نشكر ألف ريشيو عن التعليقات المفيدة وقراءة المخطوط. وأيد هذا العمل عن طريق تمويل البحوث الطبية (MRC) المجلس إلى خلية علم الأحياء وحدة الإنسان من قبل برنامج منح العلوم الحدودي (RGP0048/2009-C).

1. رد الفعل الأنزيمية -- يوم 1 (1 ساعة بعد الظهر) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الخطوة الأولى هي إعداد 10-20 التفاعلات الإنزيمية المستقلة التي IP 6 K1 أو تحويل الملكية الفكرية VIP1 6 إلى الأشكال الإسوية pyrophosphorylated. </li><li style=";text-align:right;direction:rtl"> نحن نستخدم له 6 - IP K1 والانزيمات GST - Vip1 تنقيته من E. القولونية وفقا لبروتوكول 17،18 الموصوفة سابقا. </li><li style=";text-align:right;direction:rtl"> تجهيز 50 ميكرولتر تحتوي على ردود فعل رد فعل 1X العازلة (Hepes 30 ملم 6.8 درجة الحموضة ، و 50 ملي مول كلوريد الصوديوم ، 6 مم MgSO 4 ، 1 ملم DTT) ، 6 فسفوكرياتين ملم (PCR) ، و 25 يو / مل CreatinePhosphoKinase (CPK) ، 5 مم ATP (ملغ الملح) ، 0.3 ملي IP6 ، 0،05-0،1 ميكروغرام صاحب IP - 6 K1 أو ضريبة السلع والخدمات Vip1. ضبط مستوى الصوت مع 2 MiliQ O. DDH </li><li style=";text-align:right;direction:rtl"> تدور لفترة وجيزة في رد الفعل واحتضان 37 درجة مئوية خلال الليل مع التناوب. </li></ol> 2. بولي أكريلاميد صب جل والتحميل -- يوم 2 (4 ساعات بعد الظهر) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تحضير هلام بولي أكريلاميد باستخدام 24 سم ، 18 سم لوحات زجاجية واسعة والفواصل واسعة 1.5 مم. وعادة ما يتم استخدام ممر 16 أو مشط التحضيرية حارة واحدة. </li><li style=";text-align:right;direction:rtl"> تحضير مزيج (50 مل / جل) يحتوي على ما يلي : 35.5 ٪ (W / V) الأكريلاميد : مكرر الأكريلاميد 19:01 ، 1X بيرسلفات الأمونيوم تريس / البورات / EDTA (TBE) ، 0.05 ٪ (W / V) (وكالة الأنباء الجزائرية ) ، Temed 0.05 ٪ (W / V). صب مزيج بين لوحات ما قبل محمل الزجاج ، وادخال المشط والسماح للتبلمر 30-60 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> مرة واحدة في هلام وبلمرة ، ونقل الجهاز إلى غرفة باردة وقبل تشغيله في TBE 1X لحوالي 30-60 دقيقة على 200-300 فولت. </li><li style=";text-align:right;direction:rtl"> إضافة 1X صبغة OrangeG (10 ملي تريس - pH7.0 حمض الهيدروكلوريك ، 1 ملم EDTA ، الجلسرين 30 ٪ ، 0.1 ٪ OrangeG) إلى كل التفاعل. تحضير عينة تحتوي على 2 nmol الملكية الفكرية 6 الى تحميل كعنصر تحكم القياسية. </li><li style=";text-align:right;direction:rtl"> يغسل جيدا جيدا مع كل تشغيل العازلة باستخدام المحاقن والإبر 21G لإزالة أي يعجل ، ثم تحميل الجل. تجنب التحميل على الآبار الجانبية. </li><li style=";text-align:right;direction:rtl"> تشغيل هلام بين عشية وضحاها على 450-550 فولت (7 mAmp / هلام) ، حتى الفرقة صبغ OrangeG ضمن مشاركة 10 سم من الجزء السفلي من هلام. </li></ol> 3. بمعزل عن الملكية الفكرية 7 -- يوم 3 (4 ساعات) ، ويوم 4 (6-7 عملية التجفيف SpeedVac ساعة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تفكيك جهاز هلام بعناية وإزالة إحدى الصفائح الزجاجية ترك الجل على الآخر. قطع جزء صغير من الجل من فوق الشريط صبغ OrangeG إلى القاع الذي يحتوي على 6 من IP القياسية ومسار واحد العينة ، كما هو مبين في الشكل 1. </li><li style=";text-align:right;direction:rtl"> وصمة