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

Ringraziamo A. Riccio per gli utili commenti e leggere il manoscritto. Questo lavoro è stato finanziato dal Medical Research Council (MRC) i finanziamenti per l&#39;Unità di Biologia Cellulare e da un Umano Frontier Science Grant Program (RGP0048/2009-C).

1. La reazione enzimatica - giorno 1 (1 ora al pomeriggio) <ol><li> Il primo passo è quello di preparare 10-20 indipendente reazioni enzimatiche in cui IP 6 K1 o VIP1 convertire IP 6 al isoforme pyrophosphorylated. </li><li> Utilizziamo His-IP 6 K1 e GST-VIP1 enzimi purificato da E. coli secondo il protocollo precedentemente descritto 17,18. </li><li> Preparare 50 reazioni microlitri contenente 1X tampone di reazione (30 Hepes mM pH 6,8, 50 mM NaCl, 6 mM MgSO 4, 1 mM DTT), 6 mM fosfocreatina (PCr), 25 U / mL CreatinePhosphoKinase (CPK), 5 mM ATP (Mg sale), 0,3 mM IP6, 0,05-0,1 mg suo-IP 6 K1 o GST-VIP1. Regolare il volume con MiliQ DDH 2 O. </li><li> Brevemente girare la reazione e incubare a 37 ° C durante la notte con rotazione. </li></ol> 2. Poliacrilammide colata gel e di carico - giorno 2 (4 ore al pomeriggio) <ol><li> Il gel di poliacrilammide si prepara a 24 cm di lunghezza, lastre di vetro 18 centimetri di larghezza e 1,5 mm di larghezza distanziatori. Di solito un 16 lane o un singolo pettine corsia preparativo viene utilizzato. </li><li> Preparare una miscela (50 ml / gel) contenente le seguenti: 35,5% (w / v) di acrilammide: Bis-acrilamide 19:1, 1X Tris / Borato / EDTA (TBE), 0,05% (w / v) di ammonio persolfato (APS ), 0,05% (w / v) TEMED. Versate il mix tra il pre-lastre di vetro fuso, inserire il pettine e lasciare polimerizzare per 30-60 minuti a temperatura ambiente. </li><li> Una volta che il gel è polimerizzato, trasferire l&#39;apparecchiatura alla stanza fredda e pre-run in TBE 1X per circa 30-60 minuti a 200-300 Volt. </li><li> Aggiungi 1X di OrangeG colorante (10 mM Tris-HCl pH7.0, 1 mM EDTA, 30% glicerolo, 0,1 OrangeG%) per ogni reazione. Preparare un campione contenente 2 nmol di IP da 6 a caricare come un controllo standard. </li><li> Lavare ogni pozzetto a fondo con l&#39;esecuzione buffer utilizzando una siringa e un ago 21G per rimuovere l&#39;eventuale precipitato, quindi caricare il gel. Evitare di caricare nei pozzetti laterali. </li><li> Esegui il gel notte a 450-550 Volt (7 mAmp / gel), fino a quando la band colorante OrangeG è da meno di 10 cm dal fondo del gel. </li></ol> 3. Isolamento di IP 7 - giorno 3 (4 ore) e il 4 ° giorno (6-7 ore SpeedVac processo di essiccazione) <ol><li> Smontare l&#39;apparato gel e con attenzione rimuovere una lastra di vetro lasciando il gel su l&#39;altro. Tagliare una piccola porzione del gel da appena sopra la banda colorante OrangeG al fondo contenente l&#39;IP 6 standard e una corsia campione, come mostrato nella Figura 1. </li><li> Macchiare la parte tagliata del gel con blu di toluidina (0,1% (w / v) di blu di toluidina, 20% (w / v) metanolo, 2% (w / v) glicerolo) per alcuni minuti (1-3 min) o fino a quando la band pirofosfato inositolo appare. Mettere la lastra di vetro precedentemente rimosso torna in cima al gel per evitare che il gel senza macchia si secchi. </li></ol> L&#39;IP a 7 bande dovrebbero essere visibili in quanto corre leggermente più lento del IP 6 standard. ATP, che corre più veloce di IP 6, dovrebbero essere visibili (Figura 1). Trasferire la parte macchiata del gel in un de-soluzione colorante (20% (w / v) metanolo) per alcuni minuti, lavare via ogni eccesso di blu di toluidina e riposizionare il gel con il gel senza macchia. Se la visualizzazione di più isoforme pyrophosphorylated inositolo IP (IP 8 e 9) è richiesto, macchia il gel con una soluzione di toluidina colorazione blu per 20 minuti a temperatura ambiente. Successivamente, lavare via il blu toluidina con la de-soluzione colorante per circa 15 minuti. <ol start="3"><li> Con una lametta tagliare la banda IP 7 sulla porzione senza macchia del gel utilizzando come riferimento la posizione di migrazione IP 7 determinato con il gel colorato (Figura 1). </li><li> Mettere la IP 7 band che è stata tagliata dal gel su un tubo da 15 ml e aggiungere 10 ml di MilliQ DDH 2 O. Mettere tubi in rotazione per 10 minuti a temperatura ambiente. Eliminare il liquido per rimuovere l&#39;eccesso di particelle di acrilammide TBE e microscopiche. </li><li> Successivamente, eseguire due cicli di idratazione-disidratazione. Aggiungere 5 ml di 50% (w / v) metanolo al tubo con il gel contenente 7 IP e ruotare a temperatura ambiente per 2 ore. Trasferire la fetta gel a un nuovo tubo di 15 ml contenente 5 ml di MilliQ DDH 2 O e ruotare a temperatura ambiente per 2 ore. Non eliminare il metanolo e MilliQ H 2 O dai tubi. Ripetere la disidratazione-idratazione ciclo ancora una volta da ri-trasferimento del gel di poliacrilammide in provette da 15 ml precedentemente utilizzato. Uno dei lavaggi può essere eseguita durante la notte. </li><li> Per concentrare l&#39;IP eluiti 7, asciugare il 10 mL insieme (5 ml MilliQddH 2 O e 5 ml al 50% (w / v) metanolo) utilizzando un SpeedVac riscaldata a 60 ° C. </li><li> Una volta che i campioni sono quasi a secco, trasferire il liquido residuo (300-600 mL) a A1.5 tubo ml centrifuga e gira per 2 minuti a 5000 rpm. </li><li> Raccogliere il supernatante e trasferirlo in un nuovo tubo di 1,5 ml per centrifuga; lasciareil fondo 20-30 microlitri dal momento che può contenere particelle di acrilammide. </li><li> Se necessario continuare il processo di essiccazione mediante un SpeedVac riscaldata. Il recupero di IP 7 è drasticamente ridotto, se i campioni asciugare completamente, quindi terminare il processo di essiccazione, quando i campioni di raggiungere il volume di 100-300 microlitri. </li></ol> 4. Determinazione della IP 7 concentrazione e purezza. <ol><li> Utilizzare 2-5 microlitri della recuperato IP 7 campioni per l&#39;esecuzione su un gel PAGE, in modo simile a Sezioni 2,2-2,5. Carico di diluizioni diverse di IP 6 (ovvero 0,5, 1, 2, 4 nmol) come standard la concentrazione e 4 nmol di Poly-P marcatore. Dopo aver eseguito il gel, visualizzare le isoforme pirofosfato inositolo dalla colorazione e de-colorazione il gel intero con una soluzione blu di toluidina, seguendo la procedura descritta al punto 3.2 (Figura 2A). </li><li> Dopo la colorazione toluidina, la concentrazione può essere determinata attraverso la scansione il gel e confrontando le differenze di intensità tra i 6 IP e IP 7, utilizzando software di imaging come immagine-J, come mostrato nella figura 2B. </li></ol> 5. Rappresentante dei risultati: La conversione preparativa enzimatica di IP da 6 a 7 utilizzando IP IP enzimi 6 K1 e VIP1 può essere facilmente risolto utilizzando l&#39;analisi PAGE (Figura 1). Il carico di 6 IP come controllare le dimensioni con colorazione blu toluidina gel permette l&#39;identificazione dei derivati ​​pyrophosphorylated, dal momento che corrono più lentamente a seconda del numero di gruppi fosfato presenti sull&#39;anello inositolo. La procedura descritta sopra permette la purificazione facile IP 7. L&#39;analisi del pirofosfato purificata da PAGE inositolo ha rivelato la purezza del nostro IP 7 (Figura 2A). È interessante notare che i 5 isomero 1/3PP-IP di IP 7 prodotti di VIP1 migra leggermente più lento del 5PP-IP 5 isomero di IP 7 che viene generato dal IP 6 K1. L&#39;uso di IP 6 standard consentire una facile quantificazione della concentrazione della IP purificato 7 (Figura 2B). Prima di utilizzare 7 IP per ulteriori esperimenti, la sua attività biologica può essere valutata (Figura 3). 5PP-IP 5 è incubato con VIP1 e con l&#39;IP 7 fosfatasi DDP1 (phosphohydrolase polifosfato diphosphoinositol). Di routine, l&#39;IP purificata 7 è convertito in IP 8 da VIP1 e IP 6 da DDP1 (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Figura 1:. Colorazione di toluidina PAGE e isolamento della banda IP 7 La porzione del gel contenente lo standard IP (6) è stato tagliato e colorati con una soluzione di blu di toluidina. Le tre bande rappresentano (dall&#39;alto in basso) 7 IP, IP 6 e ATP. La parte macchiata del gel è stato quindi allineato con il restante del gel. Questo permette la localizzazione della porzione del gel contenente 7 IP, che possono poi essere tagliato e purificato (casella tratteggiata). <img alt="Figura 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Figura 2: analisi PAGE di IP prodotti di reazione 6 K1 e VIP1. A) Analisi di IP 6 (4, 2, 1, 0,5 nmol) dalla colorazione blu di toluidina è stato utilizzato per determinare la concentrazione IP 7 purificato da entrambe le 6 di IP K1 (5PP-IP 5) e VIP1 (1/3PP-IP 5) reazioni. B) appezzamento di analisi a dispersione per determinare la concentrazione di IP purificato 7. Le concentrazioni sono state determinate in funzione dell&#39;intensità delle bande, calcolato utilizzando il software ImageJ, rispetto ai livelli pre-determinata quantità di IP 6. L&#39;asse X rappresenta l&#39;intensità, l&#39;asse Y rappresenta concentrazioni espresse in nmol. <img alt="Figura 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Figura 3:. Analisi di IP 7 attività biologica Per determinare la qualità della PI purificata 7 abbiamo incubato 5PP-IP 5 (IP 6 K1 generato IP 7) con VIP1 o con l&#39;IP 7 fosfatasi DDP1 e poi risolto la reazione PAGINA. La colorazione DAPI e toluidina rivelato la produzione prevista di IP 8 da VIP1 e la conversione di IP da 7 a 6 per DDP1 IP.

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Nessun conflitto di interessi dichiarati.

L&#39;uso di inositolo pirofosfato in biochimica è severamente limitato dalla indisponibilità commerciale di tali composti e la scarsa sensibilità dei metodi di rilevazione già esistenti. La combinazione di pagina, che consente la separazione di molecole in possesso di diverso numero di gruppi fosfato e blu di toluidina (Figura 1), un colorante metacromatica che si lega ai gruppi fosfato, consente la facile individuazione delle isoforme pyrophoshate inositolo aprendo nuove vie di ricerca 20. L&#39;uso della tecnologia descritta PAGE per purificare i prodotti pirofosfato inositolo della reazione enzimatica svolta outby sia IP 6 K1 o VIP1 è un metodo semplice, economico e affidabile che consente la produzione di grandi quantità di IP di alta qualità 7. Il metodo sopra descritto non si limita alla purificazione semplice IP 7, ma piccole modifiche del protocollo descritto può permettere la purificazione di una diversa gamma di pirofosfati inositolo. Superiore fosforilata isoforme pirofosfato inositolo, contenente più di otto gruppi fosfato possono essere rilevati tramite IP 7 o diverse quantità di IP 6 come substrato 20,6. Queste pirofosfati inositolo possono essere rilevati aumentando la lunghezza della procedura di colorazione e successivamente purificato (sezione 3.2). Inoltre, l&#39;uso di IP 5 come substrato per la reazione enzimatica che permette la purificazione di PP-IP 5 e altre pirofosfati inositolo contenente un gruppo idrossile sull&#39;anello inositolo. In conclusione, questo metodo permette di poco impegnativo per la purificazione affidabile di quantità milligrammo di pirofosfati inositolo con strumenti ampiamente disponibili, aprendo così nuove strade per questo campo di ricerca entusiasmante.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> L&#39;acido fitico (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poly-P (esametafosfato di sodio) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + sale </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Fosfocreatina (PCr) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho chinasi (CPK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td> Il suo-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> Il suo-Ddp1 </td><td> Disponibile in laboratorio </td><td> Disponibile in laboratorio </td></tr><tr><td> Acrilamide: Bis-acrilamide 19:01 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Di ammonio persolfato (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / Borato / 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> Blu toluidina </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Cristo </td><td> 100218 </td></tr><tr><td> Gel apparato </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Pressione di aspirazione </td><td> Cristo </td><td> Alfa 2-4 </td></tr><tr><td> Pompa del vuoto </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositolo è presente in natura sia senza modifiche o più derivati ​​complessi fosforilata. Delle più recenti, il più abbondante nelle cellule eucariotiche due sono pentakisphosphate inositolo (IP 5) e hexakisphosphate inositolo (acido fitico o IP 6). IP 5 e IP 6 sono i precursori di molecole pirofosfato inositolo che contengono uno o più legami pirofosfato 1. Fosforilazione di IP 6 genera diphoshoinositolpentakisphosphate (IP 7 o PP-IP 5) e bisdiphoshoinositoltetrakisphosphate (IP 8 o (PP) 2-IP 4). Pirofosfati inositolo sono stati isolati da tutti gli organismi eucarioti sinora studiati. Inoltre, le due classi distinte di enzimi responsabili della sintesi pirofosfato inositolo sono altamente conservati durante l&#39;evoluzione 2-4. L&#39;IP 6 chinasi (IP 6 Ks) possiede una flessibilità enorme catalizzatore, la conversione IP 5 e 6 al PP IP-IP IP 4 e 7 rispettivamente e successivamente, utilizzando questi prodotti come substrati, promuovere la generazione di molecole più complesse 5,6 . Recentemente, una seconda classe di enzimi pirofosfato generazione è stata individuata nella forma della proteina di lievito VIP 1 (noto anche come PP-IP 5 K), che è in grado di convertire IP da 6 a 7 e IP IP 8 7,8. Pirofosfati inositolo regolano molti processi cellulari disparati quali la secrezione di insulina 9, la lunghezza dei telomeri 10,11, chemiotassi 12, il traffico vescicolare 13, 14 e omeostasi fosfato di HIV-1 gag rilascio 15. Due meccanismi di azione sono stati proposti per questa classe di molecole. Essi possono influenzare le funzioni cellulari da allosterically interagendo con proteine ​​specifiche, come AKT 16. In alternativa, il gruppo pirofosfato può donare un fosfato di pre-fosforilata proteine ​​17. L&#39;enorme potenziale di questo campo di ricerca è ostacolata dalla mancanza di una fonte commerciale di pirofosfati inositolo, che impedisce molti scienziati di studiare queste molecole e questa nuova modificazioni post-traduzionali. I metodi attualmente disponibili per isolare pirofosfati inositolo richiedono sofisticate apparecchiature cromatografiche 18,19. Queste procedure utilizzano condizioni di acidità che potrebbe portare al degrado pirofosfato inositolo 20 e quindi al recupero poveri. Inoltre, l&#39;ingombrante post-colonna procedure di dissalazione limitarne l&#39;uso a laboratori specializzati. In questo studio descriviamo un metodo poco impegnativo per la generazione, l&#39;isolamento e la purificazione dei prodotti di IP 6-chinasi e PP-IP 5-chinasi reazioni. Questo metodo è stato possibile grazie alla capacità di elettroforesi su gel di poliacrilammide (PAG) per risolvere i polifosfati inositolo altamente fosforilata 20. A seguito di IP 6 K1 e PP-IP 5 K reazioni enzimatiche utilizzando IP 6 come il substrato, PAGINA è stato utilizzato per separare i pirofosfati generato inositolo che sono stati successivamente eluiti in acqua.

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

Preparation of Quality Inositol Pyrophosphates

Pirofosfati inositolo svolgono un ruolo importante nelle patologie umane come il cancro, il diabete e l&#39;obesità, ma l&#39;esatto meccanismo d&#39;azione è una questione di controversia. La mancanza di pirofosfati inositolo disponibili in commercio rende studi dettagliati problematico. Qui descriviamo un semplice protocollo per la produzione e isolare milligrammi di pirofosfati inositolo.

Preparazione di Qualità pirofosfati Inositolo

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.

DNA microarray: Esempio di controllo della qualità, ibridazione e scansione di array

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

Ricerca

Biologia

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.

Una tecnica non invasiva di campionamento dei capelli per ottenere DNA di alta qualità da Elusive piccoli mammiferi

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.

A lungo termine del test di tossicità letale con il crostaceo 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.

Cosa fare e cosa non fare di Cryo-microscopia elettronica: A Primer su preparazione del campione e raccolta dati di alta qualità per la ricostruzione macromolecolare 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.

Uno strumento web per la generazione di alta qualità Machine-readable vie biologiche

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.

Raccolta di sperma di qualità differenziale mediante centrifugazione in gradiente di densità

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.

Identificazione sul campo della camomillia Matricaria utilizzando un sistema qPCR portatile

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 di chemiotassi migliorato per la rapida identificazione dei chemioattrattivi rizobatterici negli essudati radicali

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 coltivazione, la crescita e la vitalità dei batteri lattici: una prospettiva di controllo della qualità

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

Ottimizzazione della qualità dei prodotti probiotici essiccati a spruzzo attraverso lo sviluppo del processo

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.

Autore in primo piano: Sviluppo di processi per l'essiccazione a spruzzo di batteri probiotici e valutazione della qualità del prodotto  

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

Sviluppo di processi per l'essiccazione a spruzzo di batteri probiotici e valutazione della qualità del prodotto

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