Research in the author&#8217;s laboratory is funded by the Biotechnology and Biological Sciences Research Council (BBSRC; grant references BB/N006372/1, BB/R005591/1, BB/R009333/1, BB/R016984/1).

<ol>
	<li>Jarvis, P., L&#243;pez-Juez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+homeostasis+of+chloroplasts+and+other+plastids.">Biogenesis and homeostasis of chloroplasts and other plastids.</a> Nature Reviews Molecular Cell Biology. 14 (12), 787-802 (2013).</li><li>Ling, Q., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+import+into+chloroplasts+isolated+from+stressed+plants.">Analysis of protein import into chloroplasts isolated from stressed plants.</a> Journal of Visualized Experiments. (117), (2016).</li><li>Aronsson, H., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+isolating+import-competent+Arabidopsis+chloroplasts.">A simple method for isolating import-competent Arabidopsis chloroplasts.</a> FEBS Letters. 529 (2-3), 215-220 (2002).</li><li>Fitzpatrick, L. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+isolating+a+high+yield+of+Arabidopsis+chloroplasts+capable+of+efficient+import+of+precursor+proteins.">A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins.</a> Plant Journal. 27 (1), 59-65 (2001).</li><li>Ling, Q., Jarvis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chloroplast+protein+import+by+the+ubiquitin+E3+ligase+SP1+is+important+for+stress+tolerance+in+plants.">Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants.</a> Current Biology. 25 (19), 2527-2534 (2015).</li><li>Lee, J., Kang, H., Hwang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+import+into+chloroplasts+using+protoplasts.">Studying protein import into chloroplasts using protoplasts.</a> Journal of Visualized Experiments. (142), (2018).</li><li>Asher, A., Ganesan, I., Klasek, L., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+physiologically+active+thylakoids+and+their+use+in+energy-dependent+protein+transport+assays.">Isolation of physiologically active thylakoids and their use in energy-dependent protein transport assays.</a> Journal of Visualized Experiments. (139), (2018).</li><li>Bouchnak, I., Moyet, L., Salvi, D., Kuntz, M., Rolland, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+chloroplast+sub-compartments+from+Arabidopsis+for+the+analysis+of+protein+Localization+by+immunoblotting+or+proteomics.">Preparation of chloroplast sub-compartments from Arabidopsis for the analysis of protein Localization by immunoblotting or proteomics.</a> Journal of Visualized Experiments. (140), (2018).</li><li>Salvi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AT_CHLORO:+the+first+step+when+looking+for+information+about+subplastidial+localization+of+proteins.">AT_CHLORO: the first step when looking for information about subplastidial localization of proteins.</a> Methods in Molecular Biology. 1829, 395-406 (2018).</li><li>Shanmugabalaji, V., Douet, V., Agne, B., Kessler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+chloroplast+translocon+protein+complexes+using+the+TAP+tag.">Affinity purification of chloroplast translocon protein complexes using the TAP tag.</a> Journal of Visualized Experiments. (141), (2018).</li><li>Kikuchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Ycf2-FtsHi+heteromeric+AAA-ATPase+complex+is+required+for+chloroplast+protein+import.">A Ycf2-FtsHi heteromeric AAA-ATPase complex is required for chloroplast protein import.</a> Plant Cell. 30 (11), 2677-2703 (2018).</li><li>Rantala, M., Paakkarinen, V., Aro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thylakoid+membrane+protein+complexes+by+blue+native+gel+electrophoresis.">Analysis of thylakoid membrane protein complexes by blue native gel electrophoresis.</a> Journal of Visualized Experiments. (139), (2018).</li><li>Kikuchi, S., Hirohashi, T., Nakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+preprotein+translocon+at+the+outer+envelope+membrane+of+chloroplasts+by+blue+native+PAGE.">Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE.</a> Plant and Cell Physiology. 47 (3), 363-371 (2006).</li><li>Chen, L. J., Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+megadalton+TOC-TIC+supercomplexes+as+major+mediators+of+protein+import+into+chloroplasts.">Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.</a> Plant Journal. 92 (2), 178-188 (2017).</li><li>Jarvis, R. P., JM, W. a. l. k. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 775, 432 (2011).</li><li>Jarvis, R. P., JM, W. a. l. k. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 774, 374 (2011).</li></ol>

Commercial application of the author&#8217;s research is covered by patent applications GB1803833.1, GB1803834.9, GB1815206.6 and US 16/643507.

