Journal: bioRxiv

Article Title: Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics

doi: 10.1101/2024.08.28.610020

Figure Lengend Snippet: (A) Scheme of assay: ACE2 labelled with an mCherry fluorescent protein and fused to tdPCP is incubated with a slncRNA cassette, encoding for multiple PCP-binding hairpins. 1h post incubation, an sfGFP-labelled RBD is added, facilitating FRET with the mCherry-labelled ACE2 proteins bound to the slncRNA granules. (B) (Left) Representative image of ACE2 granules in a typical field of view. Scalebar: 5µm. (Right) Enlarged images highlighted by white squares on the left. (Right-top) protein only condensate. (Right-bottom) An sRNP granule of protein co-localized with slncRNA. Scalebar for enlarged images 1µm and 2µm for top and bottom, respectively. (C) Correlation between mCherry and AF405 signals intensities of 339 mCherry-positive events. Representative events – see 1 and 2 from panel B and Supplementary Figure 2A. (D) (Left) Representative image of ACE granules together with RBD (green label). Scalebar: 5µm. (Right) Enlarged images highlighted by white squares on the left showing typical triple labelled structures. Scalebar: 5µm. (E-F) FRET intensity signal (E) and FRET efficiency (F) measured for the highlighted granule structures shown in . ( G) Putative green-red FRET excitation events obtained for various RBD concentrations. (H-I) (Left) Images of triple-labelled granules, FRET intensity and FET efficiency, and (right) putative intensity of FRET excitation events obtained for the non-dimerizing ACE2 (Δ616-726) (h) and the non-RBD-binding missense ACE2 mutant (D355A) (I).

Article Snippet: The enzyme was then heat-inactivated by incubating the restriction reaction at 65 °C for 20 min. For fluorescently labeled RNA, 1µg of the restriction product was used as template for in vitro transcription using HighYield T7 Alexafluor-405 (AF405) RNA labeling kit (Jena Bioscience, cat. RNT-101-AF405), according to the manufacturer’s instructions.

Techniques: Incubation, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics

doi: 10.1101/2024.08.28.610020

Figure Lengend Snippet: (A) Predicted AlphaFold structure for LAMP1-mCherry-tdPCP. Black, red, and green are LAMP1, mCherry, and tdPCP, respectively. Putative sialylation sites are marked by light-blue shading (asparagine residues in an N-X-S/T motif), yellow (serine residue), and magenta (threonine residue). (B) Typical field-of-view of SNA-LAMP1 granule biocondensate divided into the individual channels. (Top-left) protein (mCherry). (Top-right) SNA (AF488). (Bottom-left) slncRNA (AF405). (Bottom-right) Merged panel with all three images overlayed. (Top-set) No SNA added (scalebar: 5µm FoV, 1µm enlargement). (Bottom-set) High SNA concentration (scalebar: 5µm). (C) Box-plot distributions for D RNA for each candidate sialoprotein. The mean of each distribution is marked by red circle. Low and high SNA concentrations were set at below 1µg/ml (molar ratio of ∼1 SNA : 50 sialoproteins) and above 50µg/ml (molar ratio of ∼3 SNA : 2 sialoproteins), respectively. (D) Plot showing ΔD avg as a function of putative sialylated sited. ΔD avg is defined as the difference between the high and low mean D RNA values obtained for each protein in panel C. Color and size of dot correspond to the student t-test p-value computed for each panel in .

Article Snippet: The enzyme was then heat-inactivated by incubating the restriction reaction at 65 °C for 20 min. For fluorescently labeled RNA, 1µg of the restriction product was used as template for in vitro transcription using HighYield T7 Alexafluor-405 (AF405) RNA labeling kit (Jena Bioscience, cat. RNT-101-AF405), according to the manufacturer’s instructions.

Techniques: Residue, Concentration Assay

Journal: bioRxiv

Article Title: Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics

doi: 10.1101/2024.08.28.610020

Figure Lengend Snippet: (A) Typical field-of-view of SNA-LAMP1 granule biocondensate divided into individual channels. (Top-left) protein (mCherry). (Top-right) SNA (AF488). (Bottom-left) slncRNA (AF405). (Bottom-right) Merged panel with all three images overlayed. Scalebar: 5µm. (B) Close-up on the two features highlighted in panel a displayed via the same three separated channels and merged image. Scalebar: 5μm. (C) LAMP1 dose-response curves for RNA-displacement (blue), SNA binding to protein (green), and triple colocalized biocondensate structures (purple). (Left) decision tree used to classify the different structures. (D) Dose responses (see Supplementary Figure S7B) displayed as a normalized heatmap for SNA binding (top) and RNA displacement (bottom). Each row was normalized by its maximum value. (E) Plot of the combined agglutination score for each candidate sialoprotein as a function of the number of putative sialylation site showing a strong linear dependence. Each score is the summation of three separate values: ΔD RNA, ΔΔG SNA binding , and ΔΔG RNA displacement (see methods and text for definitions).

Article Snippet: The enzyme was then heat-inactivated by incubating the restriction reaction at 65 °C for 20 min. For fluorescently labeled RNA, 1µg of the restriction product was used as template for in vitro transcription using HighYield T7 Alexafluor-405 (AF405) RNA labeling kit (Jena Bioscience, cat. RNT-101-AF405), according to the manufacturer’s instructions.

Techniques: Binding Assay, Agglutination

Journal: Heliyon

Article Title: The unilateral involution in the thymus of a 96-year-old male leads to the preservation of structural integrity in one thymic lobe, as assessed by the expression of medullar and cortical antigens and the presence of CD3+ cells

doi: 10.1016/j.heliyon.2022.e11734

Figure Lengend Snippet: The mixed population of CD3+/CD4-and CD3+/CD4+ T cells associate with the right lobe medullar thymic epithelium in the thymus of a 96-year-old male body donor. (a & b) show the immunostaining of the thymic sections with antibodies to CD3 (a) and CK-14 (b) antigens; an overlay of (a & b) is shown in (c). (d–i) show the immunostaining of the thymic sections with antibodies to CD3 (d & g) and CD4 (e & h) antigens. The overlay of (d & e) is shown in (f); the overlay of (g & h) is shown in (i). The white and green arrows in (f) show CD3+/CD4-and CD3+/CD4+ T cells, respectively; the arrows in (i) show the co-localization of CD3 and CD4 antigens in CD3+/CD4+ cells. The inset in (h) shows the nuclear stain with Hoechst 33342. Images in (a–f) and (g–i) were obtained under 10x and 40x magnification, consecutively, and represent a tissue section from the same block as shown in Figure 3. (j-l) show respectively the isotype controls for monoclonal antibodies used in all experiments discussed in the manuscript. (j) normal mouse IgG1 conjugated to Alexa Fluor 405 represents a control to immunostaining in Fig. 4 a, d and g. (k) normal mouse IgG1 conjugated to Alexa Fluor 488 represents a control to immunostaining in Fig. 3 a & d. (l) normal mouse IgG1 conjugated to Alexa Fluor 594 represents a control to immunostaining shown in Figures b & e and b, e & h. The scale bar: 50 μm.

Article Snippet: Directly labeled mouse monoclonal antibodies to cytokeratin 14 (AlexaFluor-594), cytokeratin 8 (AlexaFluor-488), CD3 (AlexaFluor-405), CD4 (AlexaFluor-546), FoxN1 (AlexaFluor-488), and isotype control normal mouse IgG1 conjugated to Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 405 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunostaining, Staining, Blocking Assay, Bioprocessing, Control