The aggregation and propagation of pathological α-synuclein (α-Syn) are central to the pathogenesis of synucleinopathies, including Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). This study focuses on the enrichment and amplification of secreted α-Syn from cellular models and the saliva of PD patients, utilizing trichloroacetic acid (TCA) precipitation to retain the post-translational modifications (PTMs) and seeding properties of extracellular proteins. By applying TCA precipitation, we successfully concentrated α-Syn from PD patient saliva and cell culture media while preserving critical PTMs (e.g., truncation and phosphorylation) that are essential to amyloidogenicity. Subsequent Western blot (WB) and real-time quaking-induced conversion (RT-QuIC) assays revealed that TCA-enriched α-Syn retained robust prion-like seeding activity, enabling the cross-cellular propagation of pathologically misfolded species. This workflow establishes TCA precipitation as a unique tool for capturing pathologically modified α-Syn from biofluids while maintaining their native seeding capacity. By integrating enrichment and amplification strategies, our findings advance the utility of saliva-based biomarkers for synucleinopathies and provide a translational platform to evaluate therapeutics targeting α-Syn transmission.

The development of lung-specific conditional knockout animal models is pivotal in advancing lung disease research. This protocol describes the experimental procedures in detail, including the construction of loxP mice, endotracheal tube nebulization of adeno-associated virus (AAV) and whole lung clearing. Mouse lung conditional knockouts were successfully constructed. This meticulous approach allows for accurate control over the timing and location of the knockout, facilitating an in-depth investigation into the pathogenesis of lung diseases and the development of therapeutic strategies. These lung-specific conditional knockout models represent a powerful tool for the study of lung diseases, offering valuable insights and guidance for the advancement of treatment approaches and drug discovery in the future.

Telomere length (TL) is a promising biomarker for age-associated diseases and cancer. Single-molecule studies of human TL are advancing rapidly, providing unprecedented insights into the dynamics and variability of telomeres at the single-molecule level. TL is commonly reported as average TL or relative TL, depending on the methods used. However, average TL, short TL, and long TL have distinct significances: average TL serves as a general biomarker for aging, short TL indicates the risk of age-related diseases, and long TL is associated with certain cancers and all-cancer mortality. Thus, the TL distribution is more important than the average TL alone. Single-molecule techniques measure TL one telomere at a time, offering quantitative TL distributions that are crucial for understanding telomere biology. In this review, we focus on various TL measurement techniques, with particular emphasis on single-molecule methods. Single-molecule studies of human TL are poised to play a pivotal role in advancing our understanding and clinical management of age-associated diseases and cancer.

Comprehensive glycoprotein analysis is essential for exploring the role of protein glycosylation in diverse biological processes and disease mechanisms. Yet it remains challenging due to the structural complexity and heterogeneity of glycans. Bottom-up glycoproteomics utilizing liquid chromatography-mass spectrometry (LC-MS)-based techniques has emerged as a powerful tool for in-depth protein glycosylation analysis. Sample pretreatment is the first and critical step that significantly influences subsequent chromatographic separation and MS analysis. This review provides an overview of the key steps in current sample pretreatment workflows for glycoproteomics, emphasizing recent advancements in sample preparation and enrichment strategies developed over the past decade. It highlights improvements in enrichment efficiency, compatibility with high-throughput analyses, and applications to biological samples, and also discusses the remaining challenges and future directions for these technologies.

Potassium-chloride cotransporters KCCs mediate the coupled, electroneutral cotransport of K⁺ and Cl⁻ across the membrane and are involved in important physiological processes such as cell volume regulation and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Although structures of KCCs have been reported, the identification of ions bound in KCCs awaits experimental studies. Here using the cryo-electron microscopy (cryo-EM) titration methods, we present six structures of human KCC4 in different ion conditions at 2.38–2.58 Å resolutions. These structures, along with molecular dynamic simulations, allow us to assign one K⁺ and two Cl⁻ ions in the substrate-binding pocket. The K⁺ at S1 and Cl⁻ at the S2 site are tightly coupled in the binding and dissociation, suggesting that the Cl⁻ at S2 but not at S3 is the cotransported one. The S1 site provides coordination that largely matches the K⁺ dehydration radius and therefore displays higher selectivity to K⁺ over Na⁺. This study establishes the structural basis for the K⁺ selectivity of KCCs by the cryo-EM titration.