Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.
25 Related JoVE Articles!
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Institutions: UT Southwestern Medical Center.
Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.
Cancer Biology, Issue 77, Medicine, Genetics, Cellular Biology, Molecular Biology, Biochemistry, Cancer Biology, Bioengineering, Genomics, Drug Discovery, RNA Interference, Cell Biology, Neoplasms, luciferase reporters, functional genomics, chemical biology, high-throughput screening technology, signal transduction, PCR, transfection, assay
Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection
Institutions: Brigham and Women‘s Hospital.
Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile
infection (CDI). Various methods of FMT administration have been reported in the literature including nasogastric tube, upper endoscopy, enema and colonoscopy. FMT via
colonoscopy yields excellent cure rates and is also well tolerated. We have found that patients find this an acceptable and tolerable mode of delivery. At our Center, we have initiated a fecal transplant program for patients with recurrent or refractory CDI. We have developed a protocol using an iterative process of revision and have performed 24 fecal transplants on 22 patients with success rates comparable to the current published literature. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool maximizes therapeutic success. Here we detail each step of the FMT protocol that can be carried out at any endoscopy center with a high degree of safety and success.
Immunology, Issue 94, C.difficile, colonoscopy, fecal transplant, stool, diarrhea, microbiota
Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
Institutions: University of Missouri.
Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3
. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient′s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5
. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7
. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.
Bioengineering, Issue 57, cancer, circulating tumor cell, CTCs, melanoma, metastasis, optoacoustic
Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition
Institutions: University of Twente.
Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2
-Ag nanowires with a diameter of 200 nm and a length of 6-20 µm were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2
phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2
-Ag nanowires, a TiO2
gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2
gel resulted in the formation of crystalline TiO2
nanotubes. A subsequent Ag electrodeposition step inside the TiO2
nanotubes resulted in formation of coaxial TiO2
-Ag nanowires. Due to the combination of an n
-type semiconductor (ZnO or TiO2
) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2
gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min of illumination, approximately 0.2 vol% H2
gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.
Physics, Issue 87, Multicomponent nanowires, electrochemistry, sol-gel processes, photocatalysis, photochemistry, H2 evolution
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates
Institutions: University of Alberta.
In recent years π-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures
can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array
. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently π-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important π-conjugated small molecular weight organics
, such as tris-8-hydroxyquinoline aluminum (Alq3
), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.
Recently we have been able to address the above-mentioned issue by employing a novel "centrifugation-assisted" approach. This method therefore broadens the spectrum of organic materials that can be patterned in a vertically ordered nanowire array. Due to the technological importance of Alq3
, rubrene and methanofullerenes, our method can be used to explore how the nanostructuring of these materials affects the performance of aforementioned organic devices. The purpose of this article is to describe the technical details of the above-mentioned protocol, demonstrate how this process can be extended to grow small-molecular organic nanowires on arbitrary substrates
and finally, to discuss the critical steps, limitations, possible modifications, trouble-shooting and future applications.
Physics, Issue 76, Electrical Engineering, Chemistry, Chemical Engineering, Nanotechnology, nanodevices (electronic), semiconductor devices, solid state devices, thin films (theory, deposition and growth), crystal growth (general), Organic semiconductors, small molecular organics, organic nanowires, nanorods and nanotubes, bottom-up nanofabrication, electrochemical self-assembly, anodic aluminum oxide (AAO), template-assisted synthesis of nanostructures, Raman spectrum, field emission scanning electron microscopy, FESEM
Setting Limits on Supersymmetry Using Simplified Models
Institutions: University College London, CERN, Lawrence Berkeley National Laboratories.
Experimental limits on supersymmetry and similar theories are difficult to set because of the enormous available parameter space and difficult to generalize because of the complexity of single points. Therefore, more phenomenological, simplified models are becoming popular for setting experimental limits, as they have clearer physical interpretations. The use of these simplified model limits to set a real limit on a concrete theory has not, however, been demonstrated. This paper recasts simplified model limits into limits on a specific and complete supersymmetry model, minimal supergravity. Limits obtained under various physical assumptions are comparable to those produced by directed searches. A prescription is provided for calculating conservative and aggressive limits on additional theories. Using acceptance and efficiency tables along with the expected and observed numbers of events in various signal regions, LHC experimental results can be recast in this manner into almost any theoretical framework, including nonsupersymmetric theories with supersymmetry-like signatures.
Physics, Issue 81, high energy physics, particle physics, Supersymmetry, LHC, ATLAS, CMS, New Physics Limits, Simplified Models
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS
Institutions: Massachusetts Institute of Technology.
Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae
, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.
Microbiology, Issue 77, Cellular Biology, Molecular Biology, Genetics, Biophysics, Saccharomyces cerevisiae, Microscopy, Fluorescence, Cell Biology, microscopy/fluorescence and time-lapse, budding yeast, gene expression dynamics, segmentation, lineage tracking, image tracking, software, yeast, cells, imaging
A Neuroscientific Approach to the Examination of Concussions in Student-Athletes
Institutions: Elon University, Elon University, Duquesne University, Elon University.
Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement “may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an ‘impulsive’ force transmitted to the head.” Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual’s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.
Medicine, Issue 94, Concussions, Student-Athletes, Mild Traumatic Brain Injury, Genetics, Cognitive Function, Balance, Gait, Somatosensory
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
Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation
Institutions: Concordia University, Concordia University, Hôpital du Sacré-Coeur de Montréal.
Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.
Medicine, Issue 81, Data tracking, exercise, rehabilitation, adherence, patient compliance, health behavior, user-computer interface.
Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience
Institutions: The Hebrew University of Jerusalem.
The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo
experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g.
single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.
Behavior, Issue 90,
Brain, behavior, RNA, transcription, nucleus accumbens, cocaine, high-throughput qPCR, experience-dependent plasticity, gene regulatory networks, microdissection
Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Institutions: University of Wisconsin-Madison.
Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e.
, “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g.
, neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g
., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
Behavior, Issue 91,
Startle; electromyography; shock; addiction; uncertainty; fear; anxiety; humans; psychophysiology; translational
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
Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay
Institutions: The Hebrew University of Jerusalem.
Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50
values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50
Medicine, Issue 81, Inorganic Chemicals, Therapeutics, Metals and Metallic Materials, anticancer drugs, cell viability, cisplatin, metal complex, cytotoxicity, HT-29, metal-based drugs, MTT assay, titanium (IV), vanadium (V)
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, USDA-ARS Dairy Forage Research Center, USDA-ARS Pasture Systems Watershed Management Research Unit.
Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.
Environmental Sciences, Issue 90, greenhouse gas, trace gas, gas flux, static chamber, soil, field, agriculture, climate
Self-reporting Scaffolds for 3-Dimensional Cell Culture
Institutions: University of Nottingham, University of Nottingham, University of Nottingham.
Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo
microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.
Bioengineering, Issue 81, Biocompatible Materials, Nanosensors, scaffold, electrospinning, 3D cell culture, PLGA
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Institutions: Cornell University, Cornell University.
Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.
Plant Biology, Issue 15, Annual Review, Aphids, Plant Virus, Potato Leaf Roll Virus, Microinjection Technique
Functional Mapping with Simultaneous MEG and EEG
Institutions: MGH - Massachusetts General Hospital.
We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.
JoVE neuroscience, Issue 40, neuroscience, brain, MEG, EEG, functional imaging
Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
Institutions: Cornell University.
Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.
Plant Biology, Issue 15, Annual Review, Nicotine, Aphids, Plant Feeding Resistance, Tobacco
Brain Banking: Making the Most of your Research Specimens
Institutions: University of Montreal, University of Montreal.
Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1
. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20°C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.
Neuroscience, Issue 29, brain bank, systematic sampling, stereology, cryostat, antigen preserve
Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates