Journal: RSC Medicinal Chemistry

Article Title: Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection †

doi: 10.1039/d3md00724c

Figure Lengend Snippet: a) BLI sensogram of fragment hit 1 binding to PaFabF C164A. The dashed red line indicates the start of the dissociation step. b) Steady-state plot fitted to the responses of three independent experiments using exclusively repurchased material of fragment hit 1.

Article Snippet: The DNA sequence coding for Pa FabF C164A was synthesized and cloned into pET-28a-TEV vectors by Genscript (New Jersey).

Techniques: Binding Assay

Journal: RSC Medicinal Chemistry

Article Title: Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection †

doi: 10.1039/d3md00724c

Figure Lengend Snippet: Binding of fragment hit 1 (EOS102727, yellow) to Pa FabF C164A (PDB ID 8PJ0 ). a) | F o − F c | omit map of 1 binding to Pa FabF C164A, contoured at 3.0 sigma. b) Solvent accessible surface using residues 4 Å from ligand 1. c) Interactions between 1 (yellow) and Pa FabF C164A (green). Putative hydrogen bonds and π–π interactions are displayed as yellow dotted lines. d) Binding modes of 1 overlayed with the binding modes of platensimycin (magenta), PDB ID 7OC1 , and cerulenin (teal), PDB ID 4LS8 .

Article Snippet: The DNA sequence coding for Pa FabF C164A was synthesized and cloned into pET-28a-TEV vectors by Genscript (New Jersey).

Techniques: Binding Assay, Solvent

Journal: RSC Medicinal Chemistry

Article Title: Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection †

doi: 10.1039/d3md00724c

Figure Lengend Snippet: Dissociation constants and ligand efficiency of EFSL hit 1 and ECBL compounds 2 and 3

Article Snippet: The DNA sequence coding for Pa FabF C164A was synthesized and cloned into pET-28a-TEV vectors by Genscript (New Jersey).

Techniques:

Journal: RSC Medicinal Chemistry

Article Title: Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection †

doi: 10.1039/d3md00724c

Figure Lengend Snippet: Binding of ECBL compounds 2 and 3 to Pa FabF C164A (PDB ID 8R0I and 8R1V , respectively). a) | F o − F c | omit map of 2 (top) and 3 (bottom) binding to Pa FabF C164A contoured at 3.0 sigma. b) Alignment of binding modes of 1 (yellow), 2 (magenta) and 3 (cyan) together with solvent accessible surface of the binding site using residues 6 Å from ligand 3. For clarity, only selected interacting residues from the complex Pa FabF C164A-1 are shown (green). c) Interactions between 2 (magenta) and Pa FabF C164A (green). Putative hydrogen bonds and π–π interactions are displayed as yellow dotted lines. d) Binding mode of 3 (cyan) together with three water molecules that were found in the complexes with 1 and 2 but displaced by 3 (HOH392, 145 and 163, numbering from Pa FabF C164A-1).

Article Snippet: The DNA sequence coding for Pa FabF C164A was synthesized and cloned into pET-28a-TEV vectors by Genscript (New Jersey).

Techniques: Binding Assay, Solvent

Journal: Biomolecular NMR assignments

Article Title: Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1

doi: 10.1007/s12104-023-10158-y

Figure Lengend Snippet: (a) Annotated 2D 1H,15N TROSY spectrum of Adhesin P1 C3 domain (BMRB 52097) collected in an 800 MHz spectrometer at 25 °C in phosphate buffer pH 6. Resonance assignments are shown with black labels; (b) Alphafold structural model for the new, longer C3 construct with the amino acid residues that are currently assigned shown in blue; (c) C3 domain structure (PDB 3QE5) with the residues assigned using the previous, shorter C3 construct (BMRB 27935) shown in red; (d) comparison of 2D 1H,15N TROSY spectra for the previous, shorter C3 construct (red) and the new, longer construct for the AlphaFold-predicted C3 domain (blue). Disordered, poorly resolved resonances observed for the prior C3 construct are now well resolved for the new C3 construct by addition of the seven C-terminal amino acids.

Article Snippet: The DNA sequence coding for the C3 domain of adhesin P1 (residues 1328–1490; Uniprot accession number P23504) was synthesized, with codon optimization for E. coli , by Genescript and inserted into pET21a(+) plasmid to yield the pET21a-C3-His 6 plasmid with a C-terminal His tag.

Techniques: Construct, Comparison

Journal: Virology Journal

Article Title: Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus

doi: 10.1186/s12985-015-0440-z

Figure Lengend Snippet: Performance of the ORFV exo RPA assay. a Amplification curve of ORFV exo RPA assay over time using a dilution range of 10 6 to 10 1 copies/reaction of ORFV. NC represent negative control. b Reproducibility of the ORFV exo RPA assay. The threshold time is represented as the mean ± standard deviation (SD). The standard regression line was generated based on 8 data sets ( c ) Probit regression analysis using Statistics software was done on data from the eight runs of ORFV exo RPA assay. The limit of detection at 95 % probability is depicted by a triangle

Article Snippet: All ORFV DNA polymerase gene coding sequence genes were retrieved from GenBank and multiple sequence alignment of the gene sequences were manually designed based on the ORFV DNA polymerase gene recommendation by TwistDx (Cambridge, UK).

Techniques: Amplification, Negative Control, Standard Deviation, Generated, Software

Journal: Virology Journal

Article Title: Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus

doi: 10.1186/s12985-015-0440-z

Figure Lengend Snippet: Evaluation of the specificity of ORFV exo PRA assay

Article Snippet: All ORFV DNA polymerase gene coding sequence genes were retrieved from GenBank and multiple sequence alignment of the gene sequences were manually designed based on the ORFV DNA polymerase gene recommendation by TwistDx (Cambridge, UK).

Techniques: Virus

Journal: Virology Journal

Article Title: Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus

doi: 10.1186/s12985-015-0440-z

Figure Lengend Snippet: Comparison between performances of ORFV exo RPA assay and real-time ORFV qPCR assay on samples of ORFV-infected cells ( n = 15) and spiked tissues lysates ( n = 24). Linear regression analysis of the exo RPA threshold time (y axis) and qPCR cycle threshold (CT) values (x axis) were determined by Excel software

Article Snippet: All ORFV DNA polymerase gene coding sequence genes were retrieved from GenBank and multiple sequence alignment of the gene sequences were manually designed based on the ORFV DNA polymerase gene recommendation by TwistDx (Cambridge, UK).

Techniques: Comparison, Infection, Software

Journal: Virology Journal

Article Title: Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus

doi: 10.1186/s12985-015-0440-z

Figure Lengend Snippet: Comparison of ORFV exo RPA assay with qPCR assay on clinical samples a

Article Snippet: All ORFV DNA polymerase gene coding sequence genes were retrieved from GenBank and multiple sequence alignment of the gene sequences were manually designed based on the ORFV DNA polymerase gene recommendation by TwistDx (Cambridge, UK).

Techniques: Comparison

Journal: Virology Journal

Article Title: Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus

doi: 10.1186/s12985-015-0440-z

Figure Lengend Snippet: RPA primers and probes designed in this study

Article Snippet: All ORFV DNA polymerase gene coding sequence genes were retrieved from GenBank and multiple sequence alignment of the gene sequences were manually designed based on the ORFV DNA polymerase gene recommendation by TwistDx (Cambridge, UK).

Techniques: Sequencing

Journal: Molecular Biology of the Cell

Article Title: An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

doi: 10.1091/mbc.E15-11-0754

Figure Lengend Snippet: TLAP2 is conserved between T. gondii, Plasmodium spp., and C. velia and is dispensable for parasite growth. (A) Multiple sequence alignments of the C-terminal domains of TLAP2 homologues from T. gondii (TGGT1_232130), P. falciparum (-a, PF3D7_1034300 and -b, PF3D7_0517200), and C. velia (Cvel_2610). Tryptophan residues are highlighted in green. The consensus of the conserved residues is shown in the bottom row in red. (B) Deconvolved wide-field images of T. gondii transiently expressing mEmeraldFP-PfTLAP2-a from a T. gondii tubulin promoter. (C) Scheme for generating Δtlap2 parasites and Southern blotting strategy. RH Δhx parasites (parental; top) were used to generate mEmeraldFP-TLAP2 knock-in parasites (knock-in; middle) via double-crossover homologous recombination. The knock-in parasites were then transiently transfected with a plasmid expressing Cre recombinase to excise the genomic fragment between the two LoxP sites. mEmeraldFP(-) parasites were sorted by FACS to facilitate the cloning of Δtlap2 parasites (knockout; bottom). The positions of restriction sites and probe (red bar) used in Southern blotting (D) and the corresponding DNA fragment sizes are indicated. (D) Southern blotting analyses of the tlap2 locus in parental RH Δhx (P), mEmeraldFP-TLAP2 knock-in (KI), and Δtlap2 (KO) parasites generated as described in A. Genomic DNA of the parasites was digested with either Mfe I (left) or Kpn I- Sca I (right). A probe (red bar in C) hybridized to the upstream region of tlap2 gene was used for the Southern blotting. The predicted Mfe I- Mfe I fragment size recognized by the probe is 5575 base pairs for RH Δhx , 7511 base pairs for mEmeraldFP-TLAP2 knock-in, and 2967 base pairs for Δtlap2 parasites. The predicted Kpn I- Sca I fragment size recognized by the probe is 5850 base pairs for RH Δhx , 7786 base pairs for mEmeraldFP-TLAP2 knock-in, and 3242 base pairs for Δtlap2 parasites. (E) Analyses of the virulence of Δtlap2 parasites in mice. Each survival curve represents data from a group of four mice infected with an equal number of RH Δhx (parental) , mEmeraldFP-TLAP2 knock-in (mE-TLAP2 KI), or Δtlap2 parasites. Mice infected with the parental RH Δhx parasites died between days 7 and 8 postinfection, whereas mice infected with mEmeraldFP-TLAP2 knock-in or Δtlap2 parasites died between days 8 and 9 postinfection. (F) Projections of deconvolved wide-field images showing proper targeting of several coating proteins in Δtlap2 and Δspm1 parasites. Top, Δtlap2 parasites transiently expressing fluorescently tagged SPM1 or TLAP3. Bottom, Δspm1 parasites transiently expressing fluorescently tagged TLAP2 or TLAP3. The fluorescently tagged proteins were driven by the 2-kb genomic region immediately upstream of the respective genes in the pTKO2_II vector backbone. Scale bars, 2 μm. (G) Plaque assay of parental RH Δhx , Δtlap2 , Δtlap2Δspm1 , and Δtlap2Δspm1Δtlap3 parasites. HFF cultures were infected with an equal number of each line of parasites, grown for 7 d at 37°C, and then fixed and stained with crystal violet. Host cells remaining attached absorbed the crystal violet staining, whereas regions of host cells lysed by the parasites (“plaques”; arrows) were clear.

Article Snippet: Subsequently a DNA fragment containing the coding sequences for mEmeraldFP, SGLGS linker, and the N-terminus region of SPM1 was synthesized (GenScript; Supplemental Table S1), digested with Bgl II and Xma I, and subcloned into the Bgl II- Xma I sites on pTKO2-II-SPM1 to produce pTKO2-II-mEmeraldFP-SPM1 in which the coding sequence for mEmeraldFP was placed N-terminal to the SPM1 CDS. pmin-Cre-eGFP_Gra-mCherry: A Cre-eGFP expression cassette including a dhfr promoter and the dhfr 3′ untranslated region was released from the plasmid pmin-Cre-eGFP ( Heaslip et al. , 2010 ) by Apa I- Not I digestion and subcloned into the Apa I- Not I site on pTKO2-II-mCherryFP to replace the DHFR expression cassette. pmin-mEmeraldFP-TgCentrin1: The CDS of TgCentrin1 (TGGT1_247230; EuPathDB.org) was amplified by PCR using a cDNA library isolated from RH parasites as the template.

Techniques: Sequencing, Expressing, Southern Blot, Knock-In, Homologous Recombination, Transfection, Plasmid Preparation, Cloning, Knock-Out, Generated, Infection, Plaque Assay, Staining