قدم هذا الإنتاج والعمل التجريبي تعكس هذه الوثيقة مساعدة مكرسة ومتحمسة لدعم باريجا رينالدو تفضلت أغيري لينا وجيلبرتو قرطبة ، ميرا وليام ، بونيلا زيمينا وفيفيروس داريو. كما نشكر جون مايلز وثلاثة مراجعين المجهول التعليقات التي ساعدتنا على تحسين الفيديو والمخطوطة.

1. النباتات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وتستخدم قصاصات الجذعية الناضجة واحدة من محطات وحدات تجريبية. وقلص لفتات الى 10 سم لضمان اتساق المواد الزراعية. </li><li style=";text-align:right;direction:rtl"> لمنع التلوث بواسطة مسببات الأمراض النباتية ، وتغسل القطع في محلول هيبوكلوريت الصوديوم 3 ٪ لمدة 2 دقيقة ثم تشطف وthoughroughly مع مياه الحنفية. </li><li style=";text-align:right;direction:rtl"> هي التي زرعت في كل القطاعات في كاليفورنيا. 36 غرام من التربة المعقمة في أنبوب أسطواني PVC (5.3 سم خارج القطر بواسطة سم الارتفاع 6.5) مختومة في الطرف الأدنى مع فنجان الستايروفوم وتوج مع جلبة PVC. يقام في مكان القطع التي وضعت حلقة الرغوة في افتتاح المركزية للجلبة. </li><li style=";text-align:right;direction:rtl"> يتم إخصاب النباتات والماء حسب الحاجة للحفاظ على رطوبة التربة كافية. </li><li style=";text-align:right;direction:rtl"> وسوف تنمو النباتات لمدة شهر قبل أن يتم الطعن عن طريق الحشرات ، مما يسمح للجذور سطحية التنمية ، والتي تكون بمثابة تغذية مواقع لالحوريات spittlebug الجذر الزيليم الثديية. </li><li style=";text-align:right;direction:rtl"> هي مقلوب الأنابيب البلاستيكية مع قطع الجذور لمدة 8 د قبل غزو لحفز مزيد من نمو جذور سطحية ، وتعديل جزئيا عن الاختلافات الكامنة في بنية الجذر. هذه الخطوة يتطلب وجود مصدر للضوء الاصطناعي ، والتي نقدمها ليوم 24 هكتار. لتجنب الخلط يهرب من الحيوانات العاشبة مع المقاومة عاشب ، النبات الفردية يتطابق مع جذور سطحية قليلة أو معدومة بعد أن يتم القضاء على هذه المعاملة من قلب التجربة. هذا ينبغي أن يكون القضاء نادرة ، لم يؤدي إلى القضاء على النمط الجيني التي يمثلونها. </li></ol> 2. الحشرات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم جمع spittlebugs الكبار من الميدان ، والتعرف على الأنواع ، وأدخلت إلى أقفاص oviposition حيث تقدم لهم من أوراق الشجر brachiariagrasses عرضة لتتغذى على. تضع الإناث بيضها في التربة الواردة في درج قابل للإزالة الضحلة وضعها على الجزء السفلي من الأقفاص. </li><li style=";text-align:right;direction:rtl"> بعد عدة أيام ، وعلقت في مجال المياه والتربة تمريرها من خلال سلسلة من المناخل (42 و 60 و 150 شبكة) لجمع البيض. تعليق المادة منخول في محلول ملحي 30 ٪ يؤدي الى تعويم بويضات ناضجة ، مخلفين وراءهم البيض غير الناضج ، وأية مسألة العضوية المتبقية. </li><li style=";text-align:right;direction:rtl"> لمنع التلوث من قبل الممرضة للحشرات ، وتطهير البيض في محلول هيبوكلوريت الصوديوم 0.5 ٪ لمدة 5 دقائق ثم تشطف بالماء المقطر. </li><li style=";text-align:right;direction:rtl"> كل محطة اختبار (وحدة تجريبية) موبوء على سطح التربة مع ستة بويضات ناضجة. ويتم اختيار البيض فقط ناضجة على مقربة من الفقس عن المناطق الموبوءة. ويمكن التعرف عليهم من خلال وجود اثنين من النقاط الحمراء في الجزء الأمامي من الموافق عيون الحشرات ؛ علامات الوردي في جانبها الخلفي ، الموافق بطن الحشرة ، والوصاد بالكامل الموسع. </li><li style=";text-align:right;direction:rtl"> ويؤكد نجاح الفقس 4 د في وقت لاحق ، وعند هذه النقطة يتم استبدال أي بيضة لم يفقس من spittlebug الوليد من مستعمرة لدينا. </li><li style=";text-align:right;direction:rtl"> رتبت محطات الاختبار في تصميم القطع كاملة العشوائية مع ستة من تكرارها في التركيب الوراثي. </li></ol> 3. التقييمات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويتم تقييم التجربة كاليفورنيا. 30 د بعد الإصابة (مرات الدقيقة تختلف تبعا لأنواع الحشرات التي شملتها الدراسة). </li><li style=";text-align:right;direction:rtl"> وسجل الأضرار على نطاق 1-5 البصرية ، حيث 1 يناظر أي ضرر مرئية و 5 يناظر النباتية الميتة (الشكل 1). سجل الأضرار مصنع يوفر تقديرا للتسامح. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="الشكل 1" /> جدول رقم 1 الأضرار. المستخدمة لتقييم التسامح في الحيوانات العاشبة spittlebug SPP Brachiaria. 1 = أي ضرر للاكتشاف ؛ 2 = أوراق الشجر نخرية 0-25 ٪ ، 3 = أوراق الشجر نخرية 25-50 ٪ ؛ 4 = أوراق الشجر نخرية 50-75 ٪ ؛ 5 = أوراق الشجر نخرية 75-100 ٪. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويتم تقييم بقاء الحشرات عن طريق عد الحشرات الوصول بهم إما طور مرحلي أو نهائي مرحلة الكبار (الشكل 2). سجل لبقاء الحشرات يوفر تقديرا للتضاد. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="الشكل 2" /> تطور الرقم Spittlebug 2. طور مرحلي والعشرين من (اليسار) لالكبار (يمين). يتم عد الحشرات فقط التوصل على الأقل من طور مرحلي النهائي (الأسهم) في تاريخ التقييم و&quot;الناجين&quot;. 4. ممثل النتائج وتعرض النتائج من ممثل الهجينة مقايسة Brachiaria غربلة لمقاومة reducta Aeneolamia spittlebug في الشكل 3. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="الشكل 3" /> الشكل 3. نتائج الفحص الهجينة Brachiaria لمقاومة spittlebug Aeneolamia reducta. نحن نعتبر مقاومة النباتات إذا ردهم على الحيوانات العاشبة يقع في النصف السفلي من هذه المؤامرة. تصاد يزيد من اليمين إلى اليسار والتسامح يزيد من أعلى إلى أسفل. الأسهم النقطة الشيكات لدينا مقاومة.

<ol>
	<li>Painter, 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> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

يمكن التمييز بين آليات المقاومة عاشب تضيء جهود تربية المحاصيل 3. يمكن أن تقوم على تضاد المقاومة قوية ، في ظل بعض الظروف ، وخلق ضغوط لاختيار biotypes الآفات أكثر عدوانية. من ناحية أخرى ، يمكن أن المقاومة القائمة على التسامح تسمح لزيادة أعداد الآفات حتى النهاية يسود التسامح. خصبة للمقاومة دائمة ، وبالتالي ، يتطلب دراسة متأنية لآليات المقاومة ما من شأنه أن يضفي الاستقرار لمعظم محصول معين / نظام مكافحة الآفات [انظر كينيدي وآخرون. (1987) لإجراء مناقشة شاملة حول هذا الموضوع] (3). هذه التظاهرة الفيديو يبني عدة دراسات سابقة تهدف إلى تقدير مساهمات النسبية للتضاد والتسامح للمقاومة في spittlebug brachiariagrasses 6-9. في المقابل مع اختبارات اختيار أو التجارب الميدانية ، لا خيار اختبارات ضمان تلقي جميع محطات الضغط عاشب نفسه ، وبالتالي توفير الرقابة على الخلافات الناشئة عن سلوك عاشب (على سبيل المثال ، وتفضيل المضيفة ، والتجميع). لهذا السبب ، ومع ذلك ، لا خيار اختبارات غير مناسبة لتقييم مقاومة antixenosis. تربية مستعمرات الحشرات بشكل مصطنع هو الأسلوب المفضل للحصول على الحشرات اختبار لفحوصات الكشف المقاومة 10. المستعمرات توفير مصدر موثوق وموحد للspittlebugs من العمر المعروف للتجارب تجرى في أي وقت من السنة 11. غير أنه قد يكون من الضروري ضخ دوريا الأفراد البرية في مستعمرة لضمان أن لا تحيد مستعمرة وراثيا من السكان الميدانية ذات الصلة 10. لتقييم اللياقة البدنية من هذه العواشب تربى اصطناعيا ، ورصد التغيرات المحتملة في العدوانية على مر الزمن ، فمن المهم أن جميع العروض تشمل الضوابط المناسبة لمستويات المقاومة معروفة. يجب أن تكون معلمات متعددة المحددة بدقة لتصميم فحص مقاومة مناسبة مقايسة. عدد قليل من المعالم الرئيسية لدينا نظرت لتصميم الاختبار تشمل سن النباتات المضيفة ، والأنواع والمرحلة التنموية للspittlebugs ، ومستوى الإصابة ، والمدة من النبات والحشرات الاتصال 8،9،12. قد تكون هناك حاجة إلى عدة جولات من التجارب وصقل بروتوكول للوصول إلى السريع ، وفعالة من حيث التكلفة ، مقايسة وموثوق بها أن يتنبأ بشكل كاف عاشب المقاومة في الميدان.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

يمكن للنباتات مقاومة الضرر عاشب من خلال ثلاث آليات رئيسية هي : antixenosis ، تصاد والتسامح 1. Antixenosis هو الدرجة التي يتم تجنب المصنع عندما عاشب غير قادرة على تحديد النباتات الأخرى 2. تصاد هو إلى أي مدى يؤثر على النبات اللياقة البدنية للتغذية عاشب عليه 1. التسامح هو إلى أي مدى يمكن أن تصمد أمام مصنع أو إصلاح الأضرار الناجمة عن عاشب ، دون المساس بالنمو والتكاثر عاشب 1. متانة عاشب المقاومة في محيط الزراعية تعتمد الى حد كبير على آلية المقاومة يحبذ خلال جهود تربية المحاصيل 3. نظهر تجربة عدم اختيار تصميم لتقدير المساهمات النسبية للتضاد والتسامح للمقاومة في spittlebug SPP Brachiaria. عدة أنواع من الأعشاب الافريقية من جنس Brachiaria والأعلاف ونباتات المراعي قيمة في Neotropics ، ولكن يمكن أنها تواجه تحديا خطيرا بسبب العديد من الأنواع المحلية spittlebugs (نصفيات الجناح : Cercopidae) 4 تقييم مقاومتهم للspittlebugs ، والنباتات ، نشر نباتيا قصاصات الجذعية والتي سمحت لزراعة ما يقرب من شهر واحد ، والسماح للنمو الجذور السطحية التي يمكن أن تغذي spittlebugs. عند هذه النقطة ، يتم الطعن بشكل فردي لكل معمل اختبار مع البيض spittlebug six قرب الفقس. ويسمح لتفشي التقدم لمدة شهر قبل تقييم الضرر النبات والحشرات البقاء على قيد الحياة. سجل الأضرار مصنع يوفر تقدير التسامح مع بقاء الحشرات يحرز تقدم تقديرا للتضاد. وقد سهل هذا البروتوكول هدفنا تربية النباتات المقاومة لتعزيز spittlebug في brachiariagrases التجارية 5.

Watch this Scientific Journal Video about Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example at JoVE.com

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

هذا الفيديو يشرح آليات مقاومة النبات المضيف على الحيوانات العاشبة ويوضح اختبار لا للخيار أن تقديرات الإسهامات النسبية للتضاد والتسامح إلى المقاومة في spittlebug<em&gt; Brachiaria</em&gt; النيابة.

توصيف آليات المقاومة آكل النبات : Spittlebugs على Brachiaria النيابة. كمثال

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

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Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K Views.  CIAT. هذا الفيديو يشرح آليات مقاومة النبات المضيف على الحيوانات العاشبة ويوضح اختبار لا للخيار أن تقديرات الإسهامات النسبية للتضاد والتسامح إلى المقاومة في spittlebug Brachiaria النيابة.

Video: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

الدراسات الجينية البشرية البروتينات إصلاح الحمض النووي باستخدام خميرة كنظام النموذجي

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

عاشب متطايرة بفعل العنبية واشارة الى داخل المصنع

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

قياس معامل نفاذية المياه الأسموزي (P<sub&gt; و</sub&gt;) من خلايا كروية: Protoplasts محطة معزولة كمثال

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

تقنية تسلل الطرد المركزي لاستخراج Apoplastic السوائل من أوراق النبات عن طريق<em&gt; فاصيلوس الشائع</em&gt; كمثال

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

تصوير فيتك-ديكستران كمراسل للرقابة الخاضعة للتنظيم

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM Microcoil معايرة الأداء والاستخدام أظهرت على جذور طاعون Medicago في 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

استكشاف m6A و m5C Epitranscriptomes على العدوى الفيروسية: مثال مع فيروس نقص المناعة البشرية

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

توصيف الخواص الميكانيكية للجدار الخلوي الأولي في أعضاء النبات الحي باستخدام مجهر القوة الذرية

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

طريقة التثقيب الكهربائي لتحويل الريكتسيا spp. باستخدام ناقل مكوك يعبر عن البروتين الفلوري في خطوط خلايا القراد

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

التحقيق في آلية نقل الزنك في خلايا الثدييات باستخدام مقايسة FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

تسليط الضوء على المؤلف: استكشاف تنظيم الزنك الخلوي من خلال وظائف ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

توصيف ناقلات الزنك في الثدييات باستخدام مقايسة نقل الزنك في المختبر

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...