Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic monoamine oxidase B (MAOB)–gamma-aminobutyric acid (GABA) axis as a molecular brake on repair following spinal cord injury

doi: 10.1038/s41392-025-02398-2

Figure Lengend Snippet: MAOB inhibition with KDS2010 reduces astrocytic GABA levels and enhances proBDNF and TrkB expression after SCI. a Differential interference contrast (DIC) images of the dorsal horn of the spinal cord (top and middle) and a magnified view of a whole-cell patch-clamped lamina II neuron (bottom). The yellow boxes indicate the magnified region of interest. b Representative traces of GABA A receptor-mediated tonic GABA current in each group (Sham+V, SCI + V, and SCI + KDS 2w). The dashed lines (gray) and double-headed arrows (purple and brown) indicate baseline shifts (I GABA and I Tonic ) with bath application of GABA (10 μM, green bar) and bicuculline (Bic, 50 μM, orange bar). c Tonic GABA current density (top, left), GABA-induced full activation current density (top, right), frequency (bottom, left) and amplitude (bottom, right) of spontaneous inhibitory postsynaptic currents (sIPSCs) in each group. d Confocal images of the injured areas stained with anti-proBDNF (green), anti-GABA (red), anti-NeuN (magenta), and anti-GFAP (white) antibodies at PI 10w in SCI + V and SCI + KDS 2w. e Mean intensity of GABA (left), NeuN-positive proBDNF (middle), GFAP-positive proBDNF (right). f Western blotting of BDNF and TrkB in Sham+V, SCI + V, and SCI + KDS 2w at PI 10w. g Quantification of BDNF (left) and TrkB (right) expression levels in Western blotting. β-actin was used as a control for protein amount. h Experimental timeline for quantitative real-time PCR with GABA (100 μM) or Bic (50 μM) treatment of primary cultured spinal cord astrocytes at 14 days in vitro (DIV). i Relative (comparative Ct) BDNF expression level of each drug treatment condition (GABA, GABA+Bic, and Bic). j Representative images of control and GABA-treated spinal cord astrocytes. k Western blotting of proBDNF in control and GABA-treated condition. l Quantification of proBDNF in Western blotting. β-actin was used as a control for protein amount. m Confocal images of the injured areas stained with anti-TrkB (green), DAPI (blue), and each Tau, MBP, or GFAP (red) antibodies at PI 10w. KDS2010 treatment was initiated at PI 2w and continued daily until PI 10w. Group labels indicate the treatment start point. n Mean intensity of TrkB in the Tau-positive neurons (left), MBP-positive oligodendrocytes (middle), and GFAP-positive astrocytes (right) at PI 10w. All the data are expressed as the means ± S.E.M.s * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Approximately 24 h after drug treatment, primary mouse spinal cord astrocytes or spinal cord tissues were lysed in RIPA buffer (MB030-0050, ROCKLAND) containing protease and phosphatase inhibitors (1861280, Thermo Fisher Scientific).

Techniques: Inhibition, Expressing, Activation Assay, Staining, Western Blot, Control, Real-time Polymerase Chain Reaction, Cell Culture, In Vitro

Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics

Article Title: Sodium butyrate restored TRESK current controlling neuronal hyperexcitability in a mouse model of oxaliplatin-induced peripheral neuropathic pain.

doi: 10.1016/j.neurot.2024.e00481

Figure Lengend Snippet: Fig. 5. Sodium butyrate reversed oxaliplatin-induced repression of Kcnj9 and Kcnk18 expression. A, Expression levels of potassium channels encoding genes selected from the Pain Gene Database in DRG tissues of mice receiving oxaliplatin (Oxa) or vehicle (Veh) on day 14 and 21 post-injection. One-way ANOVA fol- lowed by Tukey's multiple comparisons test comparing Veh and Oxa group per gene, n ¼ 3–4 per group. p-values not shown were of no significant difference, *p < 0.05, **p < 0.01. B and C, Expression levels of Kcnj9 or Kcnk18 of sham mice followed by vehicle treatment (Veh), male animals received oxaliplatin followed by vehicle treatment (Oxa) or male animals received oxaliplatin followed by sodium butyrate treatment (NaB) on day 14. One-way ANOVA followed by Tukey's multiple comparisons test, n ¼ 3–4 per group. ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. D, Representative immunofluorescence of TRESK (Green), NeuN (Red) and DAPI (white) in L3-L6 DRG tissues collected at day 21 post first dose of oxaliplatin. Male mice were treated with sham or oxaliplatin injection followed by sodium butyrate. E. Quantification of mean fluorescence intensity of TRESK in NeuN- positive cells. One-way ANOVA followed by Tukey's multiple comparisons test, n ¼ at least 30 cells from 3 to 5 male mice per group. ns, non-significant, *p < 0.05.

Article Snippet: The samples were washed 3 times with PBS for 6 min before incubating with blocking buffer [10 % normal goat serum (Gioco, Waltham, MA, USA), 0.03 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS] for 1 h. Blocked samples were then incubated with primary antibodies against HDAC1 (#YT2145, ImmunoWay Biotechnology, Planto, TX, USA), AcH3K9 (#YK006, Immunoway, Plano, TX, USA), AcH3K14 (#07–353, Merck Millipore, Burlington, MA, USA), Kcnk18 (#APC-122, Alomone Labs, Jerusalem, Israel), NeuN (#ABN90, Merck Millipore, Burlington, MA, USA), or β3-Tubulin (#YM3165, ImmunoWay Biotechnology, Planto, TX, USA) in fresh blocking buffer at 4 C overnight.

Techniques: Expressing, Injection

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes analysis. Volcano plots showing the top twenty up- or downregulated genes for each cell type. The red dots represent upregulated genes, and the blue dots represent downregulated genes. ( P value < 0.05, and |log2foldchange| > 0.58). Bubble plot indicating the top enriched pathways for each cell type based on KEGGpathway enrichment analysis of differentially expressed genes( www.kegg.jp/kegg/kegg1.html ). The sizes of the dots represent the number of genes included in each pathway. The colour gradient of dots represents the adjusted P values of each enriched pathway. The genes Pfkfb3 , Tbc1d1 and the insulin signaling pathway; Pdgfd and the PI3K-Akt signaling pathway; Cxcr2 and PLD, Rap1 signaling pathways; Ccl21 and chemical carcinogenesis-reactive oxygen species signaling pathway; Mef2c and the calcium signaling pathway; Negr1 and leukocyte transendothelial migration signaling pathway are labelled by the black and red bars in Panels a , b , c , d , e , and f .

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Protein-Protein interactions, Migration

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: High-dimensional weighted gene coexpression network analysis (hdWGCNA). a The top left panel depicts the soft power threshold for choosing a scale-free topology model. The average connectivity of the topological network was most stable at the lowest soft threshold of 6. b Dynamic Tree Cut algorithm for gene clustering. Each leaf on the tree represents a gene, and the colour at the bottom indicates the assignment to a specific coexpression module. c Bubble plots displaying the scores obtained for eight modules in eight cell subtypes. d snRNA-seq UMAP coloured by module eigengene (ME) for eight coexpression modules. e The eight modules’ values of -log10 (Adj P value) on the Y-axis versus average log2 (fold change) on the X-axis are shown for each cell type, including endothelial cells, fibroblast 1, fibroblast 2, myocytes, neurocytes, pericytes, satellite cells and smooth muscle cells. The larger the value is, the greater the fit between the coloured module and the specific cell type. The plot shows that fibroblast 1 features a brown module, fibroblast 2 features a green module, endothelial cells feature a yellow module, and myocyte cells feature a turquoise module. f UMAP plot of the gene coexpression network with eight coloured modules and the top two hub genes labelled in each module. Nodes are coloured according to coexpression module assignment. g Hub genes in each module were identified by eigengene-based connectivity (KME). The following five hub genes associated with fibrosis were identified: Pfkfb3 and Tbc1d1 in the brown module, Pdgfd in the green module, Sema3a in the yellow module and Ryr3 in the turquoise module.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques:

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 ( a ), PDGFD ( b ), CXCR2 ( c ), CCL21 ( d ), SEMA3A ( e ), RYR3 ( f ), MEF2C ( g ), NEGR1( h ) and TBC1D1 ( i ) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 ( a ), Pdgfd ( b ), Cxcr2 ( c ), Negr1 ( d ), Sema3a ( e ), and Mef2c ( f ) normalized to that of GAPDH. Asterisks indicate significant differences ( n = 3 in each group) (* P < 0.05). Western blotting analysis of the protein expression of PFKFB3 ( g ), PDGFD ( h ), CXCR2 ( i ), NEGR1 ( j ), SEMA3A ( k ), and MEF2C ( l ) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control