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Pubmed Article
Correction: The Role of Hydrodynamic Processes on Anchovy Eggs and Larvae Distribution in the Sicily Channel (Mediterranean Sea): A Case Study for the 2004 Data Set.
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PLoS ONE
PUBLISHED: 06-09-2015
[This corrects the article DOI: 10.1371/journal.pone.0123213.].
Authors: Danilo O. Carvalho, Derric Nimmo, Neil Naish, Andrew R. McKemey, Pam Gray, André B. B. Wilke, Mauro T. Marrelli, Jair F. Virginio, Luke Alphey, Margareth L. Capurro.
Published: 01-04-2014
ABSTRACT
New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world. Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12. To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.
28 Related JoVE Articles!
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Analysis of Oxidative Stress in Zebrafish Embryos
Authors: Vera Mugoni, Annalisa Camporeale, Massimo M. Santoro.
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
51328
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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Authors: Daniel T. Claiborne, Jessica L. Prince, Eric Hunter.
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
51506
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High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays
Authors: Danika L. Hill, Emily M. Eriksson, Louis Schofield.
Institutions: Walter and Eliza Hall Institute of Medical Research, University of Melbourne.
Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.  
Immunology, Issue 89, Parasitic Diseases, malaria, Plasmodium falciparum, hemozoin, antibody, Fc Receptor, opsonization, merozoite, phagocytosis, THP-1
51590
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Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Authors: Maria J. Mazon Moya, Emma Colucci-Guyon, Serge Mostowy.
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
51601
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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Authors: Sylvie Sanschagrin, Etienne Yergeau.
Institutions: National Research Council Canada.
One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.
Molecular Biology, Issue 90, Metagenomics, Bacteria, 16S ribosomal RNA gene, Amplicon sequencing, Next-generation sequencing, benchtop sequencers
51709
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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Authors: Jin-Young Koh, Sadahiro Iwabuchi, Zhengmin Huang, N. Charles Harata.
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
51879
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A Method for Microinjection of Patiria minata Zygotes
Authors: Alys M. Cheatle Jarvela, Veronica Hinman.
Institutions: Carnegie Mellon University.
Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.
Developmental Biology, Issue 91, Embryology, Patiria miniata, sea star, echinoderm, development, gene regulatory networks, microinjection, gene expression perturbation, antisense oligonucleotide, reporter expression
51913
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Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective
Authors: Beatrix Zenger, Sabine Wetzel, Jason Duncan.
Institutions: Willamette University.
Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.
Neuroscience, Issue 92, Drosophila, behavior, coordination, crawling, locomotion, nervous system, neurodegeneration, larva
51981
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
52010
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Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Authors: Yan Wei Lim, Matthew Haynes, Mike Furlan, Charles E. Robertson, J. Kirk Harris, Forest Rohwer.
Institutions: San Diego State University, DOE Joint Genome Institute, University of Colorado, University of Colorado.
The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.
Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface
52117
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Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Authors: Andreas Florian Haas, Ben Knowles, Yan Wei Lim, Tracey McDole Somera, Linda Wegley Kelly, Mark Hatay, Forest Rohwer.
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
52131
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A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples
Authors: Michael J. Rothrock Jr., Kelli L. Hiett, John Gamble, Andrew C. Caudill, Kellie M. Cicconi-Hogan, J. Gregory Caporaso.
Institutions: USDA-Agricultural Research Service, USDA-Agricultural Research Service, Oregon State University, University of Georgia, Northern Arizona University.
The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.
Molecular Biology, Issue 94, DNA extraction, poultry, environmental, feces, litter, semi-automated, microbiomics, qPCR
52161
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A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Authors: Brandon C. Shelley, Geneviève Gowing, Clive N. Svendsen.
Institutions: Cedars-Sinai Medical Center.
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Neuroscience, Issue 88, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
51219
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High Throughput Microinjections of Sea Urchin Zygotes
Authors: Nadezda A. Stepicheva, Jia L. Song.
Institutions: University of Delaware .
Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.
Developmental Biology, Issue 83, Sea Urchins, microinjection, sea urchin embryos, treatment delivery, high throughput, mouth pipette, DNA constructs, mRNAs, morpholino antisense oligonucleotides
50841
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Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes
Authors: Kathryn N. Maher, Mary Catanese, Daniel L. Chase.
Institutions: University of Massachusetts, Amherst, University of Massachusetts, Amherst, University of Massachusetts, Amherst.
RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.
Developmental Biology, Issue 79, Caenorhabditis elegans (C. elegans), Gene Knockdown Techniques, C. elegans, dsRNA interference, gene knockdown, large scale feeding screen
50693
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The ITS2 Database
Authors: Benjamin Merget, Christian Koetschan, Thomas Hackl, Frank Förster, Thomas Dandekar, Tobias Müller, Jörg Schultz, Matthias Wolf.
Institutions: University of Würzburg, University of Würzburg.
The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution1 and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation2-8. The ITS2 Database9 presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank11 accurately reannotated10. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold12 (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling13. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold. The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST14 search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE15,16 and ProfDistS17 for multiple sequence-structure alignment calculation and Neighbor Joining18 tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure. In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.
Genetics, Issue 61, alignment, internal transcribed spacer 2, molecular systematics, secondary structure, ribosomal RNA, phylogenetic tree, homology modeling, phylogeny
3806
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Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Authors: Wei Li, Shu Zhu, Yusong Zhang, Jianhua Li, Andrew E. Sama, Ping Wang, Haichao Wang.
Institutions: North Shore – LIJ Health System.
Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3. Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-γ)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.
Medicine, Issue 62, Herbal therapies, innate immune cells, cytokines, HMGB1, experimental animal model of sepsis, cecal ligation and puncture
3926
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C. elegans Tracking and Behavioral Measurement
Authors: Jirapat Likitlersuang, Greg Stephens, Konstantine Palanski, William S. Ryu.
Institutions: University of Toronto, Vrije Universiteit, Okinawa Institute of Science and Technology, University of Toronto.
We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.
Neuroscience, Issue 69, Physics, Biophysics, Anatomy, Microscopy, Ethology, Behavior, Machine Vision, C. elegans, animal model
4094
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Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction
Authors: Avdesh Avdesh, Mengqi Chen, Mathew T. Martin-Iverson, Alinda Mondal, Daniel Ong, Stephanie Rainey-Smith, Kevin Taddei, Michael Lardelli, David M. Groth, Giuseppe Verdile, Ralph N. Martins.
Institutions: Edith Cowan University, Graylands Hospital, University of Western Australia, McCusker Alzheimer's Research foundation, University of Western Australia , University of Adelaide, Curtin University of Technology, University of Western Australia .
This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.
Basic Protocols, Issue 69, Biology, Marine Biology, Zebrafish, Danio rerio, maintenance, breeding, feeding, raising, larvae, animal model, aquarium
4196
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Appetitive Associative Olfactory Learning in Drosophila Larvae
Authors: Anthi A. Apostolopoulou, Annekathrin Widmann, Astrid Rohwedder, Johanna E. Pfitzenmaier, Andreas S. Thum.
Institutions: University of Konstanz, University of Fribourg.
In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain. Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10. Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Biochemistry, Molecular Biology, Physiology, Behavior, Drosophila, fruit fly, larvae, instar, olfaction, olfactory system, odor, 1-octanol, OCT, learning, reward, sugar, feeding, animal model
4334
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Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
Authors: Phanidhar Kukutla, Matthew Steritz, Jiannong Xu.
Institutions: New Mexico State University.
The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.
Genetics, Issue 74, Infection, Infectious Diseases, Molecular Biology, Cellular Biology, Microbiology, Genomics, biology (general), genetics (animal and plant), life sciences, Eukaryota, Bacteria, metagenomics, metatranscriptome, RNA-seq, rRNA depletion, mRNA enrichment, mosquito gut microbiome, RNA, DNA, sequencing
50093
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Neonatal Subventricular Zone Electroporation
Authors: David M. Feliciano, Carlos A. Lafourcade, Angélique Bordey.
Institutions: Yale University School of Medicine .
Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates. Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Anatomy, Biomedical Engineering, Stem Cell Biology, Genetics, Neurogenesis, Growth and Development, Surgery, Subventricular Zone, Electroporation, Neural Stem Cells, NSC, subventricular zone, brain, DNA, injection, genetic engineering, neonatal pups, animal model
50197
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Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro
Authors: Paul William Dyce.
Institutions: The University of Western Ontario, Children's Health Research Institute.
Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.
Stem Cell Biology, Issue 77, Developmental Biology, Cellular Biology, Molecular Biology, Bioengineering, Biomedical Engineering, Medicine, Physiology, Adult Stem Cells, Pluripotent Stem Cells, Germ Cells, Oocytes, Reproductive Physiological Processes, Stem cell, skin, germ cell, oocyte, cell, differentiation, cell culture, mouse, animal model
50486
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Test Samples for Optimizing STORM Super-Resolution Microscopy
Authors: Daniel J. Metcalf, Rebecca Edwards, Neelam Kumarswami, Alex E. Knight.
Institutions: National Physical Laboratory.
STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.
Molecular Biology, Issue 79, Genetics, Bioengineering, Biomedical Engineering, Biophysics, Basic Protocols, HeLa Cells, Actin Cytoskeleton, Coated Vesicles, Receptor, Epidermal Growth Factor, Actins, Fluorescence, Endocytosis, Microscopy, STORM, super-resolution microscopy, nanoscopy, cell biology, fluorescence microscopy, test samples, resolution, actin filaments, fiducial markers, epidermal growth factor, cell, imaging
50579
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Viability Assays for Cells in Culture
Authors: Jessica M. Posimo, Ajay S. Unnithan, Amanda M. Gleixner, Hailey J. Choi, Yiran Jiang, Sree H. Pulugulla, Rehana K. Leak.
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
50645
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A Method for Culturing Embryonic C. elegans Cells
Authors: Rachele Sangaletti, Laura Bianchi.
Institutions: University of Miami .
C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.
Developmental Biology, Issue 79, Eukaryota, Biological Phenomena, Cell Physiological Phenomena, C. elegans, cell culture, embryonic cells
50649
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Authors: Nikki M. Curthoys, Michael J. Mlodzianoski, Dahan Kim, Samuel T. Hess.
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
50680
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High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Authors: Jana Ninković, Sabita Roy.
Institutions: University of Minnesota, University of Minnesota, 3M Corporate Research Laboratory.
The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.
Immunology, Issue 93, Fluorometry, phagocytosis, high throughput assay, actin polymerization, immunology
52195
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