Journal: Scientific Reports

Article Title: Cardiovascular tissue regeneration system based on multiscale scaffolds comprising double-layered hydrogels and fibers

doi: 10.1038/s41598-020-77187-8

Figure Lengend Snippet: Characterization of the multiscale scaffold comprising electrospun fibers and two different hydrogel layers. Scale bar = 20 μm. ( a ) Representative cryo-SEM image revealing the compartmentalized structure of the multiscale scaffold. ( b ) Compressive modulus of scaffolds (n = 4). Data are mean ± s.d. One-way ANOVA with Tukey’s post-hoc test was used. **P < 0.01.

Article Snippet: The internal structure of multiscale scaffolds was observed using Cryo-Focused Ion Beam Scanning Electron Microscopy (cryo-FIB/SEM, Quanta 3D FEG, FEI, Netherland) with a cryo-transfer system (Alto 2500, Gatan, UK) .

Techniques:

Journal: bioRxiv

Article Title: Measuring the selective packaging of RNA molecules by viral coat proteins in cells

doi: 10.1101/2025.04.14.648219

Figure Lengend Snippet: A . A structural model of the MS2 capsid contains 180 copies of the coat protein arranged as a 28-nm icosahedral shell . B . A secondary structure model of the packaged MS2 genome contains 15 stem-loop structures (shown in red) that bind tightly to the interior surface of the capsid . One of these stem-loops is the famous TR loop (shown by a red arrowhead). A map of the primary structure shows the positions of these stem-loops relative to the viral genes. C . A schematic of the in-cellulo packaging experiment outlines the key steps of our protocol: 1 . We transform E. coli with a plasmid whose insert contains the MS2 coat protein gene flanked on either side by variable untranslated sequences. Inset: Once transformed, the plasmid insert is transcribed into RNA ( i ), which is then translated into coat protein ( ii ). When the cellular concentration of coat protein becomes sufficiently high, capsids assemble and package some of the available pool of RNA ( iii ). This pool contains a mixture of insert transcripts, plasmid vector-derived transcripts, and cellular transcripts, all of which compete for packaging by the assembling coat proteins. 2 . We lyse the cells after 24 h to release the assembled capsids, and then 3 . purify the capsids from unpackaged RNA and other cellular debris using nuclease digestion followed by ultracentrifugation. Once purified, we can infer the amount of RNA packaged per particle using a combination of electron microscopy (TEM) and interferometric scattering microscopy (iSCAT). Finally, 4 . we extract the packaged RNA from the capsids, and 5 . determine its identity using short-read high-throughput RNA sequencing (RNAseq).

Article Snippet: For packaging experiments, we used chemically competent E. coli strain BL21(DE3) (New England Biolabs).

Techniques: Plasmid Preparation, Transformation Assay, Concentration Assay, Derivative Assay, Purification, Electron Microscopy, Microscopy, High Throughput Screening Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Measuring the selective packaging of RNA molecules by viral coat proteins in cells

doi: 10.1101/2025.04.14.648219

Figure Lengend Snippet: A . The pMS2′ insert contains the full MS2 genome ( top ), modified with point mutations to prevent the production of viral proteins other than the coat protein ( SI Appendix, Fig. S1 ). Leaky expression of pMS2′ transcripts produced well-formed 28-nm capsids, as observed by negative-stain TEM ( bottom-left ), with an average mass of 3.5 MDa per particle, determined by iSCAT ( bottom-middle ). Approximately 97% (±1% across duplicate experiments) of sequencing reads from the packaged RNA aligned to the pMS2′ insert sequence ( bottom-right ). B . The pcoat′ insert contains only the MS2 coat protein gene ( top ), modified by random codon swaps to scramble the RNA sequence while preserving the amino acid sequence, codon usage, and dinucleotide frequency ( SI Appendix, Fig. S5 ). As with pMS2′, leaky expression of pcoat′ transcripts produced well-formed 28-nm capsids ( bottom-left ) with an average mass of 3.5 MDa ( bottom-middle ). However, only 3% (±1% across duplicate experiments) of sequencing reads aligned to the insert sequence, with 27% aligning to the plasmid vector and 70% to the E. coli (cellular) genome ( bottom-right ). C . Cryo-electron microscopy and single-particle reconstruction showed that the majority of particles produced by both inserts adopt identical capsid structures. The electron density maps of each capsid overlap ( left ), and their molecular models are indistinguishable ( right ).

Article Snippet: For packaging experiments, we used chemically competent E. coli strain BL21(DE3) (New England Biolabs).

Techniques: Modification, Expressing, Produced, Staining, Sequencing, Preserving, Plasmid Preparation, Cryo-Electron Microscopy, Single Particle

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: In Vitro, Motility Assay, Concentration Assay