Journal: bioRxiv

Article Title: Rapid rewiring of an archaeal transcription factor function via flexible cis-trans interactions

doi: 10.1101/2024.11.20.624553

Figure Lengend Snippet: Haloarchaeal species exhibit differential susceptibility to oxidative stress conditions. Growth is plotted as the log10 optical density at 600 nm (OD600) over time (hours). Each curve represents the generalized additive model (GAM) fit to the raw data from at least 3 biological replicate experiments (seeded from starter cultures inoculated with separate colonies), each with 3 technical replicates (aliquots from the same starter culture). Shaded grey ribbons indicate the standard error. Error is low where ribbons are not visible. Concentrations are written above the corresponding growth curve in each panel. Concentrations surrounded by a box is that condition chosen for testing Δ rosR growth in subsequent experiments. (A) Hfx. volcanii growth under a titration of peroxide (H 2 O 2 ) concentrations. (B) Hfx. mediterranei growth under H 2 O 2 . (C) Hfx. volcanii growth under varying paraquat (PQ) concentrations. (D) Hfx. mediterranei growth under PQ.

Article Snippet: Wild-type strains Halobacterium salinarum NRC-1 ( Hs) , Haloferax volcanii strain DS2 ( Hv) , and Haloferax mediterranei ( Hm) ATCC 33500 were used in this study.

Techniques: Titration

Journal: bioRxiv

Article Title: Rapid rewiring of an archaeal transcription factor function via flexible cis-trans interactions

doi: 10.1101/2024.11.20.624553

Figure Lengend Snippet: hv RosR activates arlA1 and arlA2 , encoding structural components of the archaellum in Hfx. volcanii . (A) Top, genomic region encoding the archaellum and related motility functions; middle, ChIP-seq data for the region highlighted in teal (read depth y-axis is shown at right); bottom, chromosomal coordinates for genes in the zoomed region, with genes on the forward strand depicted on top of the line and reverse strand below the line. (B) Scatterplot of normalized counts from RNA-seq data, with parental control strain counts on the X-axis and hv Δ rosR counts on the y-axis. Genes passing the significance threshold of p < 0.05 and log2 fold change >= |1| are shown in blue, those significant genes also bound in ChIP-seq data in pink (see legend). (C) ChIP-qPCR validation of ChIP-seq data. Bar height represents the mean enrichment of hv RosR binding each site relative to a control region. Error bars represent the standard deviation from the mean of triplicate samples. hv RosR-HA enrichment is shown in salmon and PyrE2-HA parent strain control in grey. Amplicons tested are shown in orange below the bar graph (“peak 1, peak 2, peak 3”) and are set relative to chromosomal position of the corresponding genes. Asterisks indicate the statistical significance of enrichment for each peak (salmon bars) relative to the parent control (grey bars) by two-sided unpaired Student’s t-test, * p < 1.02 x 10 -3 , ** p < 4.71 x 10 -4 , *** p < 6.31 x 10 -5 . (D) Logo of the consensus binding motif detected computationally from hv RosR ChIP-seq binding site sequences. Motif position in nucleotides is given on the x-axis and bit score of per-base representation in the position weight matrix is given on the y-axis. See also Supplementary Table S4 for detailed RNA-seq, ChIP-seq, and motif data.

Article Snippet: Wild-type strains Halobacterium salinarum NRC-1 ( Hs) , Haloferax volcanii strain DS2 ( Hv) , and Haloferax mediterranei ( Hm) ATCC 33500 were used in this study.

