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Past Issue in 2016
Volume: 6, Issue: 2
Cancer Biology
A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling
This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes. Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html
Microbiology
A Reliable Method for Phytophthora cajani Isolation, Sporangia, Zoospore Production and in Planta Infection of Pigeonpea
Pigeonpea (Cajanus cajan L.) is an important legume crop of rainfed agriculture. High levels of protein in pigeonpea make it a valuable protein source for developing countries. Phytophthora blight caused by Phytophthora cajani (P. cajani) is a potential threat to pigeonpea (Cajanus cajan L.) production, affecting the crop irrespective of cropping system, cultivar grown and soil types (Pande et al., 2011; Sharma et al., 2006). The primary mode of infection of P. cajani is sporangium and zoospore. Therefore, sensitive and reliable methods for zoospore production and estimating infection severity are desirable in case of Phytophthora blight of pigeonpea (Sharma et al., 2015). Here we present a protocol for isolation of P. cajani from infected plants, sporangia and zoospore production and in planta infection technique of pigeonpea seedlings. These methods will be important tool to devise a platform for rapid and reliable screening against Phytophthora blight disease of pigeonpea as well as for host x pathogen x environment interaction studies.
Preparation of Outer Membrane Vesicles from Myxococcus xanthus
Outer membrane vesicles (OMVs) represent a unique sub-cellular compartment of bacteria that may act as a scaffold for various extracellular activities, including intercellular signaling. Myxococcus xanthus (M. xanthus) is a predatory bacterium that engages in cell-cell behaviors such as fruiting body formation and contact dependent lysis of other microbes. The OMVs of M. xanthus have been shown to have an elaborate architecture of chains and tubes that can connect cells within a biofilm. These higher order OMV structures have been shown to contain proteins exchanged for community behaviors and small molecules that have antibiotic activities, and may help facilitate directed exchange. M. xanthus OMVs allow material transfer between neighboring cells for motility and predation.
HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA
Human hepatic cancer cell lines such as HepG2, Huh7, and HLE cannot get infected with Hepatitis B virus (HBV) due to lack of an HBV receptor(s). Transfection with HBV genome has so far been referred as a tool to mimic HBV infection. However, since sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HBV (Yan et al., 2012), hepatocyte cell lines that were stably transfected with a plasmid for NTCP expression have been used for HBV infection. This protocol is designed for infection with HBV in human hepatocyte cell line HepG2 expressing NTCP (HepG2-hNTCP-C4 cells; Iwamoto et al., 2014) or primary human hepatocytes (PHHs). In this section, we also describe one of the methods for the assessment of HBV infection: Quantification of the intracellular encapsidated HBV DNA.
Infection of Human Hepatocyte-chimeric Mice with HBV and in vivo Treatment with εRNA
Hepatitis B virus (HBV) can cause both acute and chronic disease in human liver with potentially high risk of cirrhosis and liver cancer. The host range of non-human primates susceptible to this virus is limited. Therefore, experimental studies with human hepatocyte-chimeric mice provide an invaluable source of information regarding the biology and pathogenesis of HBV. This section describes the protocol for infection of the human hepatocyte-chimeric mice with HBV. In addition, it has recently been shown that HBV replication can be suppressed by exogenous expression of viral epsilon RNA (εRNA; Sato et al., 2015), which serves as an encapsidation signal (Bartenschlager et al., 1992). Based upon this finding, we also describe the protocol for the liposome-mediated delivery of a plasmid encoding εRNA to liver in these chimeric mice.
Molecular Biology
Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening
We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that bind to the REQ 3’UTR, we describe the RNA ligand based cDNA expression library screening method. This protocol could be adapted to detect specific RNA-protein interactions. Following this method, we identified six positive clones in the initial round of screening and four pure clones after sib-screening. This protocol was originally published in Kim et al. (2014).
Plant Science
Cotyledon Wounding of Arabidopsis Seedlings
Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.
Agrobacterium rhizogenes-Based Transformation of Soybean Roots to Form Composite Plants
Transgenic soybean roots of composite plants are a powerful tool to rapidly test the function of genes and activity of gene promoters. No tissue culture is needed, thus avoiding loss of valuable material due to contamination. This is a simple technique that requires less training and care than tissue culture techniques. Furthermore, it takes less time to produce transgenic roots than techniques using sterile tissue culture. If the transgenic roots are to be challenged with a pathogen, there is no need to produce axenic pathogens with this technique, because sterile tissue culture medium is not used. Therefore, there is no agar medium on which contaminants may grow resulting in obscured results or diseased roots. Here, we describe the production of transgenic soybean roots on 7-day-old soybean seedlings using Agrobacterium rhizogenes. These composite plants may be grown in the greenhouse for further experimentation, such as to determine the effect of gene expression on nematode development.
Soybean Cyst Nematode, Heterodera glycines, Infection Assay Using Soybean Roots
Soybean cyst nematode (SCN; Heterodera glycines), an obligate parasite of plants, is the most damaging pathogen of soybean, causing $469 to $818 million in soybean yield losses annually in the United States. However, there are no soybean cultivars available that are resistant to all SCN populations. Therefore, much research is being conducted to develop soybean cultivars resistant to SCN (Matthews et al., 2013; Matthews et al., 2014; Youssef et al., 2013). Here we describe the rearing and harvesting of SCN, as well as how SCN can be assayed by determining the Female Index.
Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE
Starch synthases are one class of key enzymes involving in the synthesis of cereal starch, which transfer glucose from ADP-glucose to the non-reducing end of pre-existing α-(1-4)-liked glucosyl chains of amylopectin. This protocol is highly reproducible for assaying activities for starch synthase I and IIIa in wheat and barley endosperm at qualitative level and quantitative level. The protocol includes separating proteins isolated from developing endosperm with native-PAGE containing glycogen from oyster, incubating protein gels with ADP-glucose solution, and staining gels with iodine solution. The method allows researchers to compare the levels or changes of starch synthase activities.
Virus-based MicroRNA Silencing
Virus-based microRNA silencing (VbMS) is a viable and prompt method to screen and characterize the function of microRNAs (miRNAs) in plants. The Tobacco rattle virus (TRV)-based VbMS method was originally developed by the Yule Liu's group (Sha et al., 2014) using miRNA target mimic (TM) methodology. Here, we describe the TRV-based VbMS method for silencing endogenous miRNA in Nicotiana benthamiana and tomato via Agrobacterium infiltrations. For each assay, Agrobacterium cultures containing pTRV1 and specific pTRV2e derivative harboring TM fragments are mixed and infiltrated into plant tissues. Generally within 3 weeks, the target miRNAs gene will be silenced and the newly developed tissues will exhibit corresponding phenotypes.
Cytohistochemical Determination of Calcium Deposition in Plant Cells
Calcium plays important roles in maintaining plant cellular structure and also acts as a key secondary messenger in intercellular signaling. Thirty years ago, methods of detecting calcium in sub-cellular level had been established (Stockwell and Hanchey, 1982; Borgers et al., 1982) and reviewed extensively (Wick and Heplerm, 1982). We had used the method of testing calcium localization in salt tolerance improved transgenic alfalfa plant (Zhang and Wang, 2015). Here, we describe the protocol of testing calcium deposition by staining with potassium pyroantimonate (PPA) in detail, which was adapted from former reports (Stockwell and Hanchey, 1982; Borgers et al., 1982). The principle of this protocol is that the Ca2+ can react with antimonite and from black granules, which can be observed under a transmission electron microscope. The protocol includes common micromanipulation techniques of plant tissue, observation with a transmission electron microscope and photography.
LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity
Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins regulating a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. The initial Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-galactose (Figure 1). Here we describe a LC/MS-based method for the detection of HPGT activity in vitro.Figure 1. Reaction scheme for Hyp galactosylation by HPGT. HPGT catalyzes the addition of a D-galactopyranose from an UDP-α-D-Gal to the hydroxylgroup of Hyp residues.
Detection of Hydroxyproline O-galactoside by LC/MS
Hydroxyproline (Hyp) O-galactosylation is a plant-specific post-translational modification found in extracellular glycoproteins such as arabinogalactan proteins (AGPs). Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-Gal. Here we describe an LC/MS-based method for the detection of Hyp O-galactoside.