Name: preparation of quality inositol pyrophosphates
Uploaded: 2011-09-03T14:00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Мы благодарим А. Риччио за полезные комментарии и прочитать рукопись. Эта работа была поддержана Советом по медицинским исследованиям (MRC) финансирования в отдел клеточной биологии и по правам пограничной науки Программа Грант (RGP0048/2009-C).

1. Ферментативной реакции - день 1 (1 час в день) <ol><li> Первый шаг заключается в подготовке 10-20 независимых ферментативных реакций, в которых ИП 6 K1 или VIP1 преобразования IP 6 до pyrophosphorylated изоформ. </li><li> Мы используем его IP-6 К1 и GST-VIP1 ферментов, очищенный от E. палочки в соответствии с протоколом описано выше 17,18. </li><li> Подготовка 50 мкл реакции, содержащие 1X реакционного буфера (30 мМ Hepes рН 6,8, 50 мМ NaCl, 6 мМ MgSO 4, 1 мМ DTT), 6 мм фосфокреатина (ПЦР), 25 ед / мл CreatinePhosphoKinase (КФК), 5 мМ АТФ (Mg соли), 0,3 мМ IP6, 0,05-0,1 мкг Его-IP 6 K1 или GST-VIP1. Регулировка громкости с MiliQ DDH 2 O. </li><li> Кратко спина реакции и инкубировать при температуре 37 ° С в течение ночи с вращением. </li></ol> 2. Полиакриламидном геле литья и погрузка - 2-й день (4 часа в день) <ol><li> Полиакриламидном геле подготовлен с использованием 24 см в длину, 18 см шириной пластин стекла и 1,5 мм шириной прокладки. Обычно 16 полосы или препаративной одной гребенкой переулок используется. </li><li> Подготовка смеси (50 мл / гель), содержащий следующие: 35,5% (м / о) акриламид: бис-акриламид 19:1, 1X Трис / Борат / EDTA (КЭ), 0,05% (м / о) персульфата аммония (APS ), 0,05% (м / о) Temed. Вылейте смесь предварительно литых стеклянных пластин, вставьте расческу и пусть полимеризоваться в течение 30-60 минут при комнатной температуре. </li><li> Как только гель полимеризуется, передачу аппарата для холодной комнате и предварительно запустить в 1X TBE около 30-60 минут при 200-300 вольт. </li><li> Добавить 1X из OrangeG красителя (10 мМ Трис-HCl pH7.0, 1 мМ ЭДТА, 30% глицерина, 0,1% OrangeG) для каждой реакции. Подготовка образцов, содержащих 2 нмоль ИП 6 для загрузки в качестве стандартного контроля. </li><li> Промыть каждую лунку тщательно проточной буфера с помощью шприца и иглы 21G, чтобы удалить осадок, а затем загрузить геля. Избегайте нагрузки на стороне скважин. </li><li> Выполнить геля в течение ночи при 450-550 Вольт (7 MAMP / гель), пока OrangeG группы красителя в течение последних 10 см от нижней части геля. </li></ol> 3. Выделение IP-7 - день 3 (4 часа) и 4-й день (6-7 часа SpeedVac процесса сушки) <ol><li> Разберите гель аппарата и аккуратно удалить одну стеклянную пластину оставив гель на другую. Cut небольшая часть геля от чуть выше OrangeG группы красителя на дно содержащие ИП 6 стандартных и один образец полосы, как показано на рисунке 1. </li><li> Пятно вырезать часть геля толуидиновым синим (0,1% (м / о) толуидиновым синим, 20% (м / о) метанола, 2% (вес / объем) глицерин) в течение нескольких минут (1-3 мин) или до инозитол группы пирофосфат появляется. Поместите стеклянную пластинку ранее удалили обратно поверх геля для предотвращения неокрашенных гель от высыхания. </li></ol> ИП 7 полоса должна быть видна, поскольку она работает немного медленнее, чем ИП 6 стандарта. АТФ, который работает быстрее, чем IP-6, также должны быть видны (рис. 1). Передача окрашенные части геля в де-красящим раствором (20% (м / о) метанол) в течение нескольких минут, смыть любое превышение толуидиновым синим и переместить гель с неокрашенными геля. Если визуализация выше pyrophosphorylated инозитол изоформ (IP-8 и IP-9) не требуется, пятно гель с Толуидин решение окрашивания синий в течение 20 минут при комнатной температуре. Затем смыть толуидин синий с де-красящим раствором в течение приблизительно 15 минут. <ol start="3"><li> С лезвием бритвы вырезать IP 7-ми полосный на неокрашенных часть геля, используя в качестве ссылки ИП 7 миграции положение определяется с окрашенных гель (рис. 1). </li><li> Положите ИП 7 группа, которая была вырезана из геля на 15 мл трубки и добавить 10 мл MilliQ DDH 2 O. Положите труб в ротации в течение 10 минут при комнатной температуре. Удалите жидкость для удаления избытка КЭ и микроскопических частиц акриламида. </li><li> Впоследствии, выполняют две дегидратации-гидратации циклов. Добавьте 5 мл 50% (м / о) метанола в трубку с гелем, содержащим IP-7 и вращаются при комнатной температуре в течение 2 часов. Передача