Chloroplasts are the organelles that define plants1. Along with many other metabolic, developmental and signaling functions, chloroplasts are responsible for photosynthesis &#8211; the process whereby sunlight energy is harnessed to power the cellular activities of life. Consequently, chloroplasts are essential, not only for plants but also for the myriad of ecosystems that depend on plants, and for agriculture. Chloroplasts are composed of thousands of different proteins, most of which are nucleus-encoded and imported from the cytosol before being routed internally to one of a number of clearly-distinct intraorganellar compartments1. To achieve a more complete understanding of chloroplast development and functions, and to enable biotechnological strategies involving chloroplast manipulation that address global challenges linked to food security or bioenergy, it will be essential to determine the targeting, localization and interactions of important chloroplast proteins. This method collection describes a set of critically important and complementary techniques that may be used to accomplish these goals. The collection mostly focuses on the widely-used model plant Arabidopsis thaliana (thale cress), but the methods may also be adapted and applied to other organisms.
The collection includes descriptions of two different techniques for analyzing the import of nucleus-encoded proteins into chloroplasts across the double-membrane envelope. The article by Ling and Jarvis2 describes an in vitro method in which isolated chloroplasts are incubated with a radioactively-labelled precursor protein. The extent to which the chloroplasts take up the precursor protein is determined by monitoring the protein size change that occurs as a result of transit peptide (targeting leader sequence) cleavage, using SDS-PAGE and phosphor-imaging. The presented method is a development of an approach that has been used to study chloroplast protein import in vitro for several decades3,4, and incorporates additional steps that enable assessment of the responsiveness of the import machinery to stress conditions experienced by the plant5. On the other hand, the article by Lee et al.6 describes an in vivo method based on the transient expression of a chimeric precursor protein, carrying a fluorescent protein domain, in intact cells (protoplasts). In this assay, the extent of chloroplast protein import can be followed in two different ways: by monitoring the localization and intensity of the fluorescence signal using fluorescence microscopy; and, by analyzing the protein size change that occurs as a result of transit peptide cleavage using immunoblotting. These two methods are highly complementary, and can deliver compelling results when used together in parallel5.
Once a protein has been imported across the envelope into the chloroplast, and its transit peptide has been removed, it may either take on its final conformation in the stroma (the main internal aqueous compartment of the organelle), or engage one of a number of internal sorting pathways1. As the site of the highly-abundant photosynthetic complexes, the thylakoid membranes are a major destination for such internal sorting; in fact, thylakoid protein targeting involves multiple, mechanistically-distinct pathways. The article by Asher et al.7 describes a range of in vitro methods that enable different thylakoid protein translocation pathways to be studied. These methods involve the incubation of isolated thylakoids with radioactively-labelled precursor protein and, in some cases, a concentrated stromal extract. The extent to which the protein is taken up by the thylakoids is monitored by assessing cleavage of the targeting signal, and protection of the protein from exogenously-applied thermolysin protease, using SDS-PAGE and phosphor-imaging. Of course, the thylakoids are not the only subcompartment of the chloroplast, and it is often desirable to have the ability to assess other compartments as well. In this regard, the article by Bouchnak et al.8 is particularly important, as it describes methods for the subfractionation of chloroplasts to produce highly-pure samples corresponding to the envelope membranes, the stroma, and the thylakoids. Once prepared, these fractions may be analyzed by immunoblotting and/or mass spectrometry, in order to provide a wealth of information on the suborganellar localization of chloroplast proteins9.
With regard to the analysis of protein-protein interactions and multiprotein complex assemblies, two different methodologies are described in the collection. The article by Shanmugabalaji et al.10 presents a method for the affinity purification of chloroplast multiprotein complexes. This technique involves the analysis of transgenic plants expressing a component of the complex of interest that has been engineered to carry an affinity tag (the so-called tandem affinity purification tag, or TAP tag). The ability of this tag to bind strongly to an otherwise inert matrix is exploited as part of the purification strategy. The presented method focuses specifically on purification of the chloroplast protein import machinery (this comprises multiprotein complexes called TOC and TIC embedded in the envelope membranes1), but in principle it can be adapted to study any of the other multiprotein assemblies that exist in chloroplasts11. The article by Rantala et al.12 describes a complementary approach to complex characterization based on electrophoresis under native conditions. The technique, as presented here, involves the liberation of photosynthetic complexes from purified thylakoids using mild, non-ionic detergent, followed by their separation using blue native (BN)-PAGE. Primary resolution of the complexes may be followed by a second dimension of electrophoresis under denaturing conditions, and this enables visualization of the individual components of each complex identified in the first dimension. As with the TAP method, this native PAGE approach can be successfully adapted to study other protein complexes within the organelle13,14. With either approach, the purified complexes may be analyzed in a variety of ways, including by immunoblotting and mass spectrometry.
Together, the articles included in this methods collection present a powerful set of complementary techniques that, in conjunction with existing resources15,16, may be employed to greatly improve our understanding of diverse aspects of chloroplast biogenesis and function, particularly those closely linked to the organellar proteome. Because chloroplasts are responsible for the bulk of terrestrial photosynthetic primary production, and furthermore have vital roles in the responses of plants to the environment (including both biotic and abiotic stresses), these remarkable organelles will inevitably remain a major focus of fundamental and applied research across the globe for years to come.&#160;

