Nous remercions A. Riccio pour leurs commentaires et de lire le manuscrit. Ce travail a été soutenu par le Medical Research Council (MRC) de financement à l&#39;Unité de Biologie cellulaire et par un homme Frontier Science Program Grant (RGP0048/2009-C).

1. Réaction enzymatique - jour 1 (1 heure dans l&#39;après-midi) <ol><li> La première étape est de préparer les 10-20 indépendante réactions enzymatiques dans lesquels IP 6 K1 ou VIP1 conversion IP de 6 à isoformes pyrophosphorylated. </li><li> Nous utilisons Sa-IP 6 K1 et TPS-VIP1 enzymes purifiées à partir de E. coli selon le protocole décrit précédemment 17,18. </li><li> Préparer 50 réactions ul contenant 1X tampon de réaction (30 mM Hepes pH 6,8, NaCl 50 mM, 6 mM MgSO 4, 1 mM DTT), 6 Phosphocréatine mM (PCR), 25 U / ml CreatinePhosphoKinase (CPK), 5 mM d&#39;ATP (Mg sel), 0,3 mM IP6, 0,05 à 0,1 mg Sa-IP 6 K1 ou GST-VIP1. Régler le volume avec MiliQ ddH 2 O. </li><li> Centrifuger brièvement la réaction et incuber à 37 ° C pendant la nuit avec la rotation. </li></ol> 2. Coulée de gel de polyacrylamide et chargement - jour 2 (4 heures dans l&#39;après-midi) <ol><li> Le gel de polyacrylamide est préparé à partir de 24 cm de long, 18 cm de plaques de verre de large et 1,5 mm entretoises large. Habituellement une ruelle ou d&#39;un peigne 16 préparative seule voie est utilisée. </li><li> Préparer un mélange (50 ml / gel) contenant les éléments suivants: 35,5% (p / v) d&#39;acrylamide: bis-acrylamide 19h01, le persulfate d&#39;ammonium 1X Tris / borate / EDTA (TBE), 0,05% (p / v) (APS ), 0,05% (p / v) Temed. Verser le mélange entre les plaques pré-moulé en verre, insérez le peigne et laisser polymériser pendant 30-60 minutes à température ambiante. </li><li> Une fois le gel polymérisé, le transfert à l&#39;appareil de la chambre froide et pré-run en TBE 1X pendant environ 30-60 minutes à 200-300 volts. </li><li> Ajouter 1X de OrangeG colorant (10 mM Tris-HCl pH 7,0, EDTA 1 mM, glycérol 30%, 0,1% OrangeG) pour chaque réaction. Préparer un échantillon contenant 2 nmol d&#39;IP 6 à charger comme un contrôle standard. </li><li> Laver chaque puits abondamment avec de tampon en utilisant une seringue et une aiguille 21G de supprimer tout précipité, puis de charger le gel. Évitez de charger sur le puits côté. </li><li> Exécutez le gel de nuit à 450-550 volts (7 mAmp / gel), jusqu&#39;à ce que la bande de teinture OrangeG est dans les 10 derniers cm du bas du gel. </li></ol> 3. Isolement de la PI 7 - 3 jours (4 heures) et 4 jours (6-7 heures de séchage processus SpeedVac) <ol><li> Démonter l&#39;appareil de gel et soigneusement enlever une plaque de verre laissant le gel sur l&#39;autre. Couper une petite portion du gel de la bande juste au-dessus de teinture OrangeG au fond contenant l&#39;adresse IP standard de 6 et une voie de l&#39;échantillon, comme le montre la figure 1. </li><li> Tache la partie découpée du gel au bleu de toluidine (0,1% (p / v) bleu de toluidine, 20% (p / v) méthanol, 2% (p / v) glycérol) pendant quelques minutes (1-3 min) ou jusqu&#39;à ce que la bande de pyrophosphate inositol apparaît. Mettez la plaque de verre préalablement retiré de retour au sommet du gel pour éviter le gel sans tache de sécher. </li></ol> L&#39;IP 7 bandes devraient être visibles, car il tourne légèrement plus lent que l&#39;adresse IP standard de 6. ATP, qui court plus vite que 6 IP, doit également être visible (figure 1). Transférer la partie colorée du gel dans une solution de-coloration (20% (p / v) méthanol) pendant quelques minutes, laver tout excès de bleu de toluidine et de repositionner le gel avec le gel non colorées. Si la visualisation de la hausse des isoformes pyrophosphorylated inositol (IP 8 et 9 IP) est nécessaire, aux taches le gel avec une solution de toluidine coloration bleue pendant 20 minutes à température ambiante. Par la suite, laver les bleu de toluidine avec la solution de-coloration pour environ 15 minutes. <ol start="3"><li> Avec une lame de rasoir couper la propriété intellectuelle 7 bandes sur la partie non colorée du gel en utilisant comme référence la position de la migration IP 7 déterminée avec le gel coloré (figure 1). </li><li> Mettez l&#39;adresse IP 7 bandes qui a été coupé à partir du gel sur un tube de 15 ml et ajouter 10 ml de MilliQ ddH 2 O. Mettez les tubes en rotation pendant 10 minutes à température ambiante. Jeter le liquide pour enlever l&#39;excès de particules d&#39;acrylamide TBE et microscopiques. </li><li> Par la suite, effectuer deux cycles de déshydratation-hydratation. Ajouter 5 ml de 50% (p / v) méthanol pour le tube avec le gel contenant 7 IP et tournent à température ambiante pendant 2 heures. Transférer la tranche de gel d&#39;un nouveau tube de 15 ml contenant 5 ml de MilliQ ddH O 2 et tournent à température ambiante pendant 2 heures. Ne jetez pas le méthanol et l&#39;H 2 O MilliQ des tubes. Répétez le cycle de déshydratation-hydratation fois de plus en re-transférer le gel de polyacrylamide en tubes de 15 ml préalablement utilisés. Un des lavages peuvent être effectués pendant la nuit. </li><li> Pour concentrer la propriété intellectuelle élué 7, sec les 10 mL ensemble (5 ml MilliQddH 2 O et 5 ml à 50% (p / v) méthanol) en utilisant un SpeedVac chauffé à 60 ° C. </li><li> Une fois les échantillons sont presque à sec, le transfert du liquide restant (300-600 ul) à A1.5 tube à centrifuger mL et de spin 2 minutes à 5000 rpm. </li><li> Recueillir le surnageant et transférer dans un nouveau tube de 1,5 ml centrifugeuse, laisserle bas de 20 à 30 uL car il peut contenir des particules d&#39;acrylamide. </li><li> Si nécessaire de poursuivre le processus de séchage en utilisant un SpeedVac non chauffés. La reprise de la PI 7 est considérablement réduit si les échantillons sécher complètement, donc fin du processus de séchage, lorsque les échantillons atteindre le volume de 100-300 mL. </li></ol> 4. Détermination de la PI 7 concentration et la pureté. <ol><li> Utilisez 2-5 uL de la propriété intellectuelle 7 échantillon récupéré à courir sur un gel PAGE, similaire aux sections 2.2 à 2.5. Chargez plusieurs dilutions de IP 6 (soit 0,5, 1, 2, 4 nmol) comme standard de concentration et de 4 nmol de poly-P marqueur. Après l&#39;exécution du gel, de visualiser les isoformes pyrophosphate inositol par coloration et de-coloration du gel complet avec Toluidine solution bleue, suivant la procédure décrite à la section 3.2 (figure 2A). </li><li> Après coloration de toluidine, les concentrations peuvent être déterminées en analysant le gel et la comparaison des différences d&#39;intensité entre 6 IP et IP 7, en utilisant un logiciel d&#39;imagerie telles que Image-J, comme le montre la figure 2B. </li></ol> 5. Les résultats représentatifs: La conversion enzymatique préparative de la PI de 6 à 7 IP utilisant le protocole IP enzymes 6 K1 et VIP1 peut être facilement résolu en utilisant l&#39;analyse PAGE (figure 1). Le chargement de 6 IP comme un contrôle de taille avec coloration gel bleu de toluidine permet l&#39;identification des dérivés pyrophosphorylated, car ils courent plus lentement en fonction du nombre de groupes phosphates présents sur le cycle inositol. La procédure décrite ci-dessus permet la purification facile de 7 IP. L&#39;analyse de la pyrophosphate purifiée inositol par PAGE a révélé la pureté de notre IP 7 (figure 2A). Fait intéressant, le 1/3PP-IP 5 isomère de l&#39;IP 7 produit des VIP1 migre légèrement plus lent que l&#39;5PP-IP 5 isomère de 7 IP qui est généré par la propriété intellectuelle 6 K1. L&#39;utilisation de IP 6 normes permettent une quantification facile de la concentration de l&#39;IP purifiée 7 (figure 2B). Avant d&#39;utiliser 7 IP pour d&#39;autres expériences, son activité biologique peut être évaluée (Figure 3). 5PP-IP 5 est incubée avec VIP1 et avec l&#39;IP 7 phosphatase DDP1 (phosphohydrolase polyphosphate diphosphoinositol). Régulièrement, l&#39;IP purifiée 7 est converti en IP 8 par VIP1 et à IP 6 par DDP1 (figure 3). <img alt="Figure 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Figure 1:. Coloration Toluidine du PAGE et l&#39;isolement de l&#39;IP 7 bandes La portion du gel contenant le standard IP (6) a été coupées et colorées avec une solution de bleu de toluidine. Les trois bandes représentent (de haut en bas) IP 7, IP 6 et ATP. La partie colorée du gel a été ensuite aligné avec le reste du gel. Cela permet la localisation de la portion du gel contenant 7 IP, qui peut ensuite être coupé et purifié (zone en pointillés). <img alt="Figure 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Figure 2: Analyse de la PI PAGE produits de réaction 6 K1 et VIP1. A) Analyse de la propriété intellectuelle 6 (4, 2, 1, 0,5 nmol) par coloration au bleu de toluidine a été utilisée afin de déterminer la concentration de propriété intellectuelle 7 purifiés à partir de deux 6 IP K1 (5PP-IP 5) et VIP1 (1/3PP-IP 5) les réactions. B) Analyse Nuage de points pour déterminer la concentration de l&#39;IP purifiée 7. Les concentrations ont été déterminées en fonction de l&#39;intensité des bandes, calculé en utilisant le logiciel ImageJ, par rapport à des quantités prédéterminées de 6 IP. L&#39;axe X représente les intensités; l&#39;axe Y représente la concentration exprimée en nmoles. <img alt="Figure 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Figure 3:. Analyse de l&#39;activité biologique IP 7 Pour déterminer la qualité de l&#39;IP purifiée 7 nous avons incubé 5PP-IP 5 (6 K1 IP IP généré 7) avec VIP1 ou avec l&#39;IP 7 phosphatase DDP1 puis la réaction résolue sur la page. La coloration DAPI et Toluidine révélé la production attendue de 8 IP par VIP1 et la conversion de IP à IP 7 6 par DDP1.

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Aucun conflit d&#39;intérêt déclaré.

L&#39;utilisation de pyrophosphate inositol en biochimie est sévèrement limitée par l&#39;indisponibilité commerciale de ces composés et la faible sensibilité des méthodes de détection existantes. La combinaison de la page, qui permet la séparation de molécules possédant un nombre différent de groupes phosphate, et bleu de toluidine (figure 1), un colorant métachromatique qui se lie à des groupes phosphate, permet la détection facile des isoformes pyrophoshate inositol ouvrir de nouvelles avenues de recherche 20. L&#39;utilisation de la technologie décrite PAGE pour purifier les produits pyrophosphate inositol de la réaction enzymatique réalisée outby soit 6 IP K1 ou VIP1 est une méthode simple, économique et fiable qui permet la production de grandes quantités d&#39;IP de haute qualité 7. La méthode décrite ci-dessus n&#39;est pas limitée à la simple purification de la PI 7, mais de légères modifications du protocole décrit peut permettre la purification d&#39;une gamme différente de pyrophosphates inositol. Supérieur phosphorylée isoformes pyrophosphate inositol, contenant plus de huit groupes de phosphate peuvent être détectées en utilisant l&#39;IP 7 ou différents montants de 6 IP comme substrat 20,6. Ces pyrophosphates inositol peut être détecté en augmentant la longueur de la procédure de coloration et ensuite purifié (section 3.2). Par ailleurs, l&#39;utilisation de 5 IP comme substrat pour la réaction enzymatique permettant la purification de la PP-PI 5 et d&#39;autres pyrophosphates inositol contenant un groupe hydroxyle sur le cycle inositol. En conclusion, cette méthode permet peu exigeante pour la purification de quantités fiables milligramme de pyrophosphates inositol avec des instruments largement disponibles, ouvrant ainsi de nouvelles avenues pour ce domaine de recherche passionnant.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> L&#39;acide phytique (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poly-P (hexamétaphosphate de sodium) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + sel </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Phosphocréatine (PCr) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> Kinase CreatinePhospho (CPK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> TPS-VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td> Son-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> Son-Ddp1 </td><td> Disponible dans le laboratoire </td><td> Disponible dans le laboratoire </td></tr><tr><td> L&#39;acrylamide: bis-acrylamide 19h01 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Persulfate d&#39;ammonium (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / borate / EDTA (TBE) </td><td> Sigma-Aldrich </td><td> T 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Bleu de toluidine </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Le Christ </td><td> 100218 </td></tr><tr><td> Appareil de gel </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Collecteur à vide </td><td> Le Christ </td><td> Alpha 2-4 </td></tr><tr><td> Pompe à vide </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol est présent dans la nature soit intact ou en plus complexes dérivés phosphorylés. De la dernière, les deux plus abondants dans les cellules eucaryotes sont pentakisphosphate inositol (IP 5) et hexakisphosphate inositol (acide phytique ou IP 6). IP 5 et 6 du protocole IP sont les précurseurs des molécules qui contiennent pyrophosphate inositol une ou plusieurs liaisons pyrophosphates 1. La phosphorylation de propriété intellectuelle génère six diphoshoinositolpentakisphosphate IP (7 ou PP-PI 5) et bisdiphoshoinositoltetrakisphosphate IP (8 ou (PP) 2-IP 4). Pyrophosphates inositol ont été isolées de tous les organismes eucaryotes étudiées jusqu&#39;ici. En outre, les deux classes distinctes d&#39;enzymes responsables de la synthèse du pyrophosphate inositol sont très conservés au cours de l&#39;évolution 2-4. Le 6 IP kinases (IP 6 Ks) possèdent une énorme flexibilité catalytique, la conversion IP et IP 5 6 PP-PI 4 et 7, respectivement IP et ensuite, en utilisant ces produits comme substrats, promouvoir la production de molécules plus complexes 5,6 . Récemment, une deuxième classe d&#39;enzymes générant pyrophosphate a été identifié dans la forme de la protéine de levure VIP 1 (aussi appelé le PP-PI 5 K), qui est capable de convertir IP à IP 6 7 8 7,8 et IP. Pyrophosphates Inositol réguler de nombreux processus cellulaires disparates tels que 9 sécrétion d&#39;insuline, la longueur des télomères 10,11, 12 chimiotactisme, le trafic vésiculaire 13, 14 homéostasie du phosphate et du VIH-1 gag communiqué 15. Deux mécanismes d&#39;actions ont été proposées pour cette classe de molécules. Ils peuvent affecter la fonction cellulaire par allostériquement interagissant avec des protéines spécifiques comme AKT 16. Alternativement, le groupe pyrophosphate pouvez faire un don d&#39;un phosphate de pré-phosphorylée protéines 17. L&#39;énorme potentiel de ce domaine de recherche est entravée par l&#39;absence d&#39;une source commerciale de pyrophosphates inositol, qui empêche de nombreux scientifiques de l&#39;étude de ces molécules et cette nouvelle modification post-traductionnelle. Les méthodes actuellement disponibles pour isoler les pyrophosphates inositol nécessitent sophistiqués appareil chromatographique 18,19. Ces procédures utilisent des conditions acides qui pourraient conduire à la dégradation de l&#39;inositol pyrophosphate 20 et donc à une mauvaise récupération. Par ailleurs, la lourdeur des procédures dessalage post-colonne de restreindre leur utilisation à des laboratoires spécialisés. Dans cette étude, nous décrivons une méthode peu exigeante pour la génération, l&#39;isolement et la purification des produits de l&#39;IP 6-kinase et PP-PI-5 kinases réactions. Cette méthode a été possible par la capacité de l&#39;électrophorèse sur gel de polyacrylamide (PAGE) pour résoudre hautement phosphorylée polyphosphates inositol 20. Suite à IP 6 K1 et PP-PI 5 réactions enzymatiques en utilisant K 6 IP comme substrat, PAGE a été utilisé pour séparer les pyrophosphates généré inositol qui ont été ensuite élue à l&#39;eau.

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Preparation of Quality Inositol Pyrophosphates

Pyrophosphates Inositol jouer un rôle important dans les pathologies humaines telles le cancer, le diabète et l&#39;obésité; cependant, le mécanisme d&#39;action exact est un sujet de litige. Le manque de commerce pyrophosphates inositol rend études détaillées problématique. Nous décrivons ici un protocole simple de produire et d&#39;isoler milligrammes de pyrophosphates inositol.

Préparation de la qualité de pyrophosphates inositol

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 vues.  University College London. Pyrophosphates Inositol jouer un rôle important dans les pathologies humaines telles le cancer, le diabète et l'obésité; cependant, le mécanisme d'action exact est un sujet de litige. Le manque de commerce pyrophosphates inositol rend études détaillées problématique. Nous décrivons ici un protocole simple de produire et d'isoler milligrammes de pyrophosphates inositol.

Vidéo: 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.

Cellules souches: le contrôle qualité des échantillons, hybridation Array et balayage

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

Recherche

Biologie

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.

Une technique non invasive cheveux d&#39;échantillonnage pour obtenir l&#39;ADN de haute qualité provenant Elusive petits mammifères

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.

À long terme des essais de toxicité létale avec le Crustacé Artemia 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.

À faire et à ne pas faire de cryo-microscopie électronique: Notions sur la préparation de l&#39;échantillon et de collecte de données de haute qualité pour la reconstruction 3D macromoléculaire

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.

Un outil Web pour la génération de haute qualité lisible par machine voies biologiques

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.

Prélèvement du sperme de qualité différentielle en utilisant la Centrifugation en Gradient de densité

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.

Identification sur le terrain de Matricaria chamomilla à l’aide d’un système qPCR portable

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.

Un test de chimiotaxie amélioré pour l’identification rapide des chimioattractifs rhizobactériens dans les exsudats racinaires

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.

La culture, la croissance et la viabilité des bactéries lactiques : une perspective de contrôle de la qualité

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

Optimiser la qualité des produits probiotiques séchés par pulvérisation grâce au développement de procédés

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.

Pleins feux sur l’auteur : Développement de procédés pour le séchage par atomisation de bactéries probiotiques et évaluation de la qualité du produit  

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

Développement de procédés pour le séchage par atomisation des bactéries probiotiques et évaluation de la qualité du produit

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

Methods Article

Préparation de la qualité de pyrophosphates inositol