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Past Issue in 2019
Volume: 9, Issue: 2
Biochemistry
Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila
Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as ‘click chemistry’ serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.
Insulin Tolerance Test under Anaesthesia to Measure Tissue-specific Insulin-stimulated Glucose Disposal
Insulin resistance is a pathophysiological state defined by impaired responses to insulin and is a risk factor for several metabolic diseases, most notably type 2 diabetes. Insulin resistance occurs in insulin target tissues including liver, adipose and skeletal muscle. Methods such as insulin tolerance tests and hyperinsulinaemic-euglycaemic clamps permit assessment of insulin responses in specific tissues and allow the study of the progression and causes of insulin resistance. Here we detail a protocol for assessing insulin action in adipose and muscle tissues in anesthetized mice administered with insulin intravenously.
Cell Biology
A Method for Culturing Mouse Whisker Follicles to Study Circadian Rhythms ex vivo
Ex vivo tissue-culture experiments are often performed in the field of circadian biology. The major aim of these experiments is to evaluate circadian characteristics such as period length at the tissue-autonomous level by monitoring clock gene expression in real time. This culture method is also used to examine the tissue specificity of circadian entrainment factors. However, an ex vivo culture method for monitoring clock gene expression in hair follicles has yet to be established. In the present study, we developed an experimental method to analogize and evaluate circadian characteristics by performing ex vivo culture of mouse whisker follicles and monitoring clock gene expression in real time.
Immunology
Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction
Humoral alloimmunity remains a significant and unresolved problem that constrains allograft survival. Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing the effect of various drug treatments on humoral alloimmunity. In order to satisfy this demand, we developed such a protocol in which de novo alloantibodies production is induced in one-way mixed lymphocyte reaction (MLR). The amount and capacity of the generated alloantibodies in the supernatant of each one-way MLR is assessed using an antibody-mediated cell dependent cytotoxicity (CDC) assay. The principle of the assay relies on the assessment of cellular survival of resting PBMCs isolated from the same donors, supplemented with the alloantibodies from the one-way MLR supernatant. The lesser is the cellular survival, the higher the production of alloantibodies in one-way MLR and consequently the more potent the humoral alloimmunity.
Immunohistochemical Staining of TLR4 in Human Skeletal Muscle Samples
Growing evidence suggests the involvement of TLR4, a receptor in the innate immune system, in muscle loss in uremia. Recently, we have evaluated TLR4 in human skeletal muscle from chronic kidney disease patients, by immunohistochemistry and image analysis. Unlike the commonly-used Western blot method, immunohistochemistry allows for the observation of protein distribution in the intact tissue while, image analysis, its quantification. In fact, our data highlighted our hypothesis that an enhanced TLR4 skeletal muscle cell expression contributes to the activation of the downward inflammatory pathway in uremic sarcopenia. In this protocol, we describe the procedure for immunostaining TLR4 in human skeletal muscle and for quantifying it by image analysis.
Microbiology
Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure
In the presence of oxidative stress, cellular defense systems that can detoxify reactive oxygen species are activated through multiple signaling cascades and transcriptional reprogramming. The budding yeast Saccharomyces cerevisiae has served as an excellent model for genetically-identifying factors important for the response to oxidative stress. Here, we describe two assays for testing yeast gene deletion strains or strains overexpressing a gene of interest for viability following oxidative stress induced by hydrogen peroxide treatment. These include a plate-based spot assay for visualizing cell growth and a quantitative colony counting assay. As stress response assays can be highly variable depending on cell growth conditions, these protocols have been optimized for obtaining highly-reproducible results between experiments. We demonstrate the use of these protocols for genetic tests of a putative chromatin regulator implicated in regulating the transcriptional response to oxidative stress.
Intracellular Invasion and Killing Assay to Investigate the Effects of Binge Alcohol Toxicity in Murine Alveolar Macrophages
Alcohol consumption has diverse and well-documented effects on the human immune system and its ability to defend against infective agents. While pulmonary related infections can occur in healthy humans, binge alcohol use is recognized as a major health risk factor (Nelson et al., 1991). Although binge alcohol consumption has been considered as a risk factor for the development of pulmonary infections, no experimental studies have investigated the outcomes of a single binge alcohol exposure during infection. A key assay to assess the effects of a single binge alcohol exposure on the interactions between bacteria and alveolar macrophage is a binge alcohol intracellular invasion and killing assay. MH-S alveolar macrophages (AMs) are exposed to a single binge alcohol dose prior to infection for 3 h. The macrophage monolayer is then infected to allow for engulfment, followed by removal of extracellular bacteria to assess the intracellular killing capacity of infected macrophages over time. We have utilized this assay to demonstrate that low alcohol exposure significantly suppressed the uptake and killing of less virulent Burkholderia thailandensis (B. thailandensis) by AMs. More recently we found that activated AMs with interferon (IFN)-γ incubated in alcohol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h post infection, which lead to B. thailandensis survival and a ~2.5-fold replication increase compared to controls (Jimenez et al., 2017). These results provide insights into binge alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for pulmonary bacterial infections. This assay can be adapted to other bacterial species and host cell types to assess tissue specific effects of alcohol during infection.
Molecular Biology
CRISPR-Cas9 Mediated Genome Editing in Drosophila
In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.
flySAM Transgenic CRISPRa System Manual
Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.
Neuroscience
High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain
Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.
A Mouse Model of Postoperative Pain
Postoperative pain is highly debilitating and hinders recovery. Opioids are the main pain medication used for acute postoperative pain. Given the devastating opioid addiction and overdose epidemic across the US, non-opioid pain therapeutics are desperately needed. In order to develop novel, non-opioid therapies for the treatment of postoperative pain and identify the mechanisms underlying this pain, rodent models of incisional pain have been established. The protocol herein describes in detail how to create a mouse model of postoperative pain that was adapted from established protocols. This model of postoperative pain is frequently-used, highly reproducible, and results in peripheral and central nervous system alterations.