Journal: Neuron

Article Title: An Activity-Dependent Determinant of Synapse Elimination in the Mammalian Brain

doi: 10.1016/j.neuron.2021.03.006

Figure Lengend Snippet: (A–I) JAK2 inactivation suppresses eye-specific segregation. (A) Schematic diagram of RGC projections to the dLGN, labeled with AAV (EGFP; green) and CTB (CF594; pink). AAVs were injected into the left eye at E13.5. CTB was injected into the right eye at P14. dLGNs were analyzed at P15. (B and C) Projection patterns of EGFP (Control) or EGFP+JAK2DN (JAK2DN) expressing RGC axons (green) and CTB expressing axons (pink) to the right (B) and left (C) dLGN. “Overlap” shows the overlapped region between EGFP and CTB signals (15% intensity threshold). “R” shows a threshold-independent representation of the segregation of ipsi and contralateral projections to the dLGN. (D–I) Quantification of eye-specific segregation. Quantification of the variance of the R-distributions in the right (D) and left (G) dLGN. Quantification of the area encompassed by projections from the contra (E and H) and ipsilateral (F and I) eye in the right and left dLGN. JAK2DN-expressing projections (light green bars) cover larger areas than Control (dark green bars). The areas occupied by CTB-labeled axons (axons where JAK2 signaling is not altered) were not different between JAK2DN (light pink bars) and control (dark pink bars). n = 8 sections from 4 mice. Scale bar, 100 μm.

Article Snippet: Cholera Toxin Subunit B, CF594 Dye Conjugates , Biotium , Cat#00072.

Techniques: Labeling, Injection, Expressing

Journal: Neuron

Article Title: An Activity-Dependent Determinant of Synapse Elimination in the Mammalian Brain

doi: 10.1016/j.neuron.2021.03.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cholera Toxin Subunit B, CF594 Dye Conjugates , Biotium , Cat#00072.

Techniques: Virus, Recombinant, In Situ, Clone Assay, Mutagenesis, Expressing, Generated, Software

Journal: Journal of Clinical Microbiology

Article Title: Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology

doi: 10.1128/JCM.00908-18

Figure Lengend Snippet: C. difficile toxin concentrations in samples in various result categories. The Singulex Clarity C. diff toxins A/B assay concentrations (combined TcdA and TcdB concentrations) are shown for stool samples with different PCR (Xpert C. difficile/Epi), EIA (C. Diff Quik Chek Complete), and CCNA results. The dashed line shows the preliminary cutoff value for the Singulex Clarity C. diff toxins A/B assay.

Article Snippet: TcdA (CDA-TNL-100; Native Antigen Company) and TcdB (CDB-TNL-100; Native Antigen Company) were spiked at 12 different concentrations, at a TcdA-TcdB concentration ratio of 1:1, into pooled C. difficile -negative stool (range, 0 to 50,000 pg/ml) and antigen-free sample diluent (TBS-EDTA with 3% BSA) (range, 0 to 10,000 pg/ml).

Techniques: Diff-Quik

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin

doi: 10.1073/pnas.1916965117

Figure Lengend Snippet: Cell surface binding assay. TcdB (2 nM) and either 1,000 µM TCDCA or dehydro-CA were preincubated together for 30 min on ice in serum-free media before adding to HCT116 cells. After incubating for 60 min on ice, cells were harvested and lysed. (A) Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody (Sigma, T6074) as a loading control. (B) Cell-associated TcdB bands were measured by densitometry using a ChemiDoc MP Imaging System (Bio-Rad). The TcdB-binding compound TCDCA, but not dehydro-CA, prevented surface binding of TcdB to cells. Bars represent SEM of three biological replicates.

Article Snippet: Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody as a loading control.

Techniques: Binding Assay, Western Blot, Control, Imaging

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin

doi: 10.1073/pnas.1916965117

Figure Lengend Snippet: Identification of nonsteroidal bile acid mimetics. (A) Results from high-throughput DSF screening of 2,400 drugs from the Microsource Spectrum collection. A statistical cutoff of ΔT = 3 °C inhibition of increase in stabilization was based on identification of molecules that were greater than 3 SDs above the mean of the data. Green dots represent hits that were bile acids or bile acid-like molecules. (B) Titration of ethaverine and parent compound papaverine against TcdB by DSF. Ethaverine dose dependently binds and thermally stabilizes TcdB with greater potency than papaverine. Bars represent SEM of four experiments. (C) Titration of ethaverine against full-length and CROP-less TcdB by DSF. Ethaverine dose dependently binds and thermally stabilizes full-length TcdB but not CROP-truncated TcdB1–2,283. (D) Cell surface binding assay. TcdB (2 nM) and either 100 µM of positive control methyl cholate, 50 µM ethaverine, or 50 µM papaverine were preincubated together for 30 min on ice in serum-free media before adding to HCT116 cells. After incubating for 60 min on ice, cells were harvested and lysed. Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody as a loading control. Cell-associated TcdB bands were measured by densitometry using a ChemiDoc MP Imaging System (Bio-Rad). The TcdB-binding compound ethaverine and to a lesser extent papaverine, prevented surface binding of TcdB to cells. Bars represent SEM of four biological replicates. (E) Normalized transepithelial resistance measurements in human Caco-2 cells, 3 to 6 h posttreatment. Ethaverine preserved significantly increased resistance across Caco-2 monolayer cells compared to mock control values (n = 3 biological replicates). Bars represent SEM of mean. ***P < 0.0006.

Article Snippet: Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody as a loading control.

Techniques: High Throughput Screening Assay, Inhibition, Titration, Binding Assay, Positive Control, Western Blot, Control, Imaging

Journal: mBio

Article Title: Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB

doi: 10.1128/mBio.00503-17

Figure Lengend Snippet: Impact of PepB2 on TcdB enzymatic activity, thermal stability, and cell association. (A) Immunoblot analysis of intracellular Rac1 glucosylation in CHO-K1 cells exposed to TcdB (0.037 pM, 0.37 pM, and 3.7 pM) for 25 h with and without PepB1 (50 µM) or PepB2 (50 µM). (B and C) In vitro Rac1 glucosylation assay. In these assays, purified Rac1 and UDP-glucose were combined with TcdB (1.2 nM, 2.4 nM, 12 nM, and 24 nM) (B) or GTD (0.5 nM, 1 nM, 5 nM, and 25 nM) (C). These experiments were carried out in the presence and absence of 50 µM PepB1 or PepB2, and Rac1 glucosylation was analyzed by immunoblotting. In the bar graph, the immunoblot data were quantified by densitometry analysis for TcdB ( n = 3) and GTD ( n = 2). (D) In vitro autoprocessing assay. TcdB autoprocessing was triggered by incubating TcdB (37 pM) for 1 h at 37°C with 500 µM IP6 in the presence and absence of 50 µM PepB1 or PepB2. Autoprocessing activity was evaluated by immunoblotting using an antibody that recognizes the amino-terminal domain of TcdB. Densitometry analysis was performed on the bands corresponding to the GTD cleaved from full-length TcdB ( n = 4). (E) Differential scanning fluorimetry (DSF) was used to determine the thermal stability ( T m ) of TcdB (740 nM) with and without 500 µM PepB1 or PepB2. The graph depicts increases in relative fluorescence units (RFU) as SYPRO Orange binds to hydrophobic regions of proteins that undergo temperature-induced unfolding. From these data, the T m was calculated and is displayed as mean ( n = 4) and standard deviation. (F) Immunoblot analysis of TcdB associating with cells for 10 min. CHO-K1 cells were exposed at 37°C to 4 nM TcdB in the presence and absence of 50 µM peptide. The cells were then washed, and total cell lysates were examined by immunoblotting. The bar graph represents densitometry analysis of the immunoblot data ( n = 4) from the 10-min exposure to TcdB. (G) Immunoblot analysis of TcdB associating with cells for 30 min. CHO-K1 cells were exposed at 37°C to 4 nM TcdB in the presence and absence of 50 µM peptide. The cells were then washed, and total cell lysates were examined by immunoblotting. In this figure, all bar graphs represent the mean densitometry value ± standard deviation, and asterisks indicate significant change. *, P < 0.01; **, P < 0.001.

Article Snippet: For each condition, equal amounts of total protein were analyzed by immunoblot probing with antibodies to the amino-terminal domain of TcdB (R&D Systems; catalog no. AF6246) or an antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam; catalog no. AB8245).

Techniques: Activity Assay, Western Blot, In Vitro, Purification, Fluorescence, Standard Deviation