Name: fruit volatile analysis using an electronic nose
Uploaded: 2012-03-30T13:00:00
Duration: 11 min 2 s
Description: Watch this Scientific Journal Video about Fruit Volatile Analysis Using an Electronic Nose at JoVE.com

الكتاب أشكر بيل العبي (هاريس شركة البذور موران، ديفيس) لتوفير ثمار البطيخ لهذا التحليل. ويدعم هذا المشروع من قبل برنامج بحوث المحاصيل الاختصاص مبادرة المنح التنافسية لا تمنح. 2009-51181-05783 من المعهد الوطني وزارة الزراعة الأميركية للأغذية والزراعة.

<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>

ليس لدينا ما يكشف.

أنوف إلكترونية يمثل طريقة واعدة لتقييم سريع وموضوعي لمحات من رائحة الفواكه أو عينات المضطربة الغنية بالنفط. ومع ذلك، والتحولات في الوقت الاحتفاظ تمثل تحديا لتحديد الذروة ويمكن أن يؤدي إلى سوء تفسير البيانات عندما لا يتم 2 المخططات الاستشرابية متوافقة تماما. وأشار الفحص البصري من المخططات الاستشرابية أن تباين مرات الاحتفاظ بين عينات تسببت في كثير من الأحيان في ذروة نفسه إلى أن يكون المسمى مع القيم KI مختلفة قليلا (ما يقرب من ± 10). هذا يترجم إلى عدد مبالغ فيه من كيس فريدة من نوعها المكتشفة. من أجل الاستفادة من الحقائق التي (أ) مركبات مختلفة موجودة في مراحل النضج المختلفة و (ب) يعيد التقنية متطابقة تقريبا، ومخطوطات التي تعتمد على الكمبيوتر 2 (&quot;kim_merge.py&quot;، والذي يتضمن إجراءات لمعالجة البيانات وقد وضعت مجموعة، و &quot;kim_interface.py&quot;، الذي يوفر واجهة المستخدم الرسومية (GUI)) على نحو منتظممقارنة عينات بطريقة شبه آلية، والحد بشكل كبير من الوقت اللازم لتحليل اللوني مجموعات كبيرة من البيانات. هذه البرامج تسمح للتوحيد، عند الاقتضاء، من قمم المسمى مع مجموعة من القيم KI تحت تسمية واحدة كي. هذا يخدم غرضين هامين: (أ) فإنه يمكن إجراء تحليل إحصائي لعلاج قمم مثل متغير واحد، و (ب) أن تسهل تحديد الذروة وبالمقارنة مع غيرها من النظم والقيم التي تم نشرها. النتائج المعروضة هنا تشير إلى أنه يمكن تمييز عينات البطيخ على أساس الاستحقاق والتنميط رائحة باستخدام نظام zNose في تركيبة مع تحديد KI كاف. وهذا يمثل تكنولوجيا جديدة واعدة لتحليل المواد المتطايرة التي يمكن استخدامها لبرامج مراقبة الجودة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> كلوريد الكالسيوم </td><td> النائب الطبية الحيوية </td><td> 195088 </td><td></td></tr><tr><td> 2-Methylbutyl isovalerate </td><td> SAFC العالمية </td><td> W350613 </td><td> ≥ 98٪، والطبيعية، لجنة الاتصالات الفدرالية </td></tr><tr><td> الميثانول </td><td> فيشر العلمية </td><td> A411-4 </td><td></td></tr><tr><td> قارورة </td><td> سيغما / Supelco </td><td> SU860098 </td><td></td></tr><tr><td> قبعة </td><td> سيغما / Supelco </td><td> SU860101 </td><td></td></tr><tr><td> مختبر خلاط </td><td> وارنج مختبر العلوم </td><td> 7009G </td><td> 2-سرعة الخلاط: 1 - ليتر عاء زجاجي </td></tr><tr><td> زجاجة </td><td> فيشر العلمية </td><td> 06-414-1C </td><td> بيركس، 500 مل، البولي بروبلين المكونات ختم </td></tr><tr><td> إبرة </td><td> الاستشعار الالكترونية تقنية </td><td> TLC101046 </td><td> الجانب ثقب luer </td></tr><tr><td> الألكانات حل </td><td> الاستشعار الالكترونية تقنية </td><td></td><td> C6-C14 الألكانات حل في الميثانول </td></tr><tr><td> zNose </td><td> الاستشعار الالكترونية تقنية </td><td> نموذج 4500 </td><td></td></tr><tr><td> DB-5 عمود GC </td><td> الاستشعار الالكترونية تقنية </td><td> SYS4500C5 </td><td></td></tr><tr><td> MicroSense </td><td> الاستشعار الالكترونية تقنية </td><td> الإصدار 5.44.22 </td><td></td></tr><tr><td> بيثون 2.6 </td><td></td><td></td><td> متاحة مجانا على الإنترنت </td></tr><tr><td> &quot;reform_data.py&quot; و &quot;kim_interface.py&quot; مخطوطات</td><td></td><td></td><td> كما تتوفر مواد تكميلية عن مخطوطات [جوف] </td></tr></tbody></table>

