1) جمع بيض اسماك الزرد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد ظهر يوم قبل يوم من الشاشة الكيميائية ، وإعداد 10-20 الدبابات تربية الزرد. ملء كل صهريج ماء من نظام تربية الأحياء المائية. باستخدام صافي الأسماك ، نقل أحد الذكور والإناث البالغين الكبار 1-2 إلى حاوية الداخلية في كل خزان التربية. فصل الأسماك الذكور والإناث من بعضهم البعض مع المفرق. تسمية الأقفاص ووضع غطاء عليها. </li><li style=";text-align:right;direction:rtl"> في صباح يوم من الشاشة ، إزالة فواصل من الدبابات والسماح للتربية الزرد لزميله. على مدى ساعة 1 التالي ، والسماح لسقوط البويضات المخصبة من خلال الشبكة في الجزء السفلي من كل حاوية الداخلية. </li><li style=";text-align:right;direction:rtl"> بعد 1 ساعة ، والعودة مرة أخرى إلى الزرد الكبار صهاريج التخزين الدائمة ، وإزالة الحاوية الداخلية وجمع البيض بنسبة توتير المياه في كل خزان تربية من خلال مصفاة الشاي البلاستيكية. </li><li style=";text-align:right;direction:rtl"> عكس المصفاة على طبق بتري وشطف مصفاة برفق لطرد البيض في صحن بيتري باستخدام زجاجة تحتوي على غسل المتوسطة E3. </li><li style=";text-align:right;direction:rtl"> يجب إزالة كل بويضة غير مخصبة ، والتي تظهر مبهمة ، وذلك باستخدام الماصة البلاستيكية. ينبغي لكل عبر التزاوج العائد حوالي 200 الأجنة. </li></ol> 2) Arraying الأجنة في 96 - جيدا لوحات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نقل الأجنة حوالي 5 في المتوسط ​​في كل بئر E3 من لوحة 96 - جيدا باستخدام ماصة باستير الزجاج. </li><li style=";text-align:right;direction:rtl"> بمجرد المحتشدة الأجنة على طبق 96 - جيدا ، وإزالة أكبر قدر من المتوسط ​​E3 النحو المبين ممكن من الآبار باستخدام 12 قناة (3-30 ميكرولتر) ماصة ، مع الحرص على عدم ثقب الجنين. باستخدام ماصة 12 قناة ، وتقديم 250 ميكرولتر المتوسطة E3 تحتوي على الكاناميسين 0.5μg/ml إلى كل بئر في أسرع وقت ممكن حتى لا تسمح للأجنة لتجف. </li><li style=";text-align:right;direction:rtl"> وضع لوحات 96 - جيدا داخل الحاضنة 28.5 درجة مئوية حتى تصل إلى مرحلة المرجوة عندما تضاف هذه المركبات. </li></ol> 3) نقل المكتبة جزيء صغير بينما يمكن نقل مجمع الآلي مع وسائل نقل الروبوتية ، سنقوم بشرح طريقة نقل يدوي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عادة ما يتم توفيره من المكتبات جزيء صغير في شكل 96 - جيدا ، مع كل مجمع المخزنة في DMSO باعتباره المخزون 10 مم. حوالي 60 دقيقة قبل ان يصل الى مرحلة الأجنة عندما المركبات هي التي يمكن ان تضاف ، أذاب العدد المرغوب فيه من جانب 96 لوحات تحتوي على aliquots من جزيئات صغيرة (لوحة المصدر). يحيط علما رقم هوية المسلسل أو غيرها من لوحات المصدر. لتقليل التكثيف على لوحات ، ويمكن أن يحدث الذوبان في غرفة تحتوي على تجفيف Drierite (WA هاموند DRIERITE CO ، زينيا ، OH). </li><li style=";text-align:right;direction:rtl"> تدور باستمرار لفترة وجيزة في جهاز للطرد المركزي لوحات الطاولة مجهزة محول لوحة متعددة أيضا. </li><li style=";text-align:right;direction:rtl"> إزالة الشريط من لوحة الالومنيوم ختم المصدر. باستخدام ماصة 12 قناة ، ويخفف من المركبات الموجودة في لوحة مصدر للتركيز 0.5 ملم (على سبيل المثال ، إذا بدءا aliquots NL 250 من الاسهم 10MM ، إضافة 4.75 ميكرولتر من DMSO إلى كل بئر). </li><li style=";text-align:right;direction:rtl"> عند الأجنة في لوحة 96 - جيدا (لوحة المتلقي) يصل الى مرحلة المطلوبة ، استخدم لمدة 12 قناة (2-20 ميكرولتر) ماصة لنقل المركبات 2.5μL (0.5mM) من لوحات لوحات المصدر إلى المتلقي تحتوي على الأجنة. </li><li style=";text-align:right;direction:rtl"> تسجيل عدد من لوحات تحديد المصدر على لوحات الجنين المتلقية. تغطي لوحات تحتوي على المتلقي الآن الأجنة والمركبات مع الأغطية ، ومزيج بلطف بواسطة لوحات يحوم بلطف ، ووضعها في الحاضنة 28.5 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> تغطي كل لوحة تحتوي على جزيئات صغيرة مصدر غير المستخدمة (0.5 ملم) مع الالومنيوم ختم الشريط ومكان في الفريزر -80 درجة مئوية لمدة طويلة الأجل للتخزين. </li></ol> 4) الكشف عن تأثيرات الجزيئات الصغيرة عن طريق التفتيش البصري من الظواهر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل تنفيذ الشاشة ، ووضع معيار محدد لما من شأنه أن يشكل &quot;ضرب&quot;. </li><li style=";text-align:right;direction:rtl"> في بعض الأحيان في عملية التنمية المنشودة ، وإزالة لوحات تحتوي على 96 - جيدا مجمع المعالجة أجنة من الحاضنة ودراسة كل جانب تحت stereomicroscope. لأفضل تصور التغييرات الطفيفة ، مثل التغيرات في نمط الدورة الدموية ، ويمكن استخدام المجهر المرحلة على النقيض المقلوب. يمكن أن تستخدم لفحص المجهري الفلورسنت اضطراب التعبير GFP أو البروتينات DsRed المروج تحت الأنسجة محددة. </li><li style=";text-align:right;direction:rtl"> مسح سريع لوحات 96 - جيدا لأي جيدا في ما لا يقل عن 3 من أصل 5 أجنة يحمل المقررة &quot;ضرب&quot; النمط الظاهري. سجل هوية لوحة ومكان جيد للضرب كل المحتملة. </li><li style=";text-align:right;direction:rtl"> تأكيد ضربة محتملة من إعادة اختبار آثار المجمع في عدة جرعات (1 أم أم 5 ، 10 ميكرومتر وميكرومتر 50). لكل جرعة ، يتم اختبار الأجنة في 10 مل من 0.5 E3 وسائل الاعلام في شكل صفيحة ال 48 أيضا. وينبغي أن توقيت بالإضافة لمجمع إعادة الاختبار تكون مماثلة لفحص الأصلي. وأكد هو ضرب عندما يرد على النمط الظاهري أثارت إعادة الاختبار للcompounد </li><li style=";text-align:right;direction:rtl"> التعرف على مجمع ضرب من قاعدة البيانات من جزيئات صغيرة في المكتبة الكيميائية. </li></ol>

