Japan Science and Technology Agency

Effective biomolecule size discrimination is essential for bioanalysis and biomedical engineering. However, traditional techniques for discriminating large DNA molecules, such as pulsed-field gel electrophoresis, are time-consuming, while micro/nanofluidic devices that use nanostructured molecular sieves are costly and impractical for large-scale production. We hypothesized that polymer brush films could effectively separate large DNA by size, functioning as nanoscale molecular sieves. We prepared 100 nm-high hydrated polymer brush films from 2-methacryloyloxyethyl phosphorylcholine and examined the electrophoretic mobility of adsorbed λ and T4 DNA (48.5 and 166 kbp, respectively). We visualized individual DNA molecules under different electric fields using fluorescence microscopy and investigated their electrophoretic behavior at the brush–solution interface using neutron reflectivity. We found that DNA mobility in polymer brushes becomes strongly size-dependent above a critical electric field, enabling precise size discrimination. Higher voltages and larger DNA sizes result in more complex morphologies, indicating more frequent DNA–brush interactions. Neutron reflectivity confirmed that stronger electric fields draw DNA molecules closer to the substrate, enhancing DNA–brush interactions and reducing size-exclusion effects. In polymer brush films, DNA–matrix interactions are controlled by electric field strength, unlike in gel electrophoresis, where they rely on polymer density. Optimized polymer brush electrophoresis enables rapid and precise mobility-based size discrimination from single-molecule tracking and simplifies DNA recovery owing to the absence of a three-dimensional mesh structure. These observations highlight the potential of polymer brush films for biomolecular discrimination and analysis, thereby advancing their application in molecular biology and clinical diagnostics. • Polymer brush films offer a soft, scalable alternative to gel-based DNA sieving • Electric field strength regulates DNA–brush interactions • DNA mobility becomes size-dependent above a critical electric field • Larger DNA adopts complex conformations and shows reduced migration speed • Neutron reflectivity shows DNA shifts closer to the substrate under stronger fields

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues.

Abstract Understanding how molecular diversity across long-range neural circuits governs brain function remains a central challenge in neuroscience. Here, we developed a projection-specific spatial proteomics approach and revealed robust presynaptic molecular divergence across six projection-defined pathways of the medial prefrontal cortex (mPFC), including efferent projections to the basolateral amygdala (BLA), nucleus accumbens (NAc), thalamus (Thal), hypothalamus (HT) and cortex (CTX), as well as the afferent projection from the BLA to the mPFC. Among these pathways, we identify BLTP2 (KIAA0100), a previously uncharacterized transmembrane protein, as highly enriched in the projection from the mPFC to the BLA. BLTP2 localizes to excitatory presynaptic terminals and is enriched at synapses that are activated during memory formation. Loss of BLTP2 impairs synaptic structure and transmission, and reduces activity-dependent remodelling, resulting in selective deficits in contextual fear memory, anxiety-related behavior, and social behavior. Mechanistically, BLTP2 promotes presynaptic assembly by recruiting Neurexin 1. These findings reveal projection-specific presynaptic molecular diversity and provide mechanistic insights into circuit-level vulnerabilities in neuropsychiatric disorders.

Sodium taurocholate cotransporting polypeptide (NTCP) is a hepatic transmembrane (TM) protein that functions both as a bile acid transporter and as a host receptor for hepatitis B and D viruses via the viral preS1 binding. The structural and mechanistical determinants for NTCP’s dual functions remain largely undefined. In this study, using comprehensive structure-guided alanine-scanning mutagenesis based on the cryo-electron microscopy structure of the preS1/NTCP complex, we identified 13, 8 and 9 NTCP amino acid residues critical for viral infection, preS1 binding, and bile acid transport, respectively. Key residues overlappingly regulating viral receptor and transporter functions were located primarily at TM1 and TM8, whereas TM5 and outer-surface NTCP loops mediated viral receptor-specific activity. In addition to 8 amino acids key to preS1 binding, 5 residues likely acted at a post-preS1 binding step of infection. We further found naturally-occurring single nucleotide polymorphism-associated F274C/S NTCP variants abolished viral receptor function, via the potential conformational changes in bile acid tunnel and outer-surface hollow, as analyzed by molecular dynamics simulations. Our domain-specific structural-functional map of NTCP defines the mechanism how NTCP’s dual functionality is separately regulated, and provides a framework for designing selective antiviral agents that preserve bile acid transport.