عار على قطع جزء من هلام مع الأزرق طولويدين (0.1 ٪ (W / V) طولويدين الزرقاء ، و 20 ٪ (W / V) والميثانول ، 2 ٪ (W / V) الجلسرين) لبضع دقائق (1-3 دقائق) أو حتى الفرقة بيروفوسفات اينوزيتول تظهر. وضع لوحة من الزجاج الخلفي إزالة سابقا على رأس لمنع هلام هلام غير ملوثين من التجفيف. </li></ol> ينبغي أن الفرقة الملكية الفكرية 7 تكون مرئية منذ تشغيله بشكل أبطأ قليلا من مستوى 6 IP. وينبغي للاعبي التنس المحترفين ، والذي يعمل بشكل أسرع من 6 الملكية الفكرية ، وأيضا أن تكون مرئية (الشكل 1). نقل الجزء الملون من هلام في التوصل إلى حل لإزالة البقع (20 ٪ (W / V) الميثانول) لبضع دقائق ، يغسل أي فائض في الزرقاء وإعادة طولويدين الجل مع الجل غير ملوثين. إذا كان مطلوبا التصور العالي الأشكال الإسوية اينوزيتول pyrophosphorylated (IP IP 8 و 9) ، وصمة عار الجل مع الحل طولويدين تلطيخ الأزرق لمدة 20 دقيقة في درجة حرارة الغرفة. بعد ذلك ، يغسل الأزرق طولويدين مع الحل دي تلطيخ لحوالي 15 دقيقة. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مع خفض شفرة حلاقة الملكية الفكرية 7 الفرقة على الجزء غير ملوثين من هلام به كمرجع للملكية الفكرية موقف ترحيل 7 العزم مع الجل الملون (الشكل 1). </li><li style=";text-align:right;direction:rtl"> وضع الملكية الفكرية 7 الفرقة التي قطعت من الجل على أنبوب 15 مل و 10 مل من إضافة 2 MilliQ O. DDH وضع الأنابيب في التناوب لمدة 10 دقيقة في درجة حرارة الغرفة. رفض السائل لإزالة الفائض من جزيئات الأكريلاميد TBE والمجهرية. </li><li style=";text-align:right;direction:rtl"> بعد ذلك ، نفذ two - جفاف الماء دورات. إضافة 5 مل من 50 ٪ (W / V) الميثانول إلى أنبوب مع هلام يحتوي على 7 الملكية الفكرية وتدوير في درجة حرارة الغرفة لمدة 2 ساعة. نقل شريحة هلام لأنبوب جديد 15 مل تحتوي على 5 مل من 2 MilliQ O DDH وتدوير في درجة حرارة الغرفة لمدة 2 ساعة. لا يتخلص من الميثانول وMilliQ H O 2 من الأنابيب. تكرار الجفاف دورة الماء مرة أخرى ، من خلال إعادة نقل هلام بولي أكريلاميد في 15 مل من الأنابيب المستخدمة سابقا. لا يمكن أن يؤديها واحد من يغسل طوال الليل. </li><li style=";text-align:right;direction:rtl"> لتركيز IP eluted 7 ، جفاف 10 مل معا (5 مل MilliQddH 2 O و 5 مل 50 ٪ الميثانول (ث / ت)) باستخدام SpeedVac ساخنة عند 60 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> مرة واحدة على عينات جافة تقريبا ، ونقل السائل المتبقي (300-600 ميكرولتر) لa1.5 مل أنبوب الطرد المركزي وتدور لمدة 2 دقيقة في 5000 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> طاف جمع ونقل الى الطازجة 1.5 مل أنبوب الطرد المركزي ؛ مغادرةأسفل 20-30 ميكرولتر أنها قد تحتوي على جزيئات الأكريلاميد. </li><li style=";text-align:right;direction:rtl"> إذا كان ذلك ضروريا الاستمرار في عملية التجفيف باستخدام SpeedVac غير مدفأة. وخفضت بشكل كبير في انتعاش IP 7 إذا العينات يجف تماما ، ثم إنهاء عملية التجفيف عندما تصل عينات حجم 100-300 ميكرولتر. </li></ol> 4. تحديد تركيز 7 الملكية الفكرية والنقاء. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام 2-5 ميكرولتر من العينة تعافى 7 IP لتشغيل على هلام PAGE ، على غرار الأقسام 2،2-2،5. تحميل العديد من التخفيفات IP 6 (أي 0.5 ، 1 ، 2 ، 4 nmol) وفقا لمعايير وتركيز 4 nmol من بولي - P علامة. بعد تشغيل هلام ، تصور الأشكال الإسوية بيروفوسفات اينوزيتول بواسطة تلطيخ واجتثاث تلطيخ الجل كامل مع حل طولويدين الأزرق ، وبعد الإجراء الموضح في القسم 3.2 (الشكل 2A). </li><li style=";text-align:right;direction:rtl"> بعد تلطيخ طولويدين ، يمكن تحديد التركيزات عن طريق مسح الجل ومقارنة الاختلافات في كثافة تتراوح بين 6 الملكية الفكرية والملكية الفكرية 7 ، وذلك باستخدام برامج التصوير مثل J - الصور ، كما هو مبين في الشكل 2B. </li></ol> 5. ممثل النتائج : ويمكن تحويل التحضيرية الأنزيمية للملكية الفكرية من 6 إلى 7 باستخدام IP IP 6 K1 وVIP1 الانزيمات حلها بسهولة باستخدام التحليل PAGE (الشكل 1). التحميل من 6 الملكية الفكرية باعتبارها التحكم في حجم جنبا إلى جنب مع تلوين طولويدين الجل الأزرق يتيح التعرف على المشتقات pyrophosphorylated ، حيث أنها تعمل بشكل أبطأ اعتمادا على عدد من المجموعات الفوسفات الموجودة على الحلبة اينوزيتول. الإجراء المذكورة أعلاه يسمح للتنقية سهلة من 7 IP. وكشف تحليل لبيروفوسفات اينوزيتول يطهر PAGE نقاء الملكية الفكرية لدينا 7 (الشكل 2A). ومن المثير للاهتمام ، و1/3PP-IP 5 ايزومير من الناتج 7 IP من VIP1 يهاجر أبطأ قليلا من أيزومر 5 5PP - IP من 7 IP التي تم إنشاؤها بواسطة K1 6 IP. 6 استخدام معايير الملكية الفكرية تسمح الكمي سهلة للتركيز IP المنقى 7 (الشكل 2B). 7 قبل استخدام الملكية الفكرية لمزيد من التجارب ، يمكن تقييم نشاطها البيولوجي (الشكل 3). 5PP - IP هو المحتضنة 5 مع VIP1 ومع الفوسفاتيز 7 IP DDP1 (diphosphoinositol phosphohydrolase متعددة الفوسفات). بشكل روتيني ، يتم تحويل الملكية الفكرية تنقيته من 7 إلى 8 من VIP1 الملكية الفكرية والملكية الفكرية التي DDP1 6 (الشكل 3). <img alt="الشكل 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> الشكل 1 : من تلطيخ طولويدين PAGE والعزلة من الفرقة الملكية الفكرية 7 قطع جزء من الجل الذي يحتوي على معيار (IP 6) والملون باستخدام حل طولويدين الأزرق. الفرق الثلاثة تمثل (من أعلى إلى أسفل) 7 IP ، IP 6 و ATP. ثم كان متحالفا الجزء الملون من هلام مع ما تبقى من هلام. هذا يسمح للتوطين جزء من هلام يحتوي على 7 الملكية الفكرية ، والتي يمكن بعد ذلك قص وتنقيته (مربع متقطع). <img alt="الشكل 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> الشكل 2 : تحليل PAGE الملكية الفكرية منتجات التفاعل 6 K1 وVIP1. أ) تم استخدام تحليل IP 6 (4 ، 2 ، 1 ، 0.5 nmol) عن طريق تلوين طولويدين الأزرق من أجل تحديد IP 7 تركيز تنقيتها من كل 6 IP K1 (IP - 5PP 5) وVIP1 (1/3PP-IP 5) ردود الفعل. B) مبعثر تحليل مؤامرة لتحديد تركيز الملكية الفكرية المنقى 7. وتم تحديد التركيزات وفقا لكثافة الموجات ، محسوبة باستخدام البرمجيات imageJ ، مقارنة كميات محددة مسبقا من 6 IP. والمحور السيني يمثل شدة ، ويمثل المحور Y - تركيزات التي أعرب عنها في nmol. <img alt="الشكل 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> الشكل 3 : تحليل النشاط 7 IP البيولوجي لتحديد نوعية الملكية الفكرية المنقى 7 المحتضنة نحن 5PP 5 - IP (IP 6 K1 IP ولدت 7) مع VIP1 أو مع الفوسفاتيز 7 DDP1 IP وحلها ثم رد الفعل على الصفحة. كشفت وتلطيخ دابي طولويدين الإنتاج المتوقع من 8 VIP1 الملكية الفكرية وتحويل الملكية الفكرية من 7 إلى 6 من DDP1 IP.

