma) diluted 1∶10,000 in TBS-T/1% nonfat dry milk for 1 hour and with anti-rabbit IgG HRP conjugated (ECL Plus Western Blotting Reagent Pack, GE Healthcare) in the same conditions, with 2×30 min washes between antibodies incubations.

Techniques: Expressing, Labeling, SDS Page, Negative Control, Molecular Weight, Marker, Western Blot, In Vitro, Recombinant, Plasmid Preparation, Imaging, Staining

Journal: International Journal of Molecular Sciences

Article Title: Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA

doi: 10.3390/ijms22083804

Figure Lengend Snippet: Exogenous V5-tagged hRIDA was differently K-succinylated in high and low proliferating cells. ( A ) High and low proliferating HEK293 cells were transfected with pcDNA-V5-hRIDA or the empty vector pcDNA, at 4d and 7d after seeding the cells. The next day, cells were lysed, and V5-hRIDA was immunoprecipitated from protein extracts using V5 epitope antibody. Immunoprecipitated proteins were fractionated on 15% SDS-PAGE, transferred onto a nitrocellulose membrane and immunodecorated with antibodies against the V5 epitope (WB: V5-hRIDA), hRIDA (WB: hRIDA) and succinyl-lysine (WB: Succ-Lys). V5-hRIDA was in vitro desuccinylated with SIRT5, then transferred onto a nitrocellulose membrane and immunodecorated with the antibody against hRIDA (WB: hRIDA, SIRT5-treated). Chemiluminescence signals were normalized with respect to the β-actin in input protein extracts. Normalized values were reported in the histograms as fold change with respect to the cells at day 5. ( B ) Recombinant purified hRIDA was in vitro succinylated with different concentration of succinyl-CoA, fractionated on SDS-PAGE, transferred onto a nitrocellulose membrane and immunodecorated with antibodies against succinyl-lysine ( B , upper panel) and hRIDA ( B , lower panel). Values are means ± S.D. of three independent experiments. *, value significantly different from the control, p < 0.05.

Article Snippet: The immune complexes were detected using peroxidase-conjugated secondary antibodies by chemiluminescence (ECL WesternBright, Advansta, San Jose, CA, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Membrane, In Vitro, Recombinant, Purification, Concentration Assay, Control