An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.
24 Related JoVE Articles!
A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal
Institutions: MacEwan University.
Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed.
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.
Neuroscience, Issue 93, Zebrafish, Ethanol, Behavior, Anxiety, Pharmacology, Fish, Neuroscience, Drug administration, Scototaxis
Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes
Institutions: Institut Pasteur, Sorbonne Universités, Centre de Recherche en Cancérologie de Marseille, CNRS UMR5235/DAA/cc107/Université Montpellier II, CNRS-NED, Institut de Neurobiologie Alfred Fessard.
We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum
. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo
imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.
Developmental Biology, Issue 95, Amphioxus, cephalochordate, gene expression vectors, in vivo imaging, microinjection protocol, model organism
Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)
Institutions: University of Arizona.
Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.
Environmental Sciences, Issue 89, Entomology, Nematology, Steinernema, Heterorhabditis, nematodes, soil sampling, insect-bait, modified White-trap
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Modifying the Bank Erosion Hazard Index (BEHI) Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio
Institutions: Cleveland Metroparks, Case Western Reserve University.
Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream’s function and habitat it provides. Sediments from highly eroding streambanks are a major source of pollution in a stream system and have the potential to jeopardize habitat, infrastructure, and stream function. Watershed management practices throughout the Cleveland Metroparks attempt to locate and inventory the source and rate the risk of potential streambank erosion to assist in formulating effect stream, riparian, and habitat management recommendations. The Bank Erosion Hazard Index (BEHI), developed by David Rosgen of Wildland Hydrology is a fluvial geomorphic assessment procedure used to evaluate the susceptibility of potential streambank erosion based on a combination of several variables that are sensitive to various processes of erosion. This protocol can be time consuming, difficult for non-professionals, and confined to specific geomorphic regions. To address these constraints and assist in maintaining consistency and reducing user bias, modifications to this protocol include a “Pre-Screening Questionnaire”, elimination of the Study Bank-Height Ratio metric including the bankfull determination, and an adjusted scoring system. This modified protocol was used to assess several high priority streams within the Cleveland Metroparks. The original BEHI protocol was also used to confirm the results of the modified BEHI protocol. After using the modified assessment in the field, and comparing it to the original BEHI method, the two were found to produce comparable BEHI ratings of the streambanks, while significantly reducing the amount of time and resources needed to complete the modified protocol.
Environmental Sciences, Issue 96, Streambank erosion, bankfull, alluvial boundaries, sediment, geomorphic assessment, non-point source pollution, Bank Erosion Hazard Index
A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges
Institutions: Swiss Federal Research Institute WSL.
Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.
Environmental Sciences, Issue 97, Cell parameters, dendroecology, image analysis, micro sectioning, microtomes, sample preparation, wood anatomy
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Institutions: Hospital for Sick Children, University of Toronto, University of Toronto.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.
Biochemistry, Issue 97, Cystic Fibrosis, CFTR, purification, reconstitution, chloride channel, channel function, iodide efflux, potentiation
Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone
Institutions: German Rheumatism Research Center, a Leibniz Institute, German Rheumatism Research Center, a Leibniz Institute, Max-Delbrück Center for Molecular Medicine, Wimasis GmbH, Charité - University of Medicine.
Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.
Developmental Biology, Issue 98, Image analysis, neighborhood analysis, bone marrow, stromal cells, bone marrow niches, simulation, bone cryosectioning, bone histology
The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents
Institutions: University Hospital Freiburg, UMR 7364 Université de Strasbourg, CNRS, Neuropôle de Strasbourg.
Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g.,
allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; §4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; §4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.
Behavior, Issue 101, Double-H maze, spatial memory, procedural memory, consolidation, allocentric, egocentric, habits, rodents, video tracking system
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics
Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm
Institutions: University of Washington, Iowa State University, North Carolina A&T University, Iowa Geological and Water Survey.
Finding the cost-efficient (i.e.
, lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.
) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25
. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7
with a multiobjective evolutionary algorithm SPEA226
, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.
Environmental Sciences, Issue 70, Plant Biology, Civil Engineering, Forest Sciences, Water quality, multiobjective optimization, evolutionary algorithms, cost efficiency, agriculture, development
Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading
Institutions: Grand Valley State University.
Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ
measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.
Environmental Sciences, Issue 85, Limnology, internal loading, eutrophication, nutrient flux, sediment coring, phosphorus, lakes
Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity
Institutions: Case Western Reserve University.
Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
Environmental Sciences, Issue 85, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound
Institutions: German Cancer Research Center, Heidelberg, Germany, German Cancer Research Center, Heidelberg, Germany.
Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo
assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105
MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1
. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1
. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4
. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6
. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.
Cancer Biology, Issue 66, Medicine, Physiology, Physics, bone metastases, animal model, angiogenesis, imaging, magnetic resonance imaging, MRI, volumetric computed tomography, ultrasound
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
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
) (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
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
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 (http://www.proteinwisdom.org), 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
Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries
Institutions: University of Missouri, Dalton Cardiovascular Research Center.
The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.
endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+
signaling, which leads to production of diffusible autacoids (e.g.
nitric oxide and arachidonic acid metabolites)1-3
that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+
signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ
to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7
. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.
Basic Protocol, Issue 81, endothelial tubes, microcirculation, calcium signaling, resistance vasculature, Confocal microscopy
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
A Venturi Effect Can Help Cure Our Trees
Institutions: Unversity of Padova.
In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e.
pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called "trunk injection" or "trunk infusion" (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels' cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.
Environmental Sciences, Issue 80, Trunk injection, systemic injection, xylematic injection, endotherapy, sap flow, Bernoulli principle, plant diseases, pesticides, desiccants
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Design and Construction of an Urban Runoff Research Facility
Institutions: Texas A&M University, The Scotts Miracle-Gro Company.
As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2
facility was constructed which consists of 24 individual 33.6 m2
field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4
, and Ca2+
had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.
Environmental Sciences, Issue 90, urban runoff, landscapes, home lawns, turfgrass, St. Augustinegrass, carbon, nitrogen, phosphorus, sodium
A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials
Institutions: Mississippi State University, Mississippi State University.
This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g.
brain, liver, tendon, fat, etc.
) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1
. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e.
incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e.
transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e.
reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e.
optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.
Bioengineering, Issue 99, Split-Hopkinson Pressure Bar, High Strain Rate, Finite Element Modeling, Soft Biomaterials, Dynamic Experiments, Internal State Variable Modeling, Brain, Liver, Tendon, Fat
Protocols for Robust Herbicide Resistance Testing in Different Weed Species
Institutions: National Research Council (CNR), Italy.
Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro
diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.
Environmental Sciences, Issue 101, Weed science, resistant biotypes, monitoring, seed germination, weed control, herbicide efficacy, herbicide treatment, resistance level.
Electrophysiological Recording in the Brain of Intact Adult Zebrafish
Institutions: University of Georgia, University of Georgia, Oklahoma State University, University of Georgia, University of California, Davis.
Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo
electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.
Neuroscience, Issue 81, Zebrafish, adult, Electrophysiology, in vivo, craniotomy, perfusion, neural activity