геля для новых 15 мл пробирку, содержащую 5 мл MilliQ DDH 2 O и вращаться при комнатной температуре в течение 2 часов. Не выбрасывайте метанола и MilliQ H 2 O из труб. Повторите дегидратации-гидратации цикл еще раз, повторно передачи полиакриламидном геле в 15 мл трубы использовались ранее. Один из моющие средства могут быть выполнены в течение ночи. </li><li> Чтобы сосредоточиться элюировали ИП 7, сухой 10 мл вместе (5 мл MilliQddH 2 O и 5 мл 50% (м / о) метанола) с использованием SpeedVac нагревают при 60 ° C. </li><li> Как только образцы почти сухая, передача оставшейся жидкости (300-600 мкл) к A1.5 мл центрифужную пробирку и спина в течение 2 минут при 5000 оборотах в минуту. </li><li> Сбор супернатант и передачи в свежем 1,5 мл центрифужную пробирку, оставьтенижняя 20-30 мкл, поскольку она может содержать акриламида частиц. </li><li> При необходимости продолжить процесс сушки использованием неотапливаемых SpeedVac. Восстановление ИП 7 значительно сокращается, если образцы полностью высохнуть, поэтому прекращать процесс сушки, когда образцы достигают объема 100-300 мкл. </li></ol> 4. Определение IP 7 концентрации и чистоты. <ol><li> Используйте 2-5 мкл восстановленного ИП 7 образца для работы на PAGE гель, как и в разделах 2.2-2.5. Загрузите несколько разведений ИП 6 (0,5 т., 1, 2, 4 нмоль) в стандартной концентрации и 4 нмоль Поли-Р маркером. После запуска гель, визуализировать инозитол изоформ пирофосфат окрашиванием и де-окрашивание весь гель с толуидиновым синим решение, согласно процедуре, описанной в разделе 3.2 (рис. 2А). </li><li> После Толуидин окрашивания, концентрация может быть определена путем сканирования геля и сравнение различий в интенсивности между IP-6 и IP-7 с помощью обработки изображений, такие как изображения-J, как показано на рисунке 2Б. </li></ol> 5. Представитель Результаты: Препаративного ферментативного превращения IP 6 до 7 с помощью IP-IP ферменты 6 К1 и VIP1 можно легко разрешить с помощью PAGE анализа (рис. 1). Загрузка IP-6, как размер контроля совместно с Толуидин окрашивания геля Синий позволяет идентифицировать pyrophosphorylated производные, так как они работают медленнее, в зависимости от количества фосфатных групп, присутствующих на инозитол кольцо. Процедура, описанная выше, позволяет легко очистки IP 7. Анализ очищенной инозитол пирофосфат за страницей показал чистоту нашего IP-7 (рис. 2а). Интересно, что 1/3PP-IP 5 изомер ИП 7 продуктом VIP1 мигрирует немного медленнее, чем 5PP-IP 5 изомер ИП 7, порожденный IP 6 К1. Использование IP 6 стандартов разрешения легко количественная концентрация очищенного IP-7 (рис. 2Б). Перед использованием IP-7 для дальнейших экспериментов, его биологическая активность может быть оценен (Рис. 3). 5PP-IP 5 инкубируют с VIP1 и с ИП 7 фосфатазы DDP1 (diphosphoinositol полифосфат phosphohydrolase). Регулярно, очищенный ИП 7 преобразуется в IP-8, VIP1 и ИП 6 по DDP1 (рис. 3). <img alt="Рисунок 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Рисунок 1:. Толуидин окрашивание СТРАНИЦА и выделение IP-7-ми полосный часть геля, содержащего стандартные (IP-6) был вырезан и окрашивали толуидин синий раствор. Три полосы представляют (сверху вниз) IP-7, ИП 6 и АТФ. Окрашенных часть геля затем выравнивается с остальными геля. Это позволяет локализация часть геля, содержащего IP-7, которые затем можно вырезать и очищенные (пунктирная коробка). <img alt="Рисунок 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Рисунок 2: СТРАНИЦА анализ IP реакции 6 К1 и VIP1 продукции. ) Анализ IP-6 (4, 2, 1, 0,5 нмоль) по толуидиновым синим окрашивания был использован для того, чтобы определить IP 7 Концентрация очищенного от обоих ИП 6 К1 (5PP-IP 5) и VIP1 (1/3PP-IP 5) реакций. B) анализ Scatter заговор с целью определения концентрации очищенных IP 7. Концентрации определялись по интенсивности полосы, рассчитанной с использованием программного обеспечения ImageJ, по сравнению с заранее определенным количеством IP-6. X-ось представляет интенсивность, Y-ось представляет концентрации выражены в нмоль. <img alt="Рисунок 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Рисунок 3. Анализ ИП 7 биологическую активность Для определения качества очищенной ИП 7 мы инкубировали 5PP-IP 5 (ИП 6 К1 порожденных IP-7) с VIP1 или с ИП 7 фосфатазы DDP1, а затем решил реакция на странице. DAPI и Толуидин окрашивание показало, ожидается производство IP-8, VIP1 и преобразование IP-7 по IP-6, DDP1.

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Использование инозитол пирофосфата в биохимии сильно ограничена коммерческой отсутствия таких соединений и низкой чувствительности существующих методов обнаружения. Сочетание PAGE, который позволяет разделение молекул, имеющих разное количество фосфатных групп, и толуидиновым синим (рис. 1), метахроматическая краситель, который связывается с фосфатными группами, позволяет легко обнаружения инозитол изоформ pyrophoshate открывая новые направления исследований 20. Описано использование технологии СТРАНИЦА, чтобы очистить инозитол продуктов пирофосфат ферментативной реакции осуществляется outby либо ИП 6 K1 или VIP1 это простой, экономичный и надежный метод, который позволяет для производства большого количества высоких IP качества 7. Описанный выше метод не ограничивается простой очистки IP-7, но незначительные изменения описанного протокола может позволить очистки различный диапазон инозитол пирофосфаты. Высшее фосфорилированных инозитол изоформ пирофосфат, содержащие более восьми фосфатные группы могут быть обнаружены с помощью IP-7 или различное количество IP-6 в качестве подложки 20,6. Эти инозитол пирофосфаты могут быть обнаружены путем увеличения длины Процедура окраски, а затем очищенный (раздел 3.2). Более того, использование IP-5 в качестве субстрата для ферментативной реакции позволит очистки PP-IP-5 и других инозитол пирофосфаты содержащие гидроксильные группы по инозитол кольцо. В заключение этого нетребовательного метод позволяет надежной очистки миллиграмм количествах инозитол пирофосфаты с широко доступных инструментов, тем самым открывая новые перспективы для этой захватывающей области исследований.

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th></tr><tr><td> Фитиновой кислоты (IP-6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Поли-Р (гексаметафосфат натрия) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> АТФ-Mg 2 + соль </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Фосфокреатина (ПЦР) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho киназы (КФК) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td> Его-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> Его-Ddp1 </td><td> Доступный в лаборатории </td><td> Доступный в лаборатории </td></tr><tr><td> Акриламид: бис-акриламид 19:1 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Персульфата аммония (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Трис / Борат / EDTA (КЭ) </td><td> Sigma-Aldrich </td><td> T 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Толуидиновым синим </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Христос </td><td> 100218 </td></tr><tr><td> Гель аппарата </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Вакуумный коллектор </td><td> Христос </td><td> Альфа 2-4 </td></tr><tr><td> Вакуум-насос </td><td> ПРО 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

Инозитол пирофосфаты играют важную роль в человеческой патологии, такие рака, диабета и ожирения, однако точный механизм действия является предметом спора. Отсутствие коммерчески доступных инозитол пирофосфаты предоставляет детальные исследования проблематично. Здесь мы опишем простой протокол для производства и изолировать мг инозитол пирофосфаты.

Подготовка Качество Инозитол пирофосфаты

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

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.

ДНК-микрочипов: Контроль качества образцов, Array Гибридизация и сканирования

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.

Неинвазивный метод отбора проб волос для получения высоких ДНК Качество от Неуловимый мелких млекопитающих

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.

Долгосрочные Смертельное испытания токсичности с ракообразных Артемия 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.

И что нельзя делать крио-электронной микроскопии: учебник по пробоподготовки и высокое качество сбора данных для высокомолекулярных 3D реконструкции

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.

Веб-инструмент для создания высокого качества в машиночитаемой форме биологических путей

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.

Коллекция спермы дифференциальной качества с использованием градиентного центрифугирования плотность

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.

Полевая идентификация ромашки Matricaria с помощью портативной системы qPCR

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.

Улучшенный анализ хемотаксиса для быстрой идентификации ризобактериальных хемоаттрактантантов в корневых экссудатах

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.

Культивирование, рост и жизнеспособность молочнокислых бактерий: перспектива контроля качества

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

Оптимизация качества пробиотических продуктов, высушенных распылением за счет разработки технологического процесса

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.

В центре внимания автора: Разработка процесса распылительной сушки пробиотических бактерий и оценка качества продукта  

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

Разработка технологического процесса распылительной сушки пробиотических бактерий и оценка качества продукции

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