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Past Issue in 2015
Volume: 5, Issue: 9
Biochemistry
Protein Extraction from Drosophila Embryos and Ovaries
Here we provide the description of protocols to efficiently obtain protein extracts from embryos and ovaries of Drosophila melanogaster. These protocols are routinely applied in our laboratory and are based on two techniques: either embryos or ovaries are homogenized using a pestle and then the soluble proteins separated by centrifugation, or embryos are individually lysed by needle manipulation. The latter technique allows the use of small embryo numbers and the selection of specific developmental stages (Guilgur et al., 2014).
Microbiology
Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria
This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to cellular stress level; combined pigments assessment provides useful insight into cellular physiological state. The current protocol was established to minimize time and equipment requirements for the routine pigments analysis. It is important to note that this protocol is suitable only for cyanobacteria containing chlorophyll a, and is not designed for species containing other chlorophyll molecules.
Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma
Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology.
Molecular Biology
FLP/FRT Induction of Mitotic Recombination in Drosophila Germline
The FLP/FRT system is a site-directed recombination technology based on the targeting of a recombination enzyme (flipase - FLP) to specific DNA regions designated as flipase recognition target (FRT) sites. Initially identified in Saccharomyces cerevisiae, the yeast FLP-enzyme and its FRT recombination targets were successfully transferred into each major chromosome arm in Drosophila (Golic and Lindquist, 1989). This offers the ability to mediate mitotic recombination in vivo during development in a controlled manner [revised in Theodosiou and Xu (1998)]. The controlled induction of the mitotic recombination events is usually performed by expressing the FLP under the control of the heat-shock (hs) promoter. This allows the expression of high FLP levels at specific developmental time windows. Strains carrying these genetically marked FLP/FRT chromosomes have greatly enhanced our ability to study gene function in both germline and somatic Drosophila tissues. Here we describe two different protocols: One to induce and identify homozygous mutant clones in ovaries and the other to generate female germline mutants for the analysis of maternal effects on embryogenesis.
Neuroscience
ELISA Detection of Endogenous Serum Albumin in the Mouse Brain: A Measure of Extravasation Following Brain Injury
After stroke and brain contusion, serum proteins extravasate into nerve tissue through disrupted blood-brain barrier (BBB). Because extravasations of serum proteins result in vasogenic brain edema, serum albumin level in the brain is an indicator of BBB disruption and brain edema after brain insults. In this protocol, extravasation of endogenous albumin is measured in the damaged mouse brain, which would be valuable in the evaluation of vasogenic brain edema formation (Michinaga et al., 2014).
Plant Science
Chlorophyll Fluorescence Measurements in Arabidopsis Plants Using a Pulse-amplitude-modulated (PAM) Fluorometer
In this protocol, to analyze PSII activity in photosynthesis, we measure the Fv/Fm (Fv=Fm ± Fo) value (Fo and Fm are the minimum and maximum values of chlorophyll fluorescence of dark-adapted leaves, respectively). Fv/Fm is a reliable marker of photo- inhibition (Krause et al., 1988). Chlorophyll fluorescence in leaves was measured at room temperature using a photosynthesis yield analyzer (MINI- PAM, Walz, Effeltrich, Germany) and a pulse-amplitude-modulated (PAM) fluorometer (TEACHING-PAM, Walz, Effeltrich, Germany).
The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity
Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.
A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells
Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.
Quantification of Ralstonia solanacearum Colonization of Potato Germplasm Using Luminescence
We have developed an unsophisticated, non-disruptive and accurate method for evaluation of pathogen colonization in planta. In this protocol we use a Ralstonia solanacearum (R. solanacearum) UY031 strain genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. This system allows bacterial quantification in a high-throughput manner, avoiding time-consuming and tedious bacterial dilution plating and colony counting. In addition, this system could be especially useful in plant breeding programs to detect bacterial latent growth in symptomless parental lines before their inclusion in long-term disease resistance breeding programs.
MIFE Technique-based Screening for Mesophyll K+ Retention for Crop Breeding for Salinity Tolerance
Potassium is known as a rate-limiting factor for crop yield and plays an important role in plants response under abiotic stresses. Recently, cytosolic K+ retention ability in leaf mesophyll has emerged as an important component of plant salt tolerance mechanism (Wu et al., 2013; Wu et al., 2014; Wu et al., 2015). In this protocol, the procedure for screening leaf mesophyll for K+ retention by the MIFE (microelectrode ion flux estimation) technique is described in detail using wheat as an example. By measuring NaCl-induced K+ efflux in leaf mesophyll, a large number of plant accessions can be screened and categorised according to their salinity stress tolerance. The method provides a rapid and reliable tool that targets the activity of specific membrane transporters directly contributing to salinity tolerance trait and, because of this, has a competitive advantage over traditional whole-plant phenotyping. While the focus of this protocol is on wheat, the suggested method may be adopted for screening K+ retention in leaf mesophyll in any other crop species.
An Assay to Test Manganese Tolerance in Arabidopsis
Manganese (Mn) is an essential nutrient required for the catalytic or regulatory function of several cellular enzymes. However, excessive Mn concentrations in plant tissues are toxic to plant cells as they negatively affect enzymatic activities, lead to oxidative stress and disturb the uptake and distribution of other essential mineral elements (Ca, P, Mg or Fe). Plants have developed multiple mechanisms to avoid heavy metals (including Mn) toxicity, including transport across the plasma membrane or tonoplast. The genes encoding transporters involved in Mn detoxification are now being identified in different plant species, and functional characterization of genes isolated from species can be easily carried out in Arabidopsis. Here we provide a method to evaluate the tolerance to excess Mn of Arabidopsis lines transformed with empty vector pMDC43 or the same vector carrying cucumber gene CsMTP8 encoding putative manganese transporter localized in the vacuolar membrane. We analyzed the growth and developmental phenotypes of plants grown in controlled conditions (phytotrone) on sterile plates containing different concentrations of MnSO4 during a 16 days period. Mn accumulation was measured in the same plants grown in liquid medium supplemented or not (control) with toxic Mn concentration.
Arabidopsis Metabolome Analysis Using Infusion ESI FT-ICR/MS
We made the method for Arabidopsis metabolome analysis based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) (IonSpec). This method was sufficiently applied to metabolic phenotyping of Arabidopsis. This method is simple in that after homogenizing samples, powdered samples are dissolved in extraction solvents (acetone and methanol) to 20% fresh weight/volume. Extracted sample solutions are dried and dissolved in 50% (v/v) acetonitrile. Mass analysis using FT-ICR/MS (IonSpec) is performed in positive and negative ionization operation modes. Mass spectra are acquired over the 100-1,000 m/z range and accumulated to improve the S/N ratio.
Stem Cell
Glioma Associated Stem Cells (GASCs) Isolation and Culture
Glioma Associated Stem Cells (GASCs) represent a population of non-tumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma (Bourkoula et al., 2014).