Journal: Scientific Reports

Article Title: Identification of immune patterns in idiopathic pulmonary fibrosis patients driven by PLA2G7-positive macrophages using an integrated machine learning survival framework

doi: 10.1038/s41598-024-73625-z

Figure Lengend Snippet: Identification of a Robust Prognostic and Diagnostic Biomarker, PLA2G7 , Using IRS. ( A ) Identification of differentially expressed genes in high-IRS patients. ( B ) Univariate cox regression analysis of differentially expressed genes to select prognostically relevant genes. ( C ) Calculation of ROC diagnostic capabilities for each gene in distinguishing IPF samples from healthy samples in different cohorts. ( D ) Differential expression of SPP1 , MMP7 , and PLA2G7 in healthy and IPF samples in the GSE70866 dataset. ( E ) Differential expression of SPP1 , MMP7 , and PLA2G7 in healthy and IPF samples in the GSE110147 dataset. ( F ) Differential expression of SPP1 , MMP7 , and PLA2G7 in healthy and IPF samples in the GSE10667 dataset. ( G ) Survival analysis of IPF patients with high and low PLA2G7 expression in the GSE70866 dataset. ( H ) The association between PLA2G7 gene expression levels and the abundance of immune cells.

Article Snippet: The SPR technique can be used to investigate and to monitor molecular interactions in real time .The equilibrium-binding constant (KD) for the interaction between the celecoxib compound (Selleck, Cat No. S1261) and human PLA2G7 protein (MCE, Cat No. HY-P70993) was determined at 25 °C with a flow rate of 30 μl/min.

Techniques: Diagnostic Assay, Biomarker Discovery, Quantitative Proteomics, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Identification of immune patterns in idiopathic pulmonary fibrosis patients driven by PLA2G7-positive macrophages using an integrated machine learning survival framework

doi: 10.1038/s41598-024-73625-z

Figure Lengend Snippet: Validation of PLA2G7-positive macrophages driving inflammatory activation in IPF through single-cell sequencing analysis. ( A ) Heatmap depicting marker gene expression for cell annotation in different cell types. ( B ) t-SNE dimensionality reduction landscape of single-cell sequencing data. ( C ) Differential expression of PLA2G7 in IPF and healthy samples. ( D ) Differential analysis of different cell types in IPF and healthy samples. ( E ) t-SNE dimensionality reduction landscape of single-cell sequencing data samples from mice. ( F ) t-SNE dimensionality reduction landscape of cell types in the single-cell sequencing data from mice. ( G ) Heatmap displaying the marker gene expression in different cell types. ( H ) t-SNE dimensionality reduction landscape after re-extraction of macrophages in the single-cell sequencing data from mice. ( I ) Bubble plot exhibiting the expression levels of PLA2G7 and SPP1 in different samples. ( J ) t-SNE plot displaying the expression levels of PLA2G7 in different mouse samples. ( K ) In human single-cell sequencing data, violin plot depicting PLA2G7 expression in PLA2G7 -positive and PLA2G7 -negative macrophages. ( L ) In human single-cell sequencing data, differential analysis of PLA2G7 -positive and PLA2G7 -negative macrophages. ( M ) In human single-cell sequencing data, GSEA of PLA2G7 -positive and PLA2G7 -negative macrophages. ( N ) H&E, Masson’s trichrome staining and Multiplex immunofluorescence in a mouse model. ( O ) Counting of Cd68 + Pla2g7 + cells in a mouse model. ( P ) ELISA for Pla2g7 expression levels in alveolar lavage fluid and lung tissues of the mouse model.

Article Snippet: The SPR technique can be used to investigate and to monitor molecular interactions in real time .The equilibrium-binding constant (KD) for the interaction between the celecoxib compound (Selleck, Cat No. S1261) and human PLA2G7 protein (MCE, Cat No. HY-P70993) was determined at 25 °C with a flow rate of 30 μl/min.

Techniques: Biomarker Discovery, Activation Assay, Sequencing, Marker, Gene Expression, Quantitative Proteomics, Extraction, Expressing, Staining, Multiplex Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Identification of immune patterns in idiopathic pulmonary fibrosis patients driven by PLA2G7-positive macrophages using an integrated machine learning survival framework

doi: 10.1038/s41598-024-73625-z

Figure Lengend Snippet: Top five drugs that bind to PLA2G7 identified through drug sensitivity analysis.

Article Snippet: The SPR technique can be used to investigate and to monitor molecular interactions in real time .The equilibrium-binding constant (KD) for the interaction between the celecoxib compound (Selleck, Cat No. S1261) and human PLA2G7 protein (MCE, Cat No. HY-P70993) was determined at 25 °C with a flow rate of 30 μl/min.

Techniques:

Journal: Scientific Reports

Article Title: Identification of immune patterns in idiopathic pulmonary fibrosis patients driven by PLA2G7-positive macrophages using an integrated machine learning survival framework

doi: 10.1038/s41598-024-73625-z

Figure Lengend Snippet: Celecoxib exerts anti-inflammatory effects by targeting PLA2G7. ( A ) Schematic representation of the binding mode of molecules with PLA2G7. ( B ) Affinity between small molecules and PLA2G7. ( C ) Surface plasmon resonance analysis of celecoxib and PLA2G7. Celecoxib, as the ligand, was dissolved and injected at concentrations of 0 µM, 0.19 µM (purple), 0.39 µM (black), 0.78 µM (blue), 1.56 µM (orange), 3.13 µM (green), and 6.25 µM (red).

Article Snippet: The SPR technique can be used to investigate and to monitor molecular interactions in real time .The equilibrium-binding constant (KD) for the interaction between the celecoxib compound (Selleck, Cat No. S1261) and human PLA2G7 protein (MCE, Cat No. HY-P70993) was determined at 25 °C with a flow rate of 30 μl/min.

Techniques: Binding Assay, SPR Assay, Injection

Journal: bioRxiv

Article Title: Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

doi: 10.1101/2021.10.10.463806

Figure Lengend Snippet: a A conceptual schema describing rapidly inducible manipulation of intracellular organelles based on the integration of chemically- or optically-triggered hetero-dimerization and phospholipid remodeling by PLAAT enzymes. This strategy anticipates rapid switching of the PLAAT activity right at the target membrane-bound organelles (e.g., mitochondria) to induce their deformation as well as defunctionalization. b Fluorescence images of COS-7 cells expressing CFP-FRB-MoA (mitochondrial anchor, cyan) along with either mCherry-FKBP-PLAAT3-FL (red) or mCherry-FKBP-PLAAT3-FL-LD (lipase-dead PLAAT3, red) before and 35 mins after addition of 100 nM rapamycin. Enlarged images to highlight mitochondria morphology are shown on the right. Experimental timeline and schematic drawing of constructs used in this experiment are shown on top. c A summary of PLAAT3 domain structures (top), as well as C-terminal sequences of full-length PLAAT3 along with its lipase-dead mutant (LD) and a series of truncation mutants (bottom). Properties such as efficiency of inducible deformation, subcellular localization and cell viability were scored based on experiments described below, and indicated for each mutant tested (right). Pro: proline-rich domain, LRAT: Lecithin-retinol acyltransferase (also responsible for PLA 1 /A 2 activity), TM: putative transmembrane domain, CT: C-terminus domain, FL: full length of human PLAAT3, LD: lipase dead harboring C113S point mutation, 20TM, 19TM, 18TM, and 15TM: PLAAT3 mutant with truncations in TM and a full defect of CT, dTM: PLAAT3 mutant totally defective of TM and CT. d A fraction of COS-7 cells indicating fully-deformed mitochondria was calculated before (grey) and after (light brown) rapamycin treatment, and presented for cells expressing mCherry-FKBP-FL-LD, mCherry-FKBP-FL, mCherry-FKBP-2CT, mCherry-FKBP-dCT, mCherry-FKBP-18TM, or mCherry-FKBP-dTM and CFP-FRB-MoA. n = 104, 110, 121, 96, 138, 115, 103, 102, 97, 93, 125, and 111 cells from left to right; analyzed from three individual experiments. e Fluorescence images of cells expressing YFP, YFP-FL, YFP-FL-LD, or YFP-18TM, obtained at 48-hr post-transfection. These cells were stained with Hoechst. Insets are zoom-in images. Scale bar = 100 μm. f A number of YFP positive cells shown in e was calculated and used as a measure of cell viability. n = 591, 556, 499 and 601 cells from left to right; analyzed from three individual experiments. Error bars indicate means ± s.d..

Article Snippet: Human Plaat cDNAs were purchased from ORIGENE [RC208444 for PLAAT1 (NM_020386), RC212578 for PLAAT2 (NM_017878), RC200242 for PLAAT3 (NM_007069), RC201923 for PLAAT4 (NM_004585), and RC228184 for PLAAT5 (NM_054108)].

Techniques: Activity Assay, Membrane, Fluorescence, Expressing, Construct, Mutagenesis, Transfection, Staining

Journal: Gut Microbes

Article Title: Midbiotics: conjugative plasmids for genetic engineering of natural gut flora

doi: 10.1080/19490976.2019.1591136

Figure Lengend Snippet: Bacterial strains and plasmids used in the experiments and the spacer sequences of pCRISPR plasmid. Only the resistance genes relevant to the experiments are mentioned here.

Article Snippet: In this study, the so-called midbiotic system consists of the conjugative RP4 blaTEM−2∆172−714 plasmid (delivery plasmid) and mobilizable pCas9 plasmid (pCRISPR plasmid, a gift from Luciano Marraffini, Addgene plasmid # 42876) encoding the S. pyogenes CRISPR/Cas9 system with crRNA(s) targeting conservative sites of different beta-lactamase resistance genes in ESBL plasmids ( ). pCas9 was made mobilizable by cloning RP4 oriT site , (50980–51793 bps, amplified with primers RP4oriT-F and RP4oriT-R, Supplementary Table 1) into pCas9 digested with SalI (ThermoScientific; Waltham, Massachusetts, United States) into region spanning 7377–7486 bps.

Techniques: Plasmid Preparation, Sequencing