Journal: bioRxiv

Article Title: Catestatin ameliorates tauopathy and amyloidogenesis via adrenergic inhibition

doi: 10.64898/2026.01.04.697519

Figure Lengend Snippet: CST levels are reduced across Tauopathies, and AD-spectrum disorders, and CST supplementation suppresses pathological Tau accumulation. CST quantification in human Alzheimer’s disease (AD) cortex shows significantly reduced CST in the (A) prefrontal cortex (PFC) and (C) hippocampus/entorhinal cortex (HC/EC) of Braak VI (n=16) vs. Braak 0–II (n=14) subjects. CST levels positively correlate with Mini-Mental State Examination (MMSE) scores in (B) PFC and (D) HC/EC, linking CST deficiency to cognitive decline. (E&F) Immunostaining of CST in postmortem tissue reveals reduced CST level in Braak VI (n=8) hippocampus compared with Braak 0–II (n=8); quantification shown in ( F ). Scale bar = 100µm (G&H) CST levels are also significantly reduced in cortical tissue from ( G ) corticobasal degeneration (CBD; Braak 0: n=16, Braak 3: n=19) and ( H ) basal ganglia from progressive supranuclear palsy (PSP; Braak 0: n=15, Braak 3: n=19), indicating CST loss as a shared feature of 4R-Tauopathies. (I) In cortical neuron cultures transduced with AAV2–P301S hTau, CST treatment decreases hyperphosphorylated Tau species (S202/T205 and S396/S404) without altering total Tau (n=3). (J–K) Immunocytochemistry confirms CST-mediated suppression of MC1+ misfolded Tau in P301S hTau–expressing neurons; quantified as MC1/MAP2+ area in (n=4). Scale bar = 50µm. (L–N) In organotypic hippocampal slice cultures (OTSC) expressing AAV2–P301S hTau (n=4), ( L ) CST reduces pTau (S202) and pTau (S396/S404) accumulation and ( M&N ) diminishes MC1+ Tau pathology in DG and CA3 regions. Scale bar = 200µm *Data are mean ± SEM; statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

Article Snippet: HEK 293 cells expressing the human Tau RD P301S FRET biosensor (ATCC CRL-3275) were plated onto Millipore EZ chambered slides at a confluency of 1 × 10 3 cells per well in complete DMEM (DMEM supplemented with 10% Fetal bovine serum and 100μg/mL penicillin-streptomycin).

Techniques: Immunostaining, Transduction, Immunocytochemistry, Expressing

Journal: bioRxiv

Article Title: Catestatin ameliorates tauopathy and amyloidogenesis via adrenergic inhibition

doi: 10.64898/2026.01.04.697519

Figure Lengend Snippet: (A) Cortical epinephrine (EPI) levels are markedly elevated in Sal/PS19 compared with nTg controls, and CST treatment significantly lowers EPI concentrations (nTg: n=7, Sal/PS19: n=18, CST/PS19: n=15). (B) Cortical EPI levels are markedly elevated in Sal/5xFAD compared with nTg controls, and CST treatment significantly lowers EPI concentrations (nTg: n=7, Sal/5xFAD: n=6, CST/5xFAD: n=6). (C–E) CST suppresses pathological PKA signaling in the PS19 hippocampus. ( C ) Immunoblots show increased phospho-PKA substrates and elevated p-Tau (S202/T205) in Sal/PS19 mice; CST treatment reduces both, while total Tau and PKA-Cα remain unchanged. ( D&E ) Quantification confirms CST-mediated reduction of PKA activity and Tau phosphorylation (nTg: n=3, Sal/PS19: n=6, CST/PS19: n=5). (F) Schematic illustrating CST blockade ADR–driven PKA activation. In AAV-MAPT (P301S/K18)–transduced hippocampal slices, EPI strongly activates PKA signaling and enhances Tau phosphorylation, whereas CST antagonizes adrenergic/PKA hyperactivation and prevents formation of misfolded Tau species. (G–I) CST counteracts EPI-induced PKA activation and Tau pathology. ( G ) Immunoblot analysis shows that EPI markedly increases phospho-PKA substrates and p-Tau species (S202, S396/404), CST alone has minimal effect on PKA activity. Still, CST prevents EPI-induced PKA hyperactivation (PBS: n=3, CST: n=3, EPI: n=3, CST+EPI: n=3). ( H&I ) Quantification demonstrates CST’s ability to blunt adrenergic/PKA overactivation and its downstream Tau phosphorylation signatures. (J) Immunofluorescence staining (CA3, DG) reveals substantial accumulation of misfolded Tau in EPI-treated slices, which is significantly reduced by CST and nearly abolished by CST+EPI (PBS: n=3, CST: n=3, EPI: n=3, CST+EPI: n=3). Scale bar = 200µM. A.U.: Arbitrary Unit. Data shown as mean ± SEM; significance indicated as *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001; NS, not significant.

