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

Vi tackar A. Riccio för hjälpsamma kommentarer och för att läsa manuskriptet. Detta arbete stöddes av Medical Research Council (MRC) finansiering till Cell Biology Unit och en Human Frontier Science Program Grant (RGP0048/2009-C).

1. Enzymatiska reaktionen - dag 1 (1 timme på eftermiddagen) <ol><li> Det första steget är att förbereda 10-20 oberoende enzymatiska reaktioner som IP 6 K1 eller VIP1 konvertera IP 6 till pyrophosphorylated isoformer. </li><li> Vi använder oss av Hans-IP 6 K1 och GST-Vip1 enzymer som renats från E. coli Enligt protokollet beskrivits tidigare 17,18. </li><li> Förbered 50 mikroliter reaktioner som innehåller 1X reaktion buffert (30 mm Hepes pH 6,8, 50 mM NaCl, 6 mm MgSO 4, 1 mm DTT), 6 mm phosphocreatine (PCR), 25 U / mL CreatinePhosphoKinase (CK), 5 mm ATP (Mg salt), 0,3 mM IP6, 0,05-0,1 mikrogram Hans-IP 6 K1 eller GST-Vip1. Justera volymen med MiliQ DDH 2 O. </li><li> Kortfattat snurra reaktion och inkubera vid 37 ° C över natten med rotation. </li></ol> 2. Polyakrylamidgelen gjutning och lastning - dag 2 (4 timmar på eftermiddagen) <ol><li> Den Polyakrylamidgelen är beredd att använda 24 cm lång, 18 cm bred glasplåtar och 1,5 mm breda distanser. Vanligtvis en 16 körfält eller en förberedande enda körfält kam används. </li><li> Förbered en blandning (50 ml / gel) som innehåller följande: 35,5% (w / v) Akrylamid: Bis-Akrylamid 19:01, 1X Tris / Borat / EDTA (TBE), 0,05% (w / v) ammonium persulfate (APS ), Temed 0,05% (w / v). Häll blandningen mellan den gjutna före glasplåtar, sätt in kammen och låt polymerisera i 30-60 minuter vid RT. </li><li> När gelen har polymeriserade, överföring apparaten till kylrummet och pre-run i 1X TBE i ca 30-60 minuter på 200-300 volt. </li><li> Lägg 1X av OrangeG färgämne (10 mM Tris-HCl pH7.0, 1 mM EDTA, 30% glycerol, 0,1% OrangeG) till varje reaktion. Bered ett prov innehållande 2 nmol av IP 6 att ladda som en vanlig kontroll. </li><li> Tvätta varje brunn noggrant med löpande buffert med hjälp av en spruta och en 21G nål för att ta bort eventuell fällning, sedan ladda gelen. Undvik belastning på sidan brunnar. </li><li> Kör gelen övernattning på 450-550 volt (7 MAMP / gel), tills OrangeG dye bandet under de senaste 10 cm från botten av gelen. </li></ol> 3. Isolering av IP 7 - dag 3 (4 timmar) och dag 4 (6-7 timmars SpeedVac torkningen) <ol><li> Demontera gelen apparater och dra försiktigt bort en glasskiva lämnar gel på den andra. Skär en liten del av gelen från strax ovanför OrangeG färgämnet bandet till botten med IP-6 standard och ett prov körfält, som visas i figur 1. </li><li> Färga den skurna delen av gelen med toluidinblått (0,1% (w / v) toluidinblått, 20% (w / v) metanol, 2% (w / v) glycerol) under några minuter (1-3 min) eller tills inositol pyrofosfat bandet visas. Sätt glasskivan tidigare borttagna tillbaka på toppen av gelen för att förhindra att ofärgade gelen från att torka. </li></ol> IP 7-bandet ska vara synlig eftersom det går något långsammare än IP-6-standarden. ATP, som går snabbare än IP 6, bör också vara synlig (Figur 1). Överför färgade delen av gel i en de-färglösningen (20% (w / v) metanol) i några minuter, tvätta bort eventuellt överskott av toluidinblått och placera gelen med ofärgade gel. Om visualisering av högre pyrophosphorylated inositol isoformer (IP 8 och IP 9) krävs, färga gelen med toluidinblått färglösningen i 20 minuter i rumstemperatur. Därefter tvätta bort toluidinblått med de-färglösningen i ca 15 minuter. <ol start="3"><li> Med ett rakblad skär IP 7 bandet på ofärgade delen av gelen använder som referens IP 7 flyttar positionen bestämmas med färgade gel (Figur 1). </li><li> Sätt IP 7 band som klipptes från gelen på ett 15 ml rör och tillsätt 10 ml MilliQ DDH 2 O. Lägg rören i rotation i 10 minuter vid rumstemperatur. Kasta vätskan för att avlägsna överskott av TBE och mikroskopiska akrylamid partiklar. </li><li> Därefter utför två uttorkning vätskeersättning cykler. Tillsätt 5 ml 50% (w / v) metanol till röret med gel som innehåller IP-7 och rotera i rumstemperatur i 2 timmar. Överför gelen slice till ett nytt 15 ml rör som innehåller 5 ml MilliQ DDH 2 O och roterar i