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Past Issue in 2022
Volume: 12, Issue: 7
Biochemistry
A SYBR Gold-based Label-free in vitro Dicing Assay
In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.
Biological Engineering
Activation of Mitochondrial Ca2+ Oscillation and Mitophagy Induction by Femtosecond Laser Photostimulation
Ultra-precise stimulation solely to individual mitochondria, without any influence to the whole cell, is quite difficult by traditional biochemical reagents. In mitophagy research, the mitochondria and even the whole cell usually suffer irreversible and great damage caused by treatment with potent chemicals. In this protocol, we present the technical procedures of our developed noninvasive ultra-precise laser stimulation (UPLaS) technology, which introduces precise stimulation to individual mitochondria, to excite mitochondrial Ca2+ (mitoCa2+) oscillations, with little perturbation to mitochondrial membrane potential (MMP), or mitochondrial reactive oxygen species (mitoROS). The mitoCa2+ oscillation by UPLaS was able to initiate the PINK1/Parkin pathway for mitophagy. This protocol has good potential to benefit researches on mitophagy and mitochondrial diseases. Graphic abstract: Figure 1. Flowchart of the UPLaS technology. The femtosecond laser (1030 nm, 1 MHz, 220 fs) can stimulate individual mitochondria (1 μm2) for a short period (0.1 s), whereas confocal microscopy (CM) provides continuous cell imaging to monitor molecular dynamics in real time, before and after UPLaS.
Ex-vivo Skin Permeability Tests of Nanoparticles for Microscopy Imaging
Delivery of drugs through the skin is a major challenge in the field of drug delivery systems. Quantification of materials, and specifically nanoparticles, within the skin layers is essential for the development of advanced topical and transdermal delivery systems. We have developed a technique for ex-vivo segmentation and evaluation of human skin samples treated with fluorescent nanoparticles. The method is based on horizontal cryosections of skin samples, followed by confocal microscopy and image analysis. This protocol is relatively simple to perform with basic histological tools, thus it can serve for various dermatology assays.
Quantitative and Anatomical Imaging of Human Skin by Noninvasive Photoacoustic Dermoscopy
Imaging plays a vital role in the diagnosis and treatment of skin diseases. However, pure optical imaging technique is limited to the visualization of superficial skin tissues. Ultrasonic imaging technique can detect deep tissues, but it lacks detailed information on microscopic pathological structures. Photoacoustic imaging is an advanced technology that bridges the spatial-resolution gap between optical and ultrasonic techniques, by the modes of optical excitation and acoustic detection. Photoacoustic dermoscopy (PAD), based on photoacoustic technology, can noninvasively obtain high-resolution anatomical structures by endogenous absorbers, such as melanin, hemoglobin, lipids, etc. In the past years, PAD has gradually been developed in clinical dermatology for the diagnosis of melanoma, psoriasis, port-wine stains, dermatitis, skin grafting, and testing the efficacy of cosmetics. This protocol provides detailed procedures for PAD construction, including component selection, equipment setup, and system calibration. A step-by-step guide for human skin imaging is provided as an example application. Image reconstruction and troubleshooting procedures are also elaborated. PAD offers the 3D volumetric images of human skin, and quantitatively analyzes the vascular morphology in the dermis. The protocol will provide clinicians with standardized and reasonable guidance in dermatological imaging.
Cancer Biology
Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells
Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.
Conditional Human BRD4 Knock-In Transgenic Mouse Genotyping and Protein Isoform Detection
Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions. Graphic abstract:
Cell Biology
In vivo Ca2+ Imaging in Mouse Salivary Glands
Changes in intracellular calcium drive exocrine cell activity. In the salivary gland, acetylcholine released from parasympathetic neurons mobilizes endoplasmic reticulum calcium stores in acinar cells, which consequently initiates saliva secretion. However, our understanding of the signaling cascade is mainly based on ex vivo studies performed in enzymatically isolated cells. The dissociation process likely disrupts the extracellular matrix, removes neurons as the source of signal input, and disturbs the integrity of tight and gap junctional acinar connections. These alterations may affect the spatiotemporal properties of calcium signaling events. In vivo observations of calcium signals, where tissue organization is intact, are therefore important to establish the characteristics of physiological calcium signals that are crucial for the stimulation of fluid secretion. Here, we present a detailed protocol for in vivo imaging of calcium signaling events, following nervous stimulation by multi-photon microscopy in mouse salivary gland acinar cells, expressing the genetically encoded calcium indicator GCamp6F.
Developmental Biology
Isolation and Culture of Cranial Neural Crest Cells from the First Branchial Arch of Mice
Craniofacial anomalies (CFA) are a diverse group of deformities, which affect the growth of the head and face. Dysregulation of cranial neural crest cell (NCC) migration, proliferation, differentiation, and/or cell fate specification have been reported to contribute to CFA. Understanding of the mechanisms through which cranial NCCs contribute for craniofacial development may lead to identifying meaningful clinical targets for the prevention and treatment of CFA. Isolation and culture of cranial NCCs in vitro facilitates screening and analyses of molecular cellular mechanisms of cranial NCCs implicated in craniofacial development. Here, we present a method for the isolation and culture of cranial NCCs harvested from the first branchial arch at early embryonic stages. Morphology of isolated cranial NCCs was similar to O9-1 cells, a cell line for neural crest stem cells. Moreover, cranial NCCs isolated from a transgenic mouse line with enhanced bone morphogenetic protein (BMP) signaling in NCCs showed an increase in their chondrogenic differentiation capacity, suggesting maintenance of their in vivo differentiation potentials observed in vitro. Taken together, our established method is useful to visualize cellular behaviors of cranial NCCs.
Immunology
Identification of SARS-CoV-2 Neutralizing Antibody with Pseudotyped Virus-based Test on HEK-293T hACE2 Cells
Neutralizing antibodies (NAbs) are of particular importance because they can prevent binding of the receptor binding domain (RBD) of the spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) receptor present at the surface of human cells, preventing virus entry into the host cells. The gold standard method for detection of NAbs is the plaque reduction neutralization test (PRNT). Based on the measurement of cell lysis due to viral infection, this test is able to detect antibodies that prevent cell infection (Muruato et al., 2020; Lau et al., 2021). This technique requires the use of live pathogens, i.e., SARS-CoV-2 in this case, and must be done in a biosafety level 3 (BL3) laboratory. In addition, it requires expensive installations, skillful and meticulous staff, and a high workload, which prevents its wide implementation even in research laboratories. A SARS-CoV-2 pseudovirus will express the S protein responsible for cell entrance, but will not express the pathogenic genetic material of the virus, making them less dangerous for laboratory staff and the environment. Graphic abstract:
A Mouse Infection Model with a Wildtype Salmonella enterica Serovar Typhimurium Strain for the Analysis of Inflammatory Innate Immune Cells
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative intracellular bacterium, which causes gastrointestinal disorders in humans, and systemic, typhoid fever-like infections in mice. Our current knowledge regarding the involvement of cellular and humoral immunity in the defense from S. Typhimurium infections is largely based on animal models with attenuated strains. Cells of the innate immune system act as one of the first barriers in the defense from bacteria. We established a robust experimental model for the characterization of these cell types and their response during host-pathogen interactions. Therefore, this protocol focuses on the characterization of macrophages, monocytes, and neutrophils in the spleens of infected animals by employing multi-color flow cytometry.
Medicine
Preparation, Characterization, and Cell Uptake of PLGA/PLA-PEG-FA Nanoparticles
Oral administration of colon-targeting nanoformulations holds many advantages over the systemic delivery of free drugs, or traditional nontargeting formulations in the treatment of ulcerative colitis (UC). Currently, the most conventional method for constructing colon-targeting drug delivery systems (DDS) is by integrating the biocompatible materials poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) into a copolymer. This PLGA/PLA-polyethylene glycol-folic acid (PEG-FA) copolymeric nanoformulation effectively delivers the drugs for uptake by various human colon cancer cells (e.g., HT-29 and HCT-116) and mouse colon cancer cells (CT-26). There is, however, a distinct lack of comprehensive protocols for the construction of such copolymer. This protocol details an easy-to-follow single-step method for the construction of a colon-targeting PLGA/PLA-PEG-FA nanoformulation, which encapsulates a fluorescent dye and demonstrates the visualization of its cell uptake in vitro.
Molecular Biology
Plasmid and Sequencing Library Preparation for CRISPRi Barcoded Expression Reporter Sequencing (CiBER-seq) in Saccharomyces cerevisiae
Genetic networks regulate nearly all biological processes, including cellular differentiation, homeostasis, and immune responses. Determining the precise role of each gene within a regulatory network can explain its overall, integrated function, and pinpoint mechanisms underlying misregulation in disease states. Transcriptional reporter assays are a useful tool for dissecting these genetic networks, because they link a molecular process to a measurable readout, such as the expression of a fluorescent protein. Here, we introduce a new technique that uses expressed RNA barcodes as reporters, to measure transcriptional changes induced by CRISPRi-mediated genetic perturbation across a diverse, genome-wide library of guide RNAs. We describe an exemplary reporter based on the promoter that drives His4 expression in these guidelines, which can be used as a framework to interrogate other expression phenotypes. In this workflow, a library of plasmids is assembled, encoding a CRISPRi guide RNA (gRNA) along with one or more transcriptional reporters that drive expression of guide-specific nucleotide barcode sequences. For example, when interrogating regulation of the budding yeast HIS4 promoter normalized against a control housekeeping promoter that drives Pgk1 expression, this plasmid library contains a gRNA expression cassette, a HIS4 reporter driving expression of one gRNA-specific nucleotide barcode, and a PGK1 reporter driving expression of a second, gRNA-specific barcode. Long-read sequencing is used to determine which gRNA is associated with these nucleotide barcodes. The plasmid library is then transformed into yeast cells, where each cell receives one plasmid, and experiences a genetic perturbation driven by the guide on that plasmid. The expressed RNA barcodes are extracted in bulk and quantified using high-throughput sequencing, thereby measuring the effect of their corresponding gRNA on barcoded reporter expression. In the case of the HIS4 reporter described above, guides disrupting translation elongation will increase expression of the associated HIS4 barcode specifically, without changing expression of the PGK1 control barcode. It is further possible to quantify plasmid abundance by DNA sequencing, as an additional approach to normalize for differences in plasmid abundance within the population of cells. This protocol outlines the steps to prepare barcode reporter CRISPRi plasmid libraries, link guides to barcodes with long-read sequencing, and measure expression changes through barcode RNA and DNA sequencing. This method is ideal for probing transcriptional or post-transcriptional regulation, as it measures the effects of a genetic perturbation by directly quantifying reporter RNA abundance, rather than relying on indirect growth or fluorescence readouts.Graphic abstract:
A Rapid FRET Real-Time PCR Protocol for Simultaneous Quantitative Detection and Discrimination of Human Plasmodium Parasites
Malaria is the most important parasitic disease worldwide, and accurate diagnosis and treatment without delay are essential for reducing morbidity and mortality, especially in P. falciparum malaria. Real-time PCR is highly sensitive and highly specific, therefore an excellent diagnostic tool for laboratory detection and species-specific diagnosis of malaria, especially in non-endemic regions where experience in microscopic malaria diagnostics is limited. In contrast to many other real-time PCR protocols, our new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) allows the quantitative and species-specific detection of all human Plasmodium spp. in one run. Species identification is based on single nucleotide polymorphisms (SNPs) within the MalFL probe, detectable by melting curve analysis. A significant advantage of our FRET-qPCR is the short turn-around time of less than two hours, including DNA extraction, which qualifies it for routine diagnostics. Rapid and reliable species-specific malaria diagnosis is important, because treatment is species-dependent. Apart from first-line diagnosis, the quantitative results of our new FRET-qPCR can be helpful in therapy control, to detect early treatment failure. Graphic abstract:
Neuroscience
Multiplexing Thermotaxis Behavior Measurement in Caenorhabditis elegans
Thermotaxis behaviors in C. elegans exhibit experience-dependent plasticity of thermal preference memory. This behavior can be assayed either at population level, on linear temperature gradients, or at the individual animal level, by radial isothermal or microfluidic tracking of orientation. These behaviors are low-throughput as well as variable, due to the inherent sensitivity to environmental perturbations. To facilitate reproducible studies, we describe an updated apparatus design that enables simultaneous runs of three thermal preference assays, instead of single-run assays described previously. By enabling parallel runs of control and experimental conditions, this set-up enables more throughput and rigorous assessment of behavioral variability.
Stimulus-induced Robust Narrow-band Gamma Oscillations in Human EEG Using Cartesian Gratings
Stimulus-induced narrow-band gamma oscillations (20–70 Hz) are induced in the visual areas of the brain when particular visual stimuli, such as bars, gratings, or full-screen hue, are shown to the subject. Such oscillations are modulated by higher cognitive functions, like attention, and working memory, and have been shown to be abnormal in certain neuropsychiatric disorders, such as schizophrenia, autism, and Alzheimer’s disease. However, although electroencephalogram (EEG) remains one of the most non-invasive, inexpensive, and accessible methods to record brain signals, some studies have failed to observe discernable gamma oscillations in human EEG. In this manuscript, we have described in detail a protocol to elicit robust gamma oscillations in human EEG. We believe that our protocol could help in developing non-invasive gamma-based biomarkers in human EEG, for the early detection of neuropsychiatric disorders.
Plant Science
In vitro Auto- and Substrate-Ubiquitination Assays
The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto- and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.