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Baltorussus total makeover: rejuvenation and sex change in an ancient parasitoid wasp lineage.
PUBLISHED: 01-01-2014
The Orussidae is a small and rare but phylogenetically important family of parasitoid wasps. The fossil record of the family is also very poor. Baltorussus velteni was described from Baltic amber from an allegedly female specimen. This and another recently discovered specimen are examined with microCT scanning and standard microscopy. We reveal that both the holotype and the new specimen are actually males. Furthermore, the results of the microCT scanning allow us to integrate the fossils in a morphological data set assembled for extant Orussidae. Phylogenetic analyses consistently retrieve Baltorussus as a separate basal lineage within the crown group, whereas two Cretaceous fossils are placed as stem group orussids and a Dominican amber fossil in an extant genus. Based on the positions of the fossils, we estimate that the extant Orussidae radiated in the mid-Cretaceous (approx. 100 Ma ago). This is considerably younger than a previously suggested Early Jurassic date (180 Ma ago), which was primarily based on biogeographic evidence.
Authors: George Papadopoulos, Carolyn D. Kramer, Connie S. Slocum, Ellen O. Weinberg, Ning Hua, Cynthia V. Gudino, James A. Hamilton, Caroline A. Genco.
Published: 08-08-2014
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation. Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
28 Related JoVE Articles!
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A Human Fallopian Tube Model for Investigation of C. trachomatis Infections
Authors: Stefan Jerchel, Gudrun Knebel, Peter König, Michael K. Bohlmann, Jan Rupp.
Institutions: University of Lübeck, University of Lübeck, University of Lübeck, University of Lübeck.
Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9. For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.
Medicine, Issue 66, Infection, Microbiology, Physiology, Chlamydia trachomatis, human fallopian tube, tissue model, scanning electron microscopy, transmission electron microscopy
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An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response
Authors: Chiyedza Small, Indira Paddibhatla, Roma Rajwani, Shubha Govind.
Institutions: The City College of New York, CUNY, The City University of New York.
Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites. The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids. Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols. In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections. Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D). We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).
Immunology, Issue 63, Parasitoid wasps, innate immunity, encapsulation, hematopoiesis, insect, fat body, Toll-NF-kappaB, molecular biology
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Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces
Authors: Seth J. Wilks, Tom J. Richner, Sarah K. Brodnick, Daryl R. Kipke, Justin C. Williams, Kevin J. Otto.
Institutions: Purdue University, University of Wisconsin-Madison, University of Michigan , Purdue University.
Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.
Neuroscience, Issue 60, neuroprosthesis, electrode-tissue interface, rejuvenation, neural engineering, neuroscience, neural implant, electrode, brain-computer interface, electrochemistry
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Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Authors: Stacy L. Fairbanks, Rebekah Vest, Saurabh Verma, Richard J. Traystman, Paco S. Herson.
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
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2D and 3D Chromosome Painting in Malaria Mosquitoes
Authors: Phillip George, Atashi Sharma, Igor V Sharakhov.
Institutions: Virginia Tech.
Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
Immunology, Issue 83, Microdissection, whole genome amplification, malaria mosquito, polytene chromosome, mitotic chromosomes, fluorescence in situ hybridization, chromosome painting
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Generation of Transgenic Hydra by Embryo Microinjection
Authors: Celina E. Juliano, Haifan Lin, Robert E. Steele.
Institutions: Yale University School of Medicine, University of California, Irvine.
As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.
Molecular Biology, Issue 91, Hydra, transgenic, microinjection, gene overexpression, gene knockdown
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Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS
Authors: Christopher J. Zopf, Narendra Maheshri.
Institutions: Massachusetts Institute of Technology.
Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.
Microbiology, Issue 77, Cellular Biology, Molecular Biology, Genetics, Biophysics, Saccharomyces cerevisiae, Microscopy, Fluorescence, Cell Biology, microscopy/fluorescence and time-lapse, budding yeast, gene expression dynamics, segmentation, lineage tracking, image tracking, software, yeast, cells, imaging
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Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
Authors: Karen H. Martin, Karen E. Hayes, Elyse L. Walk, Amanda Gatesman Ammer, Steven M. Markwell, Scott A. Weed.
Institutions: West Virginia University .
Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.
Cellular Biology, Issue 66, Cancer Biology, Anatomy, Molecular Biology, Biochemistry, invadopodia, extracellular matrix, gelatin, confocal microscopy, quantification, oregon green
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Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Authors: Khadija Elhabazi, Safia Ayachi, Brigitte Ilien, Frédéric Simonin.
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
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Exfoliation of Egyptian Blue and Han Blue, Two Alkali Earth Copper Silicate-based Pigments
Authors: Darrah Johnson-McDaniel, Tina T. Salguero.
Institutions: The University of Georgia.
In a visualized example of the ancient past connecting with modern times, we describe the preparation and exfoliation of CaCuSi4O10 and BaCuSi4O10, the colored components of the historic Egyptian blue and Han blue pigments. The bulk forms of these materials are synthesized by both melt flux and solid-state routes, which provide some control over the crystallite size of the product. The melt flux process is time intensive, but it produces relatively large crystals at lower reaction temperatures. In comparison, the solid-state method is quicker yet requires higher reaction temperatures and yields smaller crystallites. Upon stirring in hot water, CaCuSi4O10 spontaneously exfoliates into monolayer nanosheets, which are characterized by TEM and PXRD. BaCuSi4O10 on the other hand requires ultrasonication in organic solvents to achieve exfoliation. Near infrared imaging illustrates that both the bulk and nanosheet forms of CaCuSi4O10 and BaCuSi4O10 are strong near infrared emitters. Aqueous CaCuSi4O10 and BaCuSi4O10 nanosheet dispersions are useful because they provide a new way to handle, characterize, and process these materials in colloidal form.
Chemistry, Issue 86, Nanosheets, Egyptian Blue, Han Blue, Pigment, Near Infrared, Luminescence, Exfoliation, Delamination, Two-Dimensional, Ink, Colloidal Dispersion
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Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles
Authors: John J. Beck, Douglas M. Light, Wai S. Gee.
Institutions: Agricultural Research Service.
Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8
Plant Biology, Issue 63, bioassay, chemoreception, electroantennography, electrophysiological response, high-throughput, host-plant volatiles, navel orangeworm, screening tool
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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
Institutions: Princeton University.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (, a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
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A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures
Authors: Matthew Moore, Yifan Hu, Sarah Woo, Dylan O'Hearn, Alexandru D. Iordan, Sanda Dolcos, Florin Dolcos.
Institutions: University of Illinois Urbana-Champaign, University of Illinois Urbana-Champaign, University of Illinois Urbana-Champaign.
The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal, and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.
Neuroscience, Issue 89, Anatomy, Segmentation, Medial Temporal Lobe, MRI, Manual Tracing, Amygdala, Hippocampus, Perirhinal Cortex, Entorhinal Cortex, Parahippocampal Cortex
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In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Authors: Andrew T. Jang, Jeremy D. Lin, Youngho Seo, Sergey Etchin, Arno Merkle, Kevin Fahey, Sunita P. Ho.
Institutions: University of California San Francisco, University of California San Francisco, Xradia Inc..
This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.
Bioengineering, Issue 85, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent
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Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Authors: Johnny L. Carson.
Institutions: The University of North Carolina at Chapel Hill.
Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.
Biophysics, Issue 91, Freeze-fracture; Freeze-etch; Membranes; Intercellular junctions; Materials science; Nanotechnology; Electron microscopy
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Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Authors: Marcus Cheetham, Lutz Jancke.
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2 proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness (DHL) (Figure 1). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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Assessing Differences in Sperm Competitive Ability in Drosophila
Authors: Shu-Dan Yeh, Carolus Chan, José M. Ranz.
Institutions: University of California, Irvine.
Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5. Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes. Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.
Developmental Biology, Issue 78, Molecular Biology, Cellular Biology, Genetics, Biochemistry, Spermatozoa, Drosophila melanogaster, Biological Evolution, Phenotype, genetics (animal and plant), animal biology, double-mating experiment, sperm competitive ability, male fertility, Drosophila, fruit fly, animal model
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The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Authors: Henriette A. Seichter, Felix Blumenthal, Carmen R. Smarandache-Wellmann.
Institutions: University of Cologne.
Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1. The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system. Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.
Neurobiology, Issue 93, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
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Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Authors: Alison X. Xie, Kelli Lauderdale, Thomas Murphy, Timothy L. Myers, Todd A. Fiacco.
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
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Phase Contrast and Differential Interference Contrast (DIC) Microscopy
Authors: Victoria Centonze Frohlich.
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Basic protocols, Issue 18, Current Protocols Wiley, Microscopy, Phase Contrast, Difference Interference Contrast
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Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method
Authors: Alan Daugherty, Debra Rateri, Lu Hong, Anju Balakrishnan.
Institutions: University of Kentucky.
The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.
Medicine, Issue 27, blood pressure, systolic, diastolic, tail-cuff, mouse, rat
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Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region
Authors: Kin Chiu, Wui Man Lau, Ho Tak Lau, Kwok-Fai So, Raymond Chuen-Chung Chang.
Institutions: The University of Hong Kong - HKU.
Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.
Issue 7, Neuroscience, brain, dissection
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Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
Authors: Yoonsuck Choe, David Mayerich, Jaerock Kwon, Daniel E. Miller, Chul Sung, Ji Ryang Chung, Todd Huffman, John Keyser, Louise C. Abbott.
Institutions: Texas A&M University, University of Illinois, Kettering University, 3Scan, Texas A&M University.
Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.
Bioengineering, Issue 58, Physical sectioning, serial sectioning, light microscopy, brain imaging, microtome
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Contrast Enhanced Vessel Imaging using MicroCT
Authors: Suresh I. Prajapati, Charles Keller.
Institutions: University of Texas Health Science Center at San Antonio , University of Texas Health Science Center at San Antonio , University of Texas Health Science Center at San Antonio , University of Texas Health Science Center at San Antonio .
Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360° rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.
Medicine, Issue 47, vessel imaging, eXIA 160XL, microCT, advanced visualization, 2DTF
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Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Authors: Michael Heethoff, Lukas Helfen, Peter Cloetens.
Institutions: University of Tubingen, European Synchrotron Radiation Facility.
Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning. We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.
Developmental Biology, Issue 15, Synchrotron X-ray tomography, Acari, Oribatida, micro-arthropods, non-invasive investigation
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Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging
Authors: Ed Lim, Kshitij Modi, Anna Christensen, Jeff Meganck, Stephen Oldfield, Ning Zhang.
Institutions: Caliper Life Sciences.
Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.
Medicine, Issue 50, osteolytic lesions, micro CT, tumor, bioluminescence, in vivo, imaging, IVIS, luciferase, low dose, co-registration, 3D reconstruction
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Interview: Protein Folding and Studies of Neurodegenerative Diseases
Authors: Susan Lindquist.
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
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