The care of patients with cholestatic liver diseases such as primary sclerosing cholangitis (PSC) is challenging, partly due to the lack of knowledge of disease pathogenesis and suitable in vitro models for disease modeling and drug screening. Although the pathogenesis of cholestatic liver diseases like PSC remains unknown, the importance of the vascular-biliary interface is clear. Cholangiocyte injury not only impairs barrier function such that bile leaks and damages periductal tissue, but also activates cholangiocytes to secret pro-inflammatory and pro-fibrogenic mediators to stimulate immune cells and mesenchymal cells, ultimately causing damage to the liver. Here we describe a detailed protocol for fabricating a human vascularized bile duct-on-a-chip (VBDOC) that consists of a vascular channel, biliary channel, and neighboring mesenchymal cells in a collagen gel that models the vascular-biliary interface structurally and functionally in three dimensions. This device is notable in maintaining cholangiocyte polarity and barrier function, recapitulating physiological functions and responses of the large bile ducts, and enabling manipulation of components of the mechanical microenvironment such as matrix stiffness and shear flow in the lumens. This practical workflow could help researchers manufacture the VBDOC in their own labs and apply it to studies of various cholestatic liver diseases.

The intricate structure of the brain at various scales is linked to its functional and biomechanical properties. Since understanding the biomechanical properties is vital for comprehending brain diseases, development, and injuries, measurements at different scales are necessary. Here we introduce methods to measure the biomechanical properties at both tissue and cellular levels using a mouse model. A specially designed magnetic resonance elastography system is introduced for imaging the mouse brain, enabling in vivo mapping of its shear modulus. Additionally, protocols for isolating and culturing primary neurons and astrocytes from the hippocampus and cerebral cortex of the mouse brain are presented. The nanoindentation technique using atomic force microscopy is employed to measure the biomechanical properties of individual cells. The results indicate that the storage/loss modulus of the mouse cerebral cortex and hippocampus are 8.07 ± 1.28 kPa / 3.20 ± 0.66 kPa and 6.60 ± 0.52 kPa / 2.52 ± 0.33 kPa, respectively. Meanwhile, the Young’s modulus for neurons and astrocytes is 470.88 ± 17.67 Pa and 681.13 ± 14.15 Pa, respectively. These findings demonstrate that the brain exhibits distinct biomechanical properties at different scales. The proposed methods offer general techniques for investigating the multiscale biomechanical properties of the brain.

In early pregnancy, approximately 70% of women experience nausea and vomiting, with hyperemesis gravidarum (HG) can potentially lead to severe fluid and nutritional imbalances that require hospitalization. Although HG often resolves on its own in the early stages of pregnancy, its severity is linked to ketosis and elevated serum urea levels, as well as an increased risk of neurodevelopmental disorders in children. To further investigate the immune status of HG patients, we plan to conduct single-cell transcriptomic sequencing and plasma proteomic analysis of peripheral blood samples. This approach aims to elucidate the interactions and mechanisms of PBMCs and provide new insights into potential therapeutic interventions. Our findings indicate an increased proportion of neutrophils in HG patients, along with the upregulation of interferon genes and associated pathways. Notably, the activity of interferon-related TFs, such as STAT1, IRF7, and IRF9, was significantly elevated. Additionally, we observed a decrease in T cell activity in HG patients, while the functionality of NK cells and CD14+ monocytes was enhanced. The elevated plasma levels of NDEL1 may also have implications for fetal development. We have constructed a single-cell atlas of PBMCs from pregnant women with HG, which is expected to enhance our understanding of the immune response in HG and identify potential therapeutic targets for this condition.

In bacterial DNA replication, helicase DnaB and primase DnaG form the primosome. Helicase DnaB unwinds double-stranded DNA (dsDNA) to provide templates for DNA polymerase, whereas primase DnaG supplies RNA primers to DNA polymerase for the synthesis of Okazaki fragments. How primase DnaG coordinates with helicase DnaB at the DNA replication fork remains unclear. In this study, the interactions between the helicase-binding domain of DnaG (DnaG (HBD)) and DnaB hexamer were studied. A stable ternary complex of DnaB6/dT16/DnaG(HBD) from Bacillus stearothermophilus was prepared and the homogeneity of the DnaB6/dT16/DnaG(HBD) complex was verified by dynamic light scattering. The stoichiometry of DnaG(HBD) to process DnaB6 was investigated by isothermal titration calorimetry. The results show that a single primase DnaG binds to DnaB6 in the presence of single-stranded DNA. Based on these results, a model is proposed to explain how the primase DnaG couples with the processing DnaB6 helicase during the Okazaki fragment synthesis cycle. These findings provide valuable insights into the coupling between dsDNA unwinding and RNA primer synthesis in DNA replication.

Recent advances in mass spectrometry and proteomics have revealed numerous novel lysine acylation modifications, which increase proteome functional diversity via covalent modification of lysine residues. Protein malonylation and succinylation are highly enriched in mitochondrial proteins. They use malonyl-CoA and succinyl-CoA which were primarily synthesized in mitochondria, as acyl donors and can modulate protein structure and function. However, conventional mass spectrometry-based workflows often suffer from limited coverage and may fail to detect low-abundance modification sites. Therefore, we present an organelle-centered proteomic protocol. By purifying mitochondria before enriching modified peptides, the protocol minimizes interference from non-mitochondrial proteins, thereby offering a valuable reference for investigating diverse protein acylation events in metabolic-associated fatty liver disease (MASLD) and other physiological and pathological contexts.

Sensorineural hearing loss (SNHL) remains a global health challenge with limited regenerative therapeutic options. This review delineates advancements in stem cell-based therapies, emphasizing their potential to restore auditory function through cellular regeneration and molecular modulation. Stem cells, including mesenchymal (MSCs), induced pluripotent (iPSCs), and embryonic stem cells (ESCs), exhibit multilineage differentiation and paracrine signaling capabilities, enabling hair cell (HC) regeneration, spiral ganglion neuron (SGN) repair, and inflammation modulation. Cochlear organoid models derived from stem cells have advanced mechanistic studies, though challenges persist in replicating cochlear-specific HC phenotypes. Exosomes, as nanoscale bioactive vesicles, demonstrate enhanced biocompatibility and barrier penetration, effectively mitigating oxidative stress, apoptosis, and aminoglycoside ototoxicity while promoting HC survival. Gene-editing technologies, notably CRISPR-Cas9 and adeno-associated virus (AAV) gene delivery system, synergize with stem cell platforms to correct pathogenic mutations and engineer therapeutic cells for targeted inner ear repair. Preclinical studies highlight MSC transplantation and exosome administration in improving auditory thresholds and neuronal morphology, yet clinical translation faces hurdles including delivery precision, immune responses, and scalability. Current limitations in replicating cochlear cytoarchitecture and standardizing differentiation protocols necessitate further exploration of combinatorial strategies, such as integrating stem cell therapy with cochlear implants or medicaments delivery systems. Future research need prioritize optimizing cell lineage specification, enhancing exosome targeting, and validating long-term safety to realize the transformative potential of regenerative medicine in SNHL treatment.

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.

Cryo-electron tomography (cryo-ET) enables three-dimensional imaging of cellular architecture in a near-native state, but its use in intact mammalian tissues remains technically challenging. Here, using an optimized tissuelevel cryo-ET pipeline that integrates high-pressure freezing, cryo-CLEM, and an improved serial lift-out cryo-FIB workflow, we reconstructed brush-border regions of mouse small intestine from 43 tomographic tilt series and analyzed 490 individual microvilli. Microvilli formed a quasi-regular lattice (mean inter-microvillar spacing ≈ 61 nm) with diameters of 72–114 nm and clearly resolved actin core bundles. Each protrusion bore lateral, nanobristle-like projections that were morphologically heterogeneous and, in rare cases, bridged adjacent microvilli. Importantly, we identified 27 branched microvilli (~5.5%) including Y-shaped and multi-branched forms; the organization of actin bundles in these structures is consistent with a tip-to-base division mechanism for generating daughter protrusions. These observations reveal previously underappreciated structural plasticity in the mammalian brush border and support a fission-like pathway for microvillus renewal.

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 indepth 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.