Name: measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers
Uploaded: 2014-08-22T14:00:00.000+00:00
Duration: 13 min 14 s
Description: Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1. Bitki Kültürü ve Hazırlık <ol><li> (Ayrıntılar için, 10&#39;a bakınız) iklim-kontrollü bir büyüme odasında 7 gün boyunca arpa fide hidroponokal büyütün. NOT: Bu besin gereksinimleri yaş değişecek gibi, gelişim aşamaları çeşitli tesislerini inceleyerek dikkate almak önemlidir. </li><li> Bir gün deney öncesinde, tek bir çoğaltılmasına (DI için paket başına 3 bitkiler, Cate için paket başına 6 bitkiler) yapmak için birlikte birkaç fidan paket. Sürgünlerin taban kısmı etrafında Tygon hortumunun bir 2 cm&#39;lik bir parça sararak ve bir &quot;yaka&quot; oluşturmak için bant ile boru sabitlenmesiyle Paketi fideleri. NOT: Deste başına bitki sayısı deneysel koşullar 10,13,14 göre değişebilir. Bohçalama kök kütlesi ve / veya spesifik aktivite düşük, özellikle istatistik ve ölçüm doğruluğunu iyileştirmek için yapılır. </li></ol> Deneysel Çözümleri / Malzemelerin 2. Hazırlık içerik &quot;&gt; Not: Aşağıdaki tipik deney öncesinde 1 gün yapılır. <ol><li> Ve bitki materyali ve spesifik aktivite [S o için numune şişelere ((bitki örneklerinin spin-kurutma için), santrifüj tüpleri (11 ayrıntılar için bkz) Pre-etiketleme, etiketleme ve desorpsiyon çözümleri;: DI için aşağıdaki toplamak aşağıya bakınız]). Havalandırır ve tüm çözümleri karıştırın. </li><li> , (10 ayrıntılar için bkz) elüsyonların, bitki örnekleri için akış huniler, (bitki örneklerinin spin-kurutma için) santrifüj tüpleri ve numune şişeleri (Eh-karışık, gazlı etiketleme ve elüsyon çözümleri ve: Cate için aşağıdaki toplamak S o ve seyreltme faktörü belirlenmesi [D f; aşağıya bakınız]). </li></ol> 3. radyotracer hazırlayın DİKKAT: Aşağıdaki güvenlik adımlar radyoaktivite ile çalışma öncesinde alınmalıdır. <ol><li> Emin olun Radioac gereksinimleritif malzemeler lisans anlaşılır ve takip edilmektedir. Uygun güvenlik donanımları (yani, gözlük, eldiven, laboratuvar önlüğü, kurşun yelek / yaka) ve Dozimetreleri (örneğin, TLD halka ve rozet) giyin. Koruyucu Set up (yani, pleksiglas ve kurşun tuğla) ve bunun arkasında radyoaktif çalışmayı gerçekleştirmek. Geiger-Müller sayacı rutin kontaminasyon izlemek için mevcut olduğundan emin olun. </li><li> 42 K + hazırlanması <ol><li> Dengesi üzerinde temiz, kuru bir behere koyun. Dengesini sıfır. </li><li> Ambalajından (toz halinde 42 K 2 CO 3 20 mCi) izleyicinin şişesini çıkarın ve beher içine izleyici dökün. Kitle not alın. </li><li> Beher içine H 2 SO4, 0.07 ml, ardından dH 2 O Pipet 19.93 mi,. Bu aşağıdaki