Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.
25 Related JoVE Articles!
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
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks
Institutions: Freie Universität Berlin.
Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.
Developmental Biology, Issue 87, songbird, molecular sexing, PCR, individual marking, down feather, DNA extraction, sample storage, zebra finch, buccal swabs, saliva, gender
Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing
Institutions: Georgia Regents University, New York University, Georgia Regents University, Georgia Regents University.
Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p
< 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2
= 0.38, (thin-liquid); r = 0.52, r2
= 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.
Medicine, Issue 87, videofluoroscopy, modified barium swallow studies, hyolaryngeal kinematics, deglutition, dysphagia, dysphagia research, hyolaryngeal complex
Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey
Institutions: U. S. Geological Survey, Grand Valley State University, Shedd Aquarium.
A technique for laboratory estimation of net trophic transfer efficiency (γ) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi
) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush
) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, γ was calculated for each of 75 PCB congeners in each of the eight tanks. Mean γ was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean γ could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.
Environmental Sciences, Issue 90, trophic transfer efficiency, polychlorinated biphenyl congeners, lake trout, activity, contaminants, accumulation, risk assessment, toxic equivalents
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
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
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
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
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
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Nest Building as an Indicator of Health and Welfare in Laboratory Mice
Institutions: Charles River, Tufts University, Stanford University, Stanford University.
The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.
Behavior, Issue 82, Animal Structures, Surgical Procedures, Life Sciences (General), Behavioral Sciences, Mouse, Welfare assessment, Nest building
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy
Institutions: Raymond and Beverly Sackler Foundation, University of Medicine and Dentistry of New Jersey, Institute for Advanced Study, Princeton, New Jersey.
Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100, 000 individuals per year, and they account for 0.5% of all human malignancies.1
Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo
tumor tissue organization and microenvironment.2,3
The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro
3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2
, 37 °C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd
day. The spheroids become more spherical by the 6th
day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques.
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.
Cancer Biology, Issue 66, Medicine, Neuroscience, Cell Culture, Tissue Engineering, 3D model, multicellular spheroids, therapeutic drugs, neuroendocrine tumor cell lines, agarose overlay platform, paraffin embedding
Origami Inspired Self-assembly of Patterned and Reconfigurable Particles
Institutions: The Johns Hopkins University , The Johns Hopkins University .
There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20
wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23
and (b) grippers that self-fold due to residual stress powered hinges24,25
. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26
and with a range of materials including metals21
. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 μm to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7
to possibly create even smaller nanoscale grasping devices.
Chemistry, Issue 72, Chemical Engineering, Biomolecular Engineering, Materials Science, Physics, Nanotechnology, Molecular Self-assembly, Electrochemistry, Folding, three dimensional, lithography, colloid, patchy particles, particles, nanoparticles, robotics, drug delivery, microfabrication, nanofabrication, nano, assembly, synthesis, reaction, origami
Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data
Institutions: United States Department of Agriculture-Agricultural Research Service, University of California, Davis, University of Chile, University of California, Davis, URS Corporation Australia Pty. Ltd..
Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.
Environmental Sciences, Issue 82, Conservation of Natural Resources, Engineering, Agriculture, plants, energy balance, irrigated agriculture, flux data, evapotranspiration, agrometeorology
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly
Institutions: Sunnybrook Health Sciences Centre, University of Toronto.
Obtaining in vivo
human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g.
Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2
. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.
Medicine, Issue 86, Brain, Vascular Diseases, Magnetic Resonance Imaging (MRI), Neuroimaging, Alzheimer Disease, Aging, Neuroanatomy, brain extraction, ventricles, white matter hyperintensities, cerebrovascular disease, Alzheimer disease
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
Institutions: Otto-von-Guericke University Magdeburg, Helmholtz Center for Infection Research.
After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils,
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold
and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.
Immunology, Issue 52, 2-photon microscopy, lung, neutrophil extracellular trap (NET), Aspergillus fumigatus
A Simple Way to Measure Ethanol Sensitivity in Flies
Institutions: University of Texas Southwestern Medical Center.
Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.
Neuroscience, Issue 48, Drosophila, behavior, alcohol, addiction
Using the optokinetic response to study visual function of zebrafish
Institutions: University of Science and Technology of China (USTC).
Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5
. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.
Neuroscience, Issue 36, Zebrafish, OKR, OMR, behavior, optokinetic, vision
Neutrophil Extracellular Traps: How to Generate and Visualize Them
Institutions: Max Planck Institute for Infection Biology, Max Planck Institute for Infection Biology.
Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1
. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4
. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7
and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.
JoVE Immunology, Issue 36, Neutrophil, Granulocyte, Neutrophil Extracellular Trap, NET, isolation, immunolabeling, electron microscopy
Freezing, Thawing, and Packaging Cells for Transport
Institutions: Molecular Pathology Laboratory Network, Inc.
Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.
Basic Protocols, Issue 17, Current Protocols Wiley, Freezing Cells, Cell Culture, Thawing Cells, Storage of Cells, Suspension Cells, Adherent Cells