ns309  (Alomone Labs)

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Cryo-EM densities for Ca 2+ and NS309 bound to one CaM molecule in NS309_K Ca 2.2 are shown as blue and green mesh contoured at σ = 6, respectively. b Cryo-EM densities for Ca 2+ and NS309 bound to one CaM molecule in NS309_K Ca 3.1 are shown as magenta mesh contoured at σ = 6. c Intracellular view of NS309_K Ca 2.2 (K Ca 2.2: purple cartoon/CaM: light blue surface). The CaM N-lobes are positioned far apart, and the HC helices are not visible probably due to flexibility. d Intracellular view of NS309_K Ca 3.1 (K Ca 3.1: salmon cartoon/CaM: cyan surface). The CaM N-lobes are positioned close to each other, which stabilize the HC helices in the center.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Binding energy between NS309 and amino acid residues in CaM and the S 45 A helix of K Ca 2.2. b Binding energy between NS309 and amino acid residues in CaM and the S 45 A helix of K Ca 3.1. The binding energy between NS309 and the four subunits of the activator-bound structure include van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits). c Responses of WT and mutant K Ca 2.2 channels to NS309 in whole-cell patch-clamp recordings in the presence of 0.25 μM Ca 2+ . d Responses of WT and mutant K Ca 3.1 channels to NS309 in whole-cell patch clamp recordings in the presence of 0.25 μM Ca 2+ . Data are presented as mean ± SD (n = 4 transfected cells). e Total binding energy of NS309 to binding pockets in NS309_K Ca 2.2 and NS309_K Ca 3.1, including van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits). f Chemical structure of NS309.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Binding Assay, Mutagenesis, Patch Clamp, Transfection

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Chemical structures of rimtuzalcap and CyPPA. b The binding pocket of rimtuzalcap in rimtuzalcap_K Ca 2.2_I (K Ca 2.2: green/CaM: yellow) superimposed onto the binding pocket of NS309 in NS309_K Ca 2.2 (K Ca 2.2: purple/CaM: light blue). Rimtuzalcap forms contacts with both the S 45 A and HA helices, while NS309 primarily interacts with the S 45 A helix. c Binding energy between rimtuzalcap and amino acid residues in CaM and the S 45 A helix of K Ca 2.2. The binding energy between rimtuzalcap and the four subunits of the activator-bound structure include van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits).

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: In side views, dimensions of the inner gate are measured as distances between Val391 in the transmembrane S6 helices of opposite K Ca 2.2 subunits in a apo_K Ca 2.2, b NS309_K Ca 2.2, c rimtuzalcap_K Ca 2.2_I, and d AP14145_K Ca 2.2 structures. Two opposite channel subunits are shown for clarity.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection

doi: 10.4049/jimmunol.2400250

Figure Lengend Snippet: Impact of breakthrough infection on Wuhan-Hu-1 and Omicron S-specific ADCC. (A) 51Cr release was used to assess the capacity of Ab produced after vaccination (PV3; closed circles) and breakthrough infection (post-Omicron breakthrough infection [POMI]; open circles) to elicit ADCC against Wuhan-Hu-1 and Omicron S-expressing MRC-5 cells. Lines bisecting groups (median with IQR) were calculated using Friedman test with Dunn multiple comparisons test (****p < 0.0001, ***p = 0.0001). (B) Histogram overlay of surface Wuhan-Hu-1 (green) and Omicron (purple) S expression on MRC-5 cells. Percentage of cells positive for S and geometric mean fluorescence intensity (gMFI) in comparison with nontransduced control cells (gray) are shown. Anti-Wuhan-Hu-1 and Omicron (C) FLS and (D) RBD IgG Abs in plasma (1:250) from thrice-vaccinated participants (PV3; n = 18) who later went on to experience Omicron infection (POMI; n = 18). (E) Cell-based ELISA (CELISA) was performed in parallel with 51Cr release assay to assess Ab binding (1:250) to Wuhan-Hu-1 or Omicron S-expressing live cells. Lines bisecting groups (mean ± SD) in (C–E) were calculated using repeated measures one-way ANOVA with Tukey multiple comparisons test (*p = 0.0366, **p = 0.0052, ****p < 0.0001).

Article Snippet: Peptide scan chemiluminescence ELISA Individual overlapping peptides (180 17-mers and one N-terminal 13-mer, with 10-aa overlaps) spanning the canonical Wuhan-Hu-1 S sequence (NR-52402; Biodefense and Emerging Infections Research Resources Repository [BEI Resources]) were reconstituted at 10 mg/ml in DMSO (Sigma-Aldrich), then diluted to 50 μg/ml in Dulbecco’s PBS (Sigma-Aldrich) and stored at −20°C as previously described ( 30 ).

Techniques: Infection, Produced, Expressing, Fluorescence, Comparison, Control, Clinical Proteomics, In-Cell ELISA, Release Assay, Binding Assay

Journal: The Journal of Immunology Author Choice

Article Title: Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection

doi: 10.4049/jimmunol.2400250

Figure Lengend Snippet: Distribution of anti-S IgG reactivity across S1, S2, and linear determinants in FLS. Levels of (A) anti-Wuhan-Hu-1 and Omicron S1 IgG and (B) anti-Wuhan-Hu-1 S2 IgG in plasma (1:100) from 18 participants after three vaccinations (PV3) and subsequent post-Omicron breakthrough infection (POMI) were compared, and (C) Ab reactivity against Wu-Hu-1 S1 versus S2 was contrasted. Lines bisecting groups (mean ± SD) in (A) were calculated using repeated measures one-way ANOVA with Tukey multiple comparisons test (****p < 0.0001), in (B) using paired t test, and in (C) using repeated measures one-way ANOVA with Tukey multiple comparisons test (****p < 0.0001). (D) Linear anti-S Ab epitope reactivity was determined by chemiluminescence ELISA-based peptide scanning. Compiled peptide scan data from 18 samples collected after breakthrough infection (POMI) and from 8 samples collected after one vaccination postinfection (Hy PV1) are illustrated using a heat map with Ab reactivity expressed as relative light units (RLU) aligned with known mutations in the Omicron BA.1 S sequence. The three determinants associated with ADCC are identified by asterisks. (E–G) Comparisons of Ab reactivity against the three determinants significantly associated with ADCC for persons with breakthrough infection (POMI) and hybrid immunity (Hy PV1). Lines bisecting groups (median with IQR) in (E–G) were calculated using Mann–Whitney U test (**p = 0.0079, ***p = 0.0004, ****p < 0.0001).

Article Snippet: Peptide scan chemiluminescence ELISA Individual overlapping peptides (180 17-mers and one N-terminal 13-mer, with 10-aa overlaps) spanning the canonical Wuhan-Hu-1 S sequence (NR-52402; Biodefense and Emerging Infections Research Resources Repository [BEI Resources]) were reconstituted at 10 mg/ml in DMSO (Sigma-Aldrich), then diluted to 50 μg/ml in Dulbecco’s PBS (Sigma-Aldrich) and stored at −20°C as previously described ( 30 ).

Techniques: Clinical Proteomics, Infection, Enzyme-linked Immunosorbent Assay, Sequencing, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection

doi: 10.4049/jimmunol.2400250

Figure Lengend Snippet: Ab avidity against Wuhan-Hu-1 and Omicron S after breakthrough infection. Plasma collected after third vaccination (PV3) and breakthrough infection (POMI) were diluted to obtain OD 1 (linear region of titration curve) and applied to ELISA plates coated with Wuhan-Hu-1 or Omicron FLS. Avidity was assessed in the presence of a chaotropic agent, and the relative avidity index (RAI) was calculated in relation to control conditions and expressed as a percentage. Lines bisecting groups (median and minimum to maximum) were calculated using repeated measures one-way ANOVA with Tukey multiple comparisons test (*p = 0.0450, ***p < 0.0003). Boxes outline median ± IQR.

Article Snippet: Peptide scan chemiluminescence ELISA Individual overlapping peptides (180 17-mers and one N-terminal 13-mer, with 10-aa overlaps) spanning the canonical Wuhan-Hu-1 S sequence (NR-52402; Biodefense and Emerging Infections Research Resources Repository [BEI Resources]) were reconstituted at 10 mg/ml in DMSO (Sigma-Aldrich), then diluted to 50 μg/ml in Dulbecco’s PBS (Sigma-Aldrich) and stored at −20°C as previously described ( 30 ).

Techniques: Infection, Clinical Proteomics, Titration, Enzyme-linked Immunosorbent Assay, Control