Methods in molecular biology最新文献

In recent decades, human-induced pluripotent stem cell (iPSC) technology has revolutionized in vitro disease modeling and personalized medicine, enabling the generation of patient-specific "disease-in-a-dish" systems across a wide range of tissues, including retinal pigment epithelium (RPE) and retinal organoids. Numerous protocols have been developed for iPSC-RPE and retinal organoid differentiation, typically involving prolonged culture durations, low overall efficiency, and reliance on multiple extrinsic signaling factors. Here we describe an RPE differentiation protocol over 6 weeks, incorporating three additional factors, followed by pigment isolation to isolate differentiated RPE cells for subsequent maturation. After 4 weeks of maturation, the resulting monolayer is well polarized, exhibiting hexagonal cobblestone morphology, expressing canonical RPE markers. We also describe an effective of retinal organoid differentiation protocol, using nicotinamide and a stepwise reduction of KnockOut™ Serum Replacement (KOSR) concentration over the first 9 days, that facilitates the initiation of retinal differentiation in challenging iPSC lines.

Plant adaptive responses to their environment are orchestrated by transcriptional reprogramming, regulated by sequence-specific transcription factors (TFs) recognizing specific TF-binding sites (TFBSs), and understanding their regulatory grammar is crucial for unravelling plant adaptation mechanisms. Empirical data have contributed to the elucidation of TFBS sequence motifs involved in biological processes, but comprehensive experimental approaches may not be feasible, especially in non-model species. Different computational algorithms have facilitated the elucidation of TFBS sequence motifs and the construction of predictive models, shedding light on plant gene regulatory networks. In this context, the development of straightforward computational pipelines and easy-to-use bioinformatics tools is of particular relevance to make gene expression analysis accessible to the research community. This chapter presents a methodology to infer TF regulators applicable to 60 plant species from RNA sequencing (RNA-seq) data as the starting point. It includes RNA-seq analysis and quantification, gene clustering using WGCNA to identify co-regulated gene modules, and searching for enriched TFBSs associated with these modules. The methodology is illustrated using Arabidopsis RNA-seq data related to abiotic stress. By providing a user-friendly pipeline, researchers are empowered to unravel the molecular basis of gene expression dynamics in plants.

Spiroplasma melliferum is a cultivable mollicute that can be used as a proxy model organism to test the inhibitory effects of filtrates, compounds, or substances against phytoplasma. In this protocol, we describe the use of the resazurin-based alamarBlue™ dye for the monitoring of Spiroplasma survival and growth in the presence of presumably inhibitory agents.

In this introductory chapter, we provide an overview of phytoplasma biology and outline the historical milestones that shaped the field, from their first discovery to their current taxonomic status. We also highlight how this third edition of "Phytoplasmas: Methods and Protocols" differs from previous volumes, reflecting both the adoption of new molecular and sequencing technologies and the revival of classical approaches. Taken together, these developments illustrate the evolving trajectory of phytoplasma research and set the stage for the detailed protocols presented in the following chapters.

Grapevine is among the most important economic plants that are susceptible to phytoplasma disease. Tools and procedures for genetic manipulation of grapevine are cumbersome and sometimes difficult to replicate; therefore, most of the research on plant-phytoplasma interactions focuses on species with more established transformation procedures instead, even though they might not be hosts of the studied phytoplasma. Studying the mechanisms of plant-phytoplasma interactions directly in the host plants would bring new and more accurate insights into the disease mechanisms. In this chapter, we present the protocols to obtain transformation-competent grapevine material from different tissues and the procedures for protoplast isolation and cultivation.

Immune checkpoint PD-L1 was originally identified as a cell surface transmembrane protein, but recent studies have demonstrated its presence also in the nucleus and suggested its role in transcriptional regulation. Interleukin-8 (IL-8, CXCL8) is a pro-angiogenic chemokine that promotes cancer progression. We have recently shown that in ovarian cancer (OC) cells, IFNγ induces nuclear accumulation of PD-L1, which is then recruited to IL-8 promoter, resulting in increased transcription of IL-8. Since the increased expression of IL-8 induces proliferation and invasion in OC cells, understanding the role of PD-L1 in transcriptional regulation of IL-8 is important for increasing the effectiveness of PD-L1 targeting immunotherapies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) followed by real time PCR to quantitatively measure PD-L1 recruitment to IL-8 promoter. The main points of the protocol are the ChIP analysis of PD-L1 recruitment to IL-8 promoter using antibody that specifically recognizes endogenous PD-L1, and quantitative real time PCR using primers for human IL-8 promoter spanning the transcription start site (TSS) and a region ~500 bp upstream of the TSS. Our results show that IFNγ significantly increases PD-L1 recruitment to TSS of IL-8 promoter and this recruitment is even higher to the ~500 bp upstream region containing multiple transcription factors binding sites, suggesting that PD-L1 associates with IL-8 promoter by binding to other transcription factors.

Graft-versus-host disease (GVHD) is the most serious complication limiting the clinical utility of allogeneic hematopoietic stem cell transplantation (HSCT), in which lymphocytes of donors (graft) are activated in response to the host antigen. This disease is related to increased inflammatory response through releasing inflammatory mediators and recruiting defense cells, such as leukocytes. Intravital microscopy is a technique that allows the observation of leukocyte interactions in vivo. It can be performed using conventional light microscopy in thin and transparent tissues or epifluorescence microscopy in solid and non-transparent tissues. Here, we describe the procedure to execute this method at the mesenteric and brain vessels of mice subjected to GVHD.

Nuclear accumulation of immune checkpoint PD-L1 has been associated with increased chemoresistance and cancer progression. Since nuclear PD-L1 is largely inaccessible to immunotherapies blocking cell surface PD-L1 signaling and to analytical methods measuring surface PD-L1 expression on cancer cells, it is important to develop convenient and reliable methods to measure the nuclear PD-L1 levels. Here, we describe a simple and efficient protocol that uses biochemical preparation of cytoplasmic and nuclear extracts, which are then analyzed by western blotting using PD-L1 monoclonal antibody that specifically recognizes endogenous PD-L1.

The p21 Rho family of small GTPases, RhoA, Rac1, and Cdc42, play vital roles in regulating actin dynamics and cell motility. These GTPases alternate between active (GTP-bound) and inactive (GDP-bound) states to modulate downstream signaling pathways that control cellular behavior. Monitoring their activation dynamics is essential for understanding cell morphodynamics and physiology, particularly in hematopoietic cells like monocytes and macrophages that are highly motile. Fluorescence resonance energy transfer (FRET)-based biosensors enable real-time visualization of Rho GTPase activities, but conventional ratiometric approaches can be limiting due to nonlinearity, making data interpretation challenging. Fluorescence lifetime imaging microscopy (FLIM) offers a quantitative alternative by directly measuring the change in donor fluorophore lifetime during FRET, circumventing acceptor imaging and ratiometric limitations. However, traditional FLIM methods can be technically challenging due to high photon demands and complex equipment. We discuss an alternative method of FLIM imaging using a time-domain FastFLIM system that supports rapid, sub-second imaging with reduced photon requirements, enabling visualization and quantification of FRET in a macrophage cell line. We demonstrate the utility of FastFLIM in RAW264.7/LR5 macrophages expressing a single-chain Rac GTPase FRET biosensor, showing Rac1 activation in response to mCSF1 (murine colony-stimulating factor 1) stimulation. This approach provides quantitative FRET data on GTPase dynamics, and we discuss herein practical guidance for researchers employing FastFLIM to study cell signaling.

Combining immunoassays with nucleic acid-based amplification and detection, the proximity ligation assay (PLA) is a useful tool for immunofluorescent detection, visualization, and quantification of individual proteins, protein modifications, and protein-protein interactions in fixed cells and tissue samples. Here, we present a detailed procedure for detecting protein-protein interactions in cancer cells with a commercially available Naveni® in situ proximity ligation technology to assist the researcher in successfully performing the experiments.