The authors thank Bill Copes (Harris Moran Seed Company, Davis) for providing melon fruits for this analysis. This project is supported by the Specialty Crops Research Initiative Competitive Grants Program grant no. 2009-51181-05783 from the USDA National Institute of Food and Agriculture.

1. Sample Preparation
<ol>
 <li>Harvest fruits at desired maturity stage. Rinse with tap water in order to remove dirt and dust.</li>
 <li>Select fruits for analysis based on the absence of external and internal defects, and size homogeneity.</li>
 <li>Cut fruits longitudinally into wedges to be used for volatile sampling. If applicable, remove skin, seeds, seed cavity tissue, or pit. Fruit tissue selection must be consistent throughout the experiment and take into account the variability within a single fruit (i.e., obtain sample equally from the equatorial, blossom and stem end parts).</li>
 <li>Combine the selected fruit tissue, mix it in order to randomize it, and then weigh out 200 g into a blender.</li>
 <li>Add 200 mL of saturated CaCl2 solution (372.5 g at 20 &deg;C, in 500 mL of deionized water) and 50 &mu;L of a 100 mM solution of 2-methylbutyl isovalerate in methanol. The CaCl2 is intended to act as an inhibitor of enzymatic activity, which might occur after cutting and homogenizing the fruit flesh. 2-Methylbutyl isovalerate is added as an internal standard to monitor for any possible losses of volatile compounds during the homogenization process.</li>
 <li>Homogenize mixture in a laboratory blender (Waring, USA), for 30 seconds at 18,000 rpm, then immediately pour into glass bottle and seal with Teflon lid. Keep the homogenate in the bottle at room temperature until all samples are prepared.</li>
 <li>After pouring the homogenate into the bottle, wait 10 minutes to allow the separation of the foam from the liquid, then pipette 5 mL aliquots of liquid , without foam, into 20 mL glass amber vials and seal the vials with steel screw caps fitted with Teflon/silicone septa. This procedure is suitable for melon and pear homogenate preparation. If other fruits are used for the analysis, a centrifugation step might be required. Therefore, remove the foam and then centrifuge the liquid to pellet particles that might obstruct the pipette. Prepare at least three vials per sample to serve as technical replicates.</li>
 <li>At this point, samples may be analyzed immediately or flash-frozen in liquid nitrogen and stored at ultra-low temperature (-80 &deg;C) for later analysis.</li>
 <li>For frozen samples, on the analysis day remove the samples from the freezer and allow them to thaw for one hour at room temperature. After thawing, and before analysis, replace the vial cap with a new one that has a clean, dry septum. If the septum is not replaced, water condensed on the septum during thawing can be drawn into the instrument and damage it.</li>
</ol>
2. Gas Chromatography-surface Acoustic Wave (GC-SAW) Set-up and Data Acquisition
<ol>
 <li>Load the appropriate analysis method on the zNose. 
 For analysis of the ester-rich volatile profile of melon, our parameters in the MicroSense version 5.44.22 software (Newbury Park, CA, USA) are as follows: headspace suction into the inlet for 20 seconds at 30 mL min-1 via pump; inlet temperature at 200 &deg;C; Tenax trap temperature at 225 &deg;C; carrier gas (helium purity 99.999%) flow rate of 2.9 mL min-1; column (DB-5 column, 1 m &times; 0.25mm I.D.&times; 0.25 &mu;m film thickness) temperature program from 45 &deg;C to 180 &deg;C at a rate of 10 &deg;C sec-1; sensor temperature at 40 &deg;C; valve at 165 &deg;C. The total analysis time is 1 minute per sample.</li>
 <li>Connect a stainless steel needle with non-coring tip to the zNose inlet and purge the system several times with ambient air until the baseline is stable and no peaks larger than 200 Counts (Ct) are detected.</li>
 <li>Tune the instrument using a solution of straight chain alkanes (C6-C14). The tune result is used by the instrument software to convert the retention time of the eluted peaks from time units into Kovats Index (KI) units. Consequently, after the system is tuned, the retention times are reported in KI units.</li>
 <li>Before the analysis, allow the sample to equilibrate for 30 minutes. To analyze one of the sample vials, insert a needle into the vial septum to relieve the pressure. Then insert the needle connected to the instrument inlet into the vial septum and initiate headspace sampling. Analyze at least three technical replicates per sample.</li>
 <li>Manually start the instrument by clicking on "Play" button; the pump activates and withdraws the vapors present above the sample. At the end of the analysis, a chromatogram appears on the screen, and the sensor is automatically heated to 150 &deg;C for 10 seconds to clean it. When the system status box button turns green again, the instrument is ready to analyze another sample.</li>
 <li>To ensure a stable baseline and proper system cleaning, run at least one air blank between each sample. To monitor for possible volatile contamination from the vial and cap, analyze two vial-blanks (empty vial with cap), at the beginning and end of the day.</li>
</ol>
3. Data Export and Analysis
<ol>
 <li>Export the data into a Microsoft excel file after acquisition using the "Peak logging" function in MicroSense software. Once the data are exported, add columns containing labels for variables and replicates.</li>
 <li>Transform the data format for easier manipulation using the Python (version 2.6; freely available on-line) script we developed, named "reform_data.py"(see Figure 1 for an example of the data format before and after using the script "reform_data.py"). The name of the source file (xls format) and sheet name for the input data, as well as the desired file name for output (xls format), are edited directly in the script.</li>
 <li>Start "kim_interface.py" (also written in Python 2.6; see Figure 2), and import the data from the file generated in the previous step. Specifically, the analysis is based on viewing and analyzing the number of times each KI value was detected ("KI hits"). Thus the program displays a bar graph of KI hits for each KI value.</li>
 <li>Evaluate the KI hits of specific subsets of samples, analyzing each group of technical replicates together. To do so, analyze every treatment or variable separately by checking/unchecking the corresponding boxes. See Figure 2 caption for detailed description of the graphical user interface (GUI) features.</li>
 <li>After identifying the width of each KI window using the GUI, randomly select some of the corresponding chromatograms in Microsense software and evaluate overlapping peaks among the technical replicates. See Figure 3 for an example of overlaid chromatograms of two technical replicates.</li>
 <li>Once the KI window is individuated, use the 'Merge' feature available in the GUI to merge the KIs that fall in the window, into the most populated KI. By using this feature, peaks labeled with a range of KI values are consolidated under a single KI label, allowing treating such peaks as a single variable. 
 To do so, first click on the 'Merge' button to activate the feature and select the most populated KI as the center of the window by left-clicking the corresponding bar. Once the bar has been selected, it changes color and turns green. To merge the KIs that fall within the window into the selected KI, right-click on the corresponding bars; this causes the bars to turn red, while a blue bar of the corresponding length is added on top of the central KI (See Figure 4). Once all the selected KIs have been merged into the appropriate central KI, click on the 'Merge' button again to accept the changes; this causes the 'Merge' button to turn yellow. In case of mistakes, the 'Unmerge' button is also available. To unmerge, click the 'Unmerge' button in the GUI, then right-click on the red bar you want to unmerge. From red, the bar turns blue. Click the 'Unmerge' button again to accept the changes.</li>
 <li>If one attempts to incorrectly merge two peaks in a single sample into a single KI value, an error message is printed. In such circumstances, closely check the chromatogram and redefine the KI window in that region.</li>
 <li>Once all of the merging operations have been performed, save the file.</li>
 <li>Before proceeding with statistical analysis, the chromatograms of the air and vial blanks are analyzed to monitor for possible contaminations. Once the KI of the peaks in the blanks have been identified, subtract the area of the peak detected in the air- and/or vial-blank from the area of the peak present in sample. 
 Then proceed with statistical analysis.</li>
</ol>
4. Representative Results
The electronic nose was able to detect differences in volatile profiles among melon fruit harvested at different maturity stages (Figure 5). Twenty KI windows were identified across all samples. An analysis of variance showed that 14 peaks detected by the electronic nose varied significantly between maturity stages. In Figure 6, the log of the mean peak areas of these 14 components are plotted to show differences in peak abundances between two maturity stages, early mature and fully ripe fruit.
<img alt="Figure 1" src="/files/ftp_upload/3821/3821fig1.jpg" /> 
Figure 1. Examples of data format exported from instrument software (A) and after transformation, performed using "reform_data.py" script (B). To facilitate data manipulation and analysis, all the unique KIs are identified across all the samples, then the data are reordered with sample information in rows and peak area in columns, corresponding to unique KIs. If a peak is not detected for a KI value in a sample, the corresponding cell remains empty.
<img alt="Figure 2" src="/files/ftp_upload/3821/3821fig2.jpg" /> 
Figure 2. Screen capture from the script file "kim_interface.py". The plot in the center displays number of hits per KI versus KI. 'Hit per KI' is the number of samples in which a peak with that specific KI was detected. On the left side, there are three yellow boxes controlling the selected data. They display parameters to divide the data set (treatments, replicates, qualitative variables, etc.). In this figure, they are (from top to bottom): Variety, Planting date and Maturity stage at harvest. On the bottom: by clicking on the 3 bars and moving the blue bar to the left or to the right, one can select the minimum and the maximum value of the KI range, and the minimum peak area ('Threshold'). On the right: the 'Merge' button allows merging selected KIs by manually clicking on the bars in the plot. The 'Unmerge' button allows one to reverse the process for selected cases.
<img alt="Figure 3" src="/files/ftp_upload/3821/3821fig3.jpg" /> 
Figure 3. Overlaid chromatograms (in black and red) of two technical replicates from melon volatile headspace to illustrate a shift in retention time.
<img alt="Figure 4" src="/files/ftp_upload/3821/3821fig4.jpg" /> 
Figure 4. Example of the KI merging procedure. In the central plot, the green bar (Central KI) represents the most populated KI, which has been selected as center of the KI window. KI X and KI Y are KIs falling in the window of interest and they need to be merged into the central KI. By right-clicking on KI X's bar, it turns red and, at the same time, a blue bar of the same length of KI X's bar, appears on top of the green one. By repeating the same procedure for KI Y, the length of the blue bar (Merged KIs) will increase of the corresponding length. Once all the KIs have been added, by clicking on the green 'Merge' button, the merging process ends, the changes are saved, and the button color turns yellow.
<img alt="Figure 5" src="/files/ftp_upload/3821/3821fig5.jpg" /> 
Figure 5. Two chromatograms of melon samples harvested at different maturity stages, early mature (top) and fully ripe (bottom), to illustrate the ability of the electronic nose to detect differences in volatile abundances.
<img alt="Figure 6" src="/files/ftp_upload/3821/3821fig6.jpg" /> 
Figure 6. Radar plot showing the peak area of 14 components present in two melon samples at two different maturity stages, early mature and fully ripe. The peak areas are reported in log scale to help visualize the comparison. The numbers at the end of each ray represent the corresponding Kovats Indices.