<ol>
	<li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish.">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li><li>Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors.">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li><li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery/vein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase/ERK+signaling.">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li><li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish.">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, 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=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li><li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant.">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li><li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation.">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li></ol>

الإعلان عن أي تضارب في المصالح.

عند التخطيط لشاشة الكيميائية الزرد مقرها ، يجب إيلاء اهتمام خاص لمتانة النمط الظاهري قيد النظر ، ومعدل خلفية النمط الظاهري من هذا القبيل. وهذا أمر مهم لا سيما بالنسبة للشاشات المكثفات الكيميائية من النمط الظاهري المستحث. على سبيل المثال ، عن النمط الظاهري التي تسببه...

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحد الأدنى من 20 زوجا من الزرد البالغين من النمط الجيني المطلوب. </li><li style=";text-align:right;direction:rtl"> شباك الصيد ، وخزانات تربية ، مع الحاويات الداخلية القابلة للإزالة ، والمقسمات (الموائل المائية). </li><li style=";text-align:right;direction:rtl"> أطباق بتري (10 سم). </li><li style=";text-align:right;direction:rtl"> البلاستيك مصفاة الشاي. </li><li style=";text-align:right;direction:rtl"> تغسل زجاجة ماء الجنين containg. </li><li style=";text-align:right;direction:rtl"> المتاح ماصات نقل البولي ايثيلين. </li><li style=";text-align:right;direction:rtl"> البوليسترين 96 - جيدا لوحات فحص الجولة القاع (كورنينج COSTAR ؛ ويل ، MA). </li><li style=";text-align:right;direction:rtl"> الزجاج ماصة باستير (فيشر العلمية). </li><li style=";text-align:right;direction:rtl"> دليل مضخة ماصة ، 10 مل (بلجيكا أحدث المنتجات ، Pequannock ، ونيو جيرسي). </li><li style=";text-align:right;direction:rtl"> E3 المتوسطة الجنين : 5 مم كلوريد الصوديوم ، 0.17 ملي بوكل ، 0.33 ملي CaCl 2 ، 0.33 ملي MgSO 4 ، تحتوي على 0.003 ٪ PTU (phenylthio - كارباميد ، سيغما ؛ سانت لويس ، MO). يمكن إعداد PTU كحل 10X عن طريق إذابة 0.3 جم PTU في 1 لتر من وسائل الإعلام الجنين E3. يجب حماية المحاليل المحتوية على PTU من الضوء من خلال تغطية بورق الألمنيوم. </li><li style=";text-align:right;direction:rtl"> 12 قناة ماصات ، 20-20 ميكرولتر (إيبندورف). </li><li style=";text-align:right;direction:rtl"> 12 قناة ماصات ، 300-300 ميكرولتر (إيبندورف). </li><li style=";text-align:right;direction:rtl"> البوليسترين القابل للتصرف ماصة الحوض ، 50 مل (فيشر العلمية). </li><li style=";text-align:right;direction:rtl"> مكتبة صغيرة جزيء من المركبات المتنوعة هيكليا المحتشدة في شكل 96 - WLL في الأسهم 10 ملم في DMSO. كل لوحة هي aliquoted الماجستير في 96 - جيدا لوحات تخزين البولي بروبلين (كورنينج) ، وتخزينها في -80 درجة مئوية حتى الاستخدام. </li><li style=";text-align:right;direction:rtl"> الالومنيوم ختم الشريط ل96 - جيدا لوحات (نونك ، روتشستر ، نيويورك). </li><li style=";text-align:right;direction:rtl"> DMSO (سيغما ، وسانت لويس ، MO). </li><li style=";text-align:right;direction:rtl"> الحاضنة الأساسية ، 28.5 درجة مئوية (فيشر العلمية). </li><li style=";text-align:right;direction:rtl"> Stereomicroscope مع قاعدة الضوء المرسل (لايكا مايكروسيستمز ، بانوكبورن ، IL). </li></ol>

large scale zebrafish-based in vivo small molecule screen

نظرا لحجمها الصغير الجنين ، التطور السريع ، والشفافية ، والخصوبة ، والعديد من أوجه الشبه الجزيئي ، المورفولوجية والفسيولوجية للثدييات ، برز الزرد وقوية<em&gt; في الجسم الحي</em&gt; منصة لشاشات المخدرات النمط الظاهري القائم على التحليل الكيميائي والجيني. هنا ، علينا أن نظهر بسيطة ، طريقة عملية واسعة النطاق لفحص الجزيئات الصغيرة باستخدام الأجنة الزرد.

Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

Large Scale Zebrafish-Based In vivo Small Molecule Screen

كما برز الزرد قوية<em&gt; في الجسم الحي</em&gt; منصة لشاشات المخدرات النمط الظاهري القائم على التحليل الكيميائي والجيني. هنا ، علينا أن نظهر بسيطة ، طريقة عملية واسعة النطاق لفحص الجزيئات الصغيرة باستخدام الأجنة الزرد.

اسماك الزرد نطاق واسع ، واستنادا في الجسم الحي شاشة جزيء صغير

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

large-scale-zebrafish-based-in-vivo-small-molecule-screen

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Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

13.4K Views.  Vanderbilt University School of Medicine. كما برز الزرد قوية في الجسم الحي منصة لشاشات المخدرات النمط الظاهري القائم على التحليل الكيميائي والجيني. هنا ، علينا أن نظهر بسيطة ، طريقة عملية واسعة النطاق لفحص الجزيئات الصغيرة باستخدام الأجنة الزرد.

Video: Large Scale Zebrafish-Based In vivo Small Molecule Screen

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

على نطاق واسع من شاشات مكتبات Metagenomic

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunostaining دفعي للكشف عن البروتين واسعة النطاق في الدماغ القرد الجامعة

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

على نطاق صغير مقتطفات النووية لفحوصات وظيفي من الجينات التعبير عن مكائن

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium الفحص على نطاق واسع لتطبيقات SNP التنميط الجيني

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

استراتيجية للحساسية، مقياس كبيرة الكمية الايض

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

دليل جزيء صغير شاشة تقترب عالية الإنتاجية عن طريق الزرد الأجنة

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

بروتوكول إنتاج رواية لتصنيع على نطاق صغير من بروبيوتيك المخمرة للأغذية

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

أوديلاي: طريقة على نطاق واسع لالمعلمات المتعددة الكمي للنمو الخميرة

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

تحديد المنظمين رواية نتح النبات من خلال فحص التصوير الحراري على نطاق واسع في Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

إجراء سيميومتوميد ChIP-Seq للدراسات اللاجينية على نطاق واسع

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...