Methods in molecular biology最新文献

The nematode Caenorhabditis elegans is a eukaryotic genetic model organism introduced for studies of animal development and behavior (Brenner S, Genetics 77:71-94, 1974). It is also proving useful to expedite our understanding of human diseases and to explore potential therapies (Ahringer J, Curr Opin Genet Dev 7:410-415, 1997; Culetto E, Sattelle DB, Hum Mol Genet 9:869-877, 2000). Monitoring phenotypic changes and the impact of drug candidates is particularly convenient in the case of C. elegans models of neuromuscular or neurological disorders, where changes in motility and growth are often easily observed and can be conveniently assayed. We therefore developed an Invertebrate Automated Phenotyping Platform (INVAPP) together with an algorithm (Paragon) to facilitate such work (Buckingham SD, Partridge FA, Sattelle DB, Int J Parasitol Drugs Drug Resist Int J Parasitol Drugs Drug Resist 4:226-232, 2014; Partridge FA, Brown AE, Buckingham SD, Willis NJ, Wynne GM, Forman R et al., Int J Parasitol Drugs Drug Resist 8:8-21, 2018). Similarly, in the search for novel chemicals to combat invertebrate pathogens, such as parasitic worms, and disease vectors, such as the mosquito that serves as the malaria parasite vector, the phenotyping of worms and insects in the presence of new candidate drugs and control chemicals (anthelmintics and insecticides) can be extremely useful. This is especially important in view of the current challenges in controlling the malaria vector Anopheles gambiae and the soil-transmitted helminth, the whipworm Trichuris trichiura. For example, the development of resistance to the hitherto highly successful pyrethroid insecticides threatens the impressive gains made by the deployment of insecticide-treated nets (ITNs) and indoor residual sprays (IRS) in reducing malaria cases in the period 2000-2015. Also, there is a need for new anthelmintic drugs to combat soil-transmitted helminths such as whipworm, now that the widely used benzimidazoles are becoming much less effective. In both cases, automated phenotyping assays have a role to play. Here, we describe the use of a simple invertebrate automated phenotyping system and provide some examples that illustrate its utility.

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.

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.

Sweet sorghum is one of the important crops that has been widely reported to be recalcitrant to genetic manipulation endeavors. It can serve as biofuel, food, and food per production systems toward energy sources as well as human and animal sustenance. To deploy biotechnology tools in advancement of this crop of such agronomic importance, genetic and molecular investigation for understanding is necessary. Underdeveloped cell walls are a significant physiochemical state for microspores per their developmental stages, which makes it ideal for DNA isolation, being readily lysed as natural protoplasts. After harvesting panicles from the plants grown in controlled greenhouse conditions those are separated into sections per developmental stages is the first step. During micro-gametogenesis the unicellular microspore divides asymmetrically to subsequently give rise to a mature pollen grain with a vegetative and generative cell, while the absence of the participation of the anther wall in pollen formation makes it possible to investigate these developmental events directly. Thereby, from distal to basal end along panicle length, the five stages-mid-binucleate, early-binucleate, late-uninucleate, mid-uninucleate, and early-uninucleate microspores-can be yielded toward fractionalization. Whole-genome amplification is ideally achieved through individually isolated microspores with underdeveloped exine from anthers obtained after aseptic handling of spikelets using 75% ethanol and 1% sodium hypochlorite as sterilizing agents. The free-floating microspores, thus separated from the surrounding anther wall tissues, yield single gametophyte-based high-quality genomic DNAs. Efficient breeding of sweet sorghum through genetic tools can be achieved through free microspore release after aseptic isolation and whole-genome amplification.

Single-cell whole genome sequencing (WGS) enables accurate identification and characterize circulating tumor cells (CTCs) in blood and non-blood body fluids, leading to a non- or minimally invasive liquid biopsy approach for cancer diagnosis and prognosis. Here, we describe a single-cell low-pass WGS protocol for sensitive and accurate CTCs detection in blood and non-blood body fluids by combining a metabolic function-associated marker and a Tn5 transposome-based WGS method.

The kinetic stability of peptides bound onto MHC class I molecules is a critical parameter that helps shape their interaction with immune receptors and consequently their antigenic potential. We present here a method for measuring the kinetic stability and sensitivity to proteolytic degradation of peptides bound onto MHC class I molecules after in vitro refolding, by analyzing the time-resolved MALDI-TOF Mass Spec signal from the peptide, using the whole MHC-I/peptide complex in situ. This approach can provide important information on the dynamic nature of the MHC-peptide interaction, the kinetic half-life of binding and the sensitivity of the peptide to external proteolytic digestion or other modifications.

There are various ways to activate human T cells through their T Cell Receptor (TCR) or through overexpressed Chimeric Antigen Receptors (CARs), which are one of the most promising treatments in cancer immunotherapy. Here, we describe some basic methods to activate the human TCR or CAR using Antigen Presenting Cells presenting their natural antigen or superantigen, or stimulation with soluble antibodies or antibody-covered beads. There are several methods to analyze this activation and we describe here cytokine detection by ELISA, phosphoprotein detection by Western blot and expression of activation molecules at the cell surface by flow cytometry.

T cells are key players of the cellular immune system, able to detect and fight pathogens' invasion. T cells recognize pathogen-derived peptides presented by molecules called Human Leukocyte Antigens (HLA) in humans. The interaction between the T cells and peptide-HLA (pHLA) complexes is driven by the surface T cell receptors (TCRs). This interaction is critical and the first step of T cell activation. The affinity of the TCR for the pHLA is one of the key drivers of T cell activation. In this chapter, we describe the method to determine the affinity of the TCR for the pHLA via surface plasmon resonance.

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play a critical role in antigen processing by trimming N-terminally extended peptides within the ER, thereby shaping the repertoire of peptides presented by Major Histocompatibility Complex Class I (MHC I) molecules. Polymorphic variants in these enzymes give rise to functionally distinct allotypes, influencing peptide trimming efficiency and specificity, modulating CD8+ T cell responses in both health and disease. This chapter outlines a cellular model system for assessing ERAP1 and ERAP2 peptide trimming activity, using peptide-specific T cell activation as a surrogate readout. By employing transient transfection and co-culture with either T cell hybridoma (B3Z) or cytotoxic T lymphocytes (CTL), this approach enables the evaluation of peptide processing efficiency of ERAP1/2 based on the presentation and recognition of optimally generated MHC I-restricted epitopes.

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.