Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Interferon Response Dampens the Usutu Virus Infection-Associated Increase in Glycolysis

doi: 10.3389/fcimb.2022.823181

Figure Lengend Snippet: Extracellular flux measurement indicates an increase in glycolytic activity in Vero cells after infection with USUV. A low (of 0.1) and a high (of 1) MOI were applied to address the influence of USUV load on (A) infectivity rate and (B–D) metabolic activity in Vero cells. (A) For indicated time points the number of infected Vero cells was qualitatively assessed by fluorescence microscopy after immunofluorescence analysis with pan-flavivirus antibody (shown in red as a representative for n = 3 together with nuclear counterstain DAPI in blue). (B) Quantification (mean ± SD, n = 3) of (I) OCR and (II) ECAR under basal and stressed (after co-injection of inhibitors and thus under induced energy demand) conditions. (C) The metabolic potential was determined through the percent increase in stressed OCR and ECAR over basal OCR and ECAR. (B, C) Calculations were performed by the energy phenotype test report generator software. (D) OCR/ECAR ratio was calculated based on basal OCR and ECAR as determined at measurement point 3 of extracellular flux measurements. (B–D) Data are shown as mean ± SD (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001, performed by ANOVA and Dunnett’s post-hoc multiple comparisons test.

Article Snippet: After fixation with 2% (v/v) formaldehyde solution at room temperature for 30 min immunostaining was accomplished using flavivirus-specific monoclonal primary antibody (mouse anti-pan-flavivirus 3571 antibody, Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:800 in permeabilization/wash solution containing 0.1% (w/v) BSA and 0.1% (w/v) saponine in PBS for incubation at 4°C overnight.

Techniques: Activity Assay, Infection, Fluorescence, Microscopy, Immunofluorescence, Injection, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Interferon Response Dampens the Usutu Virus Infection-Associated Increase in Glycolysis

doi: 10.3389/fcimb.2022.823181

Figure Lengend Snippet: The USUV-associated increase in glycolysis supports USUV yield and is lost in the presence of IFN β. Experiments on Vero cells were conducted at an MOI of 0.1. The dose- and application time point-dependent influence of the glycolysis inhibitor 2-DG on USUV yield was determined by (A) focus-forming assay and (B) immunofluorescence analysis of USUV-infected Vero cells with pan-flavivirus antibodies (shown in red) and nuclear counterstain (shown in blue) at 72 hpi after application of 5 mM 2-DG at 2 and 24 hpi. The impact of 1 and 10 ng/ml IFN β applied at 24 hpi to USUV-infected Vero cells was analyzed at 48 hpi by focus-forming assay (C) and immunofluorescence analysis (F) . (D) At 48 hpi basal and stressed (I) OCR and (II) ECAR values were determined through extracellular flux measurements with the cell phenotype test kit under basal and stressed conditions (co-application of 0.8 µM FCCP and 1 µM oligomycin) in the presence of 10 ng/ml IFN β applied at 24 hpi. (E) OCR/ECAR ratio was calculated based on basal OCR and ECAR as determined at measurement point 3 of extracellular flux measurements. (A, C–E) Data (n = 3) are shown as mean ± SD involving statistical analysis for (A, C) in comparison to solvent control-treated samples and (D, E) in comparison to the mock-infected control performed by ANOVA and Dunnett’s post-hoc multiple comparisons test. Statistical significance is calculated as *p < 0.05), **p < 0.01, and ***p < 0.001.

Article Snippet: After fixation with 2% (v/v) formaldehyde solution at room temperature for 30 min immunostaining was accomplished using flavivirus-specific monoclonal primary antibody (mouse anti-pan-flavivirus 3571 antibody, Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:800 in permeabilization/wash solution containing 0.1% (w/v) BSA and 0.1% (w/v) saponine in PBS for incubation at 4°C overnight.

Techniques: Focus Forming Assay, Immunofluorescence, Infection, Comparison, Solvent, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Interferon Response Dampens the Usutu Virus Infection-Associated Increase in Glycolysis

doi: 10.3389/fcimb.2022.823181

Figure Lengend Snippet: Analysis of the cell-type specificity of USUV infection rate and the associated metabolic impact. (A, B) The onset of maximum rate of infected cells was determined by immunofluorescence analysis for (A) TME-R and (B) A549 cells with pan-flavivirus antibodies for low (MOI 0.1) and high (MOI 1) infectivity rate (representatively shown in red for n = 3 with nuclear counterstain in blue). (C) At 48 hpi for TME-R and (D) at 24 hpi for A549 cells mitochondrial respiration based on (I) OCR values and glycolysis based on (II) ECAR values were determined through extracellular flux measurements under basal (without treatment) and stressed conditions (co-application of 0.8 µM FCCP and 1 µM oligomycin). (E) The metabolic potential was determined through the percent increase in stressed OCR and ECAR over basal OCR and ECAR. (F) OCR/ECAR ratio was calculated based on basal OCR and ECAR as determined at measurement point 3 of extracellular flux measurements. Data are shown as mean ± SD (n = 3). Statistical significance is indicated as *p < 0.05, performed by ANOVA and Dunnett’s post-hoc multiple comparisons test.

Article Snippet: After fixation with 2% (v/v) formaldehyde solution at room temperature for 30 min immunostaining was accomplished using flavivirus-specific monoclonal primary antibody (mouse anti-pan-flavivirus 3571 antibody, Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:800 in permeabilization/wash solution containing 0.1% (w/v) BSA and 0.1% (w/v) saponine in PBS for incubation at 4°C overnight.

Techniques: Infection, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Interferon Response Dampens the Usutu Virus Infection-Associated Increase in Glycolysis

doi: 10.3389/fcimb.2022.823181

Figure Lengend Snippet: Type I and III IFN signaling influences permissiveness of A549 cells to USUV infection-associated metabolic alterations. (A) Western blot analysis of A549 cells with KO of the type I IFN receptor IFNAR either solely or together with the type III IFN receptor (IFNLR1) and A549 control cells. Antibodies against phosphorylated STAT1 were used for analysis of samples with exogenous IFN β (10 ng/ml) or IFN λ1 (100 ng/ml) after an incubation for 24 h. (B–E) Infection with USUV strain Africa 3 and Europe 3 at MOI 0.1 was analyzed at 96 hpi. (B) Immunofluorescence analysis with pan-flavivirus antibodies (representatively shown in red for n = 3 with nuclear counterstain in blue). (C) Virus yield was determined by focus-forming assay. (D) Extracellular flux measurement with the cell energy phenotype test kit to determine (I) OCR and (II) ECAR values at basal and stressed (after co-application of 0.8 µM FCCP and 1 µM oligomycin) conditions. (E) The metabolic potential was determined through the percent increase in stressed OCR and ECAR over basal OCR and ECAR. (C–E) Data (n = 3) are shown as mean ± SD involving statistical analysis in comparison to the control performed by ANOVA and Dunnett’s post-hoc multiple comparisons test. Statistical significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001) and ****p < 0.001).

Article Snippet: After fixation with 2% (v/v) formaldehyde solution at room temperature for 30 min immunostaining was accomplished using flavivirus-specific monoclonal primary antibody (mouse anti-pan-flavivirus 3571 antibody, Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:800 in permeabilization/wash solution containing 0.1% (w/v) BSA and 0.1% (w/v) saponine in PBS for incubation at 4°C overnight.

Techniques: Infection, Western Blot, Control, Incubation, Immunofluorescence, Virus, Focus Forming Assay, Comparison

Journal: Life

Article Title: Lacticaseibacillus paracasei : Occurrence in the Human Gut Microbiota and K -Mer-Based Assessment of Intraspecies Diversity

doi: 10.3390/life11111246

Figure Lengend Snippet: List of Lacticaseibacillus genomes used for intrageneric phylogenetic analysis.

Article Snippet: 31 , AO356 (7112–2) , III , 1,310,307 , 67 , ATCC 8530 , , 1,362,386.

Techniques: