Journal: Plants

Article Title: Description of Ficus carica L. Italian Cultivars II: Genetic and Chemical Analysis

doi: 10.3390/plants14081238

Figure Lengend Snippet: Polyphenol content ( a ), flavonoid content ( b ), EC50 ( c ), and ORAC test ( d ) in leaves of fig cultivars. Values are the means (n = 3) ± SD. Data were analyzed by ANOVA, and different letters represent significant differences ( p < 0.05) according to post hoc comparison (Tukey’s HSD). PA, Paradiso; AL, Albo; VE, Verdino; PO, Portogallo; CO, Corbo; PN, Pecciolo Nero; BB, Brogiotto Bianco; GI, Gigante di Carmignano; BC, Bianco di Carmignano; PE, Perticone; PB, Pecciolo Bianco; BN, Brogiotto Nero; DO, Dottato; FI, Fiorone; SP, San Piero.

Article Snippet: Differences were tested with Tukey’s high significance difference (HSD) test at the 0.05 significance level (Statgraphics Plus, version 5.1 for Windows).

Techniques: Comparison

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A ) Immunoblots of the top ( T ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-2xFYVE protein (~35 kDa) specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of T/B bands for each liposome preparation. Significant differences (**p<0.01) as determined by one-way ANOVA with Tukey’s HSD test.( B ) Immunoblots of the three top (T 1 , T 2 , and T 3 ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 FL protein (~32 kDa) specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of (T 1 +T 2 +T 3 )/B bands for each liposome preparation. Significant differences (*p<0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( C ) Liposomes were vitrified and only representative cryo-electron microscopy images are shown. Left panel: liposomes PI3P(-) incubated even with the Ni-NTA gold particles reagent alone or pre-incubated with His-2xFYVE of His-VP3 FL. Right panel: liposomes PI3P(+) control (without protein), or incubated with His-2xFYVE- or His-VP3 FL-Ni-NTA gold particles showing gold particles decorating the membrane of the liposomes when His-2xFYVE or His-VP3 FL were present. The bar represents 50 nm. ( D ) Binding of His-Streptavidin (negative control), His-2xFYVE (positive control), or His-VP3 FL to three different concentrations of liposomes PI3P(-) or PI3P(+). Association and dissociation sensorgrams measured by bio-layer interferometry (BLI), showing the specific interaction of His-2xFYVE and His-VP3 FL with liposomes PI3P(+) in a dose-dependent manner, as indicated. ( E ) Cartoon representation of AlphaFold2 prediction of VP3 FL. Red region corresponds to the ‘ core’ region of the protein present in the experimental X-ray crystallographic model obtained by Casañas and coworkers PDB: 2R18 . Regions not present within the PDB are colored in violet and blue representing the Nt and the Ct of VP3, respectively. pLDTT values lower than 50 are a strong predictor of disorder. ( F ) Binding of His-VP3 ΔCt to three different concentrations of liposomes PI3P(-) or PI3P(+). Sensorgrams measured by BLI, showing the absence of binding to either liposomes when the VP3 lacks the Ct region blue in ( E ). ( G ) Immunoblots of the three top (T 1 , T 2 , and T 3 ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay of His-VP3 FL protein (~32 kDa) (positive control, left panel) or His-VP3 ΔCt protein (~28 kDa) (right panel) showing the lack of VP3 ΔCt binding to both liposomes. Results are representative of three independent experiments. The bar plot represents the intensity of (T 1 +T 2 +T 3 )/B bands for each liposome preparation. Significant differences (*p<0.05; ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. Figure 1—source data 1. Original membranes corresponding to . Figure 1—source data 2. Individual files corresponding to the original membranes from .

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques: Western Blot, Liposomes, Cryo-Electron Microscopy, Incubation, Control, Membrane, Binding Assay, Negative Control, Positive Control

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A ) The cartoon representation of the VP3 dimer PDB 2R18 shows each protomer in different shades of gray. The blue balls depict the residues defining the P2 region. ( B ) The close-up of P2 region showing residues K 157 , R 159 , H 198 ,and R 200 . ( C ) Electrostatic potential mapped on the surface of the structure of a VP3 dimer in the same orientations as in ( A ) structures. The close-up shows the impact of P2 residue mutations on the electrostatic potential of the binding site. P2 wild-type (WT) corresponds to the ‘UniProt code’. The color-coded electrostatic surface potential of VP3 was drawn using PyMol (blue positive, red negative). ( D ) QM7 cells transfected with pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four-point mutants (K 157 D, R 159 D, H 198 D, and R 200 D) and immunostained with anti-VP3 showing the distribution of each protein (upper panel). Images were captured using a Confocal Laser Scanning Microscopy and then the percentage of cells with punctated fluorescent signal were determined for each protein (lower panel). The red signal shows the VP3 distribution and the blue one shows the nuclei, which were Hoestch-stained. The data were normalized to the P2 WT protein. The box plot represents the percentage of cells with punctuated distribution of VP3. Significant differences (***p<0.001; ns p>0.05.) as determined by one-way ANOVA with Tukey’s HSD test. ( E ) QM7 cells co-transfected with pEGFP-Rab5 and pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four-point mutants (K 157 D, R 159 D, H 198 D, and R 200 D) and immunostained with anti-VP3 showing the distribution of each protein. Representative images, captured using a Confocal Laser Scanning Microscopy are shown where green signal represents Rab5 distribution and the red signal that of VP3. Nuclei were Hoestch-stained and are blue. White bar-scales represent 20 mm. VP3 P2 (all reversed) and R 200 D depict a cytosolic distribution of the proteins. Quantification of the co-localization of the different VP3 proteins and EGFP-Rab5 is shown in .

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques: Residue, Binding Assay, Transfection, Confocal Laser Scanning Microscopy, Staining

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: The dot plot depicts the co-localization coefficient for each protein determined as explained in the Materials and methods section. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test.

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques:

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A ) QM7 cells co-transfected with the PI3P biosensor pEGFP-2xFYVE and pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four-point mutants (K 157 D, R 159 D, H 198 D, and R 200 D) and immunostained with anti-VP3 showing the distribution of each protein. Representative images captured using a Confocal Laser Scanning Microscopy are shown where blue signal represents FYVE distribution and the magenta signal that of VP3. Nuclei were Hoestch-stained and are orange (upper panels). White bar-scales represent 20 mm. VP3 P2 (all reversed) and R 200 D depict a cytosolic distribution of the proteins with a significant lower co-localization coefficient. The dot plot in the lower panel depicts the co-localization coefficient for each protein determined as explained in the Materials and methods section. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( B ) QM7 cells were transfected with the pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four-point mutants (K 157 D, R 159 D, H 198 D, and R 200 D). The cells were fixed and then the GST-2xFYVE purified peptide and anti-VP3 antibodies were used to recognize endogenous PI3P and VP3, respectively. Additionally, anti-EEA1 antibodies were used to stain the endosomes. GST-2xFYVE was labelled with a fluorescent anti-GST antibody (blue signal), anti-VP3, and anti-EEA1 antibodies with fluorescent secondary antibodies (magenta and green signals, respectively), and Hoestch-stained nuclei in orange. White bar-scales represent 10 mm. VP3 P2 (all reversed) and R 200 D depict a cytosolic distribution of the proteins with a significant lower co-localization coefficient. The dot plot in the lower panel depicts the co-localization coefficient for each protein determined as explained in the Materials and methods section. Significant differences (***p<0.001; ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test.

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques: Transfection, Confocal Laser Scanning Microscopy, Staining, Purification

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A , left panel). Immunoblots of the top (T) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-2xFYVE protein (~35 kDa) specifically binds to liposome PI3P(+). The bar plot represents the intensity of T/B bands for each liposome preparation. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( A , right panel). Immunoblots of the three top (T 1 , T 2 , and T 3 ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 FL R 200 D protein (~35 kDa) does not bind to liposome PI3P(-) nor PI3P(+). The bar plot represents the intensity of (T 1 +T 2 +T 3 )/B bands for each liposome preparation. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( B ) Far-UV CD spectra of His-VP3 FL (red line) or His-VP3 FL R 200 D (green line). Spectral acquisitions at 50 nm/min with 0.1 nm steps at 1 s integration time, with a bandwidth of 1 nm were performed four times for the samples as well as for the buffer. The measurements were carried out with constant nitrogen gas flux of 10 ml/min. Acquisitions were averaged and buffer baseline was subtracted with Spectra Manager (JASCO). No smoothing was applied. CDtoolX was used to zero between 255–260 nm and to calibrate the signal amplitude from the fresh CSA signal . Data are presented as delta epsilon (Δε) per residue (L.mol- 1 .cm- 1 .residue 1 ) calculated using the molar concentration of protein and number of residues. ( C ) QM7 cells were grown in M24 multi-well plate for 12 hr to approximately 90–95% confluency and then 800 ng of plasmids were transfected [(SegA +SegB) or (SegA.R 200 D+SegB)] in triplicate. At 8 hr post-transfection (p.t.) the supernatants were discarded, and the monolayers were recovered for further plating on M6 multi-well plates containing non transfected QM7 cells. Avicel RC-591 (FMC Biopolymer) was added to the M6 multi-well plates. 72 hr p.i., the monolayers were fixated and stained with Coomassie R250 for revealing the foci forming units. ( D ) Partial view of amino acid alignment of the VP3 protein for nine reference members of Birnaviridae family. Multiple sequence alignment was performed with Clustal OMEGA (v1.2.4) implemented at EMBL’s European Bioinformatics Institute (The complete alignment is shown in ). Alignment visualization was done with the R ggmsa package in with assistance from the RStudio software . Amino acids are colored according to their side-chain chemistry. Protein sequence logos annotation is displayed on top of the amino acid alignment. For facilitating the view IBDV VP3 142–210 portion is shown. The black arrow indicates the K 157 , R 159 , H 198 , and R 200 . Figure 4—source data 1. Original membranes corresponding to . Figure 4—source data 2. Individual files corresponding to the original membranes from .

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques: Western Blot, Circular Dichroism, Residue, Concentration Assay, Transfection, Staining, Sequencing, Software

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A ) Left panel: Immunoblots of the three top (T 1 , T 2 , and T 3 ) and the bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 full length (FL) protein specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of (T 1 +T 2 +T 3 )/B bands for each liposome preparation. Right panel: Immunoblots of the three top (T 1 , T 2 , and T 3 ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 ΔNt protein specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of (T 1 +T 2 +T 2 )/B bands for each liposome preparation. Significant differences (***p<0.001) as determined by one-way ANOVA with Tukey’s HSD test. ( B ) Binding of His-VP3 ΔNt to three different concentrations of liposomes PI3P(-) or PI3P(+). Association and dissociation sensorgrams measured by bio-layer interferometry (BLI), showing the specific interaction of His-VP3ΔNt with liposomes PI3P(+) in a dose-dependent manner, as indicated. ( C ) Transmission electron microscope images of cryo-fixated liposomes PI3P(+) control (without protein), or incubated with His-VP3 FL- or His-VP3 ΔNt-Ni-NTA gold particles showing electrodense particles decorating the membrane of the liposomes when His-VP3 FL or His-VP3 ΔNt were present. The scale bar represents 50 nm. Figure 5—figure supplement 1—source data 1. Original western blot membranes corresponding to . Figure 5—figure supplement 1—source data 2. Individual files corresponding to the original western blot membranes from .

Article Snippet: One-way ANOVA followed by a Tukey’s HSD (honestly significant difference) test were performed using AnalystSoft Inc, StatPlus:mac - Version v8 ( ).

Techniques: Western Blot, Binding Assay, Liposomes, Transmission Assay, Microscopy, Control, Incubation, Membrane