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Past Issue in 2016
Volume: 6, Issue: 5
Cancer Biology
Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines
The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2-4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec).
Laser Microirradiation and Temporal Analysis of XRCC1 Recruitment to Single-strand DNA Breaks
The DNA molecule is exposed to a multitude of damaging agents that can compromise its integrity: single (SSB) and double strand breaks (DSB), intra- or inter-strand crosslinks, base loss or modification, etc. Many different DNA repair pathways coexist in the cell to ensure the stability of the DNA molecule. The nature of the DNA lesion will determine which set of proteins are needed to reconstitute the intact double stranded DNA molecule. Multiple and sequential enzymatic activities are required and the proteins responsible for those activities not only need to find the lesion to be repaired among the millions and millions of intact base pairs that form the genomic DNA but their activities have to be orchestrated to avoid the accumulation of toxic repair intermediates. For example, in the repair of Single Strand Breaks (SSB) the proteins PARP1, XRCC1, Polymerase Beta and Ligase III will be required and their activities coordinated to ensure the correct repair of the damage.Furthermore, the DNA is not free in the nucleus but organized in the chromatin with different compaction levels. DNA repair proteins have therefore to deal with this nuclear organization to ensure an efficient DNA repair. A way to study the distribution of DNA repair proteins in the nucleus after damage induction is the use of the laser microirradiation with which a particular type of DNA damage can be induced in a localized region of the cell nucleus. The wavelength and the intensity of the laser used will determine the predominant type of damage that is induced. It is important to note that other lesions can also be generated at the microirradiated site. Living cells transfected with the fluorescent protein XRCC1-GFP are micro-irradiated under a confocal microscope and the kinetics of recruitment of the fluorescent protein is followed during 1 min. In our protocol the 405 nm laser is used to induce SSB.
Quantification of Rae-1 Staining Intensity in Glomeruli
NKG2D is expressed on all NK cells and on subsets of NKT, CD8, CD4 and γδ T cells. NKG2D is activated by NKG2D ligands, a stress-induced family of MHC-I-like proteins. These ligands are upregulated on stressed/infected cells and are not widely expressed on healthy adult tissue. NKG2D ligands have been widely studied as potential targets for immunotherapeutic approaches in cancer and auto-immune diseases such as Systemic Lupus Erythematosus. Here we describe a method for quantifying the expression levels of the Rae-1 NKG2D ligand in the glomeruli of healthy and diseased individuals via a novel algorithm. The MRL mouse was used as a positive control strain as it spontaneously generates a lupus-like phenotype, one of the main effects of which is severe glomerulonephritis. MRL/MpJ mice develop this phenotype spon¬taneously at ~12 months of age, whereas MRL/MpJlpr mice, which have the same genetic background but which generated a spontaneous homozygous mutation in the Fas allele, develop similar but more severe symptoms by ~3-4 months of age (Spada et al., 2015).
Immunology
Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction
Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12, IL-4/IL-5 and IL-23 producing DCs induce TH1, TH2 and TH17 type of immune responses, respectively (Kumar et al., 2015). Besides cytokines, levels of co-stimulatory molecules on DCs also influence the response of T cells. The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs. A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.
Microbiology
Isolation of Genomic DNA from Mycobacterium Species
Isolation of genomic DNA from Mycobacterium species has been a tedious procedure. This can primarily be attributed to thick and waxy cell wall of mycobacteria which hampers lysis of the bacterial cell. We have tested various approaches to isolate mycobacterial DNA and based on this, an optimized protocol is presented here. This protocol involves initial incubation of mycobacteria with lysozyme, followed by SDS-proteinase K treatment to bring about cell disruption. In the case of slowly growing mycobacteria such as BCG (Bacillus Calmette–Guérin) or Mycobacterium tuberculosis (M. tuberculosis), an intermediate step of cell lysis by physical method results in significantly enhanced yield.
Testing the Effect of UV Radiation on the Survival of Burkholderia glumae
Burkholderia glumae (B. glumae) is becoming a serious threat in the major rice producing areas of the world. It was reported that Burkholderia spp., including B. glumae, are adapted to a wide range of ecological niches. Different bacterial strains show different levels of UV tolerance which may be due to the presence of different protection mechanisms. Previously we reported that pigment producing strains of B. glumae are more tolerant to UV radiation than non-pigmented strains. Here, we describe the protocol of UV tolerance assay for B. glumae in different exposure times. Using this protocol, we can calculate the survival rate of B. glumae strains, as well as other bacterial species, in exposure to UV radiation.
Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was quantified with antibodies specific to the S18-2 protein and secondary antibodies, in chemiluminescent ELISA. In this way displacement of S18-2 protein by the S18-1 protein was reported as a percentage of the full strength signal achieved through saturation of S6 with S18-2. At its foundation, this method exploits a native protein-protein interaction and could be applied to other systems where two or more proteins compete for binding to a target ligand as above.
Campylobacter jejuni γ-glutamyltranspeptidase Activity Assay
The enzyme γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) is highly conserved among eukaryotic and prokaryotic organisms (Heisterkamp et al., 2008) and has a key function in glutathione metabolism. Although the enzyme is highly conserved and found throughout organisms ranging from bacteria to plants and animals several major difference between eukaryotic and prokaryotic GGT can be noticed. They mainly concern the enzyme localization and posttranslational modification. Eukaryotic GGT is cell membrane anchored and highly glycosylated whereas prokaryotic GGT does not undergo this posttranslational modification and is a soluble periplasmic protein. GGT amino acids sequences of diverse origin exhibit high amino acid similarity (Ong et al., 2008). The prokaryotic GGT enzymes are produced as proenzyme, equipped with a typical prokaryotic signal sequence and transported through the inner membrane into the periplasm where the enzyme undergoes autocatalytic cleavage. This proteolysis yields a mature dimer which transfers the γ-glutamyl moieties from extracellular glutathione and related compounds to amino acids or peptides (Hanigan et al., 1998). The GGT enzyme activity can be easily measured as it catalyzes the transfer of a γ-glutamyl group from a colorless substrate, L-γ-glutamyl-3-carboxy-4-nitroanilide, to the acceptor, glycylglycine with leads to the production of yellow colored product, p-nitroaniline (Figure 1) which can be measured by a spectrophotometer (Figure 2). Here we describe a protocol to measure the GGT activity in the Gram-negative bacterium Campylobacter jejuni, with some minor modifications; this protocol also works for other Gram-negative bacterial species.Figure 1. Yellow colored product, p-nitroaniline formed during the GGT enzyme assayFigure 2. Spectral curve of pNA in Tris/HCl buffer, recorded on a Biodrop µLite (Isogen)
Molecular Biology
Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs
Numerous biological functions such as cytokinesis, changes in cell shape and cell migration require actomyosin-driven cellular contractility. However, the detailed mechanism of how contractile forces drive cellular processes are difficult to decipher due to the complexity of the intracellular environment. In particular, the mesoscopic description of the myosin II-dependent actin retrograde flow in cell lamellum is missing. Here, we describe a methodology for detergent extraction of cell, which preserves integrity of the actin cytoskeleton. This semi-in vitro cell model allows for the observation, using light microscopy, and quantification of changes in the actin cytoskeleton resulting from the activation of cellular contractility upon addition of ATP. This assay also allows for the evaluation of the effects of actin-associated proteins and other related factors in the modulation of the actin contractile activities. Here, we demonstrate the retrograde flow of a well-known actin-based structures- transverse arcs, which are myosin IIA-containing structures that emerge at the boundary between lamellipodium-lamellum and move centripetally in myosin II-dependent fashion.
Neuroscience
A Novel Task for Studying Memory of Occasional Events in Rats
Episodic memory has been defined in humans as the conscious recollection of unique personal past experiences often occurring singly during daily life, including remembrance of what happened, where and when it happened (Tulving, 1972). Here, we propose and describe in details a novel protocol we recently used to test the ability of rats to form and recollect episodic-like memory of previously encountered occasional episodes (Veyrac et al., 2015). During these episodes, the animals are briefly exposed to sets of specific odor–drink associations (what happened) encountered in specific locations (where it happened) within different multisensory enriched environments (in which context/occasion it happened). Memory of the episodes can be tested at relatively short (24 h) or much longer (24 d) delays in either a low or high interfering retrieval situation. This novel paradigm brought evidence for individual memory profiles of recall performance that might be correlated to different aspects of brain functional networks. More generally, it offers novel possibilities to explore cellular and network mechanisms that underlie memory of past events and memory dysfunction in brain pathologies.
Plant Science
Experimental Design to Determine Drought Stress Response and Early Leaf Senescence in Barley (Hordeum vulgare L.)
Premature leaf senescence induced by drought stress is a main factor for yield losses in barley. Research in drought stress tolerance has become more important as due to climate change the number of drought periods will increase and tolerance to drought stress has become a goal of high interest in barley breeding. However, reliable screening for drought stress tolerance is still a difficult task. This protocol describes the experimental design for the phenotyping for drought stress tolerance and early leaf senescence in the juvenile stage of barley (A) and the determination of six physiological parameters involved in drought tolerance and leaf senescence (B to G) according to Wehner et al., (2015).
Shoot Regenerative Capacity Assays in Arabidopsis and Tobacco
Plant regeneration refers to a process through which an explant is differentiated to a whole plant under certain conditions. It has been shown that two plant hormones, auxin and cytokinin, play critical roles within this process (Duclercq et al., 2011). Cytokinin induces shoot regeneration, whereas auxin promotes root production. In addition to hormones, recent study has revealed that age cue serves as a common element behind plant cell totipotency (Toledano et al., 2012). Here we present an easy protocol to assess the shoot regenerative capacity of Arabidopsis and tobacco leaves of different ages.
Semi-thin Sectioning, Light and Fluorescence Microscopy of Floral Bud to Study Microspore Development in Arabidopsis
Pollen grains are male gametophytes produced within the pollen sacs of the anthers of the flower. Recent genetic studies have revealed several components involved in microspore development (Borg et al., 2009; Berger and Twell, 2011), and yet many components controlling microspore development remain to be identified. Semi-thin sectioning of anthers and light and fluorescence microscopy of floral bud (Kim et al., 2015) are the initial key experiments to characterize Arabidopsis mutants and transgenic plants for understanding the roles of new genetic components during microspore development. Herein, we describe a protocol for semi-thin sectioning of anthers and light and fluorescence microscopy of floral bud in Arabidopsis.
Immunogold Labeling Analysis of Cell Wall Polysaccharides with Special Reference to (1;3,1;4)-β-D-glucan in Rice Cell Walls
Various types of cell wall compositions have evolved to fulfill a wide range of biological roles during the diversification of land plants. (1;3,1;4)-β-D-glucan (MLG) is a defining feature of the cell walls in the order Poales (Yokoyama and Nishitani, 2004), which has multiple functions associated with metabolic, growth, and defense systems. MLG is also a characteristic component of the matrix polysaccharides that undergo turnover and metabolism, depending on the tissue and the stage of development (Kido et al., 2015). Determining the extracellular localization of MLG is essential for elucidating its functions. Electron microscopy immunogold labeling analysis is a useful technique, which provides an accurate representation of the extracellular distribution of MLG. This strategy is also applicable to various kinds of cell wall polysaccharides, which have key roles in regulating growth and differentiation in each plant species.
Measurement of 33P-PO4 Absorption Kinetic Constants in Arabidopsis
Based on the Michaelis-Menten kinetics model (Hofstee, 1952), this method allows calculation of the kinetic parameters (Vmax, Km) of phosphate uptake by Arabidopsis roots. This method is based on the quantification of phosphate uptake by Arabidopsis roots as described in Thibaud and Marin (2016), except that a range of phosphate concentration is applied in the incubation medium. Plants are grown in high or low Pi giving access to kinetic parameters corresponding to low and high affinity respectively. In high Pi, the high-affinity transporters are not induced giving access to the low-affinity transport only. When plants are grown in low Pi, high affinity transporters are active, and the corresponding kinetic parameters can be measured. The calculation of Km and Vmax values is based on the Michaelis-Menten kinetics model.
Measurement of 33P-PO4 Absorption Capacity and Root-to-leaf Transfer in Arabidopsis
This method allows quantification of phosphate absorption capacity by Arabidopsis roots using very simple equipment, and can be scaled up or down.