Journal: Journal of Nanobiotechnology

Article Title: Carbon dot-based treatment for bacterial pneumonia by promoting a PI3K-mediated M1 polarization of macrophages

doi: 10.1186/s12951-025-03399-7

Figure Lengend Snippet: Molecular dynamics simulation and verification of interaction between CDots and PIK3CD. ( A ) Three-dimensional structure of PIK3CD. Secondary structural elements are depicted as tube helices. ( B ) Ramachandran plot of PIK3CD. ( C ) The structures of CDots binding to PIK3CD, which are extracted from the molecular dynamics simulation trajectory at 0, 60, and 120 ns, respectively. ( D ) The variation in the numbers of residues within different secondary structures of PIK3CD throughout the simulation. ( E ) RMSD of the Cα atoms of PIK3CD in PIK3CD-CDots complex against time. ( F ) RMSF values of PIK3CD and in the PIK3CD-CDots complex, respectively. ( G ) 3D diagram of the interaction between CDots and PIK3CD. Amino acid residues involved in the binding of CDots are drawn as sticks. ( H ) The Immunoprecipitation experiment of PIK3CD. Western blotting showed that there was a clear PIK3CD band after Anti-PIK3CD IP, which proved that the PIK3CD antibody had bound PIK3CD. ( I ) The fluorescence data of the immunoprecipitation complex after removing the beads were collected with a VICTOR X5 Multilabel Plate Reader. CDots (MH-S) represents immunoprecipitation obtained without antibody; IgG Isotype + CDots (MH-S) represents immunoprecipitation obtained using isotype control; Anti-PIK3CD + CDots (MH-S) represents immunoprecipitation obtained using PIK3CD antibody; Anti-PIK3CD + CDots (no cell) represents immunoprecipitation obtained using PIK3CD antibody without cells. ( J ) The fluorescence of immunoprecipitation complexes with beads was detected by confocal images. * p < 0.05, ** p < 0.01. Scale bar = 50 μm. ( K )Molecular dynamics simulation trajectory prediction of the structural domain of CDots binding to PIK3CD. ( L ) Table of amino acid residues in PIK3CD with potential interactions with CDots obtained from molecular dynamics simulations. ( M ) Laser confocal microscopy displays the binding of CDots to PIK3CD and its deletion forms, where green fluorescence represents the expression of PIK3CD and its deletion forms, and blue fluorescence represents CDots. The scale bars = 10 µ m /20 µ m, respectively. Histogram represents fluorescence intensity analysis of EGFP-C1 (green line) and CDots (blue line) in 293T cells. ( N ) The representative flow cytometry plots show the M1polarization level in MH-S cells after CDots bind to PIK3CD and its deletion

Article Snippet: PIK3CD antibody (67964-1-Ig, 1:1000, Proteintech) for immunoprecipitation and Isotype IgG antibody (Cell Signaling Technology, #2729) as control were added to the tubes containing the beads, followed by thorough mixing and incubation for 2 h. The IP antibodies were then incubated with the cell lysate at 4 °C overnight.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Fluorescence, Control, Confocal Microscopy, Expressing, Flow Cytometry

Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Inhibition of C3b and C4b deposition by r Em CRT and its functional binding regions measured by ELISA. ( A ) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of r Em CRT, r Em CRT-S, or r Em CRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b ( a ) and C4b ( b ) was detected with anti-C3 or C4 monoclonal antibodies. ( B ) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD 450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added r Em CRT).

Article Snippet: After washing with 1 × TBST containing 5 mM CaCl 2 , the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 μL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing