Biology of the Cell最新文献

During the last decade, non-invasive methods such as liquid biopsy have increasingly replaced invasive techniques for diagnosing and monitoring diseases. Liquid biopsies are easier to obtain, highly sensitive, cost-effective per sample, and can be used for real-time monitoring, such as tracking drug response. To uncover molecular and cellular characteristics today, blood, saliva, urine, or cerebrospinal fluid are typically analyzed. In this methodological proof-of-concept study, we propose that patient profiling can be facilitated by assessing surface markers on extracellular vesicles (EVs) directly in plasma without prior purification. EV composition reflects the physiological or pathological state of their parent cell, making EVs valuable tools for biomarker discovery and understanding disease mechanisms. Plasma and purified EVs from 30 ischemic stroke patients were analyzed for 17 surface markers using an in-house protein microarray method (EV Array). The findings indicate that measurements on EVs directly in plasma and on purified EVs show similar patterns in expression of surface markers, supporting the feasibility of omitting purification in EV profiling. This knowledge can provide faster monitoring of various diseases, prevent delays in patient profiling, and optimize patient care.

Exosomes are small extracellular vesicles that originate as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Upon fusion of MVBs with the plasma membrane, ILVs are released into the extracellular environment as exosomes. Although exosomes can diffuse away from their cells of origin, the expression of the antiviral restriction factor tetherin promotes their retention at the cell surface, thereby limiting their release into the extracellular milieu. Tetherin plays an analogous role in retaining other extracellular particles, including many enveloped viruses and midbody remnants. We hypothesised that tetherin physically links exosomes to the cell surface through specific structural features of the protein. To test this, we combined biochemical assays with live-cell and ultrastructural imaging approaches to determine which elements of tetherin are required for exosome retention. Our analysis shows that the formation of tetherin homodimers is essential for exosome tethering. Mutations in regions or motifs of tetherin predicted to impact tetherin traffic to ILVs have only minor impacts on exosome tethering, suggesting redundancy in the mechanisms of tetherin traffic to ILVs, and subsequently, to exosomes. Collectively, these findings provide the first molecular insights into the mechanism that govern exosome tethering.

Over the past decade, significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation, with organoids emerging as key experimental models. These three-dimensional in vitro cellular structures mimic the organization and functions of various gut regions, providing a powerful tool for research. By replicating critical stages of gut development, we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol, we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First, we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three-dimensional spheroids form spontaneously, can be collected, embedded in an extracellular matrix, and cultured over time. During this phase, the organoid epithelium develops, supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture, HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development, model diseases, and test therapeutic agents.

Sestrins are a stress-inducible family of proteins that function in cell survival and nutrient sensing through their regulation of mTORC1. Muscle wasting is associated with cellular stress, but to date, there is limited in vitro research investigating sestrins in skeletal muscle cells. Here we use C2C12 myotubes to understand how sestrin proteins (sestrin 1–3) are regulated by different forms of cellular stress linked to muscle wasting conditions. Furthermore, since sestrin2 is a well-characterised protein but is lowly expressed in muscle tissue in the absence of stress, we also aimed to determine if silencing this protein impacted parameters of muscle growth or nutrient sensing by mTORC1 under basal conditions. Incubating C2C12 myotubes with the endoplasmic reticulum (ER) stress-inducing agent tunicamycin, or a high concentration (1000 µM) of hydrogen peroxide (H₂O₂), increased sestrin2 protein levels with no change in sestrins 1 or 3. This increase was temporally associated with increased ER stress markers Ddit3 mRNA and ATF4 protein levels, and could be blocked by approximately half when myotubes were co-incubated with H₂O₂ and the ER-stress inhibitor 4-Phenylbutyrate. siRNA silencing of sestrin2 blunted the phosphorylation of the mTORC1 effector S6K1, but did not acutely influence protein synthesis or myotube size. Similarly, silencing sestrin2 did not affect mTORC1 signalling in response to nutrient deprivation. These data indicate that sestrin2 is stress-inducible and may play a role in protecting skeletal muscle from ER stress, but is less important in regulating mTORC1 and nutrient sensing in unstressed/basal conditions.