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Past Issue in 2020
Volume: 10, Issue: 24
Biochemistry
Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (2H and 15N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic components in-situ clears the way to reaching an in-depth understanding of the functional and mechanistic details of this unique cellular compartment.
Triacylglycerol Measurement in HeLa Cells
Lipid droplets store triacylglycerols (triglycerides) and sterol esters to regulate lipid and energy homeostasis. Triacylglycerol measurement is often performed during the investigation of lipid droplet formation and growth. This protocol describes a reliable method using a fluorometric lipid quantification kit to measure triacylglycerols extracted from HeLa cells, which were treated with oleic acid to trigger the formation of lipid droplets. The lipid quantification kit employs a lipid-binding molecule that emits bright fluorescence only when bound to extracted triacylglycerols, whose content can be quantified by a simple fluorescence readout.
Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening
Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of covalent fragment binding. However, robust hit identification requires characterization of the kinetics of covalent binding that can be readily achieved using quantitative irreversible tethering. This screening platform uses a non-specific cysteine-reactive fluorogenic probe to monitor the rate of reaction between covalent fragments and cysteine containing biomolecules. Fragment libraries are simultaneously screened against the target protein and glutathione, which functions as a control, to identify hit fragments with kinetic selectivity for covalent modification of the target. Screening by quantitative irreversible tethering accounts for variations in the intrinsic reactivity of individual fragments enabling robust hit identification and ranking.
Cancer Biology
Equilibrium and Kinetic Measurements of Ligand Binding to HiBiT-tagged GPCRs on the Surface of Living Cells
G-protein coupled receptors (GPCRs) remain at the forefront of drug discovery efforts. Detailed assessment of features contributing to GPCR ligand engagement in a physiologically relevant environment is imperative to the development of new therapeutics with improved efficacy. Traditionally, binding properties such as affinity and kinetics were obtained using biochemical radioligand binding assays. More recently, the high specificity of resonance energy transfer has been leveraged toward the development of homogeneous cell-based proximity assays with capacity for real-time kinetic measurements. This suite of ligand binding protocols couples the specificity of bioluminescent resonance energy transfer (BRET) with the sensitivity afforded by the luminescent HiBiT peptide. The BRET format is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent Tracers. At the same time, high affinity complementation of HiBiT with the cell impermeable LgBiT limits the bright bioluminescence donor signal to the cell surface and eliminates luminescence background from unoccupied receptors present in intracellular compartments.
Developmental Biology
Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging
Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.
Immunology
RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood
The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called ‘RNA ImmunoGenic Assay’ to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The qPCR analysis is performed to reveal the activation of specific immune pathways. The RNA ImmunoGenic Assay provides a simple and fast method to detect donor specific - immune response against mRNA therapeutics.Graphic abstract:Schematic representation of RNA ImmunoGenic Assay
Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay
Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects’ innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable method for small insects hemocyte phagocytosis assay by taking pea aphid as an example. Furthermore, we provide the protocols for pea aphid rearing and bacterial infection, which offer referential method for related research.
Microbiology
Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it is important to verify the genomic variability at primer/probe binding regions periodically. This step-by-step protocol describes a bioinformatics approach for an extensive evaluation of the sequence variability within the primer/probe target regions of the SARS-CoV-2 genome. The protocol can be applied to any molecular diagnostic assay of choice using freely available software programs and the ready-to-use multiple sequence alignment (MSA) file provided.Graphic abstract:Overview of the sequence tracing protocol. The figure was created using the Library of Science and Medical Illustrations from somersault18:24 licensed under a CC BY-NC-SA 4.0 license (https://creativecommons.org/licenses/by-nc-sa/4.0/). Video abstract: https://youtu.be/M1lV1liWE9k
Antimicrobial Sensitivity Assay for Bdellovibrio bacteriovorus
Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of treatments. In order to make this approach feasible more has to be known about the ability of B. bacteriovorus to resist antibiotics itself. Standard assays to determine the minimum inhibitory concentration (MIC) are not suitable for B. bacteriovorus, since the small size of this bacterium (0.25-0.35 by 0.5-2 μm) prevents scattering at OD600. Since these predatory bacteria require larger prey bacteria for growth (e.g., E. coli dimensions are 1 by 1-2 μm), the basis for the antimicrobial sensitivity assay described here is the reduction of the OD600 caused by prey lysis during growth. Previous studies on predatory bacteria resistance to antimicrobials employed methods that did not allow a direct comparison of antimicrobial resistance levels to those of other bacterial species. Here, we describe a procedure to determine B. bacteriovorus sensitivity to antimicrobials which can be compared to a reference organism tested as close as possible to the same experimental conditions. Briefly, minimal inhibitory concentration (MIC) values of B. bacteriovorus are determined by measuring the reduction in absorbance at 600 nm of mixed predator/prey cultures in presence and absence of different antimicrobial concentrations. Of note, this method can be modified to obtain antimicrobial MIC values of other predatory bacteria, using different conditions, prey bacteria and/or antimicrobials.
Preparation of Nippostrongylus brasiliensis Larvae for the Study of Host Skin Response
Hookworms are skin penetrating parasites, however in the laboratory the hookworm model Nippostrongylus brasiliensis, the parasite is traditionally administered subcutaneously bypassing the skin (epidermis and dermis). Here, we describe two complementary approaches for infecting mice with N. brasiliensis in order to study the skin immune responses. The first approach employs a skin percutaneous injection that is poorly efficient with the laboratory strain of the parasite in mice, but represents a natural infection. The second approach employs an intradermal injection of the parasite, allowing the controlled delivery of the parasitic larvae and leads to an infection that closely mimics the natural kinetics of parasite migration and development. Both of those infection models allow the investigator to study the skin immune response mounted against the parasite, in addition to detailed investigations of the early immunomodulatory strategies employed by the parasite during skin invasion.
Neuroscience
A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes cytoplasmic RBP concentrations sufficient for SG localization, and does not address RBP recruitment independent of SG formation. Here, we present a quantitative method to assess recruitment of recombinant RBPs into pre-formed SGs, independent of the RBP’s nuclear localization, using semi-permeabilized cells and fluorescence microscopy. In this assay, SGs are firstly induced by a stressor, and then the plasma membrane of the stressed cells is subsequently selectively permeabilized to provide access of the recombinant protein to SGs. Nuclear import of the protein-of-interest is prevented by blocking nuclear pores with wheat germ agglutinin. This assay allows one to study the molecular mechanisms underlying recruitment of RBPs into SGs quantitatively, in absence of their nuclear import and under controlled conditions. The method allows for a direct comparison of wildtype, mutant or posttranslationally modified RBPs, for addressing the influence of other proteins’ preventing or promoting SG association of RBPs, and is also applicable to synthetic peptides.Graphic abstractWorkflow overview for analysis of SG recruitment of recombinant proteins or peptides in semi-permeabilized cells
Nestlet Shredding and Nest Building Tests to Assess Features of Psychiatric Disorders in Mice
Mimicking the various facets of human psychiatric and neurodevelopmental disorders in animal models is a challenging task. Nevertheless, mice have emerged as a widely used model system to study pathophysiology and treatment strategies for these diseases. However, the corresponding behavioral tests are often elaborate and require extensive experience in behavioral testing. Here, we present protocols for two simple assays, nest building and nestlet shredding, that can serve as a starting point for the behavioral phenotyping of mouse models with (potential) features of psychiatric disorders. Both tests have been reported previously and we extend prior descriptions by including adaptations and refinements derived from our practical experience, like the use of the home cage instead of a fresh cage for nestlet shredding. Summarized, we provide ready-to-use protocols for two behavioral assays that allow the generation of robust data with minimal time and cost expenditure and enable an initial assessment of features of psychiatric or neurodevelopmental disorders in mouse models of these diseases.
Plant Science
Lipid Droplet Isolation from Arabidopsis thaliana Leaves
Lipid droplets (LDs) are neutral lipid aggregates surrounded by a phospholipid monolayer and specific proteins. In plants, they play a key role as energy source after seed germination, but are also formed in vegetative tissues in response to developmental or environmental conditions, where their functions are poorly understood. To elucidate these, it is essential to isolate LDs with good yields, while retaining their protein components. LD isolation protocols are based on their capacity to float after centrifugation in sucrose gradients. Early strategies using stringent conditions and LD-abundant plant tissues produced pure LDs where core proteins were identified. To identify more weakly bound LD proteins, recent protocols have used low stringency buffers, but carryover contaminants and low yields were often a problem. We have developed a sucrose gradient-based protocol to isolate LDs from Arabidopsis leaves, using Tween-20 and fresh tissue to increase yield. In both healthy and bacterially-infected Arabidopsis leaves, this protocol allowed to identify LD proteins that were later confirmed by microscopy analysis.
![Analysis of Isotopically-labeled Monogalactosyldiacylglycerol Molecular Species from [14C]Acetate-Labeled Tobacco Leaves](https://en-cdn.bio-protocol.org/imageup/arcimg/20201215013528186.jpg?t=1758512010)
Analysis of Isotopically-labeled Monogalactosyldiacylglycerol Molecular Species from [14C]Acetate-Labeled Tobacco Leaves
Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids (e.g., vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [14C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the individually labeled molecular species which is traditionally performed by thin layer chromatography. Here we present a reverse phase HPLC method for the separation of MGDG molecular species from tobacco leaves in under 35 min. The quantification of each 14C-labeled molecular species was accomplished by an in-line flow radio detector. This method of analysis for [14C]Acetate labeled MGDG molecular species by radio-HPLC provides a rapid, high throughput, and reliable analytical approach to identify changes in MGDG metabolism due to bioengineering or other perturbations of metabolism.
Stem Cell
Isolation of Extracellular Vesicles Derived from Mesenchymal Stromal Cells by Ultracentrifugation
Extracellular vesicles (EVs) are a heterogeneous group of membranous vesicles that differ on their biogenesis and release pathways, such as exosomes, microvesicles and apoptotic bodies. They are involved in cell-to-cell communication delivering signal molecules (proteins, nucleic acids, lipids, etc.) that can regulate different physiological processes, as well as the development and progression of several diseases. There are different methods and commercial kits to isolate EVs and depending on the methodology one could obtain EVs with different degrees of efficiency, purity and it can be more or less time-consuming. Then, the choice has to be according to the different advantages and disadvantages, and their use for downstream applications. Here, we describe the EVs isolation method from mesenchymal stromal cells by ultracentrifugation. This EVs isolation can be performed using common media and buffers, and only with the requirement of an analytical ultracentrifuge. Moreover, this method can be used to obtain large quantity of EVs with a good reproducibility for developing in vitro and in vivo experiments and studying their biological actions.
Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Osteoclasts
Defects in bone resorption by osteoclasts result in numerous rare genetic bone disorders as well as in some common diseases such as osteoporosis or osteopetrosis. The use of hiPSC-differentiated osteoclasts opens new avenues in this research field by providing an unlimited cell source and overcoming obstacles such as unavailability of human specimens and suitable animal models. Generation of hiPSCs is well established but efficient differentiation of hiPSCs into osteoclasts has been challenging. Published hiPSC-osteoclast differentiation protocols use a hiPSC-OP9 co-culture system or hiPSC-derived embryoid bodies (EBs) with multiple cytokines. Our three-stage protocol consists of 1) EB mesoderm differentiation, 2) expansion of myelomonocytic cells and 3) maturation of hiPSC-osteoclasts. We generate uniformly-sized EBs by culturing Accutase-dissociated hiPSCs on Nunclon Sphera microplates and promote EB mesoderm differentiation in a cytokine cocktail for 4 days. For Stage 2, EBs are transferred to gelatin-coated plates and cultured with hM-CSF and hIL-3 to expand the myelomonocytic population. By supplementing with vitamin D, hTGFβ, hM-CSF and hRANKL, cells collected at the end of Stage 2 are differentiated into mature osteoclasts (Stage 3). Compared to other techniques, our protocol does not require a co-culture system; induces EBs into mesoderm differentiation in a homogenous manner; uses less cytokines for differentiation; requires only a short time for osteoclast maturation and produces sufficient numbers of osteoclasts for subsequent molecular analyses.Graphic abstract