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

Journal: bioRxiv

Article Title: RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis

doi: 10.1101/2024.09.16.613344

Figure Lengend Snippet: (A) HUVECs depleted of RalA or RalB using specific siRNAs and compared with control siRNA. Western blot of total cell lysates showing RalA and RalB knockdown after 48 hours of transfection of siRNA, with β -actin as loading control. (B) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 48 hours after siRNA transfection (** P <0.01; from 3 individual experiments each with duplicates). (C) HUVECs treated with increasing concentrations of dihydroartemisinin for 2 hours or DMSO. Western blot showing degradation of RalB but not RalA by dihydroartemisinin, with β-actin as loading control. (D) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 2 hours after dihydroartemisinin treatment (** P <0.01, *** P <0.001; **** P <0.0001 from 3 individual experiments each with duplicates). (E) Total cell lysates of resting and thrombin-treated (1 U/ml for 2 minutes) HUVECs were incubated with RalBP1-RBD conjugated agarose slurry and eluates immunoblotted with anti-RalB antibody to determine total GTP-loaded RalB in each condition. Coomassie stain showing RalBP1-RBD as loading control (left). Bar graphs showing densitometric analysis (right). (** P <0.01; from 3 individual experiments).

Article Snippet: Experiments were performed on RalB and RalA knockdown cells 48 h post-transfection. pLA-CMV-N-Flag-RalBWT (plasmid#50990), pLX301 (plasmid#25895), pEGFP-C2-CD63 (plasmid#62964) were obtained from Addgene.

Techniques: Control, Western Blot, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Staining