Açıklayıcı örnekler olarak kullanılan tüm NMR spektrumları, Nestlé tarafından mali olarak desteklenen bir daha önce yayımlanan bir çalışmada 7 türetilmiştir .

1. Germ-free hayvan ve örnek toplama kolonizasyonu <ol><li> Mikrop ücretsiz hayvanlar kafeslerde denetimleri (Şekil 1) olarak görev yapacak konvansiyonel hayvanların ön filtresi ile donatılmış bir geleneksel hayvancılık oda izolatörler ve ev onları çıkarın. </li><li> Germ-free hayvanların çöp kontrolü geleneksel kafes alınan çöp Mix yarısı (eski 3 gün). Her zaman kirli geleneksel çöp 1 / 3, bir bakteri düzeyi (en az 3 gün için saklayın) korumak için, yenilemek için gerekli olan her zaman tutmak. </li><li> Tüp üzerinde fare işleme 1,5 mL mikrotüp idrar toplamak ve barsak nazikçe masaj işeme yardımcı. Snap-dondurma, sıvı azot hemen. NMR analizi kadar, en az -40 ° C&#39;de saklayın. En az 20 mcL hacmi 5 mm NMR probu ile satın alma için gerekli, ancak metabolik profil kalitesini artırmak için 30 mcL kullanılması tavsiye edilir. </li><li> Hayvanlar b olmalıdıre anestezi bileşikler (örneğin, exsanguination ölüm onayı takiben servikal dislokasyon kullanın) karaciğer metabolizması nedeniyle karıştırıcı NMR rezonanslar önlemek için herhangi bir anestezi kullanımı olmadan ötenazi </li></ol> 2. Karaciğer biyopsisi toplanması için öneriler <ol><li> Kontaminasyonu önlemek için alkol içeren herhangi bir ürün kullanmayın. Araçları sadece su veya tuzlu solüsyon kullanarak yıkayın. </li><li> Safra kesesi perfore etmeyin. Safra kaçağı durumunda, su ya da tuzlu su çözeltisi ile hemen doku yıkayın. </li><li> Şekil 2&#39;de gösterildiği gibi sol lob karaciğer biyopsisi (15-50 mg) toplayın. Tekrarlanabilir biyopsiler için, sol lob dokusu incedir periferik alanlarda kaçınarak merkezinde sürekli toplamak. </li><li> Hemen sıvı nitrojen içinde dondurarak biyopsileri çekin ve saklayın -80 ° C NMR analizi kadar. </li></ol> Idrar microvolume 3 1 H-NMR edinimi </p&gt; <ol><li> D2O (99.8%), pH 7.4, 1 mM 3 içeren 0,2 M sodyum fosfat tampon çözeltisi hazırlayın - (trimethylsilyl) propiyonik asit-d 4 (TSP) . </li><li> 30 mcL idrar sodyum fosfat tamponunda 30 mcL ile karıştırın. </li><li> Metal bir iğne (OD 0,5 mm) ile donatılmış 50 mcL cam şırınga kullanılarak 1.7 mm NMR kapiller tüp (Şekil 3 (2)) 50 mcL karma çözüm aktarın. Kabarcıkları önlemek için dikkatli olun. </li><li> Kılcal içeren idrar örneği üst Fit kapiller adaptörü (Şekil 3 (3)) ve 2.5 mm NMR mikrotüp, 5 mm NMR prob (Şekil 3 (1)) içine yerleştirin. Spektral elde etmek için düzenli olarak 5 mm NMR tüpü gibi tüplerin bu kombinasyon kullanın. </li><li> 2,5 mm NMR tüpü yavaşça çekin kapiller adaptörü vidalanarak kılcal kaldırmak için ekstraksiyon çubuk (Şekil 3 (4)) kullanın. </li></ol> 4. 1 H İK karaciğer dokusunda MAS NMR biyopsisi: numune hazırlama <ol><li> Rotor bileşenlerini ve araçları olan MASŞekil 4&#39;te açıklanmıştır. </li><li> Ekle zirkonyum rotor içine biyopsi (15-50 mg) (Şekil 4 (1)) ve NMR kilidi için saf D 2 O hacmi geri kalanını doldurun. Bu, veri toplama sonraki layneri ve kalite kalitesini değiştirmez, çünkü herhangi bir baloncuklar yapmak için dikkatli olun. </li><li> Silindirik vida (Şekil 4 (5)) kullanılarak 50 mcL Teflon ayırıcı (Şekil 4 (2)) takın. Sökün onu ve kısa kenarı (Şekil 4 (8)), derinlik ölçer kullanarak kalibre. Bu aşamada, bunun bir parçası ara delikten sızıntı olabilir çünkü örnek için özel bir dikkat etmek önemlidir. Bu durumda biyopsi parçası, daha sonra imha edilir ve numune ağırlığı güvenilir artık. Bu yüzden başından numune hazırlama tekrar başlatmak için gerekli. </li><li> Tarih katılın pin (Şekil 4 (3)) (Şekil 4 (6)) tornavida ile hafifçe yerleştirin ve vida. Bir doku parçası ile herhangi bir kalıntı su kurulayın. </li><li> Kap yerleştirin (Şekil 4 (4))rotor ve rotor packer (Şekil 4 (6)) takın. Kapağı yerine gelene kadar sıkıca bastırın. Rotor ve onun kapağı arasında kalan herhangi bir boşluk olmamalıdır. </li><li> Optik dönüş hızı algılama izin siyah bir işaretleme kalemi kullanarak rotor alt yarısı işaretleyin. </li><li> NMR spektrometresi içindeki rotor koyun ve 5 kHz dönmeye başlar. 1 H-NMR spektrumunda üreticinin kurallarına göre CPMG darbe dizisi 13 kazanırlar. </li><li> 5.22 ppm (giyilen erkek yeleği) α anomerik glikoz rezonans NMR spektrumları kalibre etmek için kullanın. </li><li> Rotor açmak için kapağını sökücü (Şekil 4 (9)) kullanarak kapağı kaldırarak geçin. Tarih katılın pin ve silindirik vida kullanarak kaldır Teflon spacer sökün. Iyice su ve deterjan kullanarak yıkayın. </li></ol> 5. Temsilcisi Sonuçlar Gut mikrobiyal aktivite idrar metabolik profil kullanılarak izlenebilir. Idrar mikrobiyal çok sayıda1 H NMR ile tanımlanabilir co-metabolitleri 7,14-17 literatürde tarif edilmiştir. Bu co-mikrobiyal metabolitler onlar yeni kurulan ekosistem kararlı tahmin etmek için non-invaziv ve hızlı bir şekilde sağlamak olarak sömürgeleştirilmesi sürecini izlemek için özellikle yararlıdır. Şekil 5A, co-metabolitleri bağırsak mikrobiyal kolonizasyon süreci üzerinde açıkça göstermektedir. Bu rakam, bir hayvan için Adım 2 açıklanan prosedürü takip ederek elde edilen bir idrar metabolik profil Adım 1 açıklanan yordamı kullanarak 20 gün kolonize gösterir. Bu hayvan, herhangi bir indoxyl sülfat ifraz ve phenylacetylglycine (PAG) ve germ-free p-kresol sülfat (günde 0-mavi) çok az miktarda vermedi. Kolonizasyon ilerledikçe, bağırsak Mikrobiyota tarafından protein metabolizması bu 3 belirteçler gün 20 (kırmızı) bir dengeye ulaşması için önemli oranda artması beklenmektedir. Bu PAG kullanarak Şekil 5B gösterildiği gibi hayvanların bir grup izlemek için özellikle kolayrezonans. Bu diyagram 7 hayvanların bir grup için PAG belirli bir rezonans (üçlü) karşılık gelen gri Şekil 5A (δ 7,40-7,43) vurgulanan rezonanslar altında kalan alan entegre ederek elde edilmiştir. 1 H Yüksek Çözünürlüklü Magic Angle İplik (İK MAS) NMR spektroskopisi 18 biyopsi her türlü metabolik profiller hızlı ve tekrarlanabilir satın almalar sağlar yıkıcı olmayan bir tekniktir. Bu protokol, önce (mavi) ve (kırmızı) kolonizasyonu (Şekil 6) sonra, 2 farelerin karaciğer metabolik profil elde etmek için bu güçlü bir tekniği kullanılmıştır. Bu rakam, iyi bir MAS NMR tabanlı metabolik profil elde edilebilir bilgiler göstermektedir. Çok sayıda amino asitlerin yanı sıra, glukoz, glikojen, laktat, trigliserid, (D)-3-hidroksi ve nicotinurate gibi enerji metabolizmasını elde edilen metabolitleri görüntülenebilir. Bu profiller aynı zamanda oksidatif stres ile ilgili bilgileri içeren (yani askorbik acid, glutatyon), (yani inozin, üridin) nükleotid metabolizması ve Methylamine metabolizma (yani kolin, trimetilamin-N-oksit). Bu örnekte, mikrop ücretsiz fare neredeyse hiç glikojen ve çok düşük miktarlarda glikoz ve trigliserid gibi daha önce 7 yayımlandı görüntüler çok açıktır. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Şekil 1."/> Şekil 1 kolonizasyon protokol Bakış. Germ-free ve konvansiyonel hayvanların yan ve kendi yavrularına geleneksel gut Mikrobiyota (1) aşamalı kolonizasyon izin alışverişinde yan filtreleri ile donatılmıştır kafeslerde ev sahipliği yapmaktadır. Gut mikrobiyal aktivite, 1 H-NMR tabanlı metabolik profil (2-3) kullanılarak izlenir . Karaciğer metabolizması 1 H İK MAS NMR tabanlı metabolik profil (4-5) ile değerlendirildi. <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Şekil 2."/> Şekil 2: Fare canlır anatomisi. Karaciğer gibi organ düz tarafı masaya yüzleri olarak görüntülenir. Tekrarlanabilir biyopsiler için, her zaman, kesik dikdörtgen ile gösterilir sol lob merkezinde örnekler toplamak için tavsiye edilir. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Şekil 3."/> Şekil 3 microvolumes çalışmak için 1.7 mm NMR kılcal kiti tuşuna: 1: 2,5 mm NMR mikrotüp, 2: 1.7 mm NMR kılcal tüp, 3: Kılcal adaptörü, 4: Ekstraksiyon çubuk. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Şekil 4."/> Şekil 4 MAS rotor ekipman Anahtar: 1: MAS rotor, 2: 50 mcL teflon ayırıcı, 3: &lt;thead&gt; pin, 4: kap, 5: silindirik vida, 6: tornavida, 7: rotor Packer, 8: derinlik göstergesi. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Şekil 5."/> Şekil 5 colonizatio sırasında idrar metabolik profiller Evrimin. <ol class="ualpha"><li> 0. günde tek bir kişi (mavi), 4 (yeşil), 15 (turuncu) ve 20 co-metabolitleri görüntülenmiştir 1 H NMR mikrobiyal 6,8-7,8 ppm arasında spektrumları aromatik bölge üzerinde Zoom elde edildi (kırmızı) post-kolonizasyon. Indoxyl sülfat eserleri şunlardır: Histidin, p-Kresol-S:;: 1-MeHistamine: Indoxyl-S 1-methylhistamine gri bölge B. Key şeması yapmak için entegre alana karşılık p-Kresol sülfat; PAG : Phenylacetylglycine. </li><li> Ortalama kolonizasyon sırasında PAG konsantrasyonu (n = 7). Student t-testi, çeşitli zaman noktalarında PAG konsantrasyon farkı karşılaştırmak için kullanılır: a: p &lt;0. günde karşılaştırıldığında 0.05; b: p &lt;0.01 günde 10 ile karşılaştırıldığında. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Şekil 6."/> Şekil 6: Tipik germ-free (mavi) ve eski-germ-free (yeniden türetilmiş karaciğer biyopsisi 600 MHz, 1 H İK MAS NMR spektrumlarıd) fareler. Kalın proton trigliserid rezonans sorumlu Anahtarı: 3-HB: 3-hidroksi, GSH: indirgenmiş glutatyon, TG: Trigliserid, TMAO: trimetilamin-N-oksit.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+spectroscopy.+Application+to+nuclear+magnetic+resonance.">Two-dimensional spectroscopy. Application to nuclear magnetic resonance.</a> J. Chem. Phys. 64, 2229-2246 (1975).</li><li>Bodenhausen, G., Ruben, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+abundance+15N+NMR+by+enhanced+heteronuclear+spectroscopy.">Natural abundance 15N NMR by enhanced heteronuclear spectroscopy.</a> Chemical. Physics. Letters. 69, 185-189 (1980).</li><li>Fan, T. W. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two-dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures.</a> Progress in nuclear magnetic resonance spectroscopy. 28, 161-219 (1996).</li><li>Fan, T., Lane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+profiling+of+metabolites+and+isotopomers+by+NMR.">Structure-based profiling of metabolites and isotopomers by NMR.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 52, 48-48 (2008).</li><li>Fonville, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

Biz ifşa etmek başka bir şey var.

Bu protokol, daha sağlam biyopsi 1 H İK MAS NMR profil ile değerlendirildi, gut Mikrobiyota, karaciğer metabolizması üzerine etkisini araştırmak için açık bir ortamda ilerici bir kolonizasyon usulü nitelendirdi . Kolonizasyonu literatürde çeşitli yöntemler tarif edilmiştir. Tanımlanmış bir Mikrobiyota hayvanlar kolonize için en yaygın yöntemlerden ağızdan sonda ile veya kontamine içme suyu 19,20. Fekal aşılama, daha önce 21 açıklandığı gibi de kullanıla...

Özel reaktifler ve ekipman Tablo: <table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> 2.5 mm mikrotüp </td><td> New Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm kılcal tüp </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Kılcal adaptörü </td><td> New Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Ekstraksiyon çubuk </td><td> New Era </td><td> KD-341-5 </td><td></td></tr><tr><td> HR-50 mcL MAS rotor BL4 küresel teflon ayırıcı kiti </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> 50 mcL uçlar için araç kiti </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Peşin III 600MHz NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H İK MAS NMR katı prob </td><td> Bruker </td><td></td><td></td></tr><tr><td> Döteryum oksit% 99,9 </td><td> Sigma-Aldrich </td><td> 530.867-1L </td><td></td></tr><tr><td> 3 - (trimethylsilyl) propiyonik asit-d 4 (TSP) </td><td> Sigma-Aldrich </td><td> 269913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

Bağırsak bakterileri bağışıklık koruma ve vitamin sentezi olarak bir dizi faydalar sağlayarak, ev sahibi homeostazı önemli ölçüde katkıda bulunduğunu çok iyi bilinmektedir. Ayrıca bu ekosistemin önemli bir metabolik organı ile ev sahipliği yaparak, hatırı sayılır bir miktarda besin kaynağı. Bağırsak florası ve metabolik sendrom arasındaki bağlantı, konak ve gut Mikrobiyota arasındaki metabolik etkileşimi anlamaya artan kanıtlar çerçevesinde modern biyolojinin önemli bir sorun haline geliyor. 1-4 Kolonizasyon (normalleşme süreci olarak anılacaktır), eski bir germ-free hayvan mikro-organizmaların kurulması atar. Doğumda meydana gelen doğal bir süreç olmakla birlikte, aynı zamanda bağırsak çiçek ekosistem kontrolü ve daha fazla ana metabolizması üzerindeki etkisini belirlemek için, yetişkin germ-free hayvanlarda kullanılmaktadır. Sömürgeleştirme sürecini kontrol etmek için yaygın bir yöntemdir, bir singl sonda yöntemi kullanmak içine veya mikro-organizmaların bir karışımı. Bu yöntem, çok hızlı bir kolonizasyon sonuçları ve 5 derece stresli olma dezavantajı sunar . Bu nedenle, stresi en aza indirmek için ve bakteriyel kurulması yavaş yavaş ev sahibi metabolizması üzerine etkisini gözlemlemek için daha yavaş bir kolonizasyon süreci elde etmek için yararlıdır. Bu yazıda, bir tahribatsız metabolik profil tekniği kullanılarak kademeli bir kolonizasyon süreci sırasında hepatik metabolizma değişiklik değerlendirmek için bir prosedür açıklanmaktadır. Biz, 1 H-NMR tabanlı metabolik profil mikrobiyal co-metabolitleri idrar atılımı tarafından yansıtılan bağırsak mikrobiyal metabolik aktivite değerlendirerek bağırsak mikrobiyal kolonizasyon izlemek için öneriyoruz . Bu bağırsak mikrobiyal aktivite, genellikle DGGE (denatüre edici gradient jel elektroforezi) fekal bakteri izlenmesi ile değerlendirildi bağırsak mikrobiyal ekosistem kararlı kurulması ötesinde istikrar takdir sağlar. 6kolonizasyonu konvansiyonel açık bir ortamda gerçekleşir ve kontrol grubu olarak hizmet verecek geleneksel hayvanlar, kirli kirli bir çöp tarafından başlatılan. Kemirgenler coprophagous hayvanlar olmak, bu, daha önce açıklandığı gibi, homojen bir kolonizasyon sağlar. 7 Karaciğer metabolik profil, 1 H NMR spektroskopisi İplik Yüksek Çözünürlüklü Magic Angle kullanarak sağlam bir karaciğer biyopsisi doğrudan ölçülür . Bu yarı-kantitatif bir tekniktir hücre yapısına zarar vermeden, hızlı bir şekilde değerlendirmek için, trigliserit, glikoz ve glikojen gibi önemli metabolitleri daha da ilerletmek için kolonizasyon süreci ve karaciğer metabolizmasını 7-10 arasındaki karmaşık bir etkileşim tahmin ediyoruz . Bu yöntem, herhangi bir doku biyopsisi 11,12 için de uygulanabilir .

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

İlerici bir kolonizasyon prosedürü daha ev sahipliği karaciğer metabolizması üzerine olan etkisini değerlendirmek için tarif edilir. Yüksek Çözünürlüklü Magic Angle İplik (İK MAS) bozulmamış biyopsi NMR profil hepatik metabolizma tarafından değerlendirilir Kolonizasyon NMR tabanlı metabolik profil mikrobiyal co-metabolitleri idrar atılımı değerlendirerek invazif olmayan izlenir.

Germ-free Mouse Aşamalı Kolonizasyon sırasında Karaciğer metabolik değişiklikler Değerlendirilmesi 1 H-NMR Spektroskopi

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

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Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.1K Görüntüleme.  The University of Reading. İlerici bir kolonizasyon prosedürü daha ev sahipliği karaciğer metabolizması üzerine olan etkisini değerlendirmek için tarif edilir. Yüksek Çözünürlüklü Magic Angle İplik (İK MAS) bozulmamış biyopsi NMR profil hepatik metabolizma tarafından değerlendirilir Kolonizasyon NMR tabanlı metabolik profil mikrobiyal co-metabolitleri idrar atılımı değerlendirerek invazif olmayan izlenir.

Video: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Çekirdek biyoanalizlerin kullanarak Fungal Kolonizasyon, Sporogenesis ve Mikotoksin Üretimi Kantitasyonu

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

İmmünoloji ve Enfeksiyon

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Kolonizasyon Euprymna scolopes Squid tarafından Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Streptococcus pneumoniae ile İntranazal Kolonizasyon Sırasında İnflamatuar Yanıtların Karakterizasyonu

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Eşodaklı Mikroskopi İzole Alveoler Makrofagların anti-fungal Hareketi değerlendirilmesi

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes Kullanarak Enfekte Farelerde Kültür ve Kalp Kolonizasyonunda Kardiyak Hücrelerin Bakteriyel İstilasının Değerlendirilmesi

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatik lökosit seçici Hasat

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Flüoresan Korelasyon Spektroskopisi Ölçülen İlköğretim Lenfositler Plazma Membranlarda moleküler difüzyon

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Germ-free Mouse Aşamalı Kolonizasyon sırasında Karaciğer metabolik değişiklikler Değerlendirilmesi¹ H-NMR Spektroskopi

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