Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Alogliptin Reduces Oxidative Stress in Cardiomyocytes and Ameliorates Diabetic Cardiomyopathy via the AURKB / NLGN2 Signaling

doi: 10.1002/kjm2.70149

Figure Lengend Snippet: AURKB transcriptionally regulates NLGN2 expression. (A) Intersection of downstream targets of AURKB and DEGs in the GSE197850 dataset. (B) ChIP dataset analyzed the binding of AURKB to the NLGN2 promoter. (C) GEPIA analyzed the expression relationship between AURKB and NLGN2. (D) RT‐qPCR detected NLGN2 expression in mouse cardiac tissues. (E) WB detected the protein expression of NLGN2 in mouse cardiac tissues. (F) RT‐qPCR detection of NLGN2 expression in mouse cardiomyocytes. (G) WB detection of NLGN2 protein expression in mouse cardiomyocytes. (H) RT‐qPCR detection of mRNA expression of AURKB and NLGN2 in mouse cardiomyocytes. (I) Dual‐luciferase assay for detection of NLGN2 luciferase activity in mouse cardiomyocytes. (J) ChIP assay to verify the binding of AURKB to the NLGN2 promoter in mouse cardiomyocytes. The cellular experiments were repeated three times independently for each group. Single‐factor comparisons were made between two groups using the t ‐test, and one‐way/two‐way ANOVA was used for multigroup comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns indicates no significant difference.

Article Snippet: The membranes were sealed with 5% skimmed milk and incubated overnight with primary antibodies against AURKB (1:500, A21918, ABclonal, Wuhan, Hubei, China), NLGN2 (1:500, A14289, ABclonal), β‐actin (1:10000, AC038, ABclonal), and with secondary antibody goat anti‐rabbit coupled to HRP (1:5000, G‐21234, Thermo Fisher).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Alogliptin Reduces Oxidative Stress in Cardiomyocytes and Ameliorates Diabetic Cardiomyopathy via the AURKB / NLGN2 Signaling

doi: 10.1002/kjm2.70149

Figure Lengend Snippet: Alogliptin ameliorates DCM by modulating the AURKB/NLGN2 signaling. (A) Measurement of EF, FS, LVIDd, IVSD, and LVPWd. (B) Representative pictures of mouse hearts and HW/BW ratios. (C) Detection of serum levels of CK‐MB and LDH in mice. (D) HE staining of cardiac lesions. (E) Masson staining of collagen fibrillation in mouse heart tissue. (F) WGA staining of the cross‐sectional area of cardiomyocytes in mouse heart tissue. (G) IHC staining of Col I and Col III contents in mouse heart tissues. (H) TUNEL to analyze apoptosis of cardiomyocytes in mouse heart tissue. (I) Relative fluorescence units of ROS in mouse heart tissues. Animal experiments were performed with five mice per group. The t‐test was used for single‐factor comparisons between the two groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were sealed with 5% skimmed milk and incubated overnight with primary antibodies against AURKB (1:500, A21918, ABclonal, Wuhan, Hubei, China), NLGN2 (1:500, A14289, ABclonal), β‐actin (1:10000, AC038, ABclonal), and with secondary antibody goat anti‐rabbit coupled to HRP (1:5000, G‐21234, Thermo Fisher).

Techniques: Staining, Immunohistochemistry, TUNEL Assay, Fluorescence

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Alogliptin Reduces Oxidative Stress in Cardiomyocytes and Ameliorates Diabetic Cardiomyopathy via the AURKB / NLGN2 Signaling

doi: 10.1002/kjm2.70149

Figure Lengend Snippet: The proposed signaling cascade of this study. Alogliptin alleviates cardiomyocyte oxidative stress injury in mice with DCM by enhancing AURKB‐mediated transcriptional activation of NLGN2.

Article Snippet: The membranes were sealed with 5% skimmed milk and incubated overnight with primary antibodies against AURKB (1:500, A21918, ABclonal, Wuhan, Hubei, China), NLGN2 (1:500, A14289, ABclonal), β‐actin (1:10000, AC038, ABclonal), and with secondary antibody goat anti‐rabbit coupled to HRP (1:5000, G‐21234, Thermo Fisher).

Techniques: Activation Assay

Journal: The Journal of Neuroscience

Article Title: Synaptic Vesicle Depletion Correlates with Attenuated Synaptic Responses to Prolonged Repetitive Stimulation in Mice Lacking α-Synuclein

doi: 10.1523/JNEUROSCI.22-20-08797.2002

Figure Lengend Snippet: Relative levels of selected synaptic proteins in Snca −/− versus Snca +/+ synaptosomes

Article Snippet: Antibodies against synapsin I, synaptophysin, synaptotagmin, rab3a, and amphiphysin were from Stressgen (Victoria, BC, Canada), antibody against vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein-25 (SNAP-25) were from Santa Cruz Biotechnology (Santa Cruz, CA), antibody against flotillin was from Transduction Laboratories, and antibody against SV2 was from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2

doi: 10.1038/s41467-023-37860-8

Figure Lengend Snippet: a A schematic diagram showing the designs of SV2A–SV2C chimeras where the sequences of SV2C-L4 and SV2A-L4 are shown as blue and red hollow bars, respectively. The structure of SV2C-L4 is shown as a blue cartoon with residues K518, I538, N559, and the N559 glycan shown as sticks (PDB: 5JLV . b The chimeric SV2Ac–G6 AA fusion protein is mono-dispersed in solution based on a gel-filtration analysis. c A pull-down assay was performed using H C E as prey and the His-tagged SV2-L4 or SV2–G6 AA fusion proteins as baits. Only SV2A–G6 AA and SV2Ac–G6 AA were able to pull down H C E (indicated by asterisks), but not other SV2–G6 AA fusion proteins or the stand-alone SV2-L4 variants. A representative result is shown ( n = 3). d and e The full-length SV2A and SV2Ac were expressed via lentiviral transduction in mouse cortical neurons cultured from SV2A/B double knockout (KO) mice. Neurons were exposed to H C E (5 min) ( d ) or BoNT/E at the indicated concentration (24 h in medium) ( e ). Cell lysates were harvested and analyzed by immunoblot assays. The synaptic vesicle protein, Synaptotagmin 1 (Syt-1), was detected as a loading control. The SNAP-25 antibody can detect both the full-length SNAP-25 and the fragment generated after cleavage by BoNT/E (marked with *). SV2A(+/+)/SV2B(−/−) neurons were analyzed in parallel as a positive control. A representative result is shown ( n = 2). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were purchased from the indicated vendors: rabbit monoclonal antibodies against β-actin (ABclonal, AC038); mouse monoclonal antibodies against SNAP-25 (Synaptic systems, Cl 71.1) or Syt-1 (Synaptic systems, #105011); rabbit polyclonal antibody against SV2C (Synaptic systems, #119202).

Techniques: Glycoproteomics, Filtration, Pull Down Assay, Transduction, Cell Culture, Double Knockout, Concentration Assay, Western Blot, Control, Generated, Positive Control

Journal: Nature Communications

Article Title: Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2

doi: 10.1038/s41467-023-37860-8

Figure Lengend Snippet: a Cartoon representation of the sialic acid (SIA)-bound H C E–SV2Ac complex with H C E colored in lemon, SV2Ac in orange, and sialic acid in red. An omit electron density map for sialic acid contoured at 1.5 σ was overlaid with the final refined model. b A close-up view of the interactions between H C E and sialic acid. One water molecule (W) that mediates the H C E–SIA binding is shown as a cyan sphere. c The H C E–SV2Ac–SIA complex and the H C A–SV2C complex ( PDB: 5JLV ) were superimposed based on H C E (lemon) and H C A (blue-white). The SIA (red), NAG (orange), and SV2C glycan (blue) are shown as sticks. The SIA-binding and the SV2C-glycan-binding pockets are highlighted in red and blue boxes, respectively. d A close-up view into the SV2C-glycan-binding pocket reveals the differences between H C A (blue-white) and H C E (lemon) in this area. H C E residue Q1250 would clash with the SV2C glycan based on the superposition. e A close-up view into the SIA-binding pocket shows that this pocket is partly conserved between H C E and H C A.

Article Snippet: The following antibodies were purchased from the indicated vendors: rabbit monoclonal antibodies against β-actin (ABclonal, AC038); mouse monoclonal antibodies against SNAP-25 (Synaptic systems, Cl 71.1) or Syt-1 (Synaptic systems, #105011); rabbit polyclonal antibody against SV2C (Synaptic systems, #119202).

