Journal: iScience

Article Title: The Drosophila connectome reveals axo-axonic synapses on descending neurons

doi: 10.1016/j.isci.2026.115624

Figure Lengend Snippet: Axcs make cholinergic axo-axonic synapses on the GFs as predicted by the MANC connectome (A) AN08B098-type neurons (green) form axo-axonic connections at distinct locations along giant fiber (red, GF) axons. The scale bars shown are 20 μm. (B) The AN08B098-GF connections reconstructed in Neuroglancer (Neuroglancer scale 530.45 μm/vh) mirror the morphology seen with fluorescence in A . (C) We validated AN08B098-to-GF connectivity using anti-bruchpilot to stain for T-bars in active zones (white) along AN08B098-type neurons and colocalized the resulting fluorescence with the GFs (red), which were filled with tetramethylrhodamine. The scale bars shown are 20 μm. (D) Neuroglancer reveals presynaptic sites at similar locations seen in colocalized fluorescent image (Neuroglancer scale 530.45 μm/vh). (E and F) XY and XZ plane views of the preparation shown in (A) and (B). The zoomed-in 6 μm (scale bars 3 μm) inlays show AN08B098 forming a single synapse with the GF in (E). The yellow arrows detail the precise location AN08B098 forms the Brp-positive chemical synapse (white) to the GFs in (F). Scale bars shown are 5 μm. (G–O) EM images showing monosynaptic connections between single GF (green) and AN08B098 neurons identified by the following MANC id: (G and H) 21041, (I) 21589, (J) 23949, (K) 152261, (L) 16900, (M) 20444, (N) 22275, and (O) 24038. Pre-and postsynaptic sites are detected in EM slices using a 3D convolutional neural network to identify T-bars (cyan dots) and postsynaptic densities (PSDs, magenta dots). (P–S) We expressed anti-choline acetyltransferase (anti-ChAT) and anti-GFP in AN08B098 neurons. Anti-ChAT colocalizes to AN08B098 cells, with particularly bright staining in the cell bodies. This finding suggests acetylcholine synthesis is present within these cells, validating connectome transmitter predictions for AN08B098. Scale bars shown are (P) 20 μm and (Q–S) 5 μm.

Article Snippet: A mouse primary antibody against bruchpilot (1:50, DSHB, NC82) was used to label presynaptic chemical active zones and coupled to secondary Goat anti-mouse Alexa Fluor 647 (1:500, 115-605-003).

Techniques: Fluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) PCR amplification results after five blind passages. ( B ) TEM observation of CPV L4. Arrows indicate spherical virus particles; scale bar, 50 nm. ( C ) Cytopathic effects of CPV L4 infection in F81 cells (MOI = 1). ( D ) Western blot analysis of VP2 expression in CPV L4-infected F81 cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of CPV L4-infected F81 cells (MOI = 1); scale bar, 100 µm. ( F ) Growth kinetics of CPV L4 in F81 cells (MOI = 0.1). Infected cells and supernatants were harvested every 12 h for 72 h. Viral genome copies are shown as log10(copies/µL). Data represent three independent experiments (triplicate each) and are presented as mean ± SEM.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Amplification, Virus, Infection, Western Blot, Expressing, Modification, Control, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Genetic and evolutionary analysis of CPV VP2. ( A ) Maximum-likelihood phylogenetic tree of the CPV VP2 gene constructed using MEGA X with the JTT+G substitution model and 1000 bootstrap replicates. Orange circles indicate isolates CPV L1–L8. ( B ) Heatmap of amino acid similarity among CPV VP2 proteins. ( C ) VP2 amino acid mutation analysis of isolates CPV L1–L8 relative to prototype CPV-2. ( D ) Visual alignment of partial VP2 sequences between CPV L4 and giant panda-derived CPV-2c strains. The orange arrow indicates the full-length VP2 sequence of CPV L4, and blue arrows indicate reported partial VP2 sequences of four panda-infecting CPV-2c strains. Sequences are aligned by VP2 amino acid positions.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Construct, Mutagenesis, Derivative Assay, Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of nanobody expression construct. Nanobody genes are expressed under the Pgrac promoter, fused with the amyQ signal peptide for secretion via the Sec pathway, and linked to a 6 × His tag for detection and purification. ( B ) SDS-PAGE analysis of five nanobodies in Bacillus subtilis culture supernatants. ( C ) Western blot identification of His tags for five nanobodies. ( D ) Indirect ELISA evaluation of binding reactivity between nanobodies and immunization VP2 protein; results are shown as OD450 values. * p < 0.05, **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Purification, SDS Page, Western Blot, Indirect ELISA, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Western blot validation of Nb10 recognition of VP2 in CPV L4-infected cells (MOI = 1); Nb10 served as the primary antibody. ( B ) Immunofluorescence detection of Nb10 recognition at the cellular level (MOI = 1); Nb10 served as the primary antibody; nuclei were stained with DAPI (blue). ( C ) Molecular docking model of Nb10 with CPV VP2 showing potential interaction sites. ( D ) Visualization of the Nb10–VP2 complex from different angles.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Western Blot, Biomarker Discovery, Infection, Immunofluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of Nb10-Fc expression construct. ( B ) Western blot validation of Nb10-Fc expression. Nb10-Fc’ indicates the non-reducing condition result; Nb10-Fc indicates the reducing condition result. Nb10-Fc was detected under both conditions but showed different migration characteristics. ( C ) Indirect ELISA evaluation of Nb10-Fc binding to immunization VP2 protein (OD450). ( D ) Western blot validation of Nb10-Fc recognition of VP2 in CPV L4-infected cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of Nb10-Fc recognition in CPV L4-infected cells (MOI = 1). Specific green fluorescence was observed in infected cells; nuclei were stained with DAPI (blue). ( F ) Molecular docking model and interface analysis of Nb10-Fc with CPV VP2. ( G ) Visualization of the Nb10-Fc–VP2 complex from different angles. **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Western Blot, Biomarker Discovery, Migration, Indirect ELISA, Binding Assay, Infection, Modification, Control, Immunofluorescence, Fluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Dynamic property analyses of CPV L4 VP2–Nb10 (left) and CPV L4 VP2–Nb10-Fc (right) complexes. ( A ) RMSD curves; ( B ) RMSF curves (green shaded regions indicate interaction interfaces); ( C ) hydrogen bond numbers; ( D ) SASA curves; ( E ) radius of gyration (Rg) curves.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques:

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging