Molecules and Cells最新文献

Advancements in single-cell analysis have facilitated high-resolution observation of the transcriptome in individual cells. However, standards for obtaining high-quality cells and data analysis pipelines remain variable. Here, we provide the groundwork for improving the quality of single-cell analysis by delineating guidelines for selecting high-quality cells and considerations throughout the analysis. This review will streamline researchers' access to single-cell analysis and serve as a valuable guide for analysis.

Cellular senescence is a crucial biological process associated with organismal aging and many chronic diseases. Here, we present a brief guide to mammalian senescence assays, including the measurement of cell cycle arrest, change in cellular morphology, senescence-associated β-galactosidase (SA-β-gal) staining, and the expression of senescence-associated secretory phenotype (SASP). This work will be useful for biologists with minimum expertise in cellular senescence assays.

The accumulation of aggregation-prone proteins in a specific neuronal population is a common feature of neurodegenerative diseases, which is correlated with the development of pathological lesions in diseased brains. The formation and progression of pathological protein aggregates in susceptible neurons induce cellular dysfunction, resulting in progressive degeneration. Moreover, recent evidence supports the notion that the cell-to-cell transmission of pathological protein aggregates may be involved in the onset and progression of many neurodegenerative diseases. Indeed, several studies have identified different pathological aggregate strains. Although how these different aggregate strains form remains unclear, a variety of biomolecular compositions or cross-seeding events promoted by the presence of other protein aggregates in the cellular environment may affect the formation of different strains of pathological aggregates, which in turn can influence complex pathologies in diseased brains. In this review, we summarize the recent results regarding cell-to-cell transmission and the molecular heterogeneity of pathological aggregate strains, raising key questions for future research directions.

Genome editing has developed rapidly in various research fields for targeted genome modifications in many organisms, including cells, plants, viruses, and animals. The clustered regularly interspaced short palindromic repeats-associated protein 9 system stands as a potent tool in gene editing for generating cells and animal models with high precision. The clinical potential of clustered regularly interspaced short palindromic repeats-associated protein 9 has been extensively reported, with applications in genetic disease correction, inhibition of viral replication, and personalized or targeted therapeutics for various cancers. In this study, we provide a guide on single-guide RNA design, cloning single-guide RNA into plasmid vectors, single-cell isolation via transfection, and identification of knockout clones using next-generation sequencing. In addition, by providing the results of insertion into mammalian cell lines through next-generation sequencing, we offer useful information to those conducting research on human and animal cell lines.

The intricate assembly process of vimentin intermediate filaments (IFs), key components of the eukaryotic cytoskeleton, has yet to be elucidated. In this work, we investigated the transition from soluble tetrameric vimentin units to mature 11-nm tubular filaments, addressing a significant gap in the understanding of IF assembly. Through a combination of theoretical modeling and analysis of experimental data, we propose a novel assembly sequence, emphasizing the role of helical turns and gap filling by soluble tetramers. Our findings shed light on the unique structural dynamics of vimentin and suggest broader implications for the general principles of IF formation.

Genetic engineering technologies are essential not only for basic science but also for generating animal models for therapeutic applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, derived from adapted prokaryotic immune responses, has led to unprecedented advancements in the field of genome editing because of its ability to precisely target and edit genes in a guide RNA-dependent manner. The discovery of various types of CRISPR-Cas systems, such as CRISPR-associated transposons (CASTs), has resulted in the development of novel genome editing tools. Recently, research has expanded to systems associated with obligate mobile element guided activity (OMEGA) RNAs, including ancestral CRISPR-Cas and eukaryotic Fanzor systems, which are expected to complement the conventional CRISPR-Cas systems. In this review, we briefly introduce the features of various CRISPR-Cas systems and their application in diverse animal models.

Vascular disease, including heart disease, stroke, and peripheral arterial disease, is one of the leading causes of death and disability and represents a significant global health issue. Since the development of human induced pluripotent stem cells (hiPSCs) in 2007, hiPSCs have provided unique and tremendous opportunities for studying human pathophysiology, disease modeling, and drug discovery in the field of regenerative medicine. In this review, we discuss vascular physiology and related diseases, the current methods for generating vascular cells (eg, endothelial cells, smooth muscle cells, and pericytes) from hiPSCs, and describe the opportunities and challenges to the clinical applications of vascular organoids, tissue-engineered blood vessels, and vessels-on-a-chip. We then explore how hiPSCs can be used to study and treat inherited vascular diseases and discuss the current challenges and future prospects. In the future, it will be essential to develop vascularized organoids or tissues that can simultaneously undergo shear stress and cyclic stretching. This development will not only increase their maturity and function but also enable effective and innovative disease modeling and drug discovery.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species−mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species−mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.