rumstemperatur i 2 timmar. Kasta inte bort metanol och MilliQ H 2 O från rören. Upprepa uttorkning vätskeersättning cykeln igen genom att åter överföra polyakrylamidgel i 15 mL rör som tidigare använts. En av de tvättar kan utföras över en natt. </li><li> Att koncentrera elueras IP-7, torr 10 ml tillsammans (5 ml MilliQddH 2 O och 5 ml 50% (w / v) metanol) med en SpeedVac uppvärmd vid 60 ° C. </li><li> När proverna är nästan torr, överföra den återstående vätskan (300-600 l) för att A1.5 ml centrifugrör och centrifugera i 2 minuter vid 5000 rpm. </li><li> Samla supernatanten och överför till en ny 1,5 ml centrifugrör, låtner från 20 till 30 mikroliter eftersom det kan innehålla akrylamid partiklar. </li><li> Om det är nödvändigt att fortsätta torkningen med en ouppvärmd SpeedVac. Återhämtningen av IP 7 dramatiskt minskar om proverna torka helt, därför avsluta torkningen när proverna når volymen 100-300 mikroliter. </li></ol> 4. Fastställande av IP 7 koncentration och renhet. <ol><li> Använd 2-5 mikroliter av det återvunna IP 7 prov att köra på ett SIDA gel, i likhet med § § 2,2-2,5. Fyll på flera spädningar av IP-6 (dvs 0,5, 1, 2, 4 nmol) som koncentration standard och 4 nmol av poly-P markör. Efter att ha kört gelen, visualisera inositol pyrofosfat isoformerna genom färgning och de-färgning hela gelen med toluidinblått lösning, enligt det förfarande som beskrivs i avsnitt 3.2 (Figur 2A). </li><li> Efter Toluidine färgning kan bestämmas genom scanning gelen och jämföra skillnader i intensitet mellan IP 6 och IP-7, med hjälp av bildbehandlingsprogram som Bild-J, som visas i figur 2B. </li></ol> 5. Representativa resultat: Den förberedande enzymatisk omvandling av IP 6 till IP 7 med hjälp av IP 6 K1 och VIP1 enzymer kan enkelt lösas med hjälp av PAGE analys (Figur 1). Lastning av IP 6 som en storlek kontroll tillsammans med toluidinblått gel färgning möjliggör identifiering av pyrophosphorylated derivat, eftersom de kör långsammare beroende på antalet fosfatgrupper finns på inositol ringen. Det förfarande som beskrivs ovan möjliggör enkel rening av IP 7. Analysen av det renade inositol pyrofosfat för sida visade renheten i våra IP-7 (Figur 2A). Intressant nog vandrar 1/3PP-IP 5 isomeren av IP 7 produkt av VIP1 något långsammare än 5PP-IP 5 isomer av IP 7 som genereras av IP 6 K1. Användning av IP 6 standarder möjliggöra en enkel kvantifiering av koncentrationen av det renade IP 7 (Figur 2B). Innan du använder IP-7 för ytterligare experiment, kan dess biologiska aktivitet ska bedömas (Figur 3). 5PP-IP 5 inkuberas med VIP1 och med IP-7 fosfatas DDP1 (diphosphoinositol polyfosfat phosphohydrolase). Rutinmässigt, är det renade IP 7 omvandlas till IP 8 av VIP1 och IP 6 med DDP1 (Figur 3). <img alt="Figur 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Figur 1:. Toluidine färgning av SIDA och isolering av IP 7 bandet Den del av gel som innehåller standarden (IP 6) skars och färgas med en toluidinblått lösning. De tre banden representerar (uppifrån och ner) IP 7, IP 6 och ATP. Den färgade delen av gelen var då i linje med resten av gelen. Detta gör att lokaliseringen av den del av gel som innehåller IP-7, som sedan kan skäras och renas (streckad ruta). <img alt="Figur 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Figur 2: PAGE analys av IP 6 K1 och VIP1 reaktionsprodukter. A) Analys av IP 6 (4, 2, 1, 0,5 nmol) genom toluidinblått färgning användes för att fastställa IP 7 koncentrationen renas från både IP 6 K1 (5PP-IP 5) och VIP1 (1/3PP-IP 5) reaktioner. B) Scatter plot analys för att fastställa koncentrationen av det renade IP 7. Koncentrationerna bestämdes av de band intensitet, beräknas med ImageJ mjukvara, jämfört med förutbestämda mängder av IP 6. X-axeln representerar intensitet, Y-axeln visar koncentrationen uttryckt i nmol. <img alt="Figur 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Figur 3:. Analys av IP 7 biologisk aktivitet för att bestämma kvaliteten på det renade IP 7 vi inkuberas 5PP-IP 5 (IP 6 K1 genererade IP-7) med VIP1 eller med IP-7 fosfatas DDP1 och sedan beslöt reaktionen på sidan. Den DAPI och Toluidine färgning visade förväntade produktionen av IP 8 av VIP1 och omvandlingen av IP 7 till IP 6 av DDP1.

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Inga intressekonflikter deklareras.

Användningen av inositol pyrofosfat i biokemi är starkt begränsad av den kommersiella avsaknad av sådana föreningar och de fattiga känslighet befintliga detektionsmetoder. Kombinationen av SIDA, som möjliggör separation av molekyler som har olika antal fosfatgrupper och toluidinblått (figur 1), en metakromatisk färgämne som binder till fosfatgrupper, möjliggör enkel detektion av inositol pyrophoshate isoformer öppna nya vägar för forskning 20. Den beskrivna användningen av SIDA-teknik för att rena inositol pyrofosfat produkter av den enzymatiska reaktionen sker outby antingen IP 6 K1 eller VIP1 är en enkel, ekonomisk och tillförlitlig metod som möjliggör för produktion av stora mängder av hög kvalitet IP 7. Metoden som beskrivs ovan är inte begränsat till den enkla rening av IP 7 men mindre ändringar av de beskrivna protokollet kan tillåta rening av olika utbud av inositol pyrophosphates. Högre fosforyleras inositol pyrofosfat isoformer, som innehåller mer än åtta fosfatgrupper kan detekteras med hjälp av IP 7 eller olika mängder av IP 6 som substrat 20,6. Dessa inositol pyrophosphates kan upptäckas genom att öka längden på färgningsproceduren och därefter renas (avsnitt 3.2). Dessutom skulle användningen av IP 5 som substrat för den enzymatiska reaktionen möjliggör rening av PP-IP-5 och andra inositol pyrophosphates som innehåller en hydroxylgrupp på inositol ringen. Sammanfattningsvis ger detta anspråkslösa metoden för tillförlitlig rening av milligram mängder inositol pyrophosphates med allmänt tillgängliga instrument, vilket öppnar nya vägar för detta spännande forskningsområde.

