Journal: Applied and Environmental Microbiology

Article Title: Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

doi: 10.1128/AEM.00982-13

Figure Lengend Snippet: Lysates of probiotic bacteria have strain-dependent effects on keratinocyte viability. Human primary epidermal keratinocytes were incubated with lysate made from 108 CFU/ml bacteria for 24 h. Following exposure, the viability of keratinocytes was measured using an MTT assay. The viability of keratinocytes incubated in the presence of lysates of B. longum (BL), L. plantarum (LP), L. reuteri (LR), or L. rhamnosus Goldwin and Gorbach (LGG) was not significantly different from that of untreated cells (CON). However, keratinocyte cultures treated with a lysate of L. fermentum (LF) had reduced viability compared to controls (∼50% reduction in viability; P < 0.01). *, statistical significance.

Article Snippet: All probiotic strains ( Bifidobacterium longum ATCC 51870, Lactobacillus plantarum ATCC 10241, Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus Goldin and Gorbach [GG] ATCC 53103, and Lactobacillus fermentum ATCC 14932) were purchased from LGC Ltd., Middlesex, United Kingdom) and were routinely grown to stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37°C in a Mark 3 anaerobic work station (Don Whitley Scientific, United Kingdom).

Techniques: Bacteria, Incubation, MTT Assay

Journal: Applied and Environmental Microbiology

Article Title: Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

doi: 10.1128/AEM.00982-13

Figure Lengend Snippet: Lysates of probiotic bacteria enhance tight-junction barrier function with strain-specific effects. Human primary keratinocytes were induced to form TJs, and the TEER of the monolayers was monitored with time in control, untreated monolayers (filled circles) and treated monolayers (open circles). In control monolayers, TEER developed with time after the calcium switch and reached a peak of around 200 ± 50.4 Ω · cm2. With the exception of L. fermentum (A), which decreased the TEER relative to control monolayers, lysates of all the probiotic bacteria increased TEER over control levels in a strain-dependent manner (L. reuteri [B] and L. plantarum [C]). B. longum (D) and L. rhamnosus GG (E) lysates produced the greatest and most sustained effects. Peptidoglycan from S. aureus (used at 0.01 μg/ml) (F) also induced increased TEER, but its effects occurred more rapidly than those of probiotic lysates and were not sustained. *, statistical significance.

Article Snippet: All probiotic strains ( Bifidobacterium longum ATCC 51870, Lactobacillus plantarum ATCC 10241, Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus Goldin and Gorbach [GG] ATCC 53103, and Lactobacillus fermentum ATCC 14932) were purchased from LGC Ltd., Middlesex, United Kingdom) and were routinely grown to stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37°C in a Mark 3 anaerobic work station (Don Whitley Scientific, United Kingdom).

Techniques: Bacteria, Control, Produced

Journal: Applied and Environmental Microbiology

Article Title: Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

doi: 10.1128/AEM.00982-13

Figure Lengend Snippet: Lysates of probiotic bacteria have dose-dependent effects on TEER in human keratinocytes. Human keratinocytes were untreated (▽) or treated with lysates made from B. longum (BL [A]) or L. rhamnosus GG (LGG [B]) at concentrations of 108 (●), 106 (○), 104 (▼), and 102 (■) CFU/ml, and the effects on TEER were measured with time. B. longum was effective only at a concentration of 108 CFU/ml. However, L. rhamnosus GG was also effective at 106 and 104 CFU/ml. *, statistical significance.

Article Snippet: All probiotic strains ( Bifidobacterium longum ATCC 51870, Lactobacillus plantarum ATCC 10241, Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus Goldin and Gorbach [GG] ATCC 53103, and Lactobacillus fermentum ATCC 14932) were purchased from LGC Ltd., Middlesex, United Kingdom) and were routinely grown to stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37°C in a Mark 3 anaerobic work station (Don Whitley Scientific, United Kingdom).

Techniques: Bacteria, Concentration Assay

Journal: Applied and Environmental Microbiology

Article Title: Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

doi: 10.1128/AEM.00982-13

Figure Lengend Snippet: Lysates of probiotic bacteria modulate tight-junction protein expression in human keratinocytes. Human keratinocytes were treated with lysates from 108 CFU/ml of either L. rhamnosus GG (LGG) or B. longum (BL) for 24 h. Subsequently, the keratinocytes were harvested, and the expression of claudin 1, claudin 4, ZO-1, and occludin was investigated using immunoblotting (A) and subsequent densitometry (B). BL increased the expression of all four TJ proteins relative to the control: claudin 1 (cld-1), 3.7× ± 0.08× (P < 0.05); claudin 4 (Cld 4), −2.15× ± 0.02× (P < 0.05); occludin (Occ), 2.53× ± 0.14× (P < 0.005); and ZO-1, 2× ± 0.024× (P < 0.05). However, LGG affected no change in claudin 4 levels but increased the expression of the other proteins: claudin 1, 3.27× ± 0.36× (P < 0.05), occludin, 2.65× ± 0.17× (P < 0.005); ZO-1, 2.22× ± 0.036× (P < 0.05). Con, control. *, statistical significance.

Article Snippet: All probiotic strains ( Bifidobacterium longum ATCC 51870, Lactobacillus plantarum ATCC 10241, Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus Goldin and Gorbach [GG] ATCC 53103, and Lactobacillus fermentum ATCC 14932) were purchased from LGC Ltd., Middlesex, United Kingdom) and were routinely grown to stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37°C in a Mark 3 anaerobic work station (Don Whitley Scientific, United Kingdom).

Techniques: Bacteria, Expressing, Western Blot, Control

Journal: Applied and Environmental Microbiology

Article Title: Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

doi: 10.1128/AEM.00982-13

Figure Lengend Snippet: Neutralization of TLR2 abolishes specific probiotic-mediated effects on TJ barrier function and protein expression. Keratinocytes were treated with a TLR2-neutralizing antibody prior to incubation with lysates from B. longum (BL) or L. rhamnosus GG (LGG). CON, control. (A) In cells treated with lysates of BL but not LGG, the probiotic-induced increase in TEER was abolished by incubation with the antibody. Similar results were also obtained using peptidoglycan (PGN) as the ligand. *, statistical significance. (B) Similarly, the increase in TJ protein expression was also abolished by neutralization of TLR-2 in BL-treated but not LGG-treated cells. Con, control.

Article Snippet: All probiotic strains ( Bifidobacterium longum ATCC 51870, Lactobacillus plantarum ATCC 10241, Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus Goldin and Gorbach [GG] ATCC 53103, and Lactobacillus fermentum ATCC 14932) were purchased from LGC Ltd., Middlesex, United Kingdom) and were routinely grown to stationary phase in Wilkins-Chalgren broth or on Wilkins-Chalgren agar at 37°C in a Mark 3 anaerobic work station (Don Whitley Scientific, United Kingdom).

Techniques: Neutralization, Expressing, Incubation, Control

Journal: ACS Central Science

Article Title: Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

doi: 10.1021/acscentsci.0c01186

Figure Lengend Snippet: Repurposing FDA-approved drugs for SARS-CoV-2 through cellular screening. (A) Summary of methodology used in this paper. (B) Screening for FDA-approved small molecule therapeutics for activities in suppressing SARS-CoV-2 replication in Vero E6 cells. Dose–response curves in the suppression of SARS-CoV-2 replication in Vero E6 cells and cytotoxicity for simeprevir (C) and remdesivir (D) are shown. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates.

Article Snippet: Entecavir (HY-13623), zidovudine (HY-17413), sofosbuvir (HY-15005), daclatasvir (HY-10466), simeprevir (HY-10241), remdesivir (HY-104077), and remdesivir triphosphate sodium (HY-126303A) were purchased from MedChemExpress (Monmouth Junction, NJ).

Techniques:

Journal: ACS Central Science

Article Title: Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

doi: 10.1021/acscentsci.0c01186

Figure Lengend Snippet: (A) Viral replication–suppression efficacies of different combinations of simeprevir and remdesivir concentrations. The numbers after S (simeprevir) and R (remdesivir) indicate the respective drug concentrations in μM. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates. (B) Bliss score analyses of synergism. (left) Diagram showing 12 combinations of simeprevir and remdesivir and their respective percentage inhibition (% inhibition, color-coded) of SARS-CoV-2 replication in Vero E6 cells compared to DMSO controls. (right) Excess over Bliss score (ΔBliss, color-coded) of different drug combinations. A positive and negative number indicates a likely synergistic and antagonistic effect, respectively, while a zero value indicates independence of action.

Article Snippet: Entecavir (HY-13623), zidovudine (HY-17413), sofosbuvir (HY-15005), daclatasvir (HY-10466), simeprevir (HY-10241), remdesivir (HY-104077), and remdesivir triphosphate sodium (HY-126303A) were purchased from MedChemExpress (Monmouth Junction, NJ).

Techniques: Inhibition

Journal: ACS Central Science

Article Title: Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

doi: 10.1021/acscentsci.0c01186

Figure Lengend Snippet: Simeprevir weakly inhibits M pro and RdRp. Assay scheme and enzyme activity of main protease (M pro ), papain-like protease (PL pro ), and RNA-dependent RNA polymerase (RdRp) under various concentrations of simeprevir. (A) For M pro , a CFP-YFP conjugate with a M pro cleavage site linker is utilized, where relative activity is determined by the residual FRET efficiency after cleavage. (B) For PL pro , rhodamine-conjugated ISG15 is used as a substrate for the enzyme, whose relative activity is determined by release of the fluorophore. (C) For RdRp, an extension assay based on the dsRNA-binding property of the intercalating agent Picogreen was established. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates.

Article Snippet: Entecavir (HY-13623), zidovudine (HY-17413), sofosbuvir (HY-15005), daclatasvir (HY-10466), simeprevir (HY-10241), remdesivir (HY-104077), and remdesivir triphosphate sodium (HY-126303A) were purchased from MedChemExpress (Monmouth Junction, NJ).

Techniques: Activity Assay, Binding Assay

Journal: ACS Central Science

Article Title: Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

doi: 10.1021/acscentsci.0c01186

Figure Lengend Snippet: RNA-seq analysis and validation of simeprevir-mediated host response and antiviral activity. (A) Schematic representation of RNA-seq sample preparation. Treatment sequence and incubation time of simeprevir and SARS-CoV-2 was indicated with arrows and legends. (B) Venn diagrams showing differentially expressed genes (DEGs) comparing simeprevir-treated (1.1 or 3.3 μM), infected, and mock-infected samples. (C) Bubble plot of top 20 hits of positively enriched reactome gene sets under simeprevir treatment using gene set analysis (GSEA). Enriched gene sets were filtered with criteria false discovery rate (FDR) q -value < 0.25 and nominal p -value < 0.05 before ranked with their normalized enrichment scores (NES). (D) Enrichment plots of GSEA results using gene ontology (GO) gene sets. (E) Clustered heatmap showing the row-normalized expression level of genes belonging to GO term “response to type I interferon”. (F) Viral titration assay using A549-ACE2 cells treated with 4 μM simeprevir and/or 1 μM JAK inhibitor I. (G) Relative RNA level of ISG15 in uninfected or infected A549-ACE2 cells, treated with 4 μM simeprevir and/or 1 μM JAK inhibitor I. Data points in all plots represent mean ± S.D. For all data points, n = 3 replicates; * p -value < 0.05, *** p -value < 0.005.

Article Snippet: Entecavir (HY-13623), zidovudine (HY-17413), sofosbuvir (HY-15005), daclatasvir (HY-10466), simeprevir (HY-10241), remdesivir (HY-104077), and remdesivir triphosphate sodium (HY-126303A) were purchased from MedChemExpress (Monmouth Junction, NJ).

Techniques: RNA Sequencing Assay, Activity Assay, Sample Prep, Sequencing, Incubation, Infection, Expressing, Titration