Methods最新文献

Multifactorial inherited disorders (MIDs) arise from complex interactions between polygenic risk and environmental exposures, presenting major challenges for mechanistic discovery, patient stratification, and targeted therapy development. While traditional approaches like genome-wide association studies (GWAS) and bulk omics profiling have identified broad associations, they often fail to resolve the cellular context in which these interactions drive pathogenesis.Emergingsingle-cell technologies now offer unprecedented resolution to dissect tissue heterogeneity, define rare or transient disease-relevant cell states, and map dynamic trajectories across tissues and disease stages. This reviewprovides a comprehensive synthesis ofcurrent single-cell methodologies including transcriptomic, epigenomic, proteomic, and spatial techniques and their application to MID research. We explore how these toolsare revealingcell-type-specific regulatory circuits,contextualizingthe functional impact of inherited risk variants, andelucidatingcellular responses to environmental perturbations.We argue thatintegrating single-cell multi-omics data is critical for illuminating the mechanistic basis of complex traits and for advancing biomarker discovery. However, significant challenges remain, including technical variability, limited cohort scalability, difficulties in multi-modal data integration, and a lack of standardized analytical workflows for polygenic diseases. Overcoming these barriers will require harmonized study designs, robust computational frameworks, and the incorporation of longitudinal and environmental exposure data.Ultimately, we conclude thatsingle-cell analysis is poised to transform MID research, offering a powerful new paradigm for mechanistic insight, therapeutic innovation, and the realization of precision medicine.

This study examines how distinct patterns of smartphone application usage relate to psychosocial indicators, specifically anger, depression, and self-esteem, while identifying user clusters based on behavioral profiles. A two-stage analytical approach was employed. K-means clustering was first used to group 523 participants based on usage patterns across 130 applications, and Principal Component Analysis (PCA) was subsequently applied to summarize covariance structures among the psychosocial variables. The PCA yielded a two-dimensional psychological space that accounted for 90.1% of the variance and provided a descriptive framework for organizing users into relative psychosocial profiles. Results indicated that participants with similar demographic backgrounds exhibited different psychosocial characteristics depending on their digital usage patterns. Users who more actively engaged with productivity, health, or analog-content applications tended to show relatively more favorable psychosocial indicators, whereas users with more limited engagement in socially interactive applications tended to report higher levels of anger and depression and lower self-esteem. These findings should be interpreted as descriptive associations rather than causal effects. Overall, the study provides an exploratory account of how heterogeneous smartphone usage patterns are associated with variation in psychosocial characteristics in a non-clinical population, and suggests that everyday digital behavior may function as an observable behavioral correlate of stable psychosocial tendencies.

The single-chain Fragment variable (scFv) antibody format is a common format used for phage display. The solubility of identified scFv clones in phage format in instances does not translate the same solubility as independent recombinant proteins. To overcome this bottleneck, we propose the fusion of scFv clones to the antibody constant domains for improved solubility. It has been reported that immunoglobulins devoid of the heavy constant domain 1 (C_H1) will have reduced solubility allowing accumulation in Russell Bodies (RB). Here we propose the introduction of C_H1 and kappa constant domain (Cκ) with and without skp chaperone to improve soluble expression. Utilizing heavy and light constant domains facilitates the spontaneous assembly of a Fab-like structure. This configuration successfully presents two distinct scFVs on each chain, thereby achieving bispecificity. This system is specifically designed to optimize the expression of the scFv-constant domain fusion necessary for bispecific formation. Crucially, its modular design allows for simple and rapid scFv swapping, enabling the seamless assessment of different antibody pairings for effective bispecific antibody generation.

The encapsulation of kidney organoids within hydrogels provides a biomimetic environment that enhances their structural and functional relevance for disease modeling and drug screening. However, the presence of hydrogel matrices poses a major challenge for molecular analysis, particularly for RNA extraction, where residual material can interfere with yield, purity, and downstream applications. The objective of this study was to systematically evaluate RNA extraction methods for kidney organoids encapsulated in alginate-norbornene hydrogels and identify an optimized protocol suitable for reliable gene expression analysis. We compared commonly used extraction methods designed for mammalian tissues and plant-derived materials, with and without prior enzymatic digestion of the hydrogel. RNA yield and purity were assessed by spectrophotometry and fluorometry, while RNA integrity was analyzed by Bioanalyzer, and performance in downstream assays was evaluated by quantitative PCR of housekeeping genes. Our results showed that RNA yield was consistently lower in encapsulated organoids compared to suspension cultures, reflecting smaller organoid size and reduced metabolic activity in encapsulated conditions. Spectrophotometric purity ratios differed between suspension and encapsulated samples, but RNA integrity was preserved across all methods, with values within the acceptable range. Quantitative PCR revealed that TRIzol-based extractions introduced significant variability between suspension and encapsulated samples. Conversely, the protocol with alginate lyase digestion followed by the Maxwell RSC RNA kit produced the most reproducible results. Ct values for control and encapsulated samples were highly consistent, with inter-condition variability remaining below 0.5 standard deviations across replicates. These findings highlight the importance of adapting RNA isolation protocols to account for the presence of hydrogels. Alginate lyase digestion combined with a plant RNA extraction kit offers a reliable strategy for obtaining high-quality RNA from encapsulated kidney organoids within alginate-based hydrogels. While this approach enabled a robust gene expression analysis providing a foundation for transcriptomic studies, different hydrogels and organoid combinations might require additional adjustments underscoring the importance of adopting methods to ensure optimal RNA quality for downstream methods.