Techniques: Binding Assay, Glycoproteomics, Residue

Journal: Nature Communications

Article Title: Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2

doi: 10.1038/s41467-023-37860-8

Figure Lengend Snippet: a Amino acid sequence alignment among human SV2A, SV2B and SV2C in the L4 region (prepared using MultAlin and ESPript 3.0 ). Identical residues are indicated with white letters on a red background, conserved residues are in red letters, and varied residues are in black letters. The SV2A residues that are recognized by H C E are indicated by orange triangles. Residue numbers of SV2A and the secondary structures of chimeric SV2Ac are shown on the top. b The WT SV2A or SV2A Y535T/Y557E mutant was expressed in SV2A/B KO mouse cortical neurons via lentiviral transduction. Neurons were exposed to the indicated toxins (14 h in medium). Cell lysates were harvested and analyzed by immunoblot assays. Actin served as a loading control. Left panel: representative immunoblots. Right panel: the percentage of SNAP-25 cleavage by BoNT/E in the left panel was quantified using ImageJ by comparing the amount of cleavage products versus the intact SNAP-25. Error bar represents SD from three independent experiments. c The WT SV2C or SV2C T521Y/E543Y mutant was expressed in SV2A/B KO neurons. Neurons were exposed to the indicated concentrations of BoNT/E (24 h in the medium). Cell lysates were harvested and analyzed by immunoblot assays. A representative result is shown ( n = 2). The cleaved SNAP-25 is marked with *. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were purchased from the indicated vendors: rabbit monoclonal antibodies against β-actin (ABclonal, AC038); mouse monoclonal antibodies against SNAP-25 (Synaptic systems, Cl 71.1) or Syt-1 (Synaptic systems, #105011); rabbit polyclonal antibody against SV2C (Synaptic systems, #119202).

Techniques: Sequencing, Residue, Mutagenesis, Transduction, Western Blot, Control

Journal: Nature Communications

Article Title: Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2

doi: 10.1038/s41467-023-37860-8

Figure Lengend Snippet: a Proposed binding modes of BoNT/E ( PDB: 3FFZ ) and BoNT/A ( PDB: 3BTA ) with the membrane-bound SV2 and gangliosides. The holotoxins are positioned based on the structures of the H C E–SV2Ac and the H C A–SV2C ( PDB: 5JLV ) complexes. A representative complex-type N-glycan (red sticks, PDB: 3QUM ) is modeled to represent the N-glycan of SV2A. The gangliosides GD1a and GT1b are modeled based on the structures of H C E–GD1a ( PDB: 7OVW ) and H C A–GT1b ( PDB: 2VU9 ). b The structures of the H C E–SV2Ac and the H C A–SV2C ( PDB: 5JLV ) complexes with the modeled gangliosides are superimposed based on H C E and H C A. The view angle on the left panel is identical to that shown in panel ( a ). A different view with a rotation of ~115° about a vertical axis is shown on the right panel. Two equivalent β-sheets (β8) on SV2Ac-L4 and SV2C-L4 are highlighted as a marker to show the similar orientations of SV2Ac and SV2C relative to the membrane in this putative model. c Surface representation of BoNT/E holotoxin in complex with SV2A (orange) and GD1a (cyan sticks). BoNT/E adopts a “closed-wing” conformation, in which H C E (lemon) and LC/E (pale cyan) are located on the same side of the long helical H N E (purple). d Surface representation of BoNT/A holotoxin in complex with SV2C (blue) and GT1b (deep teal sticks). BoNT/A has an “open-wing” conformation, in which H C A (light purple) and LC/A (yellow) are located on the opposite sides of the long helical H N A (brown).

Article Snippet: The following antibodies were purchased from the indicated vendors: rabbit monoclonal antibodies against β-actin (ABclonal, AC038); mouse monoclonal antibodies against SNAP-25 (Synaptic systems, Cl 71.1) or Syt-1 (Synaptic systems, #105011); rabbit polyclonal antibody against SV2C (Synaptic systems, #119202).

Techniques: Binding Assay, Membrane, Glycoproteomics, Marker