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Past Issue in 2020
Volume: 10, Issue: 15
Biophysics
Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.
Cancer Biology
SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3′ single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3′ single-strand DNA tails, generated from DSB processing.
Cell Biology
Measuring Intracellular Vesicle Density and Dispersion Using Fluorescence Microscopy and ImageJ/FIJI
Cell signalling, cell secretion, and plasma membrane repair are processes that critically rely on intracellular vesicles, important components of the endocytic and secretory pathways. More specifically, the strategic distribution of intracellular vesicles is important for diverse cellular processes. The method presented here is a simple, affordable, and efficient tool to analyze the distribution of intracellular vesicles such as lysosomes, endosomes, Golgi vesicles or secretory granules under different experimental conditions. The method is an accessible way to analyze the density and dispersion of intracellular vesicles by combining immunofluorescence with pixel-based quantification software (e.g., ImageJ/FIJI). This protocol can be used widely within the scientific community because it utilizes ImageJ/FIJI, an open source software that is free. By tracking fluorescent vesicles based on their position relative to cell nuclei we are able to quantify and analyze their distribution throughout the cell.
Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells
Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography–SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells.
A Quantitative Single-cell Flow Cytometry Assay for Retrograde Membrane Trafficking Using Engineered Cholera Toxin
The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.
Developmental Biology
Mechanical Tissue Compression and Whole-mount Imaging at Single Cell Resolution for Developing Murine Epididymal Tubules
Cells inside the body are subjected to various mechanical stress, such as stretch or compression provided by surrounding cells, shear stresses by blood or lymph flows, and normal stresses by luminal liquids. Force loading to the biological tissues is a fundamental method to better understand cellular responses to such mechanical stimuli. There have been many studies on compression or stretch experiments that target culture cells attached to a flexible extensible material including polydimethylsiloxane (PDMS); however, the know-how of those targeting to tissues is still incomplete. Here we present the protocol for mechanical tissue compression and image-based analysis by focusing on developing murine epididymis as an example. We show a series of steps including tissue dissection from murine embryos, hydrogel-based compression method using a manual device, and non-destructive volumetric tissue imaging. This protocol is useful for quantifying and exploring the biological mechanoresponse system at tissue level.
Immunology
Flow Cytometry of CD14, VDR, Cyp27 and Cyp24 and TLR4 in U937 Cells
Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of inflammatory and vitamin D machinery biomarkers in several disease, in the present study we used flow cytometry technique to evaluate the effect of 25 vitamin D on CD14, Toll-like receptor 4 (TLR4), vitamin D receptor (VDR), 1-α hydroxylase (CYP27), 24 hydroxylase (CYP24) in monocytes lineage (U937). The U937 culture was incubated with healthy or CKD serum and treatment with/without 25-vitamin D (50 ng/ml for 24 h) to evaluate CD14, TRL4, VDR, CYP27 and CYP24 expression. This protocol showed the advantage to investigate the effect of treatment with 25 vitamin D on the intracellular and cell membrane biomarkers expression quickly and simultaneously. In addition, this technique is not laborious, but easy to perform and to interpret compared to RT-qPCR, western blot or immunofluorescence.
Microbiology
High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR
We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a Bio-protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains.
RETRACTED: Paper Lateral Flow Biosensor for Nodavirus Reverse Transcribed RNA Detection
Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when nodavirus product was present. The proposed assay has great implications on basic research since it eliminates the need for time-consuming, cumbersome electrophoresis protocols and could be adjusted for use on the site of fish culture by fish farmers. Disease monitoring by such bioanalytical platforms without time consuming and costly procedures would have great impact on the aquaculture and environmental safety.
Molecular Biology
A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalently closed circular plasmids. It was later shown that because very little primer is used in the procedure, resuspended oligo pools, which normally require amplification before use, can be used directly in the mutagenesis procedure. Because oligo pools can contain tens of thousands of unique oligos, this enables the construction of libraries of tens of thousands of user-defined mutations in a single-pot mutagenesis reaction, which significantly improves the utility of NM as described below. Use of oligo pools afford an economically advantageous approach to mutagenic experiments. First, oligo pool synthesis is much less expensive per nucleotide synthesized than conventional synthesis. Second, a mixed pool may be generated and used for mutagenesis of multiple different genes. To use the same oligo-pool for mutagenesis of a variety of genes, the user must only quantify the fraction of the oligo-pool specific to her mutagenic experiment and adjust the volume and effective concentration of the oligo-pool for use in nicking mutagenesis.
Neuroscience
Generation of Functional Mouse Hippocampal Neurons
Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos’ isolation, culturing conditions and assessment of culture’s purity and differentiation. Evaluation of neuronal activity was performed by analysis of calcium imaging dynamics at six days in culture.
In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin
In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da fluorescein salt, 66.5 kDa bovine serum albumin, and 70 kDa dextran as well as endogenous 160 kDa mouse IgG. When administered intravenously, these substances are excluded from a healthy brain by the BBB. However, BBB dysfunction can allow entry of these tracers into the brain and this accumulation can be measured using spectrophotometry, fluorescent microscopy, and immunohistochemistry. We also describe a method to induce BBB permeability using Clostridium perfringens epsilon toxin. Finally, we include a short discussion about the advantages and disadvantages of each method and their appropriate downstream applications.
Development of the Deoxycorticosterone Acetate (DOCA)-salt Hypertensive Rat Model
According to the World Health Organization (WHO), nearly 1.13 billion people worldwide have hypertension, a major factor responsible for premature death globally. The inherent multifactorial nature of hypertension makes its study difficult since the chronic rise in blood pressure depends on the intricate connection between dietary, genetic and environmental factors. Therefore, the pathophysi-ology of hypertension is not completely understood. For these reasons, there is an ongoing search for animal models that better mimic changes resulting from this disease. Because of its complexity, the use of animal models aimed at elucidating the pathogenesis of hypertension and to evaluate new therapeutic possibilities is an important tool for understanding this disease since it enables consistent experimental strategies that are impractical in humans. Over time, many animal models have been developed for the study of chronic increases in blood pressure ranging from genetic models that include the spontaneously hypertensive rat (SHR) and genetic manipulations, such as the TGR (mRen2) rat, as well as neurogenic or endocrine models. One of the most commonly used hypertensive rat models today is that of hypertension induced by treatment with deoxycorticosterone acetate associated with high sodium intake, i.e., the DOCA-salt model. This model is known to have a neurogenic component linked to increased sympathetic nervous system activity, and as such the DOCA-salt model promotes cross-talk between endocrine and neural components that lead to increased blood pressure, and may impact the functioning of other organs.
Multiple Simultaneous Acute Stresses in Mice: Single or Repeated Induction
Stress is crucial to the survival of an organism, but excessive stress can lead to psychological disorders including depression, anxiety, substance abuse, and suicidality. The prevailing notion is that chronic stress promotes adverse outcomes on brain and body health, whereas acute stressors are generally benign. Notably, acute events such mass shootings or natural disasters are now emerging as significant sources of cognitive and emotional problems including post-traumatic stress disorder (PTSD). These events are characterized by the simultaneous occurrence of physical, emotional, and social stresses, which last minutes to hours. Hence, there is a need to model such multiple concurrent acute stresses (MAS) to uncover the mechanisms by which they lead to profound adverse outcomes. The MAS paradigm described here involves simultaneously exposing a rodent to several different stressors including restraint, crowding, and jostling alongside peers in a brightly lit and very noisy environment. Moreover, the MAS paradigm can be used once or imposed repeatedly to emulate complex, repeated modern life stresses, advancing our mechanistic understanding of consequent mental and cognitive impairments.
Plant Science
The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.