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Past Issue in 2020
Volume: 10, Issue: 20
Cancer Biology
Quantification of Salivary Charged Metabolites Using Capillary Electrophoresis Time-of-flight-mass Spectrometry
Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. i.e., the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.
Real-time Three-dimensional Tracking of Endocytic Vesicles
Endocytic trafficking and recycling are fundamental cellular processes that control essential functions such as signaling protein complexes transport and membrane identity. The small GTPase Rabs are indispensable component of the endosomal recycling machinery. The Rabs bind to effectors to mediate their functions, such as protein sorting and degradation, membrane tethering or lipid modification, and organelle motility. Due to the complex and dynamic nature of endosomal compartments and tracking route, detailed multiparametric analyses of three-dimensional data by quantitative methods are challenging. Here, we describe a detailed time-lapse imaging protocol designed for the quantitative tracking of single endosomal vesicles, using GFP-Rab4-positive recycling endosomes. This method permits automated tracking of single endocytic vesicles in three-dimensional live cell imaging, allowing the study of multiple parameters such as abundance, speed, directionality, and subcellular localization, as well as protein colocalization. This protocol can be broadly used in any kind of cellular models, under various contexts, including growth factors stimulation, gene knockdowns, drug treatments, and is suitable for high throughput screens.
Cell Biology
Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Caenorhabditis elegans Males
Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for in situ filming of spermatocyte meiosis in whole immobilized C. elegans males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe the workflow for the acquisition and processing of data to obtain quantitative information about the dynamics of chromosome segregation in spermatocyte meiosis I and II. In addition, our newly developed approach allows us to re-orient filmed spindles in silico, regardless of the initial 3D orientation in the worm, and analyze spindle dynamics in living worms in a statistically robust manner. Our live-imaging approach is also applicable to C. elegans hermaphrodites and should be expandable to other fluorescently labelled nematodes or other fully transparent small model organisms.
Microbiology
A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.
Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803
Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 μM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803.
Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily obtained through a formula that requires as parameters: chromosome size, S-phase duration, and replication rate. The chromosome size for any organism can be acquired in genomic databanks (such as NCBI), the S-phase duration can be estimated by monitoring DNA replication, and the replication rate is obtained through the DNA combing approach. The estimation of MO is a simple, quick, and easy method that provides a new methodological framework to assist studies of mapping replication origins in any organism.
Molecular Biology
Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ability to incorporate experimental data, such as NMR Chemical Shift Perturbations and biochemical protein-peptide interaction data, as restraints to guide the docking process. Based on the modelling results, a rational mutagenesis approach is used to validate the generated models. The experimental results allow to select a final structural model best representing the bona fide protein-peptide complex. The described protocol can also be applied to model protein-protein complexes. There is no size limit for the macromolecular complexes that can be characterized by HADDOCK as long as the 3D structures of the individual components are available.
Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications (2′-O-methylribonucleotide and ribonucleotide sugar substitutions), two enzymes (T4 polynucleotide kinase and T4 RNA ligase 2), and the differential susceptibility of DNA and RNA to hydroxide treatment. Radioactive probe molecules produced by this protocol are homogeneous and oxidant-compatible, and they can be used for precise cleavage-site mapping in the context of both DNase enzyme characterization and DNA footprinting assays.Graphic abstract
Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5′ end, or downstream within the body of the mRNA. The identification and location of recapping events is key to understanding the functional consequences of this process. Here we present an approach that addresses this problem, using the Lexogen TeloPrime® cDNA synthesis kit to tag recapped 5′ ends. TeloPrime uses a proprietary DNA ligase to add a double stranded DNA oligonucleotide onto the 3′ end of cDNA while it is base paired with mRNA. Specificity for capped ends is obtained by the oligonucleotide having an unpaired C residue that base pairs weakly with m7G on the mRNA 5′ end. This is followed by PCR amplification of double-stranded cDNA using primers to the appended oligonucleotide and the mRNA of interest. The resulting products are gel purified and sequenced directly (if a single band) or cloned and sequenced. The sequence at the junction between the ligated oligonucleotide and the target mRNA provides the location of the cap on the corresponding transcript. This assay is applicable to all capped transcripts. It can be used with Sanger sequencing for small numbers of transcripts or adapted for use with Illumina library sequencing.
Neuroscience
Staining and Quantitative Analysis of Myelinating Oligodendrocytes in the Mouse Grey Matter
Oligodendrocytes generate distinct patterns of myelination throughout the CNS. Variations in myelination along axons may enable neurons to fine-tune conduction velocities and alter signal synchronisation. Here we outline a staining protocol permitting the assessment of the number and length of myelin sheaths formed by oligodendrocyte in the mouse grey matter. This protocol enables the investigation of myelination without the need for reporter mice or technically challenging protocols, aiding the investigation of factors influencing myelin production in the brain.
Plant Science
Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated ‘tagmentation’. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.
Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
Acidified Blue Ink-staining Procedure for the Observation of Fungal Structures Inside Roots of Two Disparate Plant Lineages
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Identifying microscopic mycorrhizal fungal structures in roots, i.e., hyphae, vesicles and arbuscules, requires root staining procedures that are often time consuming and involves chemicals known to present health risks from exposure. By modifying established protocols, our root staining method stains roots using a safe ink- and vinegar-based staining solution, followed by a 2-16 h-long de-staining period. The entire procedure can be completed in less than 6 h (plus up to 16 h de-staining overnight) and roots are suitable for semi-permanent and permanent slide mounting for light microscopy. We tested our method on hundreds of wild-sourced roots from two different plant species: Lycopodiella inundata, a herbaceous clubmoss with tough water-resistant roots, and Sambucus nigra, a temperate woody shrub. Both plants associate with endomycorrhizae, L. inundata predominantly with Mucoromycotina fine root endophytes (MucFRE) and S. nigra with Glomeromycota arbuscular mycorrhizal fungi (AMF). Here we describe a simple, efficient, repeatable and safe method to detect the presence of fungal structures using light microscopy.
Quantification of Methylglyoxal Levels in Cowpea Leaves in Response to Cowpea Aphid Infestation
Aphids are a serious pest of crops across the world. Aphids feed by inserting their flexible hypodermal needlelike mouthparts, or stylets, into their host plant tissues. They navigate their way to the phloem where they feed on its sap causing little mechanical damage to the plant. Additionally, while feeding, aphids secrete proteinaceous effectors in their saliva to alter plant metabolism and disrupt plant defenses to gain an advantage over the plant. Even with these arsenals to overcome plant responses, plants have evolved ways to detect and counter with defense responses to curtail aphid infestation. One of such response of cowpea to cowpea aphid infestation, is accumulation of the metabolite methylglyoxal. Methylglyoxal is an α,β-dicarbonyl ketoaldehyde that is toxic at high concentrations. Methylglyoxal levels increase modestly after exposure to a number of different abiotic and biotic stresses and has been shown to act as an emerging defense signaling molecule at low levels. Here we describe a protocol to measure methylglyoxal in cowpea leaves after cowpea aphid infestation, by utilizing a perchloric acid extraction process. The extracted supernatant was neutralized with potassium carbonate, and methylglyoxal was quantified through its reaction with N-acetyl-L-cysteine to form N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine, a product that is quantified spectrophotometrically.
Stem Cell
Derivation of Induced Pluripotent Stem Cells from Human Fibroblasts Using a Non-integrative System in Feeder-free Conditions
Induced pluripotent stem cells (iPSCs) are genetically reprogrammed somatic cells that exhibit features identical to those of embryonic stem cells (ESCs). Multiple approaches are available to derive iPSCs, among which the Sendai virus is the most effective at reprogramming different cell types. Here we describe a rapid, efficient, safe, and reliable approach to reprogram human fibroblasts into iPSCs that are compatible with future iPSCs uses such as genome editing and differentiation to a transplantable cell type.