Bu araştırma kısmen desteklenen Hibeler-Aid Eğitim, Kültür, Spor, Bilim Bakanlığı keşif araştırma (JK), ve Bilimsel Araştırma (A) (JK ve SM) zorlu Bilimsel Araştırma ve Teknoloji, Japonya . Bir RIKEN FPR Bursu (RCE) ek destek sağlanmaktadır. Yazarlar Dr kendi şükranlarımı sunuyorum. Eisuke Chikayama, NMR ve istatistiksel analizler ile teknik yardım için Yasuyo Sekiyama ve Mami Okamoto.

1. Ekstrakte kaldırma Filtre Hazırlık <ol><li> 25-mm çapında 0.22 mikron gözenek boyutu Durapore PVDF hidrofilik filtreler (Millipore) kullanın. Temiz bir 500 ml Pyrex kabı koyun filtreler cımbız kullanarak. Damıtılmış su ile üç kez ön-yıkama. Eğer birbirine yapışmasını önlemek için durulama filtreleri gibi girdap. 300 ml Milli-Q (Millipore) ya da eşdeğeri yüksek kaliteli su ilave edilir. Filtreler ekstrakte tümüyle kaldırılmasını kolaylaştırmak için otoklavlayın. </li><li> Milli-Q off dökün ve tekrar üçlü Milli-Q ile, bu kez filtreleri durulayın. Temiz ve kuru bir yüzey (alüminyum folyo gibi) ve makul bir sıcaklıkta (örneğin 37 ° C) veya kuru hava ya da kuru zeminde cımbız yerde bireysel filtrelerini kullanma. Filtreleri artık kullanıma hazır. </li></ol> 2. Örnek Malzeme Filtrasyon <ol><li> Cam destekler ile 25 mm mikroanaliz filtre sütunları ile bu protokol, bir Millipore paslanmaz çelik 3 yerinde filtre manifoldu göstermek içinve mekanik bir pompa kullanılır. Aseptik teknik kullanarak, filtre sütun kaidesi üzerinde tek bir 25 mm filtre koyun, sütun uygulamak ve birbirine kenetlenmiştir. </li><li> , Sütun örneği 15 ml yükleyin filtre manifolduna stop-valf açılır ve pompa açın. Hücre kırılması (&lt;5 kPa) en aza indirmek için hafif bir basınç altında filtre. Bu eli ya da bir peristaltik pompa gibi diğer pompaları, bu protokolü için uyarlanmış olabilmektedir. Düşük yoğunluklu örnekleri için, su arda eklemeler gerekli olabilir, filtrenin su eklemeler arasında uzun bir süre kuru gitmesine izin verme. </li><li> Eğer numune filtre ettikten sonra deniz örnekleri için, isteğe bağlı bir tatlı su ürünleri örnek ve filtre üzerinde kalan paramanyetik iyonların azaltmak için durulama gerçekleştirebilirsiniz. Bu spektrometre mıknatısın daha hassas ayar ile yardımcı olabilir. Sadece hafifçe suyu küçük bir hacim kazandırmak ve sonunda toplanan örnek süzülür. </li><li> Sonra filtreleme bittiğinde pompa kapatın ve vana ope terkn böylece altında filtre negatif basınç hala. Kelepçe ve filtre sütun çıkarın. </li><li> Tek bir el ile, filtrenin beklemeye almak için temiz cımbız kullanın. Kendisi üzerinden filtre katlayın, ama kırışık yok. Diğer elinizle filtre aşağı tutmak için steril 2 ml mikrosantrifüj tüpü dudak kullanın. Filtre her iki kenarları yeniden kavramak için bunları kullanmak ve sonra cımbız bırakın. Kavuşturmak için bir 45 ° açıyla Regrip. </li><li> Içeriye bakan örnek tarafında açılır böylece 2 ml tüp ve içine serbest filtre yerleştirin. Sen steril 2 ml mikrosantrifüj tüpü bu şekilde içine iki filtreye kadar yerleştirebilirsiniz. İki filtreleri kullanarak, bunlar mümkün olduğunca az örtüşme sağlamak. Hemen (en az -30 ° C) dondurma. </li></ol> 3. Sulu-çözünür Metabolitlerinin Ekstraksiyon <ol><li> Senin örnekleri gece boyunca veya en az 10 saat Lyophilize. </li><li> Liyofilizasyon sonra, her bir tüp (Tokken) için bir paslanmaz çelik kırıcı ekleyebilir. 750 ul standart potasyum ekle2,2-Dimetil-2-silapentane-5-sülfonat (DSS) standardı (KPI ile döteryum oksit (2 H&gt;% 90) fosfat NMR tampon; 38.3 mM KH 2 PO 4, 61.7 mM K 2 HPO 4, DSS 0.1 mM, pH 7.0,% 90 D 2 O) 2. </li><li> Bir su içinde sonikatör ° C (Bioruptor, Diagenode) filtreden hücre malzemelerin ayrılması için 4, 5 dakika boyunca örnekleri sonikasyon. Temiz bir cımbız ile filtreleri çıkarın. </li><li> 5 dakika için bir değirmen kırıcı (1600 rpm) ile hücreleri bozabilir. </li><li> 15 dakika boyunca bir tezgah üst çalkalayıcı (Eppendorf) üzerine (1400 rpm) çalkalanarak 65 ° C&#39;de inkübe edin. </li><li> Temiz cımbızla metal domuz kaldırmak ve 5 dakika boyunca 13.000 g&#39;de Örnek santrifüjlenir. </li><li> NMR spektroskopisi bir NMR tüpü doğrudan süpernatant kapalı çizin. </li></ol> 4. NMR Spektroskopisi ve Veri Analizi <ol><li> (Burada, Bruker DRX-500 spektrometre ile donatılmış bir NMR spektrometresi içine örnek yükleyinXWIN-NMR çalıştıran bir bilgisayar) tarafından kontrol edilen üç eksenli gradient TXI prob. </li><li> XWIN-NMR arayüzden uygun yöntemler, daha önce yayınlanmış 2,15 kullanılarak 1D 1 H-NMR spektrası elde. Bu çalışmada 1 H NMR spektrumları 1.2 s tekrarlama zamanı ile, Watergate darbe dizisi bastırılmıştır 298 K. Artan su sinyallere 500,03 MHz&#39;de bir DRX-500 spektrometresi işletim kaydedildi. 