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Past Issue in 2021
Volume: 11, Issue: 18
Biochemistry
Methods for the Extraction of Heme Prosthetic Groups from Hemoproteins
Hemoproteins are widely researched because they contain redox-active heme prosthetic groups (iron + protoporphyrin IX) that enable them to perform a range of vital functions, acting as enzymes, participants in electron transfer reactions, or gas sensing, carrying, and storage proteins. While the heme prosthetic group is almost always essential for hemoprotein function, it is frequently desirable to remove it from the protein to enable biochemical or protein engineering studies. Obtaining high yields of the apo form of the hemoprotein can be challenging since high heme-protein binding affinities necessitate the use of harsh conditions to remove heme. In this Bio-Protocol, we present three chemical extraction methods that can be used to efficiently remove heme: methyl ethyl ketone extraction, acid-acetone precipitation, and on-column heme extraction. We also present protocols that can be used to quantitate the amount of residual heme bound to the protein after performing the extraction procedures.
Comprehensive Identification of Translatable Circular RNAs Using Polysome Profiling
Circular RNAs (circRNAs), a special type of RNAs without 5’- and 3’-ends, are widely present in eukaryotes and known to function as noncoding RNAs to regulate gene expression, including as miRNA sponges. Recent studies showed that many exonic circRNAs, generated by back-splicing of pre-mRNAs, can be translated in a cap-independent fashion through IRESs or m6A RNA methylation. However, the scope of the translatable circRNAs and the biological function of their translation products are still unclear in different cells and tissues. Ribosome footprinting and proteomic analysis were usually used to globally identify translatable circRNAs. However, both methods have low sensitivity due to the low efficiency in the discovery of circRNA specific reads or peptides (i.e., the back-splicing junctions are difficult to recover by the short reads of ribosome footprinting and the limitation of proteomic analysis). Here, we described an alternative method to identify translatable circRNAs using polysome profiling and circRNA-seq. Generally, polysome-associated RNAs were separated with sucrose gradients. Then polysome-bound circRNAs were enriched by an RNase R treatment and identified through paired-end deep sequencing. Thus, this method is more sensitive than ribosome footprint and proteomic analyses for the identification of translatable circRNAs.
Biological Engineering
Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein
Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the energy metabolism. Prokaryotes like E. coli have ISC and SUF pathways that help in the assembly and maturation of iron-sulfur proteins. These iron-sulfur proteins, especially with [4Fe-4S] clusters, are highly sensitive to molecular oxygen, and it would be advantageous if the de novo proteins and native proteins having iron-sulfur binding sites are expressed and isolated under anaerobic conditions. Bacterially assembled iron-sulfur proteins, when isolated and purified anaerobically, exhibit improved biochemical and biophysical stabilities in comparison to the counterparts expressed and purified aerobically and reconstituted under anaerobic conditions. This protocol outlines the expression and purification of the artificial protein, Coiled-Coil Iron-Sulfur (CCIS). It may be deployed to both natural and artificial [4Fe-4S] proteins when heterologously expressed in E. coli.
Cell Biology
Isolation of Nasal Brush Cells for Single-cell Preparations
Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays.Graphic abstract:Workflow of nasal digestion for brush cell isolation.
Developmental Biology
En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos
Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.
Inter-species Transplantation of Blastocysts between Medaka and Zebrafish
Transplantation of blastocysts from a donor to a host blastula constitutes a powerful experimental tool to tackle major developmental biology questions. The technique is widely implemented in diverse biological models including teleost fish, where it is typically used for intra-species blastula transplantations – i.e., labeled blastocysts into a non-labeled host to follow lineages, or mutant blastocysts into a wild-type host to address autonomous vs. non-autonomous roles of a gene of interest. We have recently implemented a protocol to transplant blastocysts between zebrafish (D. rerio) and medaka (O. latipes), two species in which blastocysts show different developmental dynamics and sizes (Fuhrmann et al., 2020). We present here a detailed protocol on how to overcome the early differences in chorion structure, blastula size, and speed of development to achieve trans-species blastocyst transplantation.
Immunology
Identification and Quantitation of Neutrophil Extracellular Traps in Human Tissue Sections
Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (e.g., DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues.Graphic abstract:Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.
Microbiology
Cell-free Translation: Preparation and Validation of Translation-competent Extracts from Saccharomyces cerevisiae
Cell-free translation is a powerful technique for in vitro protein synthesis. While cell-free translation platforms prepared from bacterial, plant, and mammalian cells are commercially available, yeast-based translation systems remain proprietary knowledge of individual labs. Here, we provide a detailed protocol for simple, fast, and cost-effective preparation of the translation-competent cell-free extract (CFE) from budding yeast. Our protocol streamlines steps combined from different procedures published over the last three decades and incorporates cryogenic lysis of yeast cells to produce a high yield of the translationally active material. We also describe techniques for the validation and troubleshooting of the quality and translational activity of the obtained yeast CFE.Graphic abstract:The flow of Cell-Free Extract (CFE) preparation procedure.
An Inexpensive Imaging Platform to Record and Quantitate Bacterial Swarming
Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion (Steager et al., 2008). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales-Soto et al., 2015); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a “homemade,” environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.
Intrathoracic Inoculation of Zika Virus in Aedes aegypti
Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers.Graphic abstract:Cartoon depiction of Aedes aegypti intrathoracic inoculation. Figure made with Biorender.com.
Glucose Starvation, Magnesium Ion Starvation, and Bile Stress Assays
Salmonella enterica serovar Enteritidis (S. Enteritidis) is a leading causative pathogen for food-borne gastroenteritis. During its course of infection, it confronts myriads of physiological barriers inside the host, such as nutrient deprivation, low micronutrient availability, and toxicity from bile salts, to promote bacterial survival and infection inside the host. The ability of the pathogen to overcome these stressful conditions determines the degree of virulence in the host. Therefore, assessment of the survival of a pathogen during different stress conditions, like glucose starvation, magnesium starvation, and bile stress, are important parameters to assess the virulence of the pathogen. Here, we describe protocols for estimating the survival of the pathogen during the above-mentioned stress conditions. We culture S. Enteritidis in an appropriate growth medium to a required O.D.600 and treat it with glucose starvation (M9 minimal culture medium containing 0.03% glucose), magnesium starvation (M9 minimal culture medium containing 20 µM MgSO4), and bile stress (bacterial cells treated with 15% bile salts in Luria Bertani (LB) culture medium) conditions. The number of surviving bacteria is obtained after the treatment by calculating the colony-forming units (CFU) of the surviving pathogen obtained on LB agar plates at relevant time intervals. The experiments are performed in biological replicates, and statistical analysis is performed to validate the experimental findings. The methodology of these stress response assays is simple and can be adapted to study the pathogenesis and stress response in other relevant and culturable enteric pathogens.
Molecular Biology
Construction of DNA/RNA Triplex Helices Based on GAA/TTC Trinucleotide Repeats
Atypical DNA and RNA secondary structures play a crucial role in simple sequence repeat (SSR) diseases, which are associated with a class of neurological and neuromuscular disorders known as “anticipation diseases,” where the age of disease onset decreases and the severity of the disease is increased as the intergenerational expansion of the SSR increases. While the mechanisms underlying these diseases are complex and remain elusive, there is a consensus that stable, non-B-DNA atypical secondary structures play an important – if not causative – role. These structures include single-stranded DNA loops and hairpins, G-quartets, Z-DNA, triplex nucleic acid structures, and others. While all of these structures are of interest, structures based on nucleic acid triplexes have recently garnered increased attention as they have been implicated in gene regulation, gene repair, and gene engineering. Our work here focuses on the construction of DNA triplexes and RNA/DNA hybrids formed from GAA/TTC trinucleotide repeats, which underlie Friedreich’s ataxia. While there is some software, such as the Discovery Studio Visualizer, that can aid in the initial construction of DNA triple helices, the only option for the triple helix is constrained to be that of an antiparallel pyrimidine for the third strand. In this protocol, we illustrate how to build up more generalized DNA triplexes and DNA/RNA mixed hybrids. We make use of both the Discovery Studio Visualizer and the AMBER simulation package to construct the initial triplexes. Using the steps outlined here, one can – in principle – build up any triple nucleic acid helix with a desired sequence for large-scale molecular dynamics simulation studies.
Neuroscience
A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin
Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Aplysia Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured Aplysia bag cell neurons (He et al., 2015; Ren et al., 2019). In order to provide in vitro evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of Aplysia cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from Yersinia enterocolitica, we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in Escherichia coli.
Plant Science
Agrobacterium-mediated Transformation of Japonica Rice Using Mature Embryos and Regenerated Transgenic Plants
Identification of novel genes and their functions in rice is a critical step to improve economic traits. Agrobacterium tumefaciens-mediated transformation is a proven method in many laboratories and widely adopted for genetic engineering in rice. However, the efficiency of gene transfer by Agrobacterium in rice is low, particularly among japonica and indica varieties. In this protocol, we elucidate a rapid and highly efficient protocol to transform and regenerate transgenic rice plants through important key features of Agrobacterium transformation and standard regeneration media, especially enhancing culture conditions, timing, and growth hormones. With this protocol, transformed plantlets from the embryogenetic callus of the japonica cultivar ‘Taichung 65’ may be obtained within 90 days. This protocol may be used with other japonica rice varieties.
Quantitative Analysis of RNA Editing at Specific Sites in Plant Mitochondria or Chloroplasts Using DNA Sequencing
Cytidine-to-uridine (C-to-U) RNA editing is one of the most important post-transcriptional RNA processing in plant mitochondria and chloroplasts. Several techniques have been developed to detect the RNA editing efficiency in plant mitochondria and chloroplasts, such as poisoned primer extension (PPE) assays, high-resolution melting (HRM) analysis, and DNA sequencing. Here, we describe a method for the quantitative detection of RNA editing at specific sites by sequencing cDNA from plant leaves to further evaluate the effect of different treatments or plant mutants on the C to U RNA editing in mitochondria and chloroplasts.
Systems Biology
Protocol for RNA-seq Expression Analysis in Yeast
Genome-wide sequencing of RNA (RNA-seq) has become an inexpensive tool to gain key insights into cellular and disease mechanisms. Sample preparation and sequencing are streamlined and allow the acquisition of hundreds of gene expression profiles in a few days; however, in particular, data processing, curation, and analysis involve numerous steps that can be overwhelming to non-experts. Here, the sample preparation, sequencing, and data processing workflow for RNA-seq expression analysis in yeast is described. While this protocol covers only a small portion of the RNA-seq landscape, the principal workflow common to such experiments is described, allowing the reader to adapt the protocol where necessary.Graphic abstract:Basic workflow of RNA-seq expression analysis.
Assessing the in vitro Binding Specificity of Histone Modification Reader Proteins Using Histone Peptide Arrays
In the field of chromatin biology, a major goal of understanding the roles of histone post-translational modifications is to identify the proteins and domains that recognize these modifications. Synthetic histone peptides containing one or more modifications are a key tool to probe these interactions in pull-down assays with recombinant proteins or cell lysates. Building on these approaches, the binding specificity of a protein of interest can be screened against many histone peptides in parallel using a peptide array. In this protocol, we describe the expression and purification of a recombinant protein of interest in bacteria, followed by an assay for binding to histone post-translational modifications using a commercially available histone peptide array. The purification uses a versatile dual-tagging and cleavage strategy and equipment commonly available in a molecular biology laboratory.Graphic abstract:Overview of protocol for purifying recombinant protein and hybridizing to a histone peptide array.
test
Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single-celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of ~2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug-selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell-specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes.Key featuresThis protocol enables the analysis of variant surface glycoproteins (VSG) switching in T. brucei without modifying the expression site sequences.It uses a streamlined computational pipeline that takes fastq DNA sequences and outputs expressed VSG genes by each parasite clone.The protocol leverages the long reads sequencing capacity of the Oxford nanopore sequencing technology, which enables accurate identification of the expressed VSGs.The protocol requires approximately eight to nine days to complete.This protocol is used in: eLife (2023), DOI: 10.7554/eLife.89331.2