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C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

غير محدودة بشدة استخدام بيروفوسفات اينوزيتول في الكيمياء الحيوية بسبب عدم توفر هذه المركبات التجارية وحساسية الفقيرة من طرق الكشف الحالية. مزيج من صفحة ، والذي تمكن من فصل الجزيئات التي تمتلك عدد مختلف من مجموعات الفوسفات ، وزرقة الطولويدين (الشكل 1) ، وصبغ متبدل اللون الذي يربط لمجموعات الفوسفات ، وتمكن من السهل اكتشاف الأشكال الإسوية pyrophoshate اينوزيتول فتح مجالات جديدة للبحث 20. نفذت استخدام التكنولوجيا PAGE وصف لتنقية المنتجات بيروفوسفات اينوزيتول رد الفعل الأنزيمية outby إما 6 أو IP K1 VIP1 هو بسيط ، طريقة اقتصادية والموثوق بها التي تسمح لإنتاج كميات كبيرة من الملكية الفكرية ذات جودة عالية 7. لا يقتصر على الطريقة الموصوفة أعلاه لعملية تنقية بسيطة للملكية الفكرية 7 ولكن تعديلات طفيفة على البروتوكول صفها قد تسمح لتنقية مجموعة مختلفة من pyrophosphates اينوزيتول. ويمكن الكشف عن أعلى فسفرته الأشكال الإسوية بيروفوسفات اينوزيتول ، تحتوي على مجموعة الفوسفات أكثر من ثمانية باستخدام الملكية الفكرية (7) أو كميات مختلفة من 6 الملكية الفكرية باعتبارها الركيزة 20،6. ويمكن الكشف عن هذه pyrophosphates اينوزيتول من خلال زيادة طول تلطيخ الداخلي وتنقيته في وقت لاحق (القسم 3.2). علاوة على ذلك ، فإن استخدام الملكية الفكرية باعتبارها الركيزة 5 للتفاعل الأنزيمي السماح للتنقية PP - IP (5) وpyrophosphates اينوزيتول أخرى تحتوي على مجموعة الهيدروكسيل على الحلقة اينوزيتول. في الختام ، وهذه الطريقة بسهولتها يسمح لتنقية موثوق بها كميات مليغرام من pyrophosphates اينوزيتول مع الأدوات المتاحة على نطاق واسع ، مما يفتح آفاقا جديدة لهذا الحقل البحوث المثيرة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> حمض الفيتيك (IP 6) </td><td> سيغما الدريخ </td><td> P8810 </td></tr><tr><td> بولي - P (فوسفات الصوديوم) </td><td> سيغما الدريخ </td><td> P8510 </td></tr><tr><td> ATP ملغ الملح + 2 </td><td> سيغما الدريخ </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> سيغما الدريخ </td><td> O3756 </td></tr><tr><td> فسفوكرياتين (PCR) </td><td> سيغما الدريخ </td><td> P7936 </td></tr><tr><td> CreatinePhospho كيناز (CPK) </td><td> سيغما الدريخ </td><td> C3755 </td></tr><tr><td> GST - Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> له ، IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> له ، Ddp1 </td><td> المتوفرة في المختبر </td><td> المتوفرة في المختبر </td></tr><tr><td> الأكريلاميد : مكرر الأكريلاميد 19:01 (40 ٪) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> بيرسلفات الأمونيوم (APS) </td><td> سيغما الدريخ </td><td> A9164 </td></tr><tr><td> تريس / البورات / EDTA (TBE) </td><td> سيغما الدريخ </td><td> تي 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> طولويدين الأزرق </td><td> سيغما الدريخ </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> المسيح </td><td> 100218 </td></tr><tr><td> جل الأجهزة </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> فراغ مشعب </td><td> المسيح </td><td> ألفا 2-4 </td></tr><tr><td> فراغ مضخة </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