kimyasal reaksiyonu izlerler: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H 2 O (l) 42 → </sK 2&gt; kadar SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> K 2 CO 3 kütlesini ve moleküler ağırlık verilen radyoaktif stok çözeltisinin konsantrasyonu, ve hacim (20 mi) hesaplayın. NOT: 13 NH 3/13 NH4 + İzleyici suyun oksijen atomunun proton bombardımanı yoluyla bir siklotron üretilir çalışma halinde (tipik olarak 100-200 mCi faaliyet ile sonuçlanan, üretim detayları için, 12 bakınız). 14. NH 3/14 NH4 + &#39;nın miktarı, bu çözeltiler içinde son derece düşük olup, stok çözeltisi N içeriği ihmal edilebilir. </li></ol></li></ol> 4. Doğrudan Akın (DI) Ölçüm <ol><li> 42 K + için, etiketleme çözeltisi içine K + arzu edilen nihai konsantrasyonuna ulaşmak için gereken radyoaktif stok çözeltisi miktarı pipetle. <ol><li> NH 3/13 13 için NH 4 +, pipet etiketleme çözümü içine küçük bir miktar (&lt;0.5 ml). Etiketleme çözümü (havalandırması yoluyla) iyice karıştırmak için izin verin. </li></ol></li><li> Bir numune şişesi içine etiketleme çözeltisi, 1 ml alt-örnek pipet ve (toplam 4 örnekleri) ile üç kez tekrarlayın. <ol><li> Bir gamma sayacı kullanılarak, (&quot;dakika başına sayımları&quot;, cpm olarak) şişelere radyoaktivite ölçülür. (Bu gibi kısa ömürlü izleyiciler için özellikle önemlidir) sayaç cpm okumaları izotop çürüme için düzeltilmiştir şekilde programlanmış olduğundan emin olun. </li><li> Dört numune sayımları (CPM mi-1) ortalama ve (umol mi -1) çözeltisi içinde alt-tabaka konsantrasyonu ile bölmek suretiyle (CPM umol -1 olarak ifade edilen) sO hesaplayın. </li></ol></li><li> (Bakınız test koşulları altında bitkiler önceden dengeye gelmesi, 5 dakika boyunca ön-etiketlenmesi (radyoaktif olmayan) çözelti içinde kökleri daldırınÖrneğin, ön etiket zamanlı olarak varyasyonlar için 10,13,14). </li><li> 5 dakika için etiketleme (radyoaktif) çözümü kökleri daldırın. NOT: Etiketleme kez deney 3,4,7-10 göre değişiklik gösterebilir. </li><li> Yüzey-yapışan radyoaktivite uzaklaştırılması için, 5 saniye boyunca yayılım çözeltisine kökleri aktarın. Hücre dışı izleyici daha açık köklerine 5 dakika boyunca desorpsiyon çözeltisinden oluşan ikinci bir kap içine aktarın kökleri. </li><li> Ayrı sürgünler, bazal sürgünler ve kökler incelemek ve. </li><li> Düşük hızda 30 saniye boyunca santrifüj tüplerine kökleri ve spin örnekleri yerleştirin, klinik dereceli santrifüj (~ 5.000 xg) yüzeyini ve interstisyel su çıkarmak için. </li><li> Kökleri (taze ağırlık, FW) tartılır. </li><li> Bitki örneklerinde radyoaktivite sayımı (ateş, bazal sürgün ve kök; adım 4.2.1). </li><li> Akı hesaplayın. Aşağıdaki formül kullanılarak bitki içerisine akışını hesaplamak Φ = Q * / S o ağırlık L burada Φ akısı(Ľmol g -1 saat -1), Q * dokusunda biriken zerresinin miktarı (genellikle kök, sürgün, kopyaya, ve, kombine bazal sürgün), S o etiketleme çözümü (CPM ľmol spesifik aktivite - 1) w kök taze ağırlığı (g) &#39;dir ve t L etiketleme süresi (saat)&#39; dir. NOT: Daha sofistike hesaplama Cate elde parametrelere dayalı, etiketleme ve desorpsiyon sırasında köklerinden eşzamanlı izleyici akışı için hesap yapılabilir (, ayrıntılar için, 4 görmek aşağıya bakın). </li></ol> Tracer efflux (CATE) Ölçümü ile 5. komparmantal Analizi <ol><li> Etiketleme çözümü ve ölçü S o (- Yukarıdaki, 4.2 adımları 4.1) hazırlayın. </li><li> Seyreltme faktörü (D f) ölçün. Not: Genellikle, gama sayacında detektöre örnek nisbetle pozisyonu miktarını etkileyebilirradyasyon ölçülür. Ayrıntılar için tartışma bakın. <ol><li> SO ölçüldükten sonra, her bir örnek için H 2 O 19 ml ilave (örneğin, bu son hacim = yıkama sıvısı hacmi = 20 ml) eklenmiştir. Her 20 ml numune radyoaktiviteyi sayın (adım 4.2.1). </li><li> 20 ml&#39;lik numunelerin ortalama cpm olarak 1 mL&#39;lik numunelerin ortalama cpm bölünmesiyle D F hesaplayın. </li></ol></li><li> 1 saat etiketleme çözümü kökleri daldırın. </li><li> Tüm kök malzeme huni dahilinde sağlanması, huni akıttığı etiketleme çözümü ve transfer bitkilerden bitkileri çıkarın. Plastik yaka üzerine bant küçük bir şerit uygulayarak akış hunisi tarafına yavaşça güvenli bitkiler. </li><li> Yavaşça huni içine ilk eluatın dökün. Sayacını başlatmak (yukarı sayma). </li><li> Musluğunu açın ve 15 sn sonra numune şişesine eluatın toplamak (not: yıkama süresi değişir; aşağıya bakınız). Musluğunu kapatın. Yavaşça hunisi içine bir sonraki eluatın dökün. </li><li> Repeaİlk elde edilen nihai eluatına, aşağıdaki yıkama seri, geri kalan kısmı için bir adım 5.6 T: 15 saniye ile (dört kez), 20 saniye (üç kez), 30 saniye için (iki defa), 40 saniye (bir defa), 50 saniye (bir defa), 29.5 dakikalık bir elüsyon süresi toplam 1 dakika (25 kez), NOT: Sızdırma serisi deneysel koşullar 7-10,13,14 göre değişiklik gösterebilir. </li><li> Yıkama protokolü tamamlandıktan sonra, bitkiler hasat (- yukarıda 4.8, 4.6 adım). </li><li> (5.2, M f her elüatın için okuma çarpılması) gama sayacında yıkama sıvılarından ve bitki örneklerinde radyoaktivite sayısı. </li><li> Yıkama süresinin bir fonksiyonu olarak Arsa izleme atışının (cpm gr (kök FW) -1 dk -1). Kararlı durum koşulları için, doğrusal gerileme ve akılarının hesaplamaları, döviz yarılanma ömürleri ve havuz boyutları (detaylar için, 6-9 bakınız) gerçekleştirin. </li></ol>

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The authors have nothing to disclose.

Yukarıdaki örneklerde gösterildiği gibi, yöntem, in planta Radyoaktif besin ve toksik maddelerin tek yönlü akıyı ölçen bir güçlü bir araçtır. 1 NH3 akışı, belki de bir 225 umol g-1 st -1, aşan ulaşabileceği göstermektedir En iyi niyetli transmembran akı Hiç bir bitki sisteminde 13 bildirilmiştir, ancak net tozları ölçüldü, bu akının büyüklüğü görünür olmaz. NH3 büyük bir a...

Besin ve Toksik maddelerin alımı ve dağıtımı kuvvetle bitki büyümesini etkilemektedir. Buna göre, yatan taşıma süreçlerinin incelenmesi, özellikle beslenme optimizasyonu ve çevresel streslere bağlamlarda, bitki biyolojisi araştırma ve tarımsal bilimler 1,2 önemli bir alanı teşkil (örneğin, tuz stresi, amonyum toksisitesi). Bitkilerde akılarının ölçülmesi için yöntemler arasında baş önemli 1950&#39;lerde geliştirilmiş radyo-izotopik izleyiciler, kullanımı ve günümüzde yaygın olarak kullanılan olmaya devam etmektedir (3, örneğin, bakınız). Bu Mife (akı tahmini iyon mikroelektrot) ve SIET (tarama iyon seçici elektrot tekniği) ve kullanımı gibi dokularda kök orta ve / veya birikim, iyon seçici titreşimli mikroelektrot kullandığı için besin tüketme ölçümü gibi diğer yöntemler, İyon seçici floresanlı boyalar, ayrıca yaygın olarak uygulanır, ancak ağ grip tespit etmek için yetenekleri açısından sınırlıdırxes (örneğin, akışı ve dışarıya akış arasındaki fark). Radyoizotopların kullanılması, diğer taraftan, araştırmacı kinetik parametreleri çözmek için kullanılabilecek tek yönlü akıları izole etmek ve ölçmek için eşsiz bir yeteneği sağlar (örneğin, K, M ve V maks) ve kapasitesi hakkında bilgi sağlar, enerjisi, ulaşım sistemlerinin mekanizmaları ve düzenlenmesi,. Radyo-ile yapılan tek yönlü akış ölçümleri ters yönde akış kadar koşullar altında özellikle yararlı olan, hücre içi ve havuzlarının devir hızla 4&#39;tür. Takip izotop, aynı elemanın diğer bir izotopun bir arka planı görülmektedir çünkü Ayrıca, Radyoaktif yöntemleri, (aşağıda, &quot;Tartışma bakınız) ölçümlerinin bir çok diğer tekniklerin aksine, oldukça yüksek bir substrat konsantrasyonlarında iletilmesine olanak tanımaktadırlar. Burada, tek yönlü ve n radyoizotopik ölçümü için ayrıntılı adım sağlayansağlam Bitkilerde ve mineral besin tozları ve toksik maddeler. Vurgu akı potasyum (K +) ölçülmesi, bir bitki makro besinin 5 ve amonyak / amonyum yapılacaktır (NH3 / NH4 +), ancak, örneğin, yüksek konsantrasyonlarda (en mevcut olduğu zaman toksik olan başka bir makro besin, 1- 10 mM) 2. Bu radyoizotoplar 42 K + (t 1/2 = 12,36 saat) ve 13 NH 3/13 NH4 + (t 1/2 = 9,98 dakika), sırası ile, bir model sistem arpa sağlam fidelerinde (Hordeum vulgare L. kullanacak .), iki temel protokollerin açıklaması: tracer efflux (CATE) tarafından doğrudan akını (DI) ve kompartman analizi. Biz bu makalede sadece her protokol gerçekleştirmek için gerekli adımları açıklar başından itibaren dikkat etmelisiniz. Her tekniğin uygun, hesaplamalar ve teorinin kısa açıklamalar sağlanan, ancak ayrıntılı edilir Fuarlarımız&#39;S arka plan ve teori konuyla 4,6-9 birkaç önemli makalelerinde bulunabilir. Daha da önemlisi, bu protokoller diğer besin maddeleri / toksik maddelerin analizi akı genel olarak aktarılabileceği (örneğin, 24, Na +, 22, Na +, Rb + &#39;86, 13 NO 3 -) ve diğer bitki türlerine, birkaç uyarılar da olsa (aşağıya bakınız) . Biz de radyoaktif maddeler ile çalışan tüm araştırmacıların kurumun iyonizan radyasyon güvenliği regülatörü ile düzenlenmiş bir lisans altında çalışması gerekir önemini vurguluyoruz.

<table><tbody><tr><td>Gamma counter</td><td>Perkin Elmer</td><td></td><td>Model: Wallac 1480 Wizard 3&quot;</td></tr><tr><td>Geiger-M&amp;uuml;ller counter</td><td>Ludlum Measurements Inc.</td><td></td><td>Model 3 survey meter</td></tr><tr><td>400 ml glass beakers</td><td>VWR</td><td>89000-206</td><td>For pre-absorption, absorption, and desorption solutions</td></tr><tr><td>Glass funnel</td><td>VWR</td><td>89000-466</td><td>For efflux funnel</td></tr><tr><td>Large tubing</td><td>VWR</td><td>529297</td><td>For efflux funnel</td></tr><tr><td>Medium tubing</td><td>VWR</td><td>684783</td><td>For bundling</td></tr><tr><td>Small tubing</td><td>VWR</td><td>63013-541</td><td>For aeration</td></tr><tr><td>Aeration manifold</td><td>Penn Plax Air Tech</td><td>vat 5.5</td><td>To control/distribute pressurized air into solutions</td></tr><tr><td>Glass scintillation vials</td><td>VWR</td><td>66022-128</td><td>For gamma counting</td></tr><tr><td>Glass centrifuge tubes</td><td>VWR</td><td>47729-576</td><td>For spin-drying root samples</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>470173-504</td><td>For spin-drying root samples</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>470001-828</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>470005-496</td><td></td></tr><tr><td>Low-speed clinical centrifuge</td><td>International Equipment Co.</td><td>76466M-4</td><td>For spin-drying root samples</td></tr><tr><td>1 ml pipette</td><td>Gilson</td><td>F144493</td><td></td></tr><tr><td>10 ml pipette</td><td>Gilson</td><td>F144494</td><td></td></tr><tr><td>1 ml pipette tips</td><td>VWR</td><td>89079-470</td><td></td></tr><tr><td>10 ml pipette tips</td><td>VWR</td><td>89087-532</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler toledo</td><td>PB403-S/FACT</td><td></td></tr></tbody></table>

measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

Şekil 1 yüksek yetiştirilen sağlam arpa fidelerinin köklerine NH 3 akını (13 N) DI tekniği kullanılarak bulundu izotermlerini gösterir (10 mM) NH 4 + ve ya (0.02 mM), düşük veya yüksek (5 mM ) K +. (, Çözelti pH 13 değişim ile ayarlanır [NH3] dahili) NH3 tozlar dış NH3 konsantrasyonunun bir fonksiyonu olarak grafiğe zaman izotermleri Michaelis-Menten kinetiğine gösterir. NH 3 tozlar...

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Radyoaktif izleyiciler ile Bitkilerde Mineral Besin ve Toksikantların Değişimler Ölçme

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Research

JoVE Journal

Environment

11.3K Görüntüleme.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Video: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Sürekli Tasarımı ve İşletilmesi<sup&gt; 13</sup&gt; C ve<sup&gt; 15</supÜniformalı veya Diferansiyel için&gt; N Etiketleme Odası, Metabolik ve Yapısal, Bitki İzotop Etiketleme

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Çevre

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

Ölçme Karbon bazlı Kirletici Mineralleştirme Kombine CO kullanma<sub&gt; 2</sub&gt; Akı ve Radyokarbon Analizleri

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Kemirgen tümör modelleri ilerlemesinde tümör izlemek için Imaging radyonüklid Floresans muhabir gen

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Kanser Araştırmaları

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Floresan Etiketli Bakteriler Sucul Ekosistemlerde Organik Karbon Akışının Yeni Yollarını Ortaya Çıkaracak Bir İzleyici Olarak

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

15Azot Analizi için Mikroplot Tasarımı ve Bitki ve Toprak Numune Hazırlama

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

Topraktaki Labile, Inorganik Bitki Besinleri ve Kirleticilerin İki Boyutlu Görselleştirilmesi ve Nicelleştirilmesi

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

Otomatik 90Sr ayrılık ve Preconcentration bir laboratuvar Vana sistemde Ppq düzeyinde

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

Kimya

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Birden çok besin kaynaklarının mikrobiyal metabolizma araştırmaya izotopik izleyici deneyler kombinasyonuna göre iş akışı

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Biyomühendislik

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Topraksız: A yönlü Sistemi Toksik Elements Besin Uygunluk ve Pozlama Besin Tahsis ve Bitki Cevapları Eğitim için

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Biyoloji

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Oak Ridge Ulusal Laboratuvarı'nın Yüksek Akı İzotop Reaktöründe Nötron Radyografisi ve Biyolojik Sistemlerin Bilgisayarlı Tomografisi

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

Radyoaktif izleyiciler ile Bitkilerde Mineral Besin ve Toksikantların Değişimler Ölçme

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Sürekli Tasarımı ve İşletilmesi

Ölçme Karbon bazlı Kirletici Mineralleştirme Kombine CO kullanma