Journal: Microbial Cell Factories

Article Title: Engineering of Saccharomyces cerevisiae as a platform strain for microbial production of sphingosine-1-phosphate

doi: 10.1186/s12934-024-02579-8

Figure Lengend Snippet: Metabolic engineering strategies to produce S1P in S. cerevisiae . Sphingolipids biosynthetic pathways for the production of S1P in S. cerevisiae . To generate a S1P-producing strain, human DES1 , ACER1 , and SPHK1 genes, encoding sphingolipid delta 4 desaturase, alkaline ceramidase, and sphingosine kinase, respectively, were introduced into the S. cerevisiae . LCB1 and LCB2 , subunits of serine palmitoyltransferase; ORM1 and ORM2 , Regulator of serine palmitoyltransferase; TSC3 , Activator of serine palmitoyltransferase; TSC10 , 3-ketosphinganine reductase; LAC1 , LAG1 , and LIP1 , Subunits of ceramide synthase; LCB4 and LCB5 , Sphingoid long-chain base kinase; SUR2 , Sphinganine C4-hydroxylase; DPL1 , Dihydrosphingosine phosphate lyase

Article Snippet: Codon optimized genes from Homo sapiens , DES1 (sphingolipid delta 4 desaturase), ACER1 (alkaline ceramidase 1), and SPHK1 (sphingosine kinase 1), were synthesized by Cosmogenetech (Korea).

Techniques:

Journal: Microbial Cell Factories

Article Title: Engineering of Saccharomyces cerevisiae as a platform strain for microbial production of sphingosine-1-phosphate

doi: 10.1186/s12934-024-02579-8

Figure Lengend Snippet: Yeast strains and plasmids used in this study

Article Snippet: Codon optimized genes from Homo sapiens , DES1 (sphingolipid delta 4 desaturase), ACER1 (alkaline ceramidase 1), and SPHK1 (sphingosine kinase 1), were synthesized by Cosmogenetech (Korea).

Techniques: Plasmid Preparation

Journal: Journal of Virology

Article Title: High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1

doi: 10.1128/jvi.01351-23

Figure Lengend Snippet: Epidemiology and Spike mutations of SARS-CoV-2 B.1.640.1. (A) Total number of B.1.640.1 variant cases by country as displayed by red circles. Also shown is total case number of B.1.640.1 variant by region of France. Data on sample number and location were obtained from GISAID EpiCoV database. Total case number = 1,107. (B) Total case number of B.1.640.1 variant per month during its circulation between January 2021 and April 2022. Data are grouped by continent. Data on sample collection date and sample location were obtained from GISAID EpiCoV database. (C) Schematic view of B.1.640.1 and B.1.640.2 Spike sequences and their respective amino acid mutations compared to the ancestral Wuhan Spike sequence (NC_045512.2) and Alpha, Delta, BA1, and BA5 variants.

Article Snippet: Human codon-optimized SARS-CoV-2 Spikes (Alpha, Delta, BA.1, BA.5, B.1.640.1, and B.1.640.2) were produced in silico (GeneArt, Thermo Fisher Scientific).

Techniques: Variant Assay, Sequencing

Journal: Journal of Virology

Article Title: High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1

doi: 10.1128/jvi.01351-23

Figure Lengend Snippet: Binding and neutralization of B.1.640.1 by monoclonal antibodies and sera from convalescent and vaccinated individuals. (A) Radial plots showing binding of a panel of mAbs that target the S2, RBD, and NTD of Spike, as depicted by color. HEK293T cells were transfected with Spike or control plasmids 24 hours before staining with mAbs. Binding is quantified by mean fluorescence intensity (MFI) of each mAb, with the y-axis showing log10(MFI). Gray circles indicate the limit of detection [MFI = log10 (3)]. N = 3. (B) Neutralization activity of therapeutic mAbs. Dose-response analysis of the neutralization of Delta, B.1.640.1, and BA.1 virus by sotrovimab and the combinations of tixagevimab + cilgavimab and casirivimab + imdevimab. Data points are a mean of two independent repeated experiments. (C) Neutralization activity of sera from convalescent individuals, 6 and 12 months after infection, and 1 month post second and third Pfizer RNA vaccine doses. Dotted line represents the limit of detection (ED50 = 30). Solid black bars represent median values. Data points are a mean of two independent repeated experiments. Mann-Whitney tests were performed to compare the respective variants, *P < 0.05, **P < 0.001, ns = not significant.

Article Snippet: Human codon-optimized SARS-CoV-2 Spikes (Alpha, Delta, BA.1, BA.5, B.1.640.1, and B.1.640.2) were produced in silico (GeneArt, Thermo Fisher Scientific).

Techniques: Binding Assay, Neutralization, Transfection, Control, Staining, Fluorescence, Activity Assay, Virus, Infection, MANN-WHITNEY

Journal: Journal of Virology

Article Title: High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1

doi: 10.1128/jvi.01351-23

Figure Lengend Snippet: Cytopathic effects of B.1.640.1 infection on A549-ACE2 cells. (A) (Top) Replication kinetics of D614G, Delta, and B.1.640.1 in A549-ACE2 cells shown by quantification of the viral E protein gene in the cell supernatant by RT-qPCR at the respective timepoints. (Bottom) Area of Spike-positive A549-ACE2-infected cells from respective variants over 72 hours. Cells were stained with anti-Spike mAb (mAb102) before staining with an Alexa-Fluor 647 conjugated secondary to allow quantification of infection. N = 4. (B) (Top) Quantification of LDH release in A549-ACE2-infected cell supernatant through a luciferase-based assay. N = 4. (Bottom) Nuclei count following infection of A549-ACE2 cells at the indicated timepoints with the respective variants. Nuclei were stained with Hoechst dye prior to quantification. (C) Confocal immunofluorescence of A549-ACE2 cells infected with MOI 0.1 D614G, Delta, and B.1.640.1 cells and control cells 48 hpi. Yellow arrows indicate the presence of syncytia. Scale bar = 200 µm. (D) Violin plot of number of nuclei per syncytia 48 hpi with D614G, Delta, and B.1.640.1 infection (MOI = 0.1). Results were taken from two independent experiments, with 12 fields analyzed per variant. Nuclei counting was performed manually using ImageJ software. Ordinary one-way ANOVA tests were performed with Tukey’s multiple comparison test to compare D614G to respective variants, *P < 0.05, ****P < 0.00001, ns = not significant. For A and B, two-way ANOVA tests were performed with Geisser-Greenhouse correction to compare Delta and B.1.640.1 to D614G, *P < 0.05. Error bars represent SD.

Article Snippet: Human codon-optimized SARS-CoV-2 Spikes (Alpha, Delta, BA.1, BA.5, B.1.640.1, and B.1.640.2) were produced in silico (GeneArt, Thermo Fisher Scientific).

Techniques: Infection, Quantitative RT-PCR, Staining, Luciferase, Immunofluorescence, Control, Variant Assay, Software, Comparison

Journal: Journal of Virology

Article Title: High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1

doi: 10.1128/jvi.01351-23

Figure Lengend Snippet: Cytopathic effects of hNEC infection with B.1.640.1. (A) LDH release from apical side of hNEC ALI culture over the time course of infection with respective SARS-CoV-2 variants (n = 3/4; left). Area under the curve (AUC) representation of LDH activity, bars represent mean values (middle). Linear regression analysis of LDH release (AUC) compared to viral copies/mL (AUC) from 96 hours of infection with respective SARS-CoV-2 variants (right). Asterisk colours represent respective variants. (B) Immunofluorescence of hNECs stained for cleavage products of caspase-3 and SARS-CoV-2 nucleoprotein. Shown is one field of each variant. Scale bar = 40 µm. (C) Quantification of total area of cleavage products of caspase-3. Each data point represents one randomly assigned field from a single biological repeat (left). An ordinary one-way ANOVA test was performed with Tukey’s multiple comparison test to compare D614G to respective variants, *P < 0.05, ***P < 0.0001, ns = not significant. Linear regression analysis of caspase-3 cleavage from two biological repeats (total number of fields = 8) normalized to Delta compared to mean viral copies/mL (AUC) 96 hpi (right). (D) Linear regression analysis of caspase-3 cleavage normalized to Delta compared to LDH activity (AUC) over 96 hours of infection. Error bars represent SD.

Article Snippet: Human codon-optimized SARS-CoV-2 Spikes (Alpha, Delta, BA.1, BA.5, B.1.640.1, and B.1.640.2) were produced in silico (GeneArt, Thermo Fisher Scientific).

Techniques: Infection, Activity Assay, Immunofluorescence, Staining, Variant Assay, Comparison