The hydrogen positions and magnetic structure of goethite α-FeOOH, a key component of iron rust, were examined through neutron diffraction. All symmetry-allowed magnetic structures under the space group Pnma with the magnetic wavevector qm = (0, 0, 0) r.l.u. were analysed using irreducible representation and magnetic space group approaches. The magnetic moments aligned along the b-axis form antiferromagnetic spin arrangements, as reproduced by first-principles calculations. Accurately determining the hydrogen positions is crucial for understanding the mechanism of catalytic reduction of CO2 in α-FeOOH. These positions were precisely identified through diffraction and calculations, highlighting the effectiveness of using both methods for undeuterated compounds.

Alteromonas stellipolaris strain 6L2 was isolated from surface seawater via enrichment culture using an ethylene-α-olefin co-oligomer, a polyolefin model substrate, as sole carbon source. We report its complete genome sequence comprising a circular chromosome and a plasmid. The genome provides insights into bacterial colonization of polyolefin surfaces in marine environments.

Impurity segregation at grain boundaries (GBs) often induces structural transformations at the atomic level and significantly influences materials’ properties, underscoring the importance of understanding the underlying mechanisms of GB segregation at the atomic scale. Here, the atomic structure of a Y‐segregated ∑13(101¯4)/[1¯21¯0] GB is thoroughly studied in α‐Al2O3 via scanning transmission electron microscopy and Monte Carlo and molecular dynamics simulations based on a neural network potential. It is found that the Y segregation at the GB involves not only the simple substitution of Y for Al atoms but also a structural adaptation with a change in GB atomic density. Such a change alters the local bonding environment at the GB so that the absolute excess volume is minimized for the lowest‐energy structure. This study offers a new insight into the atomic‐scale mechanism of GB structural transformation.

Methylmercury (MeHg) is a well-known environmental neurotoxicant that preferentially affects sensory neurons in the peripheral nervous system. While sensory-dominant neuropathy has long been described in Minamata disease, the temporal dynamics of dorsal root ganglion (DRG) injury and recovery remain incompletely understood. In this study, Wistar rats were exposed to MeHg for five consecutive days, followed by a two-day treatment-free period; this regimen was repeated once. The DRG and peripheral sensory fibers were analyzed up to 70 days after exposure. Histological and immunohistochemical analyses, DNA microarrays, and mercury quantification and distribution mapping were performed. The A-fiber density was significantly reduced at Day 14 but recovered by Day 70, whereas C-fibers showed no significant change. The total number of DRG neurons remained stable. Immunohistochemical analyses demonstrated that subtype marker-selected neurons (NF, TrkA, FAM19A1, TAC1, SST) decreased at Day 14 and gradually recovered thereafter. DNA microarray analysis at Day 14 revealed a broad downregulation of DRG neuronal subtype marker genes. The mercury concentration in the DRG peaked at Day 14 and declined to the control level by Day 70, with in situ imaging confirming preferential accumulation in DRG neurons. These data suggest that the short-term MeHg exposure caused a transient functional suppression of DRG neurons without widespread neuronal loss. The selective and reversible downregulation of neuronal phenotypes, coupled with preferential Hg accumulation in DRG neurons, underlies the sensory-dominant and potentially reversible features of MeHg neurotoxicity.

Patients with pulmonary metastasis from primary colorectal carcinoma often undergo sublobar pulmonary metastasectomy. To prevent local cancer recurrence, it is important to ensure an appropriate resection margin. For this purpose, rapid and accurate visualization of cancer lesions by fluorescence imaging would be an effective tool to guide surgeons. Thus, the aim of this study was to find an efficient fluorescence probe for visualizing pulmonary metastases from colorectal cancer. Screening of our aminopeptidase-reactive fluorescence probe library with lysates of metastatic tissues from rectal cancer identified a specifically activated puromycin-sensitive aminopeptidase (PSA)-reactive probe. Based on this finding, we evaluated the utility of our previously reported PSA-reactive red fluorescence probe, QA-2OMeSiR, for fluorescence imaging of pulmonary metastases. QA-2OMeSiR detected pulmonary metastasis from rectal cancer with 83.3% sensitivity and 91.7% specificity (AUC = 0.875). Pulmonary metastasis from colon cancer was detected with 100% sensitivity and 88.9% specificity (AUC = 0.951). Inhibitor experiments and immunohistochemical staining confirmed that the probe targets both PSA and dipeptidyl peptidase IV (DPP-IV). Furthermore, QA-2OMeSiR rapidly visualizes the boundaries of pulmonary metastatic lesions in surgical specimens. These results suggest that QA-2OMeSiR is a promising candidate as a fluorescence probe for rapid intraoperative visualization of pulmonary metastases from colorectal cancer.