chloroplast research methods: probing the targeting, localization and interactions of chloroplast proteins

Watch this Scientific Journal Video about Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins at JoVE.com

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins

Chloroplasts are the organelles that define plants1. Along with many other metabolic, developmental and signaling functions, chloroplasts are responsible ...

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Biochemistry

3.1K ビュー.  University of Oxford. Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins

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preparation of chloroplast sub-compartments from arabidopsis for the analysis of protein localization by immunoblotting or proteomics

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and...

affinity purification of chloroplast translocon protein complexes using the tap tag

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass...

using changes in leaf transmission to investigate chloroplast movement in arabidopsis thaliana

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Many plant species change the positioning of chloroplasts to optimize light absorption. This protocol describes how to use a straightforward, home-built instrument to investigate chloroplast movement in Arabidopsis thaliana leaves using changes in the transmission of light through a leaf as a...

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

localization of sumo-modified proteins using fluorescent sumo-trapping proteins

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell...

microfluidic tools for probing fungal-microbial interactions at the cellular level

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular...

an immunolabeling technique to visualize the subcellular localization of proteins

This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron...

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

methods for rapid transfer and localization of lyme disease pathogens within the tick gut

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks.  Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours.  We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within...

After osmication, immerse the tissue in 2% sodium acetate solution for 10 minutes at room temperature. Next, immerse the tissue in 2% uranyl acetate solution for 1 hour at room temperature. After uranyl acetate staining, dehydrate the tissue sequentially through the graded alcohols and...

transient expression and cellular localization of recombinant proteins in cultured insect cells

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also...

identification of plasmodesmal localization sequences in proteins in planta

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

probing high-density functional protein microarrays to detect protein-protein interactions

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in...

study of protein-protein interactions in autophagy research

Study of Protein-protein Interactions in Autophagy Research

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.<br...

in vivo immunofluorescence localization for assessment of therapeutic and diagnostic antibody biodistribution in cancer research

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining...

super-resolution imaging to study co-localization of proteins and synaptic markers in primary neurons

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal...

multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18...

fission yeast as a platform for antibacterial drug screens targeting bacterial cytoskeleton proteins

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence...

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

targeting gfp-tagged bacterial cytoskeletal proteins expressed in schizosaccharomyces pombe and microscopic imaging of the polymers

Targeting GFP-Tagged Bacterial Cytoskeletal Proteins Expressed in Schizosaccharomyces pombe and Microscopic Imaging of the Polymers

cell-free production of proteoliposomes for functional analysis and antibody development targeting membrane proteins

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody...

December 2011: This Month in JoVE

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.

Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).

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 ...

Biology

October 2012: This Month in JoVE

Here are some highlights from the October 2012 issue of Journal of Visualized Experiments (JoVE). Gr&oslash;nlund et al. demonstrate how to isolate and analyze specific cell types in plant leaves expressing green fluorescent protein (GFP) using fluorescence-activated cell sorting (FACS). This method overcomes the interfering fluorescence of the chlorophyll found in leaves, and distinguishes GFP-expressing protoplasts from non-GFP protoplasts. In JoVE Neuroscience, Babona-Pilipos et al. demonstrate how to construct a chamber for measuring galvanotaxis, or cell migration within an electric field. Through time-lapse imaging and image analysis, the authors can study the migratory behavior of neural precursor cells in an electric field, which may lead to the use of electrical stimulation to direct neural precursors to sites of injury or disease. Articles involving microfluidic platforms have a significant publication history in JoVE. Harris et al. have released three articles, which involve a microfluidic device for separating axons from neuronal cell bodies. This month in JoVE Neuroscience, Higashimori et al. use this microfluidic platform to investigate the interactions between neuronal axons and glial cells, which are critical for the physiological function of the nervous system. In JoVE Bioengineering, two research groups demonstrate the novel bioadhesive properties of chitosan-a polymer derived from chitin, which is found in fungal cell walls or in the exoskeletons of crustaceans and insects. Chitosan is used in many industrial and agricultural applications, and bioengineers are also finding uses for it in surgical applications. Foster et al. have developed a laser-activated surgical film called Surgilux, which combines chitosan with indocyanine green ICG), a photosensitive dye. This surgical film binds strongly to tissue, such as muscle, after laser irradiation. Lauto et al. developed a surgical film that combines chitosan with the photoactive dye, rose Bengal. This novel film also binds strongly to tissues, such as intestine, after it is irradiated. These adhesive films are biocompatible and may potentially be used in various surgical procedures in place of sutures. In JoVE Clinical and Translational Medicine, Fiema et al. demonstrate a high-throughput technique for validating biomarkers in graft vs. host disease, a common and life-threatening complication of cell or tissue transplantation. The authors use commercially available ELISAs to analyze multiple proteins in sequential fashion. In JoVE Immunology and Infection, Keyel et al. demonstrate how to measure the real-time kinetics of immune cell responses to bacterial toxins using high-speed live cell microscopy. This method can be used to show how immune cells respond to bacterial toxins. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response. In JoVE Applied Physics, Borisenko et al. determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy in a synchotron radiation facility. By combining recent advances in synchotron radiation, surface science, and cryogenics, this method can gain a precise picture of the energy and momentum of electrons inside a solid, and address key questions in the field of condensed matter physics. This preview summarizes just a few of the notable video-articles available in the October 2012 issue of JoVE. For additional videos, please visit <a href="http://www.jove.com">www.jove.com</a>.

Here are some highlights from the October 2012 Issue of Journal of Visualized Experiments (JoVE).

Here are some highlights from the October 2012 issue of Journal of Visualized Experiments (JoVE).  Gr&oslash;nlund et al. demonstrate how to isolate and ...