<ol>
	<li>Kader, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavor+quality+of+fruits+and+vegetables.">Flavor quality of fruits and vegetables.</a> Journal of the Science of Food and Agriculture. 88, 1863-1868 (2008).</li><li>Persaud, K., Dodd, G. <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+discriminant+mechanisms+in+the+mammalian+olfactory+system+using+a+model+nose.">Analysis of discriminant mechanisms in the mammalian olfactory system using a model nose.</a> Nature. 299, 352-355 (1982).</li><li>Gardner, J. W., Bartlett, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief-history+of+electronic+noses.">A brief-history of electronic noses.</a> Sensors and Actuators. 18, 211-220 (1994).</li><li>Rock, F., Barsan, N., Weimar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+nose:+Current+status+and+future+trends.">Electronic nose: Current status and future trends.</a> Chem. Rev. 108, 705-725 (2008).</li><li>Li, C., Heinemann, P. H., Irudayaraj, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+apple+deterioration+using+an+electronic+nose+and+zNose.">Detection of apple deterioration using an electronic nose and zNose.</a> Transactions of the Asabe. 50, 1417-1425 (2007).</li><li>Li, Z. F., Wang, N., Raghavan, G. S. V., Vigneault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ripeness+and+rot+evaluation+of+'Tommy+Atkins'+mango+fruit+through+volatiles+detection.">Ripeness and rot evaluation of 'Tommy Atkins' mango fruit through volatiles detection.</a> J. Food Eng. 91, 319-324 (2009).</li><li>Oh, S. Y., Ko, J. W., Jeong, S. Y., Hong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+and+exploration+of+fast+gas+chromatography-surface+acoustic+wave+sensor+to+the+analysis+of+thymus+species.">Application and exploration of fast gas chromatography-surface acoustic wave sensor to the analysis of thymus species.</a> J. Chromatogr. A. 1205, 117-127 (2008).</li><li>Watkins, P., Wijesundera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zNose+for+the+analysis+of+selected+grape+aroma+compounds.">Application of zNose for the analysis of selected grape aroma compounds.</a> Talanta. 70, 595-601 (2006).</li><li>Gan, H. L., Man, Y. B. C., Tan, C. P., NorAini, I., Nazimah, S. A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+vegetable+oils+by+surface+acoustic+wave+sensing+electronic+nose.">Characterisation of vegetable oils by surface acoustic wave sensing electronic nose.</a> Food Chem. 89, 507-518 (2005).</li><li>Marina, A. M., Man, Y. B. C., Amin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+SAW+Sensor+Electronic+Nose+for+Detecting+the+Adulteration+of+Virgin+Coconut+Oil+with+RBD+Palm+Kernel+Olein.">Use of the SAW Sensor Electronic Nose for Detecting the Adulteration of Virgin Coconut Oil with RBD Palm Kernel Olein.</a> Journal of the American Oil Chemists Society. 87, 263-270 (2010).</li><li>Evans, M. B., Haken, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+the+gas-chromatographic+index+scheme.">Recent developments in the gas-chromatographic index scheme.</a> Journal of Chromatography. 472, 93-127 (1989).</li></ol>

We have nothing to disclose.