fruit volatile analysis using an electronic nose

التغيرات الفسيولوجية عديدة ومتنوعة تحدث أثناء نضج الثمار، بما في ذلك تطوير مزيج متقلب المحددة التي تميز رائحة الفاكهة. النضج عند الحصاد هو واحد من العوامل الرئيسية التي تؤثر على جودة نكهة الفواكه والخضروات 1. المصادقة على وسائل قوية التي تقيم بسرعة نضج الثمار ونوعية رائحة سيسمح تحسين إدارة برامج التربية المتقدمة، وممارسات الإنتاج والمناولة بعد الحصاد. على مدى العقود الثلاثة الماضية، وقد أجريت الكثير من البحوث لتطوير ما يسمى الأنوف الإلكترونية، والتي هي أجهزة قادرة على الكشف عن الروائح والنكهات بسرعة 2-4. حاليا هناك العديد من أنوف الإلكترونية المتاحة تجاريا قادرا على أداء تحليل متقلبة، تعتمد على تقنيات مختلفة. الأنف الالكترونية المستخدمة في عملنا (zNose، بتوقيت شرق الولايات المتحدة، نيوبري بارك، كاليفورنيا، الولايات المتحدة الأمريكية)، ويتألف من اللوني للغاز فائقة السرعة إلى جانب وجود موجة استشعار سطح الصوتية (UFGC-ص). وقد تم بالفعل هذه التكنولوجيا لاختبار قدرته على رصد نوعية السلع المختلفة، بما في ذلك الكشف عن تدهور في التفاح 5؛ النضج وتقييم تعفن في المانجو 6؛ التنميط رائحة الأنواع الغدة الصعترية (7)؛ C 6 مركبات طيارة في التوت العنب 8؛ توصيف الزيوت النباتية (9) وكشف الغش في زيت جوز الهند 10. هذا النظام يمكن تنفيذ الخطوات الرئيسية الثلاثة لتحليل رائحة: فراغ الرأس لأخذ العينات، والفصل بين مركبات طيارة، والكشف. في حوالي دقيقة واحدة، ويتم إنتاج الإخراج، واللوني، وبعد دورة تطهير، صك على استعداد لمزيد من التحليل. ويمكن مقارنة النتائج المتحصل عليها مع zNose لتلك النظم الغاز الكروماتوغرافي الأخرى عن طريق حساب الأرقام القياسية لKovats (كي). مرة واحدة تم ضبطها الصك مع حل معيار ألكان، يتم تحويلها تلقائيا إلى الاحتفاظ مراتكيس. ومع ذلك، يتوقع تغييرات طفيفة في درجة الحرارة ومعدل تدفق للتحدث مع مرور الوقت، مما تسبب في مرات الاحتفاظ إلى الانحراف. أيضا، اعتمادا على قطبية المرحلة عمود ثابت، لا يمكن للاستنساخ من حسابات KI تختلف من وحدة مؤشر عدة 11. وقد وضعت لذلك سلسلة من البرامج واجهات رسومية لمقارنة بين عينات KIS محسوبة بطريقة شبه آلية. هذه البرامج تقليل الوقت اللازم لتحليل اللوني مجموعات كبيرة من البيانات وتقليل احتمالات سوء تفسير البيانات عندما لا تكون متوافقة تماما المخططات الاستشرابية. نحن نقدم طريقة لتحليل مركب سريع التقلب في الفاكهة. وتناقش أيضا إعداد نموذج، الحصول على البيانات والتعامل مع الإجراءات.

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

Fruit Volatile Analysis Using an Electronic Nose

ووصف طريقة سريعة لتحليل مركب متطاير في الفاكهة. ويفصل بسرعة المتطايرة موجودة في فراغ الرأس من جناسة من العينة والكشف مع اللوني للغاز فائقة السرعة (GC) إلى جانب وجود سطح الموجة الصوتية (SAW) الاستشعار. ويناقش أيضا إجراء لمعالجة البيانات وتحليلها.

تحليل الفاكهة المتطايرة باستخدام الأنف الالكتروني

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

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

دراسة العدوان في ذبابة الفاكهة (ذبابة الفاكهة)

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.

الموقع محلية الصنع تطفير موجه من البلازميدات الجامعة

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.

والخلايا العصبية ونجمية المشارك الثقافة الفحص لتحليل محتوى عال من العصبية

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.

بروتوكول الأمثل لتربية Fopius arisanus ، وهو الطفيل من الفاكهة Tephritid الذباب

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.

العزلة ودراسة البيوفيزيائية البشرة من الفاكهة

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.

تحليل الانصهار الغشاء بوساطة كمين باستخدام الانصهار خلية الفحص الانزيمي

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.

بروتوكول مختبر لتحليل محتوى الأمعاء الوراثية ماكروينفيرتيبراتيس المائية استخدام مجموعة محددة المتاشب كبسولة تفجير

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.

أدوات بسيطة محلية الصنع للتعامل مع ذباب الفاكهة -دروسوفيلا ميلانوجاستر

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.

الفحص الحيوي للتطبيق الموضعي لتحديد سمية المبيدات الحشرية للبعوض وذباب الفاكهة

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.

بروتوكولات كريسبر / كاس 9 طفرات ذبابة الفاكهة الشرقية باكتروسيرا الظهرية

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 ...