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Past Issue in 2018
Volume: 8, Issue: 9
Biochemistry
The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw conclusions regarding autophagic cargo flux. Here, we provide a protocol to quantitatively assess bulk autophagic flux by employing the long-lived protein degradation assay. Our procedure, which traces the degradation of 14C valine-labelled proteins, is simple and quick, allows for processing of a relatively large number of samples in parallel, and can in principle be used with any adherent cell line. Most importantly, it enables quantitative measurements of endogenous cargo flux through the autophagic pathway. As such, it is one of the gold standards for studying autophagic activity.
Cancer Biology
An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells
In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death.
Cell Biology
Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining
In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.
Immunology
Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation
We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
Nab Escaping AAV Mutants Isolated from Mouse Muscles
Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.
Microbiology
Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
Time-of-addition and Temperature-shift Assays to Determine Particular Step(s) in the Viral Life Cycle that is Blocked by Antiviral Substance(s)
Viruses infect their host cells to produce progeny virus particles through the sequential steps of the viral life cycle, such as viral attachment, entry, penetration and post-entry events. This protocol describes time-of-addition and temperature-shift assays that are employed to explore which step(s) in the viral life cycle is blocked by an antiviral substance(s).
Infection Process Observation of Magnaporthe oryzae on Barley Leaves
Rice blast and wheat blast caused by Magnaporthe oryzae is a serious threat to rice and wheat production. Appropriate methods for observing M. oryzae infection process are important for study of the fungal infection mechanisms, plant resistance reactions, and host-M. oryzae interactions. The rice leaf sheath is commonly used to inoculate M. oryzae for observing the infection process, however, this method is a time-consuming and high technical work. Here, we describe an easier solution to observe M. oryzae infection process on barley leaves.
Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803
Cyanobacteria, which have the extraordinary ability to grow using sunlight and carbon dioxide, are emerging as a green host to produce value-added products. Exploitation of this highly promising host to make products may depend on the ability to modulate the glucose metabolic pathway; it is the key metabolic pathway that generates intermediates that feed many industrially important pathways. Thus, before cyanobacteria can be considered as a leading source to produce value-added products, we must understand the interaction between glucose metabolism and other important cellular activities such as photosynthesis and chlorophyll metabolism. Here we describe reproducible and reliable methods for measuring extracellular glucose and glycogen levels from cyanobacteria.
Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta
MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript.
Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection
Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To study the inhibition of persistent norovirus replication by small molecule antivirals in vivo, we used a murine norovirus CR6 strain (MNV.CR6). We demonstrated earlier that efficient small molecules can reduce viral shedding in the stool of infected mice. Here we present how to generate the MNV.CR6 virus stock, infect type I and II interferon receptor knockout AG129 mice via oral gavage, administer antivirals to mice, and quantify viral genome copies in the stool of these mice.
A Modified Low-quantity RNA-Seq Method for Microbial Community and Diversity Analysis Using Small Subunit Ribosomal RNA
We propose a modified RNA-Seq method for small subunit ribosomal RNA (SSU rRNA)-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10-100 ng) and does not require a DNA removal step. Using this method, we could obtain more 16S rRNA sequences of the same regions (variable regions V1-V2) without the interference of DNA in order to analyze OTU (operational taxonomic unit)-based microbial communities and diversity. The generated SSU rRNA sequences are also suitable for the coverage evaluation for bacterial universal primer 8F (Escherichia coli position 8 to 27), which is commonly used for bacterial 16S rRNA gene amplification. The modified RNA-Seq method will be useful to determine potentially active microbial community structures and diversity for various environmental samples, and will also be useful for identifying novel microbial taxa.
Molecular Biology
Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies
Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.
Plant Science
Functional Evaluation of the Signal Peptides of Secreted Proteins
Here, we describe a method that can be used to evaluate the function of predicted signal peptides. This method utilizes the yeast Saccharomyces cerevisiae YTK12 strain and pSUC2 vector in which the pSUC2 vector with fused predicted signal peptide is transformed into yeast. The function of the signal peptides can be evaluated by using different selective media and color reaction. In this protocol, we provide the detailed description of manipulation in order to implement easily.
Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria
Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis. Furthermore, chlorophyll fluorescence from cyanobacteria reflects the condition of respiration, since cyanobacterial photosynthesis shares several components of electron transport chain with respiration. This protocol presents the method to monitor the condition of both photosynthesis and respiration in cyanobacteria simply by measuring chlorophyll fluorescence in the dark and in the light with pulse amplitude modulation (PAM) chlorophyll fluorometer.
Stem Cell
Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.