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Past Issue in 2016
Volume: 6, Issue: 14
Biochemistry
Ubiquitination Assay for Mammalian Cells
Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum
To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.
Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum
Glycolysis provides metabolites for energy production via oxidative phosphorylation during vegetative growth of Fusarium oxysporum. Therefore, determination of intracellular ATP levels might be of valuable help to analyze regulation of glycolysis/gluconeogenesis pathways. The protocol described here can be applied to other filamentous fungi.
Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.
Developmental Biology
Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.
Immunology
House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis
Asthma is a complex disease of the airways primarily mediated by T helper 2 cells and innate lymphoid type 2 cells (Licona et al.,2013). Mice do not develop spontaneous asthma and therefore models have been developed for the assessment of key processes that underlie human pathology (Nial et al.,2008). Exposure to House Dust Mite (HDM) extract induces many key features of acute airway inflammation including elevated IgE levels, eosinophilia, goblet cell metaplasia, epithelial hypertrophy and airway hyperresponsiveness (AHR) in response to methacholine (Hammad et al., 2009; Dullaers et al., 2012; Coquet et al., 2015). The exact dose and duration of exposure to HDM can affect the type and extent of inflammation. In our case, we start with a low sensitizing dose that is increased on challenge, while others use differing schedules or a higher antigen concentration during sensitization of mice (Hondowicz et al., 2016; Trompette et al., 2014; Zaiss et al., 2015). We believe that using a low sensitizing dose more accurately separates the primary and secondary immune responses and reduces the possibility that HDM given during sensitization continues to fuel the immune response during challenge (Coquet et al., 2015; Plantinga et al., 2013). Here, we outline in text, pictures and video how to administer HDM extracts or cytokines via the intranasal route and briefly touch upon the subsequent analysis of inflammation in the airways [covered otherwise in (Han et al., 2013)].
Microbiology
Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the polyP values obtained. Figure 1. Enzymatic hydrolysis of polyP
A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii
Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and Keshavan, 2006; Steindler and Venturi, 2007), or lethal gene fusions downstream QS-controlled promoters (Weiland-Bräuer et al., 2015). With three parallel QS circuits, the bioluminescent marine bacterium Vibrio campbellii (formerly harveyi, Lin et al., 2010) constitutes a complex Gram-negative model for which an extensive body of knowledge exists, including an array of mutant biosensors. In V. campbellii, bioluminescence is regulated by QS. However, bioluminescence is the result of complex biochemical networks that converge with cell respiration and fatty acid metabolism. It is also an energy-demanding reaction that strongly depends on the overall metabolic state of the bacterium, consuming up to 1/5 of the cell resources (Munn, 2011). Thus, disruption of QS-controlled phenotypes might be the result of toxic side effects or interference with the above-mentioned biochemical pathways rather than QS signaling. Therefore, adequate control experiments should be included. The protocol described herein provides a method and workflow for the identification of putative QS-disrupting compounds in Vibrio. It can also be easily adapted for other QS studies (e.g., detection of AI molecules).
Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection
Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear cells (PBMCs).
Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM diacetate). This compound diffuses into cells and intracellular cleavage by esterase enzymes liberates weakly-fluorescent DAF-FM, which reacts with NO or other specific RNS to become highly fluorescent (Kojima et al., 1999). Although quantification of fluorescence is performed using a fluorescent plate reader, it is envisioned that this protocol could be adapted for intracellular NO/RNS imaging of S. aureus biofilms by confocal microscopy. Likewise, this technique could be optimized for the detection of intracellular NO/RNS in other growth conditions (i.e., planktonic cultures) and/or in other bacteria/archaea.
Separation of Free and Bound cAMP in Mycobacteria
Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of intracellular cAMP may be bound to protein. The methods typically employed to measure cAMP are radioimmunoassay (RIA) and ELISA. However, these procedures include prior acidification of samples that would dissociate cAMP ‘bound’ to protein, and therefore represent the ‘total’ cAMP present in the sample. In this protocol, we describe a method to separate the fraction of cAMP ‘bound’ to protein from what is ‘free’ or not associated with protein. This is performed by subjecting the cytosolic fraction or the culture supernatant to filtration through a membrane with a 3 kDa cut-off. Only ‘free’ cAMP is able to pass through the membrane. Therefore, cAMP concentrations in the filtrate represent the ‘free’ cAMP in the sample. Cyclic AMP levels in the original cytosolic fraction or the culture supernatant represent the ‘total’ cAMP concentration. Subtracting the ‘free’ from the ‘total’ provides the amount of cAMP bound to protein.
Neuroscience
Mouse Subependymal Zone Explants Cultured on Primary Astrocytes
Neuroblast migration is a major component in the process of neuronal circuit assembly. In the rodent postnatal brain, the subependymal zone (SEZ) is the largest neurogenic niche where new neurons are born throughout life. These cells migrate several millimeters following a specific pathway called the rostral migratory stream (RMS) towards the olfactory bulb. Once they reach their final destination, they differentiate mainly as interneurons and integrate into already established neuronal circuits. Along the RMS, neuroblasts use a particular mode of migration known as chain migration. They stay attached to each other while migrating and are intimately associated with adjacent astrocytic processes. To dissect the molecular and cellular signals involved in neuroblast migration, we developed an in vitro system consisting of SEZ-derived explants co-cultured on top of an astrocyte monolayer.
Plant Science
Isolation of Flavonoids from Piper delineatum Leaves by Chromatographic Techniques
The genus Piper (Piperaceae) is widely distributed in the tropical and subtropical regions of the world, and species belonging to this genus are included in the Ayurvedic system of medicine and in folklore medicine of Latin America. Phytochemical investigations of Piper species have led to the isolation of several classes of physiologically active compounds such as alkaloids, amides, pyrones, dihydrochalcones, flavonoids, phenylpropanoids, lignans and neolignans. In an ongoing investigation of bioactive secondary metabolites from Piper species, herein, we describe the isolation procedure of nine flavonoids, including two chalcones and two flavanones from the leaves of Piper delineatum Trel. (Piperaceae), a shrub native to tropical regions of the Americas. All compounds were elucidated by spectroscopic and spectrometric methods, and comparison with data reported in the literature.
Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues
Fusarium graminearum (Fg) is the causal agent of Fusarium head blight disease of wheat (Triticum aestivum), oats (Avena sativa) and barley (Hordeum vulgare), which targets the floral tissues and thereby adversely impacts grain yield and quality. Mycotoxins produced by F. graminearum further limit the consumability of infected grain. In the laboratory, F. graminearum also has the ability to colonize both leaves and inflorescence tissues of Arabidopsis thaliana. The interaction between A. thaliana and F. graminearum makes available a large array of genetic and molecular tools to study the interaction between plants and F. graminearum to elucidate plant genes and pathways that contribute to resistance, as well as study how the fungus targets plant genes and mechanisms to promote disease. The methods described below allow for efficient infection of Arabidopsis leaves and inflorescence, and evaluation of disease progress and fungal growth. Disease spread in Arabidopsis can be readily monitored by the visual observations of chlorosis of leaf tissue and disease phenotype of inflorescence tissue including fungal mass on surface of the inflorescence tissue. Fungal growth can be further monitored by measuring the relative amount of Fg DNA in the host tissue by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR).