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Past Issue in 2016
Volume: 6, Issue: 7
Biochemistry
Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy
This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers.
Cancer Biology
Colon Cancer-associated Fibroblast Establishment and Culture Growth
Cancer-associated fibroblasts (CAFs) are one of the major players in tumor-stroma crosstalk. Findings in experimental studies suggest important roles for CAFs in regulation of tumor growth, metastasis and drug response (Hanahan and Coussens, 2012). Furthermore, their clinical relevance is supported by new findings from tumor analyses, demonstrating the prognostic and response-predictive significance of CAF-derived markers or gene signatures (Berdiel-hacer et al., 2014; Finak et al., 2008; Navab et al., 2011; Paulsson and Micke, 2014). CAFs are a heterogeneous pool of cell subsets with distinct functions which needs to be better defined by their marker expressions. The development of a methodology for the establishment of fibroblast primary cultures derived from human colon tumors allowed us to characterize their functional and molecular properties (Herrera et al., 2013). In addition, the different molecular mechanisms through which CAFs affect tumor growth and metastasis are still to be clarified. Therefore, functional and molecular characterization of the cancer-associated fibroblasts is essential to fully understand their role in tumor progression.
Extraction and Quantification of Tryptophan and Kynurenine from Cultured Cells and Media Using a High Performance Liquid Chromatography (HPLC) System Equipped with an Ultra-sensitive Diode Array Detector
Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.
Microbiology
Rust Removal Experiments
Iron oxidation, known as rust formation, causes enormous loss in term of property damages and associated economic risks. Depending on the degree of formation, rust consists of several layers of iron in different oxidation states. The brownish top layer is mostly iron (III) oxide-hydroxide [FeO(OH), Fe(OH)3] while the deepest black layers possess iron oxide (Fe2O3.nH2O). The flaky nature of surface rust meditates diffusion of oxygen and water to inner material sections which can lead to total disintegration of iron mass. As a result, it is desirable to remove rust and protect fresh surface from oxidizers. The common rust removal reagents are mainly based on complex formation of ferric ion with organic and inorganic acids such as citric acid, oxalic acid, and phosphoric acid. Rust removal ability is typically a qualitative observation which makes direct comparison between treatment options cumbersome if not impractical. In our recent work (Ahmadi et al., 2015), we have developed a colorimetric assay to measure ferric concentration in rust removal treatment media using a bacterially-produced siderophore (yersiniabactin) in comparison to a commercial rust removal reagent. In this approach, ferric concentration is correlated to the mass of rust being dissolved in the presence of different removal agents. This assay is based on a modification of the 1, 10-phenanthroline assay (Skoog and West, 1979) to enable detection using a 96-well plate format for higher throughput screening and assessment.
Procedure for Rhamnolipids Quantification Using Methylene-blue
Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g., bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric acid and orcinol based quantification (Helbert and Brown, 1957; Chandrasekaran and BeMiller, 1980), are laborious and operationally hazardous because of the strong acid/chemical emanation which can cause deterioration of instruments measurements (e.g., spectrophotometer). Therefore, the methylene-blue-based analysis appears as a promising alternative to safely quantify whole rhamnolipid molecules based on chemical complexation reaction (Pinzon and Ju, 2009). Indeed, methylene blue and rhamnolipids form a complex in a water-chloroform phase system. The rhamnolipids-methylene blue complex is partitioned into the chloroform phase which will develop a blue color that can be quantified at 638 nm to deduce rhamnolipids concentration. Here, we describe a variant of methylene-blue-based rhamnolipids quantification procedure that allows spectrophotometric quantification on standard 96-well plastic microplate contrarily to original methylene blue procedure that requires specific and expensive microplate due to chloroform chemical properties.
Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography
Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures.
Molecular Biology
Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers
Despite the great promise that short interfering RNA (siRNA) induced RNAi responses hold as a therapeutic modality, due to their size (~15 kDa) and high negative charge (Bumcrot et al., 2006), siRNAs have no bioavailability and require a delivery agent to enter cells (Figure 1). TAT peptide transduction domain (PTD) has been developed as an agent that mediates cellular delivery of macromolecular therapeutics that otherwise lack bioavailability, making it a tantalizing candidate for siRNA delivery (Farkhani et al., 2014). Unfortunately, when conjugated to TAT PTD, the presence of 40 negative phosphodiester backbone charges on siRNA neutralizes the cationic PTD resulting in aggregation and poor cellular delivery (Meade and Dowdy, 2007). In light of this, we synthesized a neutral RNAi trigger, termed siRiboNucleic Neutrals, for conjugation to TAT PTD (Meade et al., 2014). In brief, the negatively charged phosphodiester backbone was neutralized by synthesis with bio-reversible phosphotriester protecting groups which are specifically converted into charged phosphodiester bonds inside of cells by the action of cytoplasmic restricted thioesterases resulting in a wild type siRNA that can induce RNAi responses. Here we describe the conjugation and cellular delivery of siRNN oligonucleotides with TAT PTD delivery domain (DD) HyNic peptides.
Neuroscience
Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons
We here provide a detailed protocol for the isolation and culture of primary mouse sensory neurons. The cell bodies of sensory afferent pseudounipolar neurons are located in dorsal root ganglia (DRGs) along the vertebral column. Dissected mouse DRGs can be dissociated into single cells by enzymatic digestion to obtain primary cultures of mouse sensory neurons as performed in the studies reported by Khaminets et al. (2015).
Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices
Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc.) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.
Plant Science
Preparation of Mitotic and Meiotic Metaphase Chromosomes from Young Leaves and Flower Buds of Coccinia grandis
Somatic chromosomes are usually studied from the root tip cells of the plants for cytological investigations. In dioecious plant species like Coccinia grandis, it is very difficult to get meristematic root tip cells from the mature plants of the respective sex forms. In this report, young leaves of the respective sexual phenotypes were used as tissue samples for mitotic chromosome analysis. For meiotic preparation, flower buds of appropriate size were selected for chromosomal studies. Following protocols could be effectively used for routine chromosome preparations in other plant species as well.
Strategies for Performing Dynamic Gene Perturbation Experiments in Flowers
Dissecting the gene regulatory networks (GRNs) underlying developmental processes is a central goal in biology. The characterization of the GRNs underlying flower development has received considerable attention, however, novel approaches are required to reveal temporal and spatial aspects of these GRNs. Here, we provide an overview of the options available to perform dynamic gene perturbations to identify downstream response genes at specific stages of development in the flowers of Arabidopsis thaliana.
Expression, Purification and Enzymatic Assay of Plant Histone Deacetylases
Histone deacetylases (HDACs) catalyzing the removal of acetyl groups from lysine residues of histone and non-histone proteins play vital roles in regulation of gene transcription. In plants, HDACs can be grouped into three families, including RPD3-type, SIR2-type and plant specific HD2-type HDACs. Here we describe a method to determine plant HDAC enzymatic activity. This protocol includes expression, purification and enzymatic activity assay of recombinant plant HDACs expressed in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).
Analysis of Telomeric G-overhangs by in-Gel Hybridization
Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini by the lagging strand mechanism and post-replicative nucleolytic processing. The G-overhang is important for telomere protection as it serves as a binding platform for specific proteins and is required for t-loop formation. Hence, structure of telomeric G-overhang is an important indicator of telomere maintenance and functionality. Here we describe a method for analysis of G-overhangs in a model plant Arabidopsis thaliana by in-gel hybridization technique. This method allows relative quantification of the amount of single stranded telomeric DNA. Short telomeric probes are radioactively labeled and hybridized to DNA under non-denaturing conditions to specifically detect ssDNA. Total telomeric DNA can be measured using denaturing conditions in the same gel and this procedure usually follows the non-denaturing in-gel hybridization. Terminal nature of the ssDNA is verified by exonuclease treatment. This technique was originally developed in yeast and now is used as a major tool for G-overhang analysis in a variety of organisms ranging from human to plants.
Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).
Stem Cell
Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells
Circulating endothelial progenitor cells (EPCs) have been the focus of many clinical trials due to their roles in revascularisation following ischemic events such as acute myocardial infarction as well as their contribution to vascular repair during organ transplantation. Research on EPCs has been controversial due to the lack of distinct markers expressed at the cell surface and varying methods for isolation and culture have resulted in the identification of a multitude of cell types, with differing phenotype and function, all falling under the label of “EPCs”. The most widely documented EPCs isolated for cell therapy are adherent in nature and lacking the progenitor markers such as CD133 and therefore unlikely to represent a true circulating EPC, the cells mobilised in response to a vascular injury. We recently published the isolation and extensive characterisation of a population of non-adherent endothelial forming cells (naEFCs) (Appleby et al., 2012) (Figure 1). These cells expressed the progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ EPCs, the ‘late outgrowth EPC’ [more recently known as endothelial colony forming cells (ECFCs)] as well as mature endothelial cells (ECs). Figure 2A exemplifies the surface expression profile of the naEFCs. Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin (Figure 2A), (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature ECs increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human ECs and have contributed to various advances in scientific knowledge (Appleby et al., 2012; Barrett et al., 2011; Moldenhauer et al., 2015; Parham et al., 2015). Here, we describe the isolation and enrichment of a non-adherent CD133+ endothelial forming population of cells from human cord blood.