One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete’s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.
20 Related JoVE Articles!
Experimental Approaches to Tissue Engineering
Institutions: Brigham and Women's Hospital.
Issue 7, Cell Biology, tissue engineering, microfluidics, stem cells
A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat
Institutions: Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke.
We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which 11
C-AcAc and 18
F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11
C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11
C-AcAc and 18
F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.
Neuroscience, Issue 82, positron emission tomography (PET), 18F-fluorodeoxyglucose, 11C-acetoacetate, magnetic resonance imaging (MRI), kinetic modeling, cerebral metabolic rate, rat
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
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
Measurement of Metabolic Rate in Drosophila using Respirometry
Institutions: Max Planck Institute for Biophysical Chemistry.
Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila
a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila
simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2
output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.
Physiology, Issue 88, Insects, Diptera, Metabolism, Drosophila, energy homeostasis, respiration, carbon dioxide (CO2), oxygen (O2)
Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue
Institutions: Massachusetts General Hospital/Harvard Medical School, China Pharmaceutical University, Perkin Elmer.
Brown adipose tissue (BAT), widely known as a “good fat” plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18
F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18
F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18
F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.
Medicine, Issue 92, Cerenkov luminescence imaging, brown adipose tissue, 18F-FDG, optical imaging, in vivo imaging, spectral unmixing
Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Institutions: University of New Mexico Health Sciences Center.
Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, model
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Institutions: San Diego State University, DOE Joint Genome Institute, University of Colorado, University of Colorado.
The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.
Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface
Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models
Institutions: University of Missouri, University of Missouri, University of Missouri.
This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e.,
high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.
Medicine, Issue 97, mouse, murine, rodent, swallowing, deglutition, dysphagia, videofluoroscopy, radiation, iohexol, barium, palatability, taste, translational, disease models
Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for C
omputer tomography-guided f
radiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
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
Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools
Institutions: Brigham and Women's Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, capture, blood, HIV, bioengineering
Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM
Institutions: Brigham and Women's Hospital and Harvard Medical School, Erasmus University Medical Center, Brigham and Women's Hospital and Harvard Medical School.
In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool. Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants.
Cell biology, Issue 16, yolk sac, aorta-gonad-mesonephros, AGM, stem cell, dissection, embryo
Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research
Institutions: Karlsruhe Institute of Technology.
A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.
Cellular Biology, Issue 15, Interview, bioreactors, cell culture systems, 3D cell culture, stem cells
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
An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)
Institutions: University of Washington School of Medicine.
Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in São Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery.
A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1
Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2
With constant increase in cesarean rates in the US 3
and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4
Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time.
In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5
Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.
JoVE Medicine, Issue 35, diffuse noxious inhibitory control, DNIC, temporal summation, TS, psychophysical testing, endogenous analgesia, pain modulation, pregnancy-induced analgesia, cesarean section, post-operative pain, prediction
Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology
Institutions: University of Texas Health Science Center at Houston .
Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1
. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2
) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2
, aberrant neuronal signaling 3
and hormonal changes 4
. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7
(and reviewed in 8
). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p<0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p<0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9
. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.
Medicine, Issue 58, spinal cord injury, bone, osteoporosis, x-ray, femur, tibia, longitudinal
Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil
Institutions: Oxitec Ltd, Universidade de São Paulo, Universidade de São Paulo, Moscamed Brasil, University of Oxford, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).
New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3
. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4
, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8
. A RIDL strain of Ae. aegypti
was successfully tested in the field in Grand Cayman9,10
; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12
To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14
. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8,
for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.
Basic Protocol, Issue 83, Aedes aegypti, mass rearing, population suppression, transgenic, insect, mosquito, dengue
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
Institutions: Vanderbilt University, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Vanderbilt University.
Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18
F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18
F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 °C) or cold-activated (CA) (17 °C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.
Medicine, Issue 96, magnetic resonance imaging, brown adipose tissue, cold-activation, adult human, fat water imaging, fluorodeoxyglucose, positron emission tomography, computed tomography