pyrophosphates اينوزيتول تلعب دورا هاما في أمراض السرطان مثل الإنسان ، والسكري والسمنة ، إلا أن الآلية الدقيقة لعمل هو موضع خلاف. عدم وجود pyrophosphates اينوزيتول المتاحة تجاريا يجعل دراسات تفصيلية للمشاكل. نحن هنا وصف بروتوكول بسيطة لإنتاج وعزل ملليغرام من pyrophosphates اينوزيتول.

إعداد Pyrophosphates إينوسيتول الجودة

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Research

JoVE Journal

Biology

13.0K Views.  University College London. pyrophosphates اينوزيتول تلعب دورا هاما في أمراض السرطان مثل الإنسان ، والسكري والسمنة ، إلا أن الآلية الدقيقة لعمل هو موضع خلاف. عدم وجود pyrophosphates اينوزيتول المتاحة تجاريا يجعل دراسات تفصيلية للمشاكل. نحن هنا وصف بروتوكول بسيطة لإنتاج وعزل ملليغرام من pyrophosphates اينوزيت...

Video: Preparation of Quality Inositol Pyrophosphates

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

ميكروأرس] DNA : نموذج مراقبة الجودة ، وتهجين صفيف والمسح الضوئي

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

عينات الشعر نونينفاسيفي تقنيات الحمض النووي للحصول على ارتفاع الجودة من الثدييات الصغيرة المراوغ

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

على المدى الطويل اختبار السمية المهلكة مع القشريات الأرتيميا franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

ما يفعل وما يترك من المجهري البرد الإلكترون: وهو أول كتاب عن تحضير العينة وجمع البيانات عالية الجودة للتعمير الجزيئات 3D

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

أداة الويب لتوليد عالية الجودة آلة قابل للقراءة البيولوجية مسارات

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

جمع الحيوانات المنوية فرق الجودة باستخدام الكثافة المتدرجة الطرد المركزي

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

التعرف الميداني على Chamomilla Matricaria باستخدام نظام qPCR المحمولة

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

فحص كيميائي محسن للتعرف السريع على الجاذبات الكيميائية الريزوبكتيرية في إفرازات الجذر

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

زراعة ونمو وصلاحية بكتيريا حمض اللاكتيك: منظور مراقبة الجودة

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

تحسين جودة منتجات البروبيوتيك المجففة بالرش من خلال تطوير العمليات

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

تسليط الضوء على المؤلف: تطوير عملية التجفيف بالرش لبكتيريا البروبيوتيك وتقييم جودة المنتج  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

تطوير عملية التجفيف بالرش لبكتيريا البروبيوتيك وتقييم جودة المنتج

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...