Journal: bioRxiv

Article Title: A bioelectric neural interface towards intuitive prosthetic control for amputees

doi: 10.1101/2020.09.17.301663

Figure Lengend Snippet: Experimental paradigm and hardware components. (A) Diagram of the experiment setup including the Scorpius front-end neural recording system, implanted FAST-LIFE microelectrode arrays, a computer for data acquisition and back-end AI models implementation, and a data glove for obtaining ground-truth movements during mirrored bilateral training. (B) Photo of the transradial amputee during an experiment session (C) The Scorpius is a miniaturized prototype designed toward implantable applications. The head-piece contains a fully-integrated Neuronix neural recording chip, an electrode connector, and passive components while the auxiliary-piece includes voltage regulators and data relaying circuits. (D) Microphotograph of the Neuronix chip that has ten fully-integrated, high-performance neural recording channels based on the frequency-shaping (FS) architecture.

Article Snippet: shows the overview of the Scorpius system – a miniaturized front-end recorder equipped with the fully-integrated Neuronix neural recording chip.

Techniques:

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ca 2+ dependency of the interaction of the rat complex. Size exclusion chromatograms after assemblies: (i) in Ca 2+ free conditions (grey), (ii) with Ca 2+ preloaded NCS-1ΔH10 (magenta) and (iii) with a dialysis from EGTA to Ca 2+ (green). 12 % SDS-PAGE gels analyzing elution of NCS-1ΔH10 (N) and rRic-8A-452 (R) after assembly (i) and the NCS-1/rRic-8A-452 complex (C) after assembly (iii). B) Nano-DSF curves of the different samples during assembly (iii). The ratio between the emission fluorescence at 350 and 330 nm is shown vs the temperature. Curves corresponding to the EGTA purified NCS-1 (NCS-1 2 mM EGTA), the fully Ca 2+ saturated protein (NCS-1 2 mM Ca 2+ ) and rRic-8A-452 are shown as references in yellow, brown and grey, respectively. The mixture of proteins before dialysis (0.6 mM EGTA) and afterwards (assembled complex, 2 mM CaCl 2 ) are shown in magenta and blue respectively. NCS-1 refers to NCS-1ΔH10, while Ric-8A to Ric-8A-452 construct. C) Co-IP protein-protein interaction assay in HEK293 cells of full-length human NCS-1 and V5-tagged hRic-8A constructs: full-length (hRic-8A-FL) and C-terminally truncated hRic-8A-424 (residue 1-424) and hRic-8A-433 (residue 1-433). D) Nano-DSF curves of hNCS-1 bound to different Ric-8A peptides. NCS-1 refers to NCS-1ΔH10, P2 and P3 refers to Ric-8A peptides P2 (purple) and P3 and (green).

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: SDS Page, Fluorescence, Purification, Construct, Co-Immunoprecipitation Assay, Protein Protein Interaction Assay

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ribbon representation of the hNCS-1ΔH10/Ric-8A-P3 complex. Two views are displayed. The NCS-1 structure is shown in light purple, while Ric-8A-P3 is shown in light pink. The N- and C-termini are indicated. Ca 2+ , Na + and Cl - ions are shown in hotpink, yellow and cyan, respectively. R1 and R2 helices, and EF-hands 2, 3 and 4 are indicated. The orange square represents a zoomed view of the R1-R2 loop in stick mode, Cl - coordination and H-bonds are displayed as yellow and grey dashes, respectively. Residues participating in R1-R2 contacts are displayed in hot pink (Triad 1: I407-T410-A415), magenta (F406-L418) and purple (Triad 2: K408-Y409-N414). B) rRic-8A sequence from 400 to 442 residues. The helix boundaries of Ric-8A sequence encompassing a9 and b9 in different structural contexts (NCS-1/Ric-8A-peptide (PDB: 8AHY), Ric-8A/Gα (PDB: 6UKT, ) and uncomplexed Ric-8A (PDB: 6NMG, ) are indicated as pink boxes and labelled. Coiled regions are shown in pink. Disordered regions are shown in grey, while phosphorylated sites are shown as red spheres. The interacting residues shown in panel (A) are indicated with dots in the same color code as in (A). C) Electrostatic surface potential of rRic-8A-P3. NCS-1 is shown as yellow ribbons. Positive and negative potential are represented in blue and red. On the right, the Ric-8A region that faces and contacts NCS-1 is shown with NCS-1 removed for proper visualization.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Sequencing

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Identification of Ca 2+ , Mg 2+ and Na + ions in the hNCS-1ΔH10/Ric-8A-P3 complex (Structure 2, see ). Top: Electron density at EF-hands EF-2, 3 and 4. The 2F o -F c electron density map ( contoured at 1.0 σ) and the anomalous difference map (contoured at 6.0 σ) are shown in green and blue respectively. NCS-1 is shown in stick mode (light purple), Ca 2+ and Na + ions as hot-pink and yellow spheres, respectively, and water molecules (w) as red spheres. Bottom: The Mg 2+ ion (green sphere) found in Structure 1 and 2 (see ). NCS-1 symmetry related molecule is depicted in yellow. B) The binding of Na + to hNCS-1 in solution. Representation of the normalized fluorescence emission (mean + SEM; n =3) of full-length hNCS-1 at increasing concentrations of NaCl or KCl. The curves are the least squares fitting of the experimental data to a 1:1 stoichiometry equilibrium. Na + and K + titrations are shown in blue and magenta respectively. C) Assembly of the NCS-1ΔH10/rRic-8A-452 complex in the presence of 200 mM Na + (blue) or K + (magenta). Size exclusion chromatograms indicating the elution of the assembled complexes (C). D) ITC binding isotherm at 25 ºC for Ca 2+ to NCS-1 in 20 mM Tris pH 7.9 supplemented with 150 mM Na + (blue) or 150 mM K + (magenta). Solid lines show the best fits of the titration data in terms of a three-site sequential binding model using the thermodynamic parameters shown in . Θ is the faction of sites available for each class of Ca 2+ sites.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Binding Assay, Fluorescence, Titration

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Co-IP protein-protein interaction assay of hNCS-1 and V5-tagged full-length hRic-8A WT (hRic-8A-WT) and a non-phosphorylatable mutant (Ric-8A-P-Mut; S436A, T441A) in HEK293 cells. B) Anionic exchange chromatograms of CK2 treated samples eluted in a salt gradient. On the left, phosphorylated and unphosphorylated rRic-8A-452 (prRic-8A-452 (orange) and urRic-8A-452 (green), respectively. On the right, CK2 treated (pink) or untreated (blue) NCS-1ΔH10/rRic-8A samples. Conductivity (mS/cm) is shown as grey lines. C) Size exclusion chromatograms of the resulting samples after the assembly of NCS-1ΔH10 with unphosphorylated (green) and phosphorylated (orange) rRic-8A-452. C stands for assembled complex.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Co-Immunoprecipitation Assay, Protein Protein Interaction Assay, Mutagenesis