Techniques: ChIP-sequencing, RNA Sequencing, Control, ChIP-qPCR, Biomarker Discovery, Binding Assay, Standard Deviation

Journal: Frontiers in Neuroscience

Article Title: The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

doi: 10.3389/fnins.2019.01133

Figure Lengend Snippet: Treatment with DS2 reduces infarct volume and improves functional outcomes post-stroke. Mice were subjected to focal stroke and injected intraperitoneally with either DS2 (0.01, 0.1, 1 or 4 mg/kg) or vehicle 1 and 24 h after the surgery. The brains were collected 7 days later, stained with cresyl violet and the stroke volume was calculated. (A) Representative cresyl violet-stained sections of animals treated with vehicle or three different concentrations of DS2. (B) Mice injected with 0.1 mg/kg DS2 had significantly smaller strokes than untreated animals. Baseline motor function was established by grid-walking and cylinder test 1 week prior stroke surgery. Mice treated with either vehicle or DS2 (0.01 0.1, 1, and 4 mg/kg) at 1 and 24 h post-stroke induction were assessed in the (C) grid-walking (foot-faults) and (D) cylinder task (forelimb asymmetry). Data are expressed as mean ± SD for n = 5-7 per group. @ P < 0.01 compared with vehicle control.

Article Snippet: Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin TM from Invitrogen (Auckland, New Zealand) and Quantiblue TM from InvivoGen (CA, United States).

Techniques: Functional Assay, Injection, Staining, Control

Journal: Frontiers in Neuroscience

Article Title: The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

doi: 10.3389/fnins.2019.01133

Figure Lengend Snippet: DS2 dampens activation of innate immune cells after an inflammatory stimulus. (A,B) Expression of GABA receptor subunits by RAWblue macrophages was analyzed by qPCR. Relative mRNA levels of (A) GABA receptor subunits and (B) transporters involved in GABA signaling on unstimulated RAWblue macrophages in relation to the reference gene succinate dehydrogenase complex subunit A (SDHA) are shown. (C) RAWblue macrophages (5 × 10 4 cells/well) were either pre-treated with DS2 (10 –3 to 10 –6 M) or DMSO (2.5%) for 30 min or left un-treated before stimulation with LPS (2.5 ng/mL) for 6 h. Cell supernatants were collected and the extent of NF-κB activation was determined by quantifying the reporter protein SEAP with the Quantiblue TM assay. (D–F) BMDCs were incubated with decreasing concentrations of DS2 (10 –4 to 10 –8 M) for 30 min prior to the addition of LPS (100 ng/mL) to the cultures. BMDCs were harvested 24 h later and expression of cell surface markers CD40, CD80 and CD86 on live, CD11c + MHCII + BMDCs was determined by flow cytometry. MFI shown as n-fold increase over LPS-treated cells. Results are the mean ± SD of three independent experiments with three replicates each. ∗∗∗ P < 0.001 and ∗∗ P < 0.01.

Article Snippet: Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin TM from Invitrogen (Auckland, New Zealand) and Quantiblue TM from InvivoGen (CA, United States).

Techniques: Activation Assay, Expressing, Incubation, Flow Cytometry

Journal: Frontiers in Neuroscience

Article Title: The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

doi: 10.3389/fnins.2019.01133

Figure Lengend Snippet: DS2 decreases pro-inflammatory cytokine production post-stroke. Mice were injected i.p. with vehicle or DS2 (0.1, 1 or 4 mg/kg) 1 and 24 h after induction of stroke and peripheral blood collected from the tail vein of mice 3-days post-stroke. Sera were analyzed for (A) IL-β, (B) TNF-α, (C) IL-6, and (D) IL-17 concentrations using a BioPlex kit. Data are expressed as mean ± SD for n = 5 per group. ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05 compared to vehicle. +++ P < 0.001, ++ P < 0.01, + P < 0.05 compared to sham control.

Article Snippet: Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin TM from Invitrogen (Auckland, New Zealand) and Quantiblue TM from InvivoGen (CA, United States).

Techniques: Injection, Control

Journal: Frontiers in Neuroscience

Article Title: The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

doi: 10.3389/fnins.2019.01133

Figure Lengend Snippet: Brain and plasma concentrations following DS2 administration and co-treatment with or without elacridar.

Article Snippet: Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin TM from Invitrogen (Auckland, New Zealand) and Quantiblue TM from InvivoGen (CA, United States).

Techniques: Clinical Proteomics

Journal: Frontiers in Neuroscience

Article Title: The Delta-Subunit Selective GABA A Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism

doi: 10.3389/fnins.2019.01133

Figure Lengend Snippet: Delayed administration of DS2 from day-3 post-stroke improves functional outcomes when continuously administered. Mice were subjected to focal stroke or sham surgery and DS2 (10 mM, red) or vehicle (blue) were loaded into ALZET 1002 mini-pumps and implanted subcutaneously from 3-days post-stroke. Mini-pumps were replaced after 2 week with new pumps to allow for continuous treatment for 28-days after stroke. Mice were assessed for functional deficits using the (A) grid-walking (foot-faults) and (B) cylinder task (forelimb asymmetry). Data are expressed as mean ± SD for n = 7 per group. +++ P < 0.001, ++ P < 0.01 compared to stroke vehicle control. @ P < 0.001 compared to sham controls.

Article Snippet: Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin TM from Invitrogen (Auckland, New Zealand) and Quantiblue TM from InvivoGen (CA, United States).

Techniques: Functional Assay, Control

Journal: NPJ Vaccines

Article Title: DS2 designer pre-fusion F vaccine induces strong and protective antibody response against RSV infection

doi: 10.1038/s41541-024-01059-9

Figure Lengend Snippet: A The pre-fusion and post-fusion conformational states of the RSV F glycoprotein are depicted, with colored antigenic sites (Ø, V, IV, III, II, and I) recognized by distinct groups of monoclonal antibodies (mAbs). B Specific mutations introduced to maintain the prefusion conformation in the three designer pre-F glycoproteins DS2, DS-Cav1, and SC-TM. C A schematic diagram illustrates the recombinant AdC68 vector expressing the three designer pre-F glycoproteins of RSV. Each glycoprotein was presented with signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) from RSV A2 strain. The coding sequences of each designer pre-F were inserted into the E1 region of the AdC68 vector, controlled of the cytomegalovirus (CMV) promoter, and terminated by a bovine growth hormone (BGH) polyadenylation signal sequence. D A comparison of the expression and epitope display of various designer pre-F on the surface of infected HEK 293T cells. Following infection with recombinant AdC68, HEK 293T cells were analyzed by flow cytometry using a panel of mAbs targeting various epitopes in the pre- and post-F glycoproteins of RSV. The names and sites specificity of each mAb in the F glycoprotein of RSV are indicated. Motavizumab and Palivizumab, two clinically evaluated antibody drugs, recognize the identical epitope within site II. The X-axis indicates the PE fluorescence intensity while Y-axis denotes the number (counts) of the events. The percentage in each of the flow cytometry graph represents the percentage of PE-positive cells.

Article Snippet: Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins – , were synthesized by GenScript Biotech Corporation, China.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Comparison, Infection, Flow Cytometry, Fluorescence

Journal: NPJ Vaccines

Article Title: DS2 designer pre-fusion F vaccine induces strong and protective antibody response against RSV infection

doi: 10.1038/s41541-024-01059-9

Figure Lengend Snippet: A Timeline and grouping for vaccination and monitoring of antibody response among five groups of vaccinated mice. Fifty 6-week-old BALB/c mice, ten in each group, were immunized with indicated dose and route of designer vaccines or controls at week 0 and again at week 3. Blood samples were collected every two weeks to monitor the binding and neutralizing titers to RSV pre-F and live RSV, respectively. B , C Comparison of serum neutralizing activity to live RSV and binding activity to each of the designer pre-F and WT F recombinant glycoproteins among the five groups of vaccinated mice (n = 10). Bar graphs depict the week 10 neutralizing titers (ID 50 ) and binding activities (ED 50 ) to recombinant DS2, DS-Cav1, SC-TM, and WT. Each dot represents one animal and color-coded according to the vaccinated group as shown in Fig. . Dotted lines represent assay limit of detection. D Experimental design for evaluating the protective activity of AdC68-DS2 in mice. Ten weeks after immunization with either AdC68-DS2 or AdC68 empty vector, mice were intranasally challenged with 3 × 10 5 PFU of live RSV Long strain. E , F All animals were carefully monitored for body weight changes, for viral loads in the lungs by qPCR and plaque assays, and for pathological analysis of lung tissue by H&E staining (n = 5). The scale bar is 200 μm for 5× and 50 μm for 20×. VL vascular lumen, BL bronchiolar lumen. All data are presented as median ±interquartile range. The Mann–Whitney U test is used for comparisons between two independent groups and Kruskal–Wallis H test for comparisons among multiple independent groups, as the data sets are not uniformly normally distributed. The results shown are representatives of two independent experiments. The p -values are marked in the graphs.

Article Snippet: Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins – , were synthesized by GenScript Biotech Corporation, China.

Techniques: Vaccines, Binding Assay, Comparison, Activity Assay, Recombinant, Plasmid Preparation, Staining, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: DS2 designer pre-fusion F vaccine induces strong and protective antibody response against RSV infection

doi: 10.1038/s41541-024-01059-9

Figure Lengend Snippet: A Overall experimental procedure for isolating and characterizing DS2-specific mAbs from bone marrow antibody-secreting cells (ASCs). The image was created with BioRender.com, with permission. B Representative bone marrow ASCs secreting DS2-specific antibodies were captured by the surrounding DS2-coated polystyrene particles, detected using a secondary anti-mouse IgG antibody conjugated with Alexa Fluor™ 488, and visualized under a fluorescence microscope. C Phylogenetic analysis of the heavy and light chains of 29 DS2-specific mAbs, and their relative genetic relatedness to control mAbs (D25, CR9501, 101F, MPE8, Motavizumab, and 4D7) targeting six major antigenic sites (Ø, V, IV, III, II and I) in the F glycoprotein of RSV. Nirsevimab, a commercially available antibody drug derived from D25, specifically recognizes the site Ø antigenic site. The epitope specificity for each of the 29 DS2-specific mAbs, defined by the competitive SPR with the six control mAbs, is indicated along with their binding (ED 50 ) and neutralizing (ID 50 ) abilities. The results shown are representatives of two independent experiments. The branch length is drawn to scale to assess genetic relatedness among the mAbs.

Article Snippet: Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins – , were synthesized by GenScript Biotech Corporation, China.

Techniques: Fluorescence, Microscopy, Control, Derivative Assay, Binding Assay

Journal: NPJ Vaccines

Article Title: DS2 designer pre-fusion F vaccine induces strong and protective antibody response against RSV infection

doi: 10.1038/s41541-024-01059-9

Figure Lengend Snippet: Phenotypic and genotypic characterization of the DS2-specific neutralizing mAbs

Article Snippet: Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins – , were synthesized by GenScript Biotech Corporation, China.

Techniques: Sequencing, Control

Journal: NPJ Vaccines

Article Title: DS2 designer pre-fusion F vaccine induces strong and protective antibody response against RSV infection

doi: 10.1038/s41541-024-01059-9

Figure Lengend Snippet: A Lateral and top view of mAb60 Fab in complex with DS2 pre-fusion F trimer. The heavy chain of mAb60 Fab is shown in red, and the light chain in magenta. The DS2 pre-fusion F trimer is depicted in dark gray as a molecular surface, with one of the protomers depicted in green as a ribbon diagram. B Modeling analysis of mAb60 Fab binding to both pre- and post-fusion F monomers. mAb60 binds to the antigenic site II, similar to Motavizumab, but distinct from antibodies to antigenic site Ø, which are only present in the pre-fusion F and are bound by D25 (in teal) and Nirsevimab (in coral). C The epitope and the paratope residues involved in mAb60 Fab binding to the DS2 pre-fusion F. Epitope residues are shown in green and depicted as a molecular surface, while paratope residues are highlighted with yellow circles and represented as ribbon diagrams. The epitope of Motavizumab is outlined by a dark orange dashed line. D Interactions between mAb60 Fab and DS2 pre-fusion F at the binding interface. Hydrogen bonds are shown as pure-blue dotted lines, and salt bridges as light-yellow lines. E Superimpose of mAb60 Fab onto the post-fusion F (PDB: 5J3D).

Article Snippet: Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins – , were synthesized by GenScript Biotech Corporation, China.

Techniques: Binding Assay