Electronic noses represent a promising method for the rapid, objective evaluation of aroma profiles from fruits or volatile-rich samples. However, shifts in retention time represent a challenge for peak identification and might lead to misinterpretation of the data when two chromatograms are not perfectly aligned. Visual inspection of chromatograms indicated that the variability of retention times among samples frequently caused the same peak to be labeled with slightly different KI values (approximately &plusmn;10). This translated into an exaggerated number of unique KIs detected. In order to take advantage of the facts that (a) different compounds are present at different maturity stages and (b) technical replicates are approximately identical, two computer-based scripts ("kim_merge.py", which contains the routines for handling the data set, and "kim_interface.py", which provides a graphical user interface (GUI)) were developed to systematically compare samples in a semi-automated fashion, greatly reducing the time needed for chromatogram analysis of large data sets. These programs allow the consolidation, where appropriate, of peaks labeled with a range of KI values under a single KI label. This serves two important purposes: (a) it enables a statistical analysis to treat such peaks as a single variable, and (b) it facilitates peak identification and comparison to other systems and published values. Results presented here indicate that melon samples could be discriminated based on maturity and aroma profiling using the zNose system in combination with adequate KI identification. This represents a promising new technology for analysis of volatiles that may be used for quality control programs.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>MP Biomedical</td>
 <td>195088</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-Methylbutyl isovalerate</td>
 <td>SAFC Global</td>
 <td>W350613</td>
 <td>&ge; 98%, natural, FCC</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher Scientific </td>
 <td>A411-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vial</td>
 <td>Sigma/Supelco</td>
 <td>SU860098</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cap</td>
 <td>Sigma/Supelco</td>
 <td>SU860101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laboratory blender</td>
 <td>Waring Laboratory Science</td>
 <td>7009G</td>
 <td>2-speed blender;
 &nbsp;1- Liter glass container</td>
 </tr>
 <tr>
 <td>Bottle</td>
 <td>Fisher Scientific</td>
 <td>06-414-1C</td>
 <td>Pyrex, 500 mL; polypropylene plug-seal </td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Electronic Sensor Technology</td>
 <td>TLC101046</td>
 <td>Side hole luer</td>
 </tr>
 <tr>
 <td>Alkanes solution</td>
 <td>Electronic Sensor Technology </td>
 <td>&nbsp;</td>
 <td>C6-C14 alkanes solution in methanol</td>
 </tr>
 <tr>
 <td>zNose</td>
 <td>Electronic Sensor Technology</td>
 <td>Model 4500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DB-5 GC column</td>
 <td>Electronic Sensor Technology</td>
 <td>SYS4500C5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MicroSense</td>
 <td>Electronic Sensor Technology</td>
 <td>Version 5.44.22 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Python 2.6</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Freely available on-line</td>
 </tr>
 <tr>
 <td>"reform_data.py" and "kim_interface.py" scripts</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Scripts available as supplementary material on JoVE</td>
 </tr>
 </tbody>
</table>

fruit volatile analysis using an electronic nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

Watch this Scientific Journal Video about Fruit Volatile Analysis Using an Electronic Nose at JoVE.com

Fruit Volatile Analysis Using an Electronic Nose

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

fruit-volatile-analysis-using-an-electronic-nose

Research

JoVE Journal

Biology

21.2K vues.  University of California, Davis. Une méthode rapide pour l'analyse des composés volatils dans les fruits est décrite. Les composés volatils présents dans l'espace de tête d'un homogénat de l'échantillon sont séparés et détectés rapidement à la chromatographie gazeuse ultra-rapide (GC) couplée à une onde acoustique de surface (SAW) du capteur. Une procédure de traitement des données et l'analyse est également discuté.

Vidéo: Fruit Volatile Analysis Using an Electronic Nose

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Simple Homemade Tools to Handle Fruit Flies&amp;#8212;&lt;em&gt;Drosophila melanogaster&lt;/em&gt;

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...