Journal: Computational and Structural Biotechnology Journal

Article Title: Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus

doi: 10.1016/j.csbj.2024.03.013

Figure Lengend Snippet: APA visualization and experimental validation in the GOM vs NOM group. (a) RNA-seq tracks for two representative APA-related genes (LRRC25, UBXN8). Red tracks represent samples from the GOM group, while blue tracks represent samples from the NOM group. (b) PDUI levels of LRRC25 (left) and UBXN8 (right) between the GOM and NOM groups. (c) Schematic diagram of primer design for lengthening and shortening APA isoforms. (d) qRT-PCR validations for lengthening and shortening APA isoforms of LRRC25 and UBXN8. Results are presented as the relative proportion of 3'UTR lengthening isoform in GDM compared with normal group. Significance levels: * p < 0.05, * * p < 0.01, * ** p < 0.001, the same applies to all figures.

Article Snippet: The primary antibodies used in Western blot included LRRC25 (Bioss, bs-12339R), CSTF3 (Proteintech, 24290–1-AP) and PPP1CB (Proteintech, 10140–2-AP).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR

Journal: Computational and Structural Biotechnology Journal

Article Title: Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus

doi: 10.1016/j.csbj.2024.03.013

Figure Lengend Snippet: Comparative analyses of the transcriptome and proteome from different adipose groups. (a) PCA of the transcriptome (left) and proteome (right) clearly distinguished different groups. (b) Bar graphs showing the number of DEmRNAs (top) and DEPs (bottom) in different group comparisons. (c) Bubble chart displaying functional enrichment analysis of DEPs in different group comparisons. The size of the points represents the number of related genes. (d) Venn diagrams illustrating gene expression differences between the transcriptome and proteome. (e) Violin plots showing the distribution of FC ratios for non-common DEGs (left) and common DEGs (right) in different group comparisons. The y-axis values are represented as log 10 (FC ratio) because the FC ratio for some genes is extremely large. (f) mRNA and protein expression levels of two representative genes (LRRC25, SEMA3C) in the GOM and NOM groups indicate large differences between the transcriptome and proteome.

Article Snippet: The primary antibodies used in Western blot included LRRC25 (Bioss, bs-12339R), CSTF3 (Proteintech, 24290–1-AP) and PPP1CB (Proteintech, 10140–2-AP).

Techniques: Functional Assay, Expressing

Journal: Computational and Structural Biotechnology Journal

Article Title: Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus

doi: 10.1016/j.csbj.2024.03.013

Figure Lengend Snippet: 3'UTR elongation leads to reduced protein translation due to increased availability for miRNA binding. (a) Bar chart showing the distribution of mRNA-protein matched genes based on log2(FC) and PDUI values. The chi-squared test results indicate a significant correlation between PDUI and mRNA as well as protein levels in the GOM vs NOM group. (b) APA-regulated genes associated with metabolism further enriched in energy metabolism, immune regulation, and nucleic acids and proteins regulation. (c) The number of genes gained or lost potentially binding miRNAs due to APA regulation in the GOM vs NOM groups. (d) Calculated miRNA binding density per kb for 3’UTR lengthening and shortening genes in the GOM vs NOM groups. (e) tSNE projection of all 28513 sequenced cells in the GOM and NOM groups. Cells were categorized into 26 clusters (left) and further annotated into 11 cell types based on cell markers (right). (f-g) The predominant cell population for LRRC25 (f) and mRNA expression levels in the corresponding populations (g). (h) Expression levels of LRRC25 3'UTR lengthening and total isoforms detected by long and short primers indicate successful transfection of both overexpression plasmids with no significant differences in transfection efficiency. (i) Western blot results showing protein levels of LRRC25 in diferent groups, suggesting that the 3'UTR shortening isoform has higher protein translation efficiency. (j) LRRC25 functional validation schematic. Control group: no treatment; LPS-treated group: treated with 100 ng/ml LPS for 48 h; LRRC25 overexpression + LPS-treated group: transfected with LRRC25 overexpression plasmids for 48 h, then treated with 100 ng/ml LPS for 48 h. (k-l) Flow cytometry results showing that overexpressing LRRC25 reduced the ratio of pro-inflammatory phenotype (CD86 + CD206 - ) to anti-inflammatory phenotype (CD86 - CD206 + ) in response to LPS treatment.

Article Snippet: The primary antibodies used in Western blot included LRRC25 (Bioss, bs-12339R), CSTF3 (Proteintech, 24290–1-AP) and PPP1CB (Proteintech, 10140–2-AP).

Techniques: Binding Assay, Expressing, Transfection, Over Expression, Western Blot, Functional Assay, Control, Flow Cytometry

Journal: Computational and Structural Biotechnology Journal

Article Title: Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus

doi: 10.1016/j.csbj.2024.03.013

Figure Lengend Snippet: The decrease in APA-related trans-acting factors causes 3'UTR elongation and reduced protein translation. (a) Differential protein levels of APA-related trans-acting factors in different group comparisons, with 10 out of 23 factors not detected. (b) Pearson correlation coefficients between the protein levels of trans-acting factors and the mean PDUI in OM adipose tissues show a strong correlation between trans-acting factors and APA. (c) Pearson correlation coefficients of protein levels between trans-acting factors (CSTF3 and PPP1CB) and other genes in OM adipose tissue indicate a strong positive correlation between trans-acting factors and protein translation levels. (d) Cell population distribution of CSTF3 (top) and PPP1CB (bottom). (e) Expression levels of CSTF3 (top) and PPP1CB (bottom) in various cell populations. (f) Experimental verification of protein expression levels of CSTF3 and PPP1CB in OM adipose tissue. (g) The knockdown efficiency of CSTF3 and PPP1CB in TDMs, HUVECs and ADSCs. (h) qRT-PCR validations for lengthening and shortening APA isoforms of LRRC25 and RAI14. Results are presented as the relative proportion of 3'UTR lengthening isoforms in different groups. Fig. S1 Distribution and functional enrichment of APA events across different groups. (a) Heatmap of dynamic APA events in adipose tissue. (b) PCA plots for samples originating from the same tissue source. (c) Scatter plots of mean PDUI values for genes in different group comparisons, with a cutoff value of ∆PDUI = ± 0.1. (d) Functional enrichment networks of significantly different APA events in each group comparison, with clusters of functions connected by edges. The top 20 clusters by P-value are shown. Fig. S2 RNA-seq tracks and PDUI levels for representative genes in different group comparisons: GSC vs NSC group (a), NOM vs NSC group (b), and GOM vs GSC group (c). Fig. S3 Distribution of genes in different group comparisons: PCA plots of mRNA (a) and protein (b) from the GDM and normal groups. DEmRNA expression heatmaps (c) and volcano plots (d), DEP expression heatmaps (e), and volcano plots (f). The cut-off values were │FC│≥ 1.2 and adj. P < 0.01 for DEmRNAs, │FC│≥ 1.2 and adj. P < 0.05 for DEPs. Fig. S4 Gene distribution differences between the transcriptome and proteome. (a) Distribution of Pearson correlation coefficients for protein-mRNA matched genes with the proportion of genes above and below ± 0.5 indicated. (b-d) mRNA and protein expression levels of representative genes in the GSC vs NSC (b), NOM vs NSC (c), and GOM vs GSC (d) groups. Fig. S5 Partial results from the snRNA-seq analysis: Distribution of cells from GOM and NOM groups in tSNE projection (a) and heatmap of cell markers for different cell populations (b). Additional material information. Additional file 1: Table S1 The clinic characteristics of GDM patients and controls. Additional file 2: Table S2 The primers for qRT-PCR and overexpression vectors and the sequence of siRNA. Additional file 3: Table S3 The dynamic APA events calculated by Dapars algorithm. Additional file 4: Fig. S1 Distribution and functional enrichment of APA events across different groups. Additional file 5: Fig. S2 RNA-seq tracks and PDUI levels for representative genes in different group comparisons. Additional file 6: Fig. S3 Distribution of DEGs in different group comparisons. Additional file 7: Table S4 The transcriptomics and proteomics data analysis in different group comparisons. Additional file 8: Table S5 The functional enrichment analysis of DEPs in different group comparisons. Additional file 9: Fig. S4 Gene distribution differences between the transcriptome and proteome. Additional file 10: Table S6 The protein-per-mRNA FC ratio analysis for mRNA-protein matched genes in different group comparisons. Additional file 11: Table S7 The GO biological process enrichment results for potentially APA-regulated genes. Additional file 12: Table S8 Predicted binding miRNAs between proximal and distal PAS of mRNA-Protein matched genes and their relation with GDM. Additional file 13: Fig. S5 Partial results from the snRNA-seq analysis.

Article Snippet: The primary antibodies used in Western blot included LRRC25 (Bioss, bs-12339R), CSTF3 (Proteintech, 24290–1-AP) and PPP1CB (Proteintech, 10140–2-AP).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Functional Assay, Comparison, RNA Sequencing Assay, Over Expression, Sequencing, Binding Assay

Journal: Theranostics

Article Title: Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice

doi: 10.7150/thno.49199

Figure Lengend Snippet: SCI decreases transcriptional activity in the cortex related to neuronal synaptic function while long-term PLX depletion in the context of SCI increases astrocyte-related gene expression. ( A ) Table showing the number of differential expressed (DE) genes in the somatosensory cortex by pairwise comparison using NanoStringDiff. N = 4 mice/group. The three comparisons were as follows: ( 1) Sham/PLX vs. Sham/Veh; (2) SCI/Veh vs. Sham/Veh; and (3) SCI/PLX vs. SCI/Veh . About two-thirds of DE genes in the cortex after SCI were downregulated ( SCI/Veh vs. Sham/Veh ). PLX treatment in both Sham and SCI animals resulted in largely decreased expression of genes. ( B ) Distribution of DE genes in the cortex for the SCI/Veh vs. Sham/Veh comparison by pathway annotation. The neurons/neurotransmission and apoptosis pathways had the highest number of DE genes, with eight out of ten decreased in each category. Microglia function had the next highest number of DE genes as well as the highest number of genes with increased expression of any pathway (five). ( C ) Heatmap of DE genes in the cortex after SCI that are either downregulated (left; 30 genes) or upregulated (right; 14 genes). Color coding was based on z-score scaling. Individual tables show lists of DE genes by specific pathway. The “†” sign denotes genes that are upregulated. ( D ) Venn diagram showing the overlap of the DE gene lists for the three pairwise comparison. The central overlap area identifies five genes that are both modified by injury as well as PLX treatment after SCI and shown in the corresponding heatmap (color coding based on z-score scaling.) Two genes, Il1a and Lrrc25 , were increased with injury in the cortex and decreased with PLX treatment. 68 genes were modified by PLX only in both Sham and SCI animals, and only two of these genes had increased expression ( C4a , Slc2a1 ). A group of 17 genes were modified by PLX only in SCI animals. 11 out of 17 genes were increased in these animals relative to the SCI Veh group, and five of these upregulated genes were specifically associated with astrocyte function.

Article Snippet: Real-time PCR for target mRNAs was performed using TaqMan gene expression assays for Itgam (CD11b), Mm00434455_m1, TNFα, Mm00443258_m1; Csfr1, Mm01266652_m1; P2ry12, Mm00446026_m1; Trem2, Mm04209424_g1; Mfge8, Mm00500549_m1; Casp1, Mm00438023_m1; Casp6, Mm01321726_g1; Casp8, Mm01255716_m1; Bax, Mm00432051_m1; Bcl2, Mm00477631_m1; Gfap, Mm01253033_m1; Il-1a, Mm00439620_m1; Lrrc25, Mm00462019_m1; Gbp2, Mm00494576_g1; Osmr, Mm01307326_m1; Entpd2, Mm00515450_m1; Vim, Mm01333430_m1; C4a, Mm01132415_g1; GAPDH, Mm99999915_g1 (Applied Biosystems, Carlsbad, CA] on an QuantStudioTM 5 Real-Time PCR System (Applied Biosystems). qPCR reactions were run in duplicates to eliminate mistakes of pipetting, which was composed of 3 stages, 50 °C for 2 min, 95 °C for 10 s for each cycle (denaturation), and finally, the transcription step at 60 °C for1 min. Gene expression was normalized by GAPDH and compared to the control sample to determine relative expression values by the 2-ΔΔCt method.

Techniques: Activity Assay, Gene Expression, Comparison, Expressing, Modification

Journal: Theranostics

Article Title: Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice

doi: 10.7150/thno.49199

Figure Lengend Snippet: qPCR analysis in RNA isolated from the cerebral cortex. ( A ) PLX treatment caused a significant reduction of microglial receptors (Csf1r, P2ry12, Trem2, and Itgam) in both Sham and SCI groups. ( B ) Inflammatory cytokine IL-1α and Lrrc25 expression. ( C-D ) Astrocytes marker Gfap and the genes that are associated with astrocyte function including Vim, Gbp2, Osmr, C4a, and Entpd2 remained unchanged in both SCI and PLX groups. All data are presented as independent data points. N = 4 mice/group. * p < 0.05, ++++ p < 0.0001, vs. Sham/Veh group; #### p < 0.0001 vs. SCI/WT. Two-way ANOVA following Tukey's multiple comparisons test.

Article Snippet: Real-time PCR for target mRNAs was performed using TaqMan gene expression assays for Itgam (CD11b), Mm00434455_m1, TNFα, Mm00443258_m1; Csfr1, Mm01266652_m1; P2ry12, Mm00446026_m1; Trem2, Mm04209424_g1; Mfge8, Mm00500549_m1; Casp1, Mm00438023_m1; Casp6, Mm01321726_g1; Casp8, Mm01255716_m1; Bax, Mm00432051_m1; Bcl2, Mm00477631_m1; Gfap, Mm01253033_m1; Il-1a, Mm00439620_m1; Lrrc25, Mm00462019_m1; Gbp2, Mm00494576_g1; Osmr, Mm01307326_m1; Entpd2, Mm00515450_m1; Vim, Mm01333430_m1; C4a, Mm01132415_g1; GAPDH, Mm99999915_g1 (Applied Biosystems, Carlsbad, CA] on an QuantStudioTM 5 Real-Time PCR System (Applied Biosystems). qPCR reactions were run in duplicates to eliminate mistakes of pipetting, which was composed of 3 stages, 50 °C for 2 min, 95 °C for 10 s for each cycle (denaturation), and finally, the transcription step at 60 °C for1 min. Gene expression was normalized by GAPDH and compared to the control sample to determine relative expression values by the 2-ΔΔCt method.

Techniques: Isolation, Expressing, Marker

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: Increase in the transcription and translation of LRRC25 in H37Rv-infected BV2 cells. ( a ) The laser scanning confocal images GFP-H37Rv infects BV2 cells with MOIs of 1, 5, and 10 (n = 3). ( b ) Relative expression of LRRC25 mRNA after H37RV infection of BV2 cells for 4 h (n = 4), Student’s t test. ( c ) Western blot images of LRRC25 after H37RV infection of BV2 cells for 4 h (n = 3). ( d ) Cellular immunofluorescence image of LRRC25 H37RV-infected BV2 cells for 4 h. Scale bar, 100 μm. ( e ) H37Rv CFU count of mice brain tissue at 2 weeks, 4 weeks, and 6 weeks (n = 3), Kruskal–Wallis test. ( f ) HE staining images of the brain regions of mice infected with tuberculosis at 0, 2, 4, and 6 weeks (red arrows show the inflammatory cell infiltration areas. There were three mice in each group. Scale bar, 50 μm). ( g ) Immunofluorescence image of cerebellum of mice infected with H37Rv at 6 weeks; the nucleus is blue, IBA-1 is green, LRRC25 protein is red, and the scale is 50 μm (n = 3). The figure shows a representative result. *** p < 0.001.

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with LRRC25 antibody (LRRC25 antibody: Santa Cruz; 1:200).

Techniques: Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: Interference of the translation of LRRC25 using siRNA of LRRC25: L1, L2, and L3. ( a ) Relative expression of LRRC25 mRNA after transfection of BV2 cells for 48 h and 72 h (n = 4), One-way ANOVA. ( b ) Western blot images of LRRC25 after transfection of BV2 cells for 48 h and 72 h (n = 3). ( c ) Relative expression of LRRC25 mRNA in BV2 cells after being transfected for 48 h and 72 h and infected by GFP-H37Rv for 4 h at an MOI of 5 (n = 4), Student’s t test. ( d ) Western blot images of LRRC25 in BV2 cells after being transfected for 48 h and 72 h and infected by H37Rv for 4 h at an MOI of 5 (n = 3). ( e ) Histogram and percentage statistics of uninfected BV2 cells in flow cytometry (n = 3), One-way ANOVA, Ex post facto comparison -LSD. ( f ) Confocal images of infected BV2 cells. Among them, the nucleus was stained and marked with Hoechst (blue), and GFP-H37Rv was marked with green fluorescent protein (green). Scale bar, 50 μm. BV2 cells were transfected with L1 for 72 h and infected with GFP-H37Rv for 4 h at an MOI of 5. GA is the abbreviation for GAPDH, and NC is the abbreviation for Negative Control. *** p < 0.001.

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with LRRC25 antibody (LRRC25 antibody: Santa Cruz; 1:200).

Techniques: Expressing, Transfection, Western Blot, Infection, Flow Cytometry, Comparison, Staining, Negative Control

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: LRRC25 prevents BV2 cells from releasing ISG15 and IFN-γ. ( a ) Relative expression of IL-1β mRNA of BV2 and secretion level of IL-1β in the cell supernatant of each group. ( b ) Levels of IFN-α, IFN-γ, and ISG15 in the cell culture supernatant before and after H37Rv infection of BV2 cells. ( c ) Relative expression of ISG15 mRNA and the expression of ISG15 in the cell supernatant in each group. ( d ) Expression of IFN-γ in the cell supernatant in each group. ( e ) Expression of IFN-α in the cell supernatant in each group. Bar graphs show the mean ± SD from four independent experiments (n = 4). Student’s t test for figure ( b ) and Two-way ANOVA test for figure ( a , c , d ). BV2 cells were transfected with L1 for 72 h and infected with GFP-H37Rv for 4 h at an MOI of 5. ** p < 0.01, and *** p < 0.001.

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with LRRC25 antibody (LRRC25 antibody: Santa Cruz; 1:200).

Techniques: Expressing, Cell Culture, Infection, Transfection

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: Illustration of the negative regulation of anti-tuberculosis immunity in microglia by the LRRC25-ISG15-IFN-γ axis.

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with LRRC25 antibody (LRRC25 antibody: Santa Cruz; 1:200).

Techniques: