The Journal of Visualized Experiments (JoVE) closes 2011 with a December issue that begins by honoring World's AIDS Day with an article that deals with visualizing the structure of HIV envelope glycoproteins, which are a critical component of the viral infection pathway. One key method for determining virion envelope structure is cryo-electron tomography. Prior to using this technique, soluble virus must be vitrified on a gold grid - a process that involves adding gold to a viral suspension, transferring that suspension to the grid, and plunging the grid into liquid ethane. The virus particles, present in a thin film of liquid atop the grid, are frozen at a rate greater than 100,000 K per second. Following vitrification, collected samples are loaded into the electron microscope and precisely defined regions on the grid are hit with a focused electron beam, while the grid is tilted. The tilt series that results from these imaging sessions is comprised of hundreds of individual images, which must be aligned and averaged to resolve individual envelope proteins. This computationally intense process often requires computer clusters, capable of remote-access parallel processing, like NIH's Biowulf. Once completed, 3D models can not only provide insight into the structure of envelope glycoproteins themselves, but can also reveal the manner in which they interact other proteins, like the neutralizing antibody B12, which is information that can greatly influence vaccine design. This article, from the laboratory of Dr. Sriram Subramaniam involves contributions from twenty authors, including not only graduates students and postdocs, but also middle and highschool students, showing that you are never too young to make an impact in the fight against a disease that has infected over 60 million people, worldwide. In Clinical and Translational Medicine, JoVE presents an article from the University College London and the University of Oxford that investigates the physiological correlates of nociception, or pain sensation, in infants. Typically, infants respond to noxious stimuli by crying and changing their facial expressions - reactions considered to be autonomic and reliant on subcortical regions of the brain. In order to understand the involvement of the cerebral cortex and spinal cord in infant nociception our authors first prepare infants for EEG and EMG recordings. They also set up physiological monitoring and video recording, so that the infant's facial expressions can be linked to physiological data. Following assessment of infant responses to neutral tactile stimuli, the baby is subjected to heel lancing - a clinical procedure used to obtain blood samples from infants. After data processing, electrical potentials can be compared between noxious and non-noxious stimuli in these infants, which will hopefully lead to meaningful conclusions, regarding how nociceptive information is interpreted by newborns. JoVE's Bioengineering section takes a more clinical focus in December with an article from the University of Wisconsin, Madison, which involves the fabrication of a compartmentalized microfluidic device for studying cancer stem cell migration. Following device design, master molds are created by photolithography for the application of poly dimethyl siloxane (PDMS)- an organosilicon compound that is transparent and biologically inert - making it suitable for microfluidics applications with live cells. Specifically, our authors load tumor stem cells derived from multiform glioblastoma, a highly malignant brain cancer, into the compartmentalized devices and then place them in a specialized imaging system that acquires data over a few days. Time lapse video from these authors show that cancer stem cells are able to regulate their morphology in the size-constrained microfluidic channels much like they would in the interstitial space, and fluidic isolation made possible by chambers like these facilitates drug screening for potential therapeutics that can affect cancer stem cell migration. Late on in the month, JoVE travels to France, where investigators from the Institute Pasteur and the Centre National de la Recherche Scientifique, illustrate a method for inducing expression of channel-rhodopsin, a light-activated ion channel, into the olfactory bulb of mice, as well as subsequent neuronal stimulation using a miniature light emitting diode (LED). Our authors give detailed step-by-step instructions for preparing injection needles, and for precise injection of virus into the rostral migratory stream, a site of migration for adult born neuroblasts. Furthermore, they show how miniature LED stimulation devices are fabricated, calibrated, and implanted atop a cranial window prepared over the bulb. In particular, our authors are interested in the way that adult born neurons integrate into existing olfactory neuronal circuits, and they show that LED stimulation in awake behaving animals, can lead to activation of virally-tranduced neuroblasts in the olfactory bulb, which have migrated from the rostral migratory stream. These video-articles comprise JoVE's highlights for the month of December. Other noteworthy upcoming articles illustrate methods for patterning conductive inks into microelectrodes, measuring cytosolic calcium in contractile lymphatics, and imaging neuronal responses to pheromones in the vomeronasal organ.
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Mar 27, 2024
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