Journal: Parasites & Vectors

Article Title: Leishmania braziliensis prostaglandin F 2α synthase impacts host infection

doi: 10.1186/s13071-020-3883-z

Figure Lengend Snippet: Comparative analysis of the Lbr PGF2S amino acid sequence. a Multiple sequence alignment of putative PGF2S proteins from L. braziliensis (LbrM.31.2410), L. major (LmjF.31.2150), L. mexicana (LmxM.30.2150 ) , L. infantum (LinJ.31.2210) and L. tarentolae (LtaP.31.2590). b Sequence alignment of L. braziliensis PGF2S and human putative ortholog (AKR1C3, NP_003730.4, gi|24497583) using the ClustalW algorithm. Aldo/keto reductase domains are shown in red and the blue square indicates a I247V mutation. c 3D sequence alignments of protein sequences of Lmj PGF2S (PBD ID 4G5D, in grey) and human ortholog AKR1C3 (PDB ID 4YVV in blue), using RCSB PDB’s online comparison tool (rcsb.org/pdb)

Article Snippet: The parasites were then incubated in anti- Lbr PGF2S and anti-tubulin (Millipore, Massachusetts, USA) antibodies at 1:10,000 in 1% BSA-PBS for 1 h and washed three times in PBS-T.

Techniques: Sequencing, Mutagenesis

Journal: Parasites & Vectors

Article Title: Leishmania braziliensis prostaglandin F 2α synthase impacts host infection

doi: 10.1186/s13071-020-3883-z

Figure Lengend Snippet: Lbr PGF2S expression and secretion in L. braziliensis promastigotes. a Evaluation of Lbr PGF2S protein levels in L. braziliensis promastigotes grown in axenic culture for 8 days using polyclonal anti- Lbr PGF2S. Anti-Tubulin antibody was used for protein loading control. b Secretion of Lbr PGF2S by promastigotes in the axenic medium. Immunoblots were used to detect Lbr PGF2S in logarithmic (L) and stationary (St) phase promastigotes, 3 and 7 days of culture, respectively, or in the supernatant of the stationary phase culture (S) using anti- Lbr PGF2S. Parasite-free M199 medium (M) was used as a negative control. Blots were stained with Ponceau S (lower panel) for protein loading control. c Immunofluorescence imaging used to detect Lbr PGF2S in wild type promastigotes (at stationary phase). Lbr PGF2S (CF488A, green); tubulin (Alexa555, red); DNA was stained with DAPI (blue). An image of a single promastigote is shown bottom right; a strong fluorescence signal appears at the flagellar pocket (white arrow). Ab control: parasites submitted to the same labelling protocol without primary antibodies. d Dosage of prostaglandin F 2a synthesized by promastigotes in the presence (+) or absence (−) of 66 µM arachidonic acid (AA), measured by EIA assay. Quantification was performed with promastigotes at day 3 (logarithmic phase) or 7 (stationary phase), at 26 °C or 37 °C, as indicated below the x-axis. Data are means ± standard deviation from three replicates. Scale-bars : c , 5 µm

Article Snippet: The parasites were then incubated in anti- Lbr PGF2S and anti-tubulin (Millipore, Massachusetts, USA) antibodies at 1:10,000 in 1% BSA-PBS for 1 h and washed three times in PBS-T.

Techniques: Expressing, Western Blot, Negative Control, Staining, Immunofluorescence, Imaging, Fluorescence, Synthesized, Enzyme Immunoassay, Standard Deviation

Journal: Parasites & Vectors

Article Title: Leishmania braziliensis prostaglandin F 2α synthase impacts host infection

doi: 10.1186/s13071-020-3883-z

Figure Lengend Snippet: Lbr PGF2S detection in L. braziliensis -infected macrophages. Immunofluorescence imaging of uninfected bone marrow-derived macrophages (BMDM, top row), macrophages infected with wild type promastigotes (middle row) and Lbr PGF2S-overexpressing transfectants (OE, BA778[ pXLbrPGF2S ]) (bottom row), for 48 h. DNA was stained with DAPI (blue); Lbr PGF2S (CF488A, green); tubulin (Alexa555, red). Scale-bars : 5 μm

Article Snippet: The parasites were then incubated in anti- Lbr PGF2S and anti-tubulin (Millipore, Massachusetts, USA) antibodies at 1:10,000 in 1% BSA-PBS for 1 h and washed three times in PBS-T.

Techniques: Infection, Immunofluorescence, Imaging, Derivative Assay, Staining