Feng Han

A single substituent can flip a ligand from an agonist to an inhibitor, yet the underlying conformational dynamics remain elusive. Here, multiscale simulations calibrated against Yoda1 binding data resolve the ligand-dependent gating free-energy landscapes of the mechanosensitive channel Piezo1. In the unliganded state, the free-energy landscape supports a gating mechanism involving axial pore dislocation, lateral dilation, and realignment. The agonist Yoda1 stabilizes a widely dilated pore with persistent axial asymmetry, whereas the inhibitor Dooku1 arrests pore expansion after initial dislocation. This divergence reveals a geometrically selective relay between the intracellular Beam and transmembrane helical unit 9 (THU9). Productive coupling to pore dilation, rather than Beam rotation alone, dictates the pharmacological response. Guided by this mechanism, we designed Yoda1 analogs bearing an amino or nitro substitution at the same position, whose free-energy landscapes predicted contrasting functions. Intracellular calcium fluorescence assays were consistent with these predictions: the amino-substituted analog 188 elevated intracellular calcium levels, whereas the nitro-substituted analog 187 attenuated the Yoda1-evoked calcium response. These findings link single-site chemical modifications to divergent gating outcomes via symmetry breaking and pore dilation, identifying productive conformational coupling as a key molecular design parameter alongside binding affinity.

Introduction: Human adenovirus type 3 (HAdV-3) is a major adenovirus type associated with respiratory infections in China. Despite its clinical importance, the biological characteristics and potential biomedical applications of contemporary circulating HAdV-3 strains remain incompletely understood. Naturally occurring species B adenoviruses also remain relatively underexplored as potential starting backbones for oncolytic virotherapy. This study aimed to characterize a contemporary clinical HAdV-3 isolate and evaluate its viral genome amplification, apoptosis induction, and antitumor activity. Methods: A clinical HAdV-3 isolate, designated HAdV-3-TZ01, was recovered from a throat swab specimen from a patient with adenovirus-associated respiratory disease. Viral identity and genomic characteristics were determined using multiplex PCR, Sanger sequencing, whole-genome sequencing, phylogenetic analysis, and recombination analysis. Viral genome amplification kinetics were evaluated in human respiratory-related and tumor-derived cell lines and a murine cell line by quantitative PCR. Comparative apoptosis induction by HAdV-3-TZ01 and HAdV-5 was assessed in 293F cells infected at a low multiplicity of infection using Annexin V/PI flow cytometry. Antitumor activity was evaluated following repeated intratumoral administration of HAdV-3-TZ01 in an A549-luciferase xenograft model in BALB/c-nu nude mice, with tumor growth and bioluminescence monitored longitudinally. Results: Genomic and phylogenetic analyses confirmed that HAdV-3-TZ01 belonged to HAdV-3 within species B and exhibited a genomic backbone highly conserved with contemporary circulating HAdV-3 strains. A short putative HAdV-7-derived recombinant fragment was identified. HAdV-3-TZ01 showed efficient viral genome amplification in multiple human respiratory-related cell lines, including A549, H3122, BEAS-2B, MRC-5, and HEK293FT cells, whereas genome amplification was markedly restricted in murine L929 cells. HAdV-3-TZ01 induced a significantly higher proportion of late apoptotic cells than HAdV-5 in 293F cells under low-multiplicity infection conditions, although the absolute increase in total apoptosis was modest. In the A549-luciferase xenograft model, intratumoral administration of HAdV-3-TZ01 significantly suppressed tumor progression. By day 43, mean tumor volume was reduced by approximately 54% and tumor-associated bioluminescence by approximately 50% compared with mock-treated controls. No significant body-weight loss or overt signs of distress were observed under the experimental conditions. Conclusion: HAdV-3-TZ01 retains the characteristic human epithelial tropism of HAdV-3 and exhibits efficient viral genome amplification in human epithelial-derived cells, apoptosis induction, and measurable antitumor activity in vivo. The apoptosis difference between HAdV-3-TZ01 and HAdV-5 should be interpreted cautiously because the analysis was limited to 293F cells and did not establish a tumor-specific pro-apoptotic mechanism. As an unmodified respiratory pathogen, HAdV-3-TZ01 should not be considered a directly translatable therapeutic agent. Rather, these findings support HAdV-3-TZ01 as a potential starting backbone for future genetic engineering aimed at improving tumor selectivity and safety.

Visual hallucination (VH) is a common nonmotor symptom of Parkinson's disease (PD). However, the lack of reliable animal models and quantitative assessment tools poses significant challenges for mechanism and intervention research in this area. Here, we developed a novel PD-related visual hallucination (PDVH) model by administering benzhexol hydrochloride, which can induce hallucinatory phenotypes in PD model mice. On the basis of this model, we used a multidimensional behavioral analysis framework to identify a hallucination-related hunching state (HHS) that is strongly associated with hallucinatory episodes. This state is characterized by a sustained hunching posture accompanied by prolonged staring and embedded head twitching. By constructing a spontaneous behavior transition map, we found that the emergence of spontaneous behaviors is highly dependent on the immediately preceding state, revealing that the core hallucinatory features are structured and predictable at the level of spontaneous behavior. The relative fractions and transition probabilities of staring and head twitching allow high-throughput identification of PDVH mice and reflect temporal dynamics of the hallucination process. Our findings demonstrate that this approach enables high spatiotemporal resolution acquisition and analysis of behaviors associated with PDVH, offering a robust experimental framework for investigating PDVH mechanisms at the circuit level and developing targeted therapeutic strategies.

The transmembrane complex formed by TREM2 (Triggering Receptor Expressed on Myeloid cells 2) and DAP12 (DNAX-activating protein of 12 kDa) constitutes a pivotal therapeutic target for Alzheimer's disease. However, the intrinsic plasticity of its interface poses a formidable challenge for structure-based discovery of protein-protein interaction stabilizers (PPI stabilizers). Conventional approaches, ranging from static molecular docking to geometric deep learning, fail to capture the subtle interfacial energetics required for stabilizing this assembly, often leading to erroneous activity predictions. Here, we present MechMemDyn, a novel predictive framework that uniquely integrates protein frustration analysis with membrane-embedded molecular dynamics (MD) simulations. To our knowledge, this is the first systematic application of frustration analysis to rationalize PPI-stabilizing activity and guide ligand design. By mapping the local frustration landscape, we identify critical, minimally frustrated contact networks at the protein interface, which are essential for stabilizing the PPI. We demonstrate that the ability of a ligand to dampen distance fluctuations within these key networks, a metric rooted in thermodynamic rigor, correlates strongly with experimental potency. This method outperforms conventional static docking, AI-driven dynamic docking approaches, and standard molecular simulations, providing a more accurate and reproducible basis for cross-ligand comparison. This work not only resolves the intractability of the TREM2-DAP12 complex but also establishes a physics-driven paradigm for targeting dynamic transmembrane interfaces via frustration-optimized PPI stabilizers.

14 April 2026 The authors have requested that this preprint be removed from Research Square due to a pending patent application.

Efficient homologous recombination, homology-directed repair (HDR), remains a major hurdle for precise genome editing in mammalian cells, particularly for kilobase-scale insertions. Bacterial recombineering proteins, such as RecE and RecT, offer potential solutions, but their activity in eukaryotic systems has been largely uncharacterized. Here, we identify Escherichia coli RecE (EcRecE) as a potent enhancer of HDR in mammalian cells. Targeted recruitment of EcRecE via CRISPR/Cas9 significantly increased HDR efficiency at multiple genomic loci across different cellular contexts, including human embryonic stem cells, achieving a 3–6-fold enhancement in the integration efficiency of kilobase-scale sequences. Furthermore, in combination with RecT and a catalytically inactive Cas9 (dCas9), applying functional domain engineering, we developed a dCas9-miniRecTE editor that enhances large-fragment integration without introducing double-strand breaks in human cells and primary mouse neurons, achieving ∼20% kilobase-scale knock-in efficiency. These results establish EcRecE as a versatile tool for improving precision genome engineering, with potential applications in therapeutic gene editing.

Parkinson's disease (PD)‐associated cognitive decline is heralded by olfactory dysfunction, but the network mechanisms bridging sensory and cognitive impairments remain poorly defined. Combining chronic multisite electrophysiology, behavioral tracking, and machine learning in PD models, a hierarchical disintegration of oscillatory dynamics across the olfactory network that mechanistically drives disease progression is uncovered. Early‐stage PD mice are identified to show attenuated odor discrimination, accompanied by hyperexcitability of mitral/tufted (M/T) cells. Causally linking these deficits, aberrant gamma oscillation in the cross‐olfactory network is identified as a causal factor underlying olfactory deficits. Notably, cognitive impairment emerged at later stages, correlating with abnormal theta oscillations in the cross‐olfactory network. Pharmacological modulation of the olfactory bulb (OB)‐lateral entorhinal cortex (LEC) pathway ameliorated cognitive deficits and restored cross‐network theta oscillation. Collectively, the findings establish cross‐olfactory network oscillations as dual diagnostic and therapeutic targets for PD cognitive impairment, providing a mechanism‐guided framework for early intervention.