128 geçici spektrum başına 32.000 veri noktası elde etmek için toplandı. </li><li> NMRPipe yazılımı 16 yüklü bir PC&#39;ye NMR verileri dizinleri aktarın. 0 ppm referans sonra el spektrumları faz olarak ham veri ve set DSS işleyin. Sayısallaştır entegre veya &#39;binning&#39; onlar gibi rNMR, Automics üzere yazılım veya ECOMICS web sitesi (gelen halka açık FT2B paketini kullanarak ayrık değerler kümesi içine spektral verileri <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> ) 3,17. Th, spektrumları 0.032 ppm ayrılmaz ECOMICS kullanarak bölgeler ve DSS veya toplam sinyal yoğunluğu ya normalize üzerinden 0,5 ile 10,5 ppm arasında örneğin entegre edildi edilir. Çıkış verileri şimdi bu tür R 18 gibi ücretsiz yazılım paketleri kullanılarak temel bileşenler analizi (PCA) olarak aşağı istatistiksel analiz için kullanılabilir. </li></ol> 5.. Temsilcisi Sonuçlar 1 H NMR spektrumları bir örneği, yukarıdaki yöntemlerin Şekil 1 &#39;de gösterilmiştir kullanılarak elde edilmiştir. Bu örnekler, bir evren deney iki kez puan alg metabolik faaliyetleri nedeniyle belirgin farklılıklar göstermektedir. 4 gün spektrumu gün 1 örnek karşılaştırıldığında özellikle 3-4 ppm aralığında tepe önemli bolluk, gösterir. Bu zirveleri mikrokozmos içinde diatom çiçeklenme tarafından üretilen şekerler atfedilebilir. Yapay veya doğal deniz suyu ile doğal plankton topluluklarının büyümesini karşılaştırarak benzer bir deneyde, istatistiksel yaklaşımlar syükleme araziler spektrumu içinde zirveleri tespit olurken sağlıklı bir yer NMR türetilen temel bileşenler analizi (PCA) puanı komplo olarak uch, iki tedavi (Şekil 2) arasında belirgin metabolik farklılıklar göstermek için kullanılabilecek şekil veri dağıtım . Bu sonuçlar gibi genomik parmak izi yöntemleri gibi başka omik seviyeleri, (Şekil 3) gelen veri ile mukayese edilebilir. Bunlar NMR pikleri (BMRB örn. bireysel olarak sorgulanabilir <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> 19, ya da tüm spektrumu (at SpinAssign ile örneğin istatistiksel analiz edilebilir) <a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken. jp /? action = nmr_search</a> ) 2. Bu örnekte, tedaviler arasındaki farklılıkları doğal plankton topluluğunun metabolitlerden spektrumlarının şekerler bölgede zirveleri bir bolluk (3.39 ppm 4.04 ppm) sonucu ortaya çıkmış ve yapay deniz suyu topluluklara karakteristik birkaç tepe geçici ide vardılaktat ve SpinAssign kullanarak format gibi ntified. <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="Şekil 1" /> Şekil 1. Bu prosedür kullanılarak elde edilen işlenmiş örnekleri Örnek 1 H-NMR. Microcosm örnekleri öncesi (1. gün) ve (4 gün), yoğun bir diatom çiçeklenme sırasında alınmıştır. NMR deneyleri internal standart pik yüksekliği (; 0 ppm DSS) normalize sinyalleri ile bir Bruker DRX-500 üzerinde yapıldı. <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="Şekil 2" /> Şekil 2. Doğal olan mikrokosmlarından yetiştirilen doğal kaynaklı mikrobiyal planktonik topluluklar (açık karo) veya yapay (siyah daireler) deniz suyu metabolomes gelen sağlıklı bir yer NMR için temel bileşenler analizi (PCA) skoru arsa. Açık bir metabolik farklılıklar saçılım görülebilir. Böyle bir analizden bir yükleme arsa daha sonra da önemini farklı zirveleri tanımlamak için kullanılabilirSistem; gerektiği gibi, bu zirveleri daha analiz edilebilir. <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="Şekil 3" /> Şekil 3. Çok-omik analizi için bir örnek genomik NMR verileri ile birleştirilmesi. Topluluk kompozisyon denatüre edici gradient jel elektroforezi 18S (solda) ve Şekil 2&#39;de analiz aynı örneklerde 16S (sağda) rRNA genlerinde göre de doğal (açık elmas) ve yapay (siyah daireler) deniz suyu mikrokosmlarından arasındaki farklı mikrobiyal toplum modellerini gösterir. Doğal sistemlerinden metabolome ve genom arasındaki bu yazışmalar bu yaklaşımın yararlılığı tanıtılmakta.

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Çıkar çatışması ilan etti.

Burada gösterildiği filtrasyon ve metaboliti ekstraksiyon yöntemi NMR metabolomik için yeterli miktarda toplanabilir üzere mikrobik planktonik biyokütle sağlar. KPI ve 1D 1 H-NMR kullanarak sulu bir çözünür metabolitlerinin, sadece ekstraksiyon gösterilmiştir birlikte, başka bir ekstraksiyon çözücüsü ve spektroskopik yaklaşımlar kullanılabilir. Bir yararlı örneğin heterojen örnekleri üstün NMR spektrumları üretmek için gösterilen ve paramanyetik iyonları tarafından kirlenmeye...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 0,22 um hidrofilik Durapore PVDF filtreleri, 25 mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Mikroanaliz Filtre Tutucu, 25 mm, cam destek fritli </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3-yer manifoldu, 47 mm, paslanmaz çelik </td><td> Millipore </td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Wako </td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Wako </td><td> 164-04295 </td><td></td></tr><tr><td> Döteryum oksit, 2 H&gt;% 90 </td><td> Campridge İzotop Laboratoties </td><td> DLM-4 </td><td></td<html> &gt; </tr><tr><td> DSS </td><td> Fluka </td><td> 92754 </td><td></td></tr><tr><td> Automill </td><td> Tokken </td><td> TK-AM4 </td><td> Paslanmaz çelik kırıcılar dahil </td></tr><tr><td> Thermomixer comfort </td><td> Eppendorf </td><td> 5355 000.011 </td><td></td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 </td><td></td></tr><tr><td> Vakum evaporatör </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> NMR </td><td> Bruker </td><td> 5 mm-TXI prob ile DRX-500 </td><td></td></tr><tr><td> Spektral binning aracı </td><td> Başlangıçta gelişmiş </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Metaboliti açıklama aracı ve veritabanı </td><td> Başlangıçta gelişmiş </td><td> SpinAssign </td><td>= Nmr_search &quot;&gt; http://prime.psc.riken.jp/?action=nmr_search </td></tr></tbody></table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

Çevre metabolomik organizmalar cevap ve biyokimyasal 1. düzeyde çevre ve birbirleri ile nasıl etkileşime yeni bir anlayışın geliştirilmesinde bir gelişmekte olan bir alandır. Nükleer manyetik rezonans (NMR) spektroskopisi gibi çalışmalar için önemli vaadi ile, gaz kromatografisi-kütle spektrometresi (GC-MS) dahil olmak üzere çeşitli teknolojileri biridir. NMR avantajları, hedefsiz analizler için uygundur yapısal bilgi sağlar ve spektrumları bireysel metaboliti spektrumları 2,3, son zamanlarda mevcut veri tabanları karşı niceliksel ve istatistiksel davranış içinde sorgulanabilir olmasıdır. Buna ek olarak, NMR spektral veriler birbirine taksonların fizyolojik yanıtları ve çevre 4,5,6 daha kapsamlı bir anlaşılmasını sağlamak için başka bir omik seviyeleri (örn. transkriptomik, genomik) veriler ile kombine edilebilir. Ancak, NMR zor yapma, diğer metabolomic yöntemlerine göre daha az duyarlı olduğu aphasta populasyonu düşük iyi tanımlanmış ve bu bütün dokuları, biofluids veya hücre kültürleri gibi kolayca ekstrakte kaynakları. metabolitlerin kıyasla düşük yoğunluklu ve metabolit konsantrasyonları olabilir doğal mikrobiyal sistemlerine kat Sonuç olarak, bugüne kadar yapılan mikropların birkaç doğrudan çevresel metabolomic çalışmalar, ana-symbiont sistemleri gibi kültür-tabanlı veya kolayca tanımlanan yüksek yoğunluklu ekosistemler, kararlı izotop etiketleme olabilir bağırsak çevre ko-kültür veya manipülasyonlar inşa sınırlı olmuştur Buna ek olarak NMR sinyalleri 7,8,9,10,11,12 artırmak için kullanılır. NMR için uygun konsantrasyonda çevresel metabolitlerinin konsantrasyonu ve toplanmasını kolaylaştırmak yöntemler eksiktir. Son dikkatini çok enerji ve malzeme akışının planktonik topluluk 13,14 aracılık su ortamında, içindeki organizmaların çevre metabolomik verilmiş olduğundan, biz konsantrasyon için bir yöntem geliştirdiTION ve süzülerek planktonik mikrobiyal sistemlerinden tam topluluk metabolitlerin çıkarma. Piyasada bulunan hidrofilik poli-1 ,1-difluoroethene (PVDF) filtreleri özel olarak tamamen aksi takdirde daha sonraki analizlerde kirleticiler gibi görünebilir özleri, kaldırmak için tedavi edilir. Bu tedavi filtreler daha sonra ilgi, çevresel ya da deney örnekleri filtrelemek için kullanılır. Islak numune filtreleri içeren liyofilize ve sulu-çözünür metabolitleri standart bir potasyum fosfat tampon ekstraksiyon 2 kullanılarak konvansiyonel NMR spektroskopisi için doğrudan ayıklanır. Bu yöntemlerden elde edilen veriler istatistiksel olarak anlamlı desenleri tanımlamak için analiz, ya da topluluk ve ekosistem fonksiyonlarının kapsamlı bir anlayış için diğer omik seviyeleri ile entegre edilebilir.

Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Mikrobiyal planktonik toplulukların metaboliti çıkarımı için bir yöntem sunulmaktadır. Tüm topluluk örnekleme özel hazırlanmış filtreler üzerine filtrasyon sağlanır. Liyofilizasyon sonra, sulu çözünür metabolitleri ekstre edilir. Bu yaklaşım doğal ya da deneysel mikrobiyal toplulukların trans-omik araştırmalar çevre metabolomik uygulaması için izin verir.

Nükleer Manyetik Rezonans Spektroskopisi ile Çevre metabolomik için düşük yoğunluklu Planktonik topluluklardan Metabolitlerinin Konsantrasyon

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

concentration-metabolites-from-low-density-planktonic-communities-for

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Biology

12.7K Görüntüleme.  RIKEN Advanced Science Institute. Mikrobiyal planktonik toplulukların metaboliti çıkarımı için bir yöntem sunulmaktadır. Tüm topluluk örnekleme özel hazırlanmış filtreler üzerine filtrasyon sağlanır. Liyofilizasyon sonra, sulu çözünür metabolitleri ekstre edilir. Bu yaklaşım doğal ya da deneysel mikrobiyal toplulukların trans-omik araştırmalar çevre metabolomik uygulaması için izin verir.

Video: Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Microbial Communities in Nature and Laboratory - Interview

Doğa ve Laboratuvar Mikrobiyal Toplulukları - Röportaj

Biyoloji

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

İnsan Embriyonik Kök Hücreleri Manyetik Rezonans Görüntüleme için in vitro Etiketleme

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Çevre transkriptomik kullanarak Deniz Mikrobiyal Toplulukları Gen İfade Analizi

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Nitrik Oksit ve hücreler arası Elektron Paramanyetik Rezonans Spektroskopisi ile süperoksit Radikal Anyon tespiti Spin Tuzakları kullanarak

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Manyetik Rezonans Görüntüleme kullanarak Karışım miktarının

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Transkript ve Metabolitler gelen saflaştırılması<em&gt; Drosophila</em&gt; Kafaları

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Antik Biyolojik Topluluklar barındırmıştır İç Araştırma Dondurulmuş Çekirdek dış bölümünden Eksojen Malzeme Kaldırma

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Drosophila toksisite laboratuvarda test etmek için bir omurgasız Model sistemi olarak kullanmak için deneysel protokol

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

68Ga çekirdek katkılı demir oksit nano tanecikleri çift pozitron emisyon tomografi için sentezi / (TRT1) manyetik rezonans görüntüleme

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;