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Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.

To reveal gene regulation mechanisms, it is essential to understand the role of regulatory elements, which are possibly distant from gene promoters. Integrative analysis of epigenetic and transcriptomic data can be used to gain insights into gene-expression regulation in specific phenotypes. Here, we discuss STITCHIT, an approach to dissect epigenetic variation in a gene-specific manner across many samples for the identification of regulatory elements without relying on peak calling algorithms. The obtained genomic regions are then further refined using a regularized linear model approach, which can also be used to predict gene expression. We illustrate the use of STITCHIT using H3k27ac ChIP-seq and RNA-seq data from the International Human Epigenome Consortium (IHEC).

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host’s immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host’s immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.

Dysregulations of cholesterol biosynthesis are known to be associated with several pathologies. Due to the rapid growth of clinical investigations in this research area, a specific, fast, and valid method for analyzing cholesterol, its precursors, and metabolites is required. Here, we describe a rapid method for sample preparation, separation, and quantification of sterols in blood-derived samples using polymeric solid phase extraction followed by gas chromatography-mass spectrometry. The validated method demonstrates a reliable quantification of cholesterol, its precursors, and metabolites.

Over a decade has passed since the development of the Hi-C method for genome-wide analysis of 3D genome organization. Hi-C utilizes next-generation sequencing (NGS) technology to generate large-scale chromatin interaction data, which has accumulated across a diverse range of species and cell types, particularly in eukaryotes. There is thus a growing need to streamline the process of Hi-C data analysis to utilize these data sets effectively. Hi-C generates data that are much larger compared to other NGS techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) or RNA-seq, making the data reanalysis process computationally expensive. In an effort to bridge this resource gap, the 4D Nucleome (4DN) Data Portal has reanalyzed approximately 600 Hi-C data sets, allowing users to access and utilize the analyzed data. In this chapter, we provide detailed instructions for the implementation of the common workflow language (CWL)-based Hi-C analysis pipeline adopted by the 4DN Data Portal ecosystem. This reproducible and portable pipeline generates standard Hi-C contact matrices in formats such as .hic or .mcool from FASTQ files. It enables users to output their own Hi-C data in the same format as those registered in the 4DN Data portal, facilitating comparative analysis using data registered in the portal. Our custom-made scripts are available on GitHub at https://github.com/kuzobuta/4dn_cwl_pipeline .

Up to 90% of patients with Parkinson’s disease (PD) experience neuropsychiatric symptoms (NPSs) including depression, anxiety, psychosis, impulse control disorders, and cognitive impairment. NPSs impact a patient’s quality of life and are more delicate and distressing than the motor aspects of PD. Treatment options include pharmacological and non-pharmacological interventions such as deep brain stimulation (DBS), repetitive transcranial magnetic stimulation (rTMS), and electroconvulsive therapy (ECT). Depression is the most common NPS in PD, with mild to moderate depressive symptom severity and impairments in the subcortical nuclei, prefrontal cortex, and monoamine systems. Depression in PD (DPD) may precede the onset of motor symptoms by years or even decades, leading to poor cognitive performance, worse functional status, and an increased risk of death. It is essential to diagnose and treat DPD early in patients. Cognitive behavioral therapy (CBT) is recommended for DPD psychotherapy. Common clinical depression scales are recommended for assessments of the severity of depressive symptoms in PD. The treatment decision-making process must consider both the potential benefit and adverse effects in PD and NPS.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety. This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.