2012: A Year In Review

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE reached a major milestone by publishing our 1500th article. In this publication, in JoVE Bioengineering, Yu et al. described a method for analyzing the free radical composition of cigarette smoke. This method simulated cigarette puffing with a single-port smoking device, and combined it with electron spin resonance (ESR) spectroscopy to measure the degree to which antioxidant compounds can scavenge free radicals in cigarette smoke. A possible outcome of this research is the creation of less harmful cigarettes.
JoVE Bioengineering also contained the most-viewed article of 2012. Owczarczak et al. described a method for hacking a standard inkjet printer, loading ink cartridges with a cell-suspension "bioink," and printing lines of live cells onto glass slides. The thermal inkjet printing process creates temporary pores in the cell membrane, visualized by the incorporation of fluorescently labeled actin monomers. This concept, called "bioprinting," has many potential applications in cell and tissue engineering.
Also in 2012, JoVE Bioengineering featured a protocol by Hsia et al. for isolating and purifying spider silk protein, spinning it into fibers, and assessing the fiber strength. This protocol for laboratory-scale production of spider silk, which is stronger than tensile steel, can potentially be extended to large-scale manufacturing.
In JoVE Clinical and Translational Medicine, we featured a number of articles related to stroke, one of which. In one article, M&ouml;bius-Winkler et al. demonstrated placement of the WATCHMAN left atrial appendage occlusion device from Boston Scientific. This device is designed to prevent strokes by trapping clots within the left atrial appendage of the heart before they exit. Our authors demonstrated how to insert the guide wire through the femoral artery into the heart, advance the device into the left atrium, and deploy the device in the left atrial appendage.
Another article focused on stroke, Hamel et al. described the use of a driving simulator to study how patients compensate for visual field defects following stroke. By analyzing the compensatory gaze behavior of patients as they navigated through virtual driving courses with varying degrees of complexity, our authors see great potential for the use of driving simulators in stroke rehabilitation.
In JoVE Immunology and Infection, we published an article by Keyel et al. describing the real-time kinetics of immune cell responses to bacterial toxins using live cell microscopy. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response.
Also in JoVE Immunology and Infection, we featured an article by Yap et al. describing methods for diagnosing helminth infections in children. Helminths are parasitic worms that can infect the intestinal tract of humans and animals, and may be distinguished based on their morphology. The same article demonstrated how to measure the impact of helminth infections on physical fitness in children, which is a measure of overall health.
In JoVE Neuroscience, we featured a method by Hoffmann et al., who used tiny headphones to alter the auditory feedback in songbirds, and analyzed the computational and neurophysiological basis of vocal learning in birds when they adjust their singing in response to altered acoustic signals.
Also in JoVE Neuroscience, we moved from birds to bees with a method that assesses associative and non-associative tactile learning in honeybees. This method, by Mujagi&#263; et al., allowed bees to scan different metal surfaces with their antennae, then conditioned them to expect sugar water when their antennae touched particular surfaces, and analyzed the corresponding changes in antennal movement.
In 2012, we launched a brand-new section: JoVE Applied Physics, which features articles on subjects ranging from plasma physics to material science. The Applied Physics section also contained JoVE's 2000th video article, filmed in a synchrotron radiation facility, where Borisenko et al. showed how to determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy.
In the JoVE General section, we published an article by Artioli et al. demonstrating the use of a portable gas analyzer to measure oxygen consumption during complex exercise - such as judo. By analyzing oxygen consumption and collecting blood for measuring plasma lactate concentration, this method can determine the relative contributions of different energy systems to specific aspects of complex exercise.
Finally, JoVE followed Dolhi et al. to a permanently ice-covered saline lake in Antarctica. There, scientists drilled through the ice to study single-celled microorganisms called protists that live in the harsh conditions of Antarctica.
This Year in Review was just a brief glimpse of a few of over 600 video articles that JoVE offered in 2012. Browse the JoVE archives to see thousands of other videos, and stay tuned for what's coming up this year in JoVE: The Journal of Visualized Experiments.

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).

 
 
Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE ...