The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.
27 Related JoVE Articles!
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
Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Institutions: McMaster University, Hamilton, University of Toronto.
Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2
. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4
. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5
. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7
. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.
Immunology, Issue 65, Medicine, Physiology, Dendritic Cells, allergic airway inflammation, ovalbumin, lymph nodes, lungs, dendritic cell maturation, dendritic cell migration, mediastinal lymph nodes
Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses
Institutions: Donald Danforth Plant Science Center, Boyce Thompson Institute.
is an emerging model system for C4
grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica,
has been sequenced 1,2
and a 25x coverage genome sequence of the weedy relative S. viridis
is in progress. S. viridis
has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3
and developed systems for both transient and stable transformation 4
. However, the genetics of S. viridis
is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis
, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 °C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis
X S. viridis
or S. viridis
X S. italica
, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.
Environmental Sciences, Issue 80, Hybridization, Genetics, plants, Setaria viridis, crosses, emasculation, flowering, seed propagation, seed dormancy
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Institutions: The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill.
models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens.
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc.
models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo
Cellular Biology, Issue 80, Epithelium, Cell culture models, ciliated, air pollution, co-culture models, nasal epithelium
Dry Oxidation and Vacuum Annealing Treatments for Tuning the Wetting Properties of Carbon Nanotube Arrays
Institutions: California Institute of Technology.
In this article, we describe a simple method to reversibly tune the wetting properties of vertically aligned carbon nanotube (CNT) arrays. Here, CNT arrays are defined as densely packed multi-walled carbon nanotubes oriented perpendicular to the growth substrate as a result of a growth process by the standard thermal chemical vapor deposition (CVD) technique.1,2
These CNT arrays are then exposed to vacuum annealing treatment to make them more hydrophobic or to dry oxidation treatment to render them more hydrophilic. The hydrophobic CNT arrays can be turned hydrophilic by exposing them to dry oxidation treatment, while the hydrophilic CNT arrays can be turned hydrophobic by exposing them to vacuum annealing treatment. Using a combination of both treatments, CNT arrays can be repeatedly switched between hydrophilic and hydrophobic.2
Therefore, such combination show a very high potential in many industrial and consumer applications, including drug delivery system and high power density supercapacitors.3-5
The key to vary the wettability of CNT arrays is to control the surface concentration of oxygen adsorbates. Basically oxygen adsorbates can be introduced by exposing the CNT arrays to any oxidation treatment. Here we use dry oxidation treatments, such as oxygen plasma and UV/ozone, to functionalize the surface of CNT with oxygenated functional groups. These oxygenated functional groups allow hydrogen bond between the surface of CNT and water molecules to form, rendering the CNT hydrophilic. To turn them hydrophobic, adsorbed oxygen must be removed from the surface of CNT. Here we employ vacuum annealing treatment to induce oxygen desorption process. CNT arrays with extremely low surface concentration of oxygen adsorbates exhibit a superhydrophobic behavior.
Chemistry, Issue 74, Chemical Engineering, Materials Science, Nanotechnology, Engineering, Nanotubes, Carbon, Oxidation-Reduction, Surface Properties, carbon nanotubes (synthesis and properties), Carbon nanotube, Wettability, Hydrophobic, Hydrophilic, UV/ozone, Oxygen Plasma, Vacuum Annealing
Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues
Institutions: Université de Mons.
It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro
. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro
cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.
Bioengineering, Issue 90, hydrogels, mechanotransduction, polyacrylamide, microcontact printing, cell shape, stiffness, durotaxis, cell-ligand density
In vitro Cell Culture Model for Toxic Inhaled Chemical Testing
Institutions: University of Colorado, Colorado School of Mines.
Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.
Bioengineering, Issue 87, air-liquid interface, chlorine exposure, toxic inhaled chemicals, Transepithelial Electrical Resistance,Immunocytochemistry
Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees
Institutions: University of Ottawa.
We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification (RFID) readers built into artificial flowers that display various visual cues, and RFID tags (i.e.
, passive transponders) glued to the thorax of bumblebee workers. The novelty in our implementation is that RFID readers are built directly into artificial flowers that are capable of displaying several distinct visual properties such as color, pattern type, spatial frequency (i.e.
, “busyness” of the pattern), and symmetry (spatial frequency and symmetry were not manipulated in this experiment). Additionally, these visual displays in conjunction with the automated systems are capable of recording unrewarded
choice behavior. The second method consists of recording choice behavior at artificial flowers using motion-sensitive high-definition camcorders. Bumblebees have number tags glued to their thoraces for unique identification. The advantage in this implementation over RFID is that in addition to observing landing behavior, alternate measures of preference such as hovering and antennation may also be observed. Both automation methods increase experimental control, and internal validity by allowing larger scale studies that take into account individual differences. External validity is also improved because bees can freely enter and exit the testing environment without constraints such as the availability of a research assistant on-site. Compared to human observation in real time, the automated methods are more cost-effective and possibly less error-prone.
Neuroscience, Issue 93, bumblebee, unlearned behaviors, floral choice, visual perception, Bombus spp, information processing, radio-frequency identification, motion-sensitive video
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
Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants
Institutions: Purdue University, Purdue University, Jiangsu Academy of Agricultural Sciences, Zhejiang University, Shanxi Academy of Agricultural Sciences, Chinese Academy of Sciences.
Developmental and environmental cues induce Ca2+
fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+
patterns are sensed by cellular Ca2+
binding proteins that initiate Ca2+
signaling cascades. However, we still know little about how stimulus specific Ca2+
signals are generated. The specificity of a Ca2+
signal may be attributed to the sophisticated regulation of the activities of Ca2+
channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+
signals at both the tissue and cellular levels. Genetically encoded Ca2+
indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+
signals. Here we describe instructions for the use of two Ca2+
detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+
imaging and case12 based live cell confocal fluorescence Ca2+
imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+
signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+
signals at a high resolution.
Plant Biology, Issue 91, Aequorin, Case12, abiotic stress, heavy metal stress, copper ion, calcium imaging, Arabidopsis
Murine Model of Allergen Induced Asthma
Institutions: Emory University and Atlanta VA Medical Center.
Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1
More than 8% of the US population has asthma, with the prevalence increasing.2
As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3
Availability of a variety of transgenic strains further increases the attractiveness of these models.4
Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6
and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7
techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.
Immunology, Issue 63, Allergy, airway hyperresponsiveness, pulmonary function, eosinophil, ovalbumin, methacholine, airway resistance, plethysmography, flexiVent, bronchoalveolar lavage, physiology
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans
Institutions: Deakin University.
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
Neuroscience, Issue 88, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies
Institutions: Flemish Institute for Technological Research (VITO), Hasselt University, Hasselt University, Leuven University.
The microcirculation consists of blood vessels with diameters less than 150 µm. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.
Medicine, Issue 92, retina, microvasculature, image analysis, Central Retinal Arteriolar Equivalent, Central Retinal Venular Equivalent, air pollution, particulate matter, black carbon
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Training Synesthetic Letter-color Associations by Reading in Color
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2
. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
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
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
Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient
Institutions: Dynamac, Inc., University of Cincinnati, McMicken College of Arts and Science, Agricultural Research Service, U.S. Department of Agriculture, US Environmental Protection Agency.
is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii
cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii
cysts in meat1, 2
. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5
. To date, research on understanding the prevalence of T. gondii
oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6
. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii
oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii
oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7
Jove Infectious Diseases, Microbiology, Issue 33, Toxoplasma gondii, cesium chloride, oocysts, discontinuous gradient, apicomplexan
Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain
Institutions: University of Calgary.
Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia
which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia
is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia
refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3).
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.
Medicine, Issue 28, Chronic pelvic pain, cutaneous allodynia, trigger points, dysmenorrhea, endometriosis, dyspareunia
Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging
Institutions: University of Alabama at Birmingham.
Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral symptoms displayed by individuals with autism. While structural neuroimaging findings have provided some insights into brain abnormalities in autism, the consistency of such findings is questionable. Functional neuroimaging, on the other hand, has been more fruitful in this regard because autism is a disorder of dynamic processing and allows examination of communication between cortical networks, which appears to be where the underlying problem occurs in autism. Functional connectivity is defined as the temporal correlation of spatially separate neurological events1. Findings from a number of recent fMRI studies have supported the idea that there is weaker coordination between different parts of the brain that should be working together to accomplish complex social or language problems2,3,4,5,6
. One of the mysteries of autism is the coexistence of deficits in several domains along with relatively intact, sometimes enhanced, abilities. Such complex manifestation of autism calls for a global and comprehensive examination of the disorder at the neural level. A compelling recent account of the brain functioning in autism, the cortical underconnectivity theory,2,7
provides an integrating framework for the neurobiological bases of autism. The cortical underconnectivity theory of autism suggests that any language, social, or psychological function that is dependent on the integration of multiple brain regions is susceptible to disruption as the processing demand increases. In autism, the underfunctioning of integrative circuitry in the brain may cause widespread underconnectivity. In other words, people with autism may interpret information in a piecemeal fashion at the expense of the whole. Since cortical underconnectivity among brain regions, especially the frontal cortex and more posterior areas 3,6
, has now been relatively well established, we can begin to further understand brain connectivity as a critical component of autism symptomatology.
A logical next step in this direction is to examine the anatomical connections that may mediate the functional connections mentioned above. Diffusion Tensor Imaging (DTI) is a relatively novel neuroimaging technique that helps probe the diffusion of water in the brain to infer the integrity of white matter fibers. In this technique, water diffusion in the brain is examined in several directions using diffusion gradients. While functional connectivity provides information about the synchronization of brain activation across different brain areas during a task or during rest, DTI helps in understanding the underlying axonal organization which may facilitate the cross-talk among brain areas. This paper will describe these techniques as valuable tools in understanding the brain in autism and the challenges involved in this line of research.
Medicine, Issue 55, Functional magnetic resonance imaging (fMRI), MRI, Diffusion tensor imaging (DTI), Functional Connectivity, Neuroscience, Developmental disorders, Autism, Fractional Anisotropy
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Improving IV Insulin Administration in a Community Hospital
Institutions: Wyoming Medical Center.
Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4
The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5
It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6
Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p<0.001). From this pivotal trial and two others,7,8
Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia.
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG < 40 mg/dL), clinical hypoglycemia (BG < 70 mg/dL), and hyperglycemia (BG > 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG < 70 mg/dL) with the use of the computer glucose management system was well under 1%.
Medicine, Issue 64, Physiology, Computerized glucose management, Endotool, hypoglycemia, hyperglycemia, diabetes, IV insulin, paper protocol, glucose control