Macrophage activation has emerged as a key area in immunology, in which they are polarized to adopt different functional phenotypes in response to various environmental signals. Here, we describe the procedure of isolating human primary CD14+ monocytes and polarization toward M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. Relevant cytokine levels can be detected by manual and automated enzyme-linked immunosorbent assays (ELISA).

The vast amount of genomic data available for various plant species has enabled a greater understanding of genomic regions associated with phenotypic variation, mainly through approaches such as Genome-Wide Association Studies (GWAS). However, relying solely on GWAS results limits our understanding of trait variation, as it focuses on individual SNPs without considering the linked variants that may also influence the trait. As a follow-up to GWAS, haplotype analysis focusing on local regions of the genome can provide detailed insights into the genetic variants associated with a trait. crosshap, a local haplotyping R-package, identifies and visualises haplotype structures, allowing users to explore allelic diversity and associated phenotypic variation in targeted genomic regions. This chapter demonstrates local haplotyping analysis using crosshap to understand the genetic control of flowering time in domesticated and wild soybean, informing breeding strategies and crop improvement efforts; however, crosshap can be applied to any species where suitable data is available.

The detection of dystrophin and the proteins of the dystrophin-associated protein complex (DAPC) by immunostaining allows for the detection of the presence and localization of these proteins in slide-mounted sections of skeletal muscle or heart tissue. This technique has been used historically for the diagnosis and characterization of muscle disorders including Duchenne muscular dystrophy and many of the limb girdle and congenital muscular dystrophies. The extensive usefulness of this technique has allowed the development of numerous antibodies that can be used in immunostaining assays. As an increasing number of research and translational science studies have recently focused on the restoration of dystrophin as a therapeutic strategy, high-quality immunostaining techniques and analysis remain sought-after scientific practices. This chapter describes useful procedures to perform the immunofluorescence staining for dystrophin and several other useful DAPC proteins, while also providing guidance based on our clinical diagnostic and translational science research experience from working in the muscular dystrophy field.

Capillary electrochromatography is a miniaturized technique offering some advantages over conventional ones in separation science. It combines the best features of capillary zone electrophoresis (high efficiency of separation) and liquid chromatography (high selectivity). Because of the low flow rates, minute volumes of mobile phases can be applied, and therefore, it can be considered a green analytical technique. Capillary electrochromatography has been successfully used to separate many classes of compounds, including peptides, proteins, drugs, antibiotics, pollutants, enantiomers, etc. In this overview chapter, the main features of this technique are summarized. Its potential for the separation of enantiomers of chiral compounds and the main chiral stationary phases employed are presented. Finally, some selected applications in this field, reported in the literature in the period 2020–2024, are presented.

Komagataella phaffii is an efficient system for secreted recombinant protein expression. However, this host also expresses native proteases whose activity can degrade the target proteins, thus impacting native yield and quality. This proteolysis creates impurities considered critical quality attributes (CQAs) by regulatory agencies. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an ideal analytical method for characterizing these impurities due to its high resolution, sensitivity, and ability to separate protein fragments, making it valuable for the control strategy and characterization of a biopharmaceutical product.

Genomic medicine, which provides diagnosis and treatment decisions based on genomic variations, has been implemented in clinical practice and become more accessible. The clinical interpretation of genomic variations detected through genome analysis is crucial in genomic medicine. Clinical interpretation often utilizes genomic databases and effects predictions by machine learning. However, it has been noted that the genomic data accumulated thus far exhibit biases toward certain ethnic groups, leading to efforts to enhance genomic data diversity across various regions. While diverse and highly accurate machine learning methods have been developed for coding region variants, prediction methods for noncoding region variants still need to be improved in accuracy and applicable region. This chapter outlines the basic flow of genomic medicine and how detected variants are interpreted, introducing disease variant databases to advance genomic medicine and machine learning methods for predicting the effect of noncoding region variants.

Genomics, the study of an organism’s full set of genes, is often enhanced by gene expression data from RNA analysis, reflecting the correlation between gene function and expression profiles. This is particularly important for understanding complex traits regulated by multiple genes. RNA sequencing (RNA-Seq) has become a standard technique for studying gene expression, especially in crops like soybean (Glycine max), which is valued for its high protein and oil content. Despite the availability of soybean genomic data, processing raw RNA-Seq data into usable formats remains a resource-intensive task, often requiring expensive hardware and bioinformatics expertise. The Soybean Expression Atlas (SEA) simplifies this process by providing access to transcript- and gene-level abundance data from 5481 RNA-Seq samples through a user-friendly interface. SEA’s features include tools like the Global Expression Viewer for visualizing gene expression profiles and the Search Gene List for generating lists across selected tissues. Users can also download gene expression matrices by BioProject and explore gene metadata, including tissue-specific indices and protein domain filters. SEA has facilitated studies of key metabolic pathways in soybean, such as oil biosynthesis, nodulation, and responses to environmental factors. It has also supported the development of additional tools in G. max and inspired similar initiatives in other plant species. This chapter demonstrates SEA’s application in pathogen research, using examples of the fungal species Colletotrichum truncatum and the soybean cyst nematode Heterodera glycines, showcasing SEA’s utility for narrowing down genes of interest for specific traits.

Once dismissed as nonfunctional transcriptional noise, small open reading frames (sORFs) and their encoded microproteins have rapidly emerged as key players in diverse biological processes. Ranging from just a few to about 150 amino acids in length, microproteins are now recognized for their ability to modulate cellular functions, often acting as dominant-negative regulators, scaffolds, or signaling intermediates. Initially overlooked due to technical and conceptual limitations, they are increasingly being detected thanks to advances in high-resolution proteomics, ribosome profiling, and integrative bioinformatics. In this chapter, we provide a concise overview of the discovery, origins, functions, and biological significance of microproteins. We also introduce the structure of this methods book, which captures the latest experimental and computational tools used to identify, characterize, and functionally dissect microproteins across a wide range of organisms and research disciplines. This emerging field exemplifies the power of cross-disciplinary collaboration, and we hope this volume will support and inspire further research in this exciting and rapidly expanding area.

Mushroom cultivation has been growing as a promising field to generate additional income in the public and private sectors. Several government schemes are helping farmers with cultivation and good financial outcomes from mushroom farming. Cordyceps mushrooms are known for their high medicinal value and cost. The optimized methods for the production of mushrooms with high contents of desirable compounds such as cordycepin, cordycepic acid, and adenosine have been discovered but are less known. The current protocol summarizes the basic procedure of the production strategy with cheaper raw materials and high production efficiency in a simplified manner. The protocol aims to provide a simple approach to the cultivation of Cordyceps militaris, suggesting a cost-effective and sustainable methodology including liquid- and solid-state cultures. Optimization methods of fruit body formation for better yield using grain-based cheaper substrate and controlled culture conditions such as temperature, humidity, light exposure, and so on, are explained in the protocol. The Cordyceps growers must ensure to check the quality of the mushroom product. The cultures with higher cordycepin and adenosine are considered the best for commercial usage in the medicinal sector. Thus, the quality test for it might ensure good financial gain. The protocol is useful in addressing the economic aspects of large-scale cultivation, with significant cost benefits and market opportunities.