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th></tr><tr><td> Fytinsyra (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poly-P (natriumhexametafosfat) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + salt </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Phosphocreatine (PCR) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho kinas (CK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> Hans-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> Hans-Ddp1 </td><td> Finns i lab </td><td> Finns i lab </td></tr><tr><td> Akrylamid: Bis-Akrylamid 19:01 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Ammonium Persulfate (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / Borat / 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> Toluidinblått </td><td> Sigma-Aldrich </td><td> 198.161 </td></tr><tr><td> SpeedVac </td><td> Kristus </td><td> 100.218 </td></tr><tr><td> Gel apparater </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Vakuum grenrör </td><td> Kristus </td><td> Alpha 2-4 </td></tr><tr><td> Vakuumpump </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol är närvarande i naturen antingen oförändrade eller i mer fosforylerade komplexa derivat. Av de senaste, de två mest förekommande i eukaryota celler inositol pentakisphosphate (IP 5) och inositol hexakisphosphate (fytinsyra eller IP 6). IP-5 och IP 6 är föregångare till inositol pyrofosfat molekyler som innehåller en eller flera pyrofosfat obligationer 1. Fosforylering av IP 6 genererar diphoshoinositolpentakisphosphate (IP-7 eller PP-IP 5) och bisdiphoshoinositoltetrakisphosphate (IP-8 eller (PP) 2-IP 4). Inositol pyrophosphates har isolerats från alla eukaryota organismer hittills studerat. Dessutom är de två olika klasser av enzymer som ansvarar för inositol pyrofosfat syntes mycket bevarad genom evolutionen 2-4. IP-6-kinaser (IP 6 KS) besitter en enorm katalytisk flexibilitet, konvertera IP 5 och IP 6 till PP-IP 4 och IP respektive 7 och därefter, genom att använda dessa produkter som substrat, främja uppkomsten av mer komplexa molekyler 5,6 . Nyligen var en andra klass av pyrofosfat generera enzymer identifierats i form av jästen protein VIP-1 (även kallat PP-IP 5 K), som kan konvertera IP 6 till IP 7 och IP 8 7,8. Inositol pyrophosphates reglera många olika cellulära processer såsom insulinutsöndring 9, telomerlängd 10,11, kemotaxi 12, vesikulär människohandel 13, fosfat homeostas 14 och HIV-1 gag släpper 15. Två mekanismer åtgärder har föreslagits för denna klass av molekyler. De kan påverka cellulära funktion genom allosterically interagera med specifika proteiner som AKT 16. Alternativt kan pyrofosfat gruppen donera en fosfat i förväg fosforylerade proteiner 17. Den enorma potential detta forskningsområde är hämmas av avsaknaden av en kommersiell källa inositol pyrophosphates, som hindrar många forskare från att studera dessa molekyler och denna nya post-translationell modifiering. De metoder som för närvarande finns att isolera inositol pyrophosphates kräver avancerade kromatografiska apparater 18,19. Dessa förfaranden använder sura förhållanden som kan leda till inositol pyrofosfat nedbrytning 20 och därmed dålig återhämtning. Dessutom besvärliga efter kolumnen avsaltning förfaranden begränsa deras användning till specialiserade laboratorier. I denna studie beskriver vi en krävande metod för generering, isolering och rening av produkter för IP 6-kinas och PP-IP 5-kinaser reaktioner. Denna metod var möjligt genom förmåga polyakrylamidgelelektrofores (SIDA) för att lösa mycket fosforyleras inositol polyfosfater 20. Efter IP 6 K1 och PP-IP 5 K enzymatiska reaktioner med hjälp av IP 6 som substrat, var PAGE används för att separera genererade inositol pyrophosphates som därefter elueras i vatten.

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

Preparation of Quality Inositol Pyrophosphates

Inositol pyrophosphates spelar en viktig roll i mänskliga sjukdomar såsom cancer, diabetes och fetma, men det är den exakta verkningsmekanismen en tvistefråga. Bristen på kommersiellt tillgängliga inositol pyrophosphates gör detaljerade studier problematiska. Här beskriver vi ett enkelt protokoll för att producera och isolera milligram inositol pyrophosphates.

Beredning av kvalitet 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 ...

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 Microarrays: Exempel Kvalitetskontroll, Array hybridisering och skanning

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.

En icke-invasiv Hair provtagningsteknik för att med hög kvalitet DNA från Elusive små däggdjur

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.

Långsiktig Dödligt toxicitetstest med Crustacean 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.

Göra och inte göra av Cryo-elektronmikroskopi: en primer på Provberedning och High Quality Data Collection för Macro 3D återuppbyggnad

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.

En webbverktyg för att generera hög kvalitet maskinläsbara biologiska Pathways

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.

Spermier samling av differentiell kvalitet med hjälp av täthet lutning centrifugering

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.

Fält Identifiering av Matricaria chamomilla med hjälp av en Bärbar qPCR System

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.

En förbättrad Chemotaxis-analys för snabb identifiering av Rhizobacterial Chemoattractants i Root Exudates

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.

Odling, tillväxt och livskraft hos mjölksyrabakterier: Ett kvalitetskontrollperspektiv

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

Optimera kvaliteten på spraytorkade probiotiska produkter genom processutveckling

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.

Författare i fokus: Processutveckling för spraytorkning av probiotiska bakterier och utvärdering av produktkvalitet  

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

Processutveckling för spraytorkning av probiotiska bakterier och utvärdering av produktkvalitet

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