Article Snippet: HEK 293 cells expressing the human Tau RD P301S FRET biosensor (ATCC CRL-3275) were plated onto Millipore EZ chambered slides at a confluency of 1 × 10 3 cells per well in complete DMEM (DMEM supplemented with 10% Fetal bovine serum and 100μg/mL penicillin-streptomycin).

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Activation Assay, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Multiobjective V H H discovery through integrated high-throughput screening and AlphaFold3-guided structural prioritization

doi: 10.1126/sciadv.aef5325

Figure Lengend Snippet: ( A ) Overview of LNTxs used for the screenings, including a cLNTx (structure predicted with AF3), αCbtx [PDB ID: 1YI5 ], and αBgtx [PDB ID: 1HC9 ] with RMSD between structures annotated. ( B ) Yeast display screenings were performed using biotinylated cLNTx with streptavidin-PE to detect antigen binding and SpyCatcher-GFP to detect V H H surface display. Yeast cells were sorted with FACS into cLNTx binder and nonbinder populations on the basis of these two signals. ( C ) Poly- and monospecific selections were performed using biotinylated αBgtx detected with streptavidin-PE and αCbtx directly labeled with AF488. On the basis of these two signals, three populations were FACS sorted: αBgtx monospecific V H Hs, αCbtx monospecific V H Hs, and αBgtx/αCbtx polyspecific V H Hs. ( D ) Each yeast display selection was deep sequenced, and sequences were clustered by hierarchical clustering using pairwise distances across CDR1-CDR3. Clusters were visualized using t -distributed stochastic neighbor embedding ( t -SNE) plots, enabling selection of relevant variants for each sample. Coloring of populations is the same as in (B) and (C).

Article Snippet: All measurements were performed in Hepes buffer (pH 7.2), and sensors were preincubated in Hepes buffer for 30 min. Biotinylated αBgtx and αCbtx (200 nM final) were immobilized on streptavidin biosensors (Sartorius) to a loading response of ∼1.0 nm.

Techniques: Yeast, Binding Assay, Labeling, Selection

Journal: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 (W45A) biosensor , Addgene , Cat#120808.

Techniques: Mutagenesis

Journal: Science Advances

Article Title: Chemically encoded pH-tunable covalent adhesion by a bacterial thioester domain

doi: 10.1126/sciadv.aeg7646

Figure Lengend Snippet: TEDs were immobilized on the biosensor through the SpyTag003-SpyCatcher003 reaction. (A) Experimental procedure for the “Assay 1: Binding analysis” ( and ). Biotinylated SpyCatcher003 was immobilized on streptavidin-coated SAX2 biosensors through the biotin-streptavidin interaction, followed by conjugation of SpyTag003-fused TED. (B) Experimental procedure for the “Assay 2: Covalent cross-linking analysis” ( and ). SpyCatcher003 was immobilized on AR2G biosensors through amine-coupling chemistry, followed by conjugation of SpyTag003-fused TED.

Article Snippet: Streptavidin-coated SAX2 biosensors (Sartorius) were hydrated for 10 min and loaded with 2 μM biotinylated SpyCatcher003-His-Avi for 5 min. To remove loosely bound proteins, the biosensors were regenerated using 10 mM glycine (pH 2.0).

Techniques: Binding Assay, Analysis, Conjugation Assay, Covalent Coupling

Journal: bioRxiv

Article Title: Protein Deimmunization with a Generative Diffusion Model

doi: 10.64898/2026.10.09.758136

Figure Lengend Snippet: (A) Fixed amino acid residues for MESSI-based deimmunization of LuxSit-i. The structure of LuxSit-i (shown in blue and red) with its luciferin substrate (shown in green) is generated by Alphafold-3. The color on LuxSit-i denotes the number of overlapping NMP-predicted HLA-DRB1*15:01 epitopes at each amino acid position. Fixed amino acid residues are depicted as yellow-colored spheres. (B) Sequence identity of MESSI-generated LuxSit-i variants relative to the parental LuxSit-i. (C) Structural metrics of MESSI-generated LuxSit-i variants. (D) Comparison of epitope density between parental LuxSit-i and a MESSI-generated variant, MESSI-D2. All structures were generated by AlphaFold-3. Color denotes the number of overlapping NMP-predicted HLA-DRB1*15:01 epitopes at each amino acid position. (E) Experimental validation of luminescence from MESSI-generated LuxSit-i variants. Gene fragments encoding the parental and MESSI-designed proteins were synthesized and used as linear DNA template for in vitro RNA transcription and protein translation (IVTT). Afterwards, the reaction product was purified with a nickel spin column to remove the His-tagged IVTT machinery. A final concentration of 25 µM of diphenylterazine (DTZ) was added to the purified proteins. Luminescence was recorded on a plate reader every minute over 2 hr. (F) Peak luminescence recorded within 2 hours after DTZ addition. “No template” denotes the negative control condition where no DNA template was added to the IVTT reaction. Error bars, standard deviation. n = 3 replicates. A two sided unpaired t-test with welch’s correction is calculated between no-target condition and each of the MESSI designs and parent. **, p < 0.01; ****, p < 0.0001. (G) Computational workflow for designing a deimmunized binder against PD-1 using MESSI coupled with BindCraft. In particular, MESSI is inserted at the end of the original BindCraft workflow to replace the soluble MPNN inverse folding model to design sequences against the optimized structures. (H) Structural metrics of MESSI-generated variants of anti-PD-1 binder. (I) Comparison of epitope density between the parental anti-PD-1 binder and a MESSI-generated variant, MESSI-D4. All structures were generated by AlphaFold-3. Blue colors denote a binder region that does not contain NMP predicted epitopes. Red color denotes binder regions with NMP-predicted HLA-DRB1*15:01 epitopes greater than 0. White color denotes the PD-1, the target protein for the designed binder. (J) Strep-tagged minibinders are produced with Genscript IVTT kit. After the production, the binders are purified with streptavidin beads and eluted in 1 mL of PBS. The dot plots show the concentration (expression) fold-change (FC) of the MESSI-generated binders relative to the parental binder designed by BindCraft. (K) Bio-Layer Interferometry (BLI) binding curves for measuring the binding parameters of MESSI-designed anti-PD-1 binders against the target PD-1 protein. The assay was performed at 30 °C and at 1,000 rpm. His-tagged PD-1 protein was firstly immobilized onto HIS1K biosensor on Octet RED384. Binders was applied at a concentration of 1 µM as an analyte for association and dissociation steps for 150 seconds and 30 seconds respectively. PA denotes the parent binder. (L) Equilibrium dissociation constant (K d ) of anti-PD-1 binder variants. K d was derived from the association (K a ) and dissociation (K dis ) rate constants measured from the five concentration scan BLI curves. Error bars represent the 95% confidence interval of the fitted K d . Differences in K d were assessed by a two-sided z-test using moving-block bootstrap standard errors of the global fit (85 seconds blocks, 1,000 resamples; Holm-corrected for comparisons). **, p<0.01

Article Snippet: Binding kinetics were measured by biolayer interferometry (BLI) on an Octet RED384 instrument (Sartorius, Cat. No. FB-20493) using anti-penta-HIS (HIS1K) biosensors (Sartorius, Cat. No. 18-5120) and tilted-bottom 384-well microplates (Sartorius, Cat. No. 18-5166).

Techniques: Generated, Sequencing, Comparison, Variant Assay, Biomarker Discovery, Synthesized, In Vitro, Purification, Concentration Assay, Negative Control, Standard Deviation, Produced, Expressing, Binding Assay, Affinity Constant, Derivative Assay, Blocking Assay