Onların teknik destek için uygulanan Biosciences, Toyo Üniversitesi ve Bay Koudai Taniguchi Gunma tarım teknoloji merkezinin Anabilim Dalı için Bay Akira Murakami minnettarız. Ayrıca onların önerilerini Profesör Shosaku Kashiwada ve Dr Uma Maheswari Rajagopalan, Toyo üniversite için sana şükrediyoruz. Bu çalışmada kısmen Araştırma Merkezi tarafından desteklenmiştir yaşam ve Çevre Bilimleri, Toyo üniversite için.

Not: Bu, endojen etki analizi kolaylaştırır çünkü ipeka (C. ipecacuanha) Bu çalışmada kullanılmıştır.
1. büyüme koşulları adventif Ipecac Shoots ikna etmek için
<ol><li>Phytohormone-Alerjik B5 orta pH 5,710' a ayarlanmış hazırlamak ve % 0.2 gellan sakız ekleyin. Tarafından ısıyla sterilize.</li>
	<li>Autoclaved Orta 25 mL steril Petri kabına (90 mm × 20 mm) dökün.</li>
	<li>Ipecac Bitkicik steril bir akrilik plaka üzerinde bir bıçak No. 22 ile cerrahi neşter kullanarak 8 mm internodal parçalarını keser ve orta (şekil 1) yerleştirir.</li>
	<li>Kültür phytohormone ücretsiz B5 orta 24 ° c 15 µmol m−2 s– 1 14-h ışık/10-h karanlık döngüsü 5 hafta için ışığın altında.</li>
	<li>Bölgeleri tanımlamak (apikal) IV (her segmentte (şekil 1B) Bazal) ben. Adventif sürgünler > 0.3 mm uzunluğu bir mikroskop altında her bölgedeki haftada bir kez saymak.</li>
</ol>2. çıkarma ve arıtma etki
<ol><li>5 mm Zirkon boncuk her dört 2 mL örnek tüplerinin koymak.</li>
	<li>Segmentleri bölgelere kesmek ben cerrahi neşter steril akrilik tabağa kullanarak IV (her 2 mm içinde süre).</li>
	<li>Sekiz ayrı örnek tüpler (10-30 mg taze ağırlık) her bölgede parçalarını toplamak.</li>
	<li>Tartmak ve sıvı azot örnekleri dondurma.</li>
	<li>Donmuş numuneler boncuk tabanlı homogenizer kullanarak ezmek.</li>
	<li>Auxin iç her standart 500 pg ve CKs (d5-IAA, d5- tZ, d5- tZR, d6- IP, d6- IPR) içeren 1 mL Asetonitril içinde ezilmiş örnekleri askıya bir girdap Mikser kullanarak.</li>
	<li>1s için 4 ° C'de basılı tutun, sonra 3500 × g oda sıcaklığında 5 min için de santrifüj kapasitesi.</li>
	<li>Pelet %80 (v/v) Asetonitril (v/v) % 1 Asetik asit içeren yıkayın. Santrifüj tekrar 3500 × g de oda sıcaklığında 5 min için. (Kimden adım 2.7 ve 2.8) supernatants tek kullanımlık cam tüp içinde birleştirir.</li>
	<li>600 eklemek µL içeren %1 (v/v) Asetik asit her kombine süpernatant için su ve vakum yoğunlaştırıcı kullanarak Asetonitril buharlaşır.</li>
	<li>Hidrofilik lipofilik-dengeli (HLB) sütun kartuşları Asetonitril, metanol ve su (v/v) % 1 Asetik asit içeren her 1 mL uygulayarak equilibrate.</li>
	<li>Dengelenmiş HLB kartuş başına bir örnek çözümü uygulayın.</li>
	<li>Kartuşları (v/v) % 1 Asetik asit içeren 1 mL su ile yıkayın.</li>
	<li>2 mL % 80'i (v/v) Asetonitril (v/v) % 1 Asetik asit bir cam tüp içeren ile tüm hormon elute.</li>
	<li>Asetonitril özü (v/v) % 1 Asetik asit vakum yoğunlaştırıcı kullanarak içeren su elde etmek için eluate içinde buharlaşır. Not: Bu tamamen kuru değil.</li>
	<li>Karma mod, güçlü katyon değişim (MCX) sütun kartuşları 1 mL Asetonitril, 1 mL metanol, 0.5 mL uygulayarak equilibrate 0.1 M HCl ve 1 mL su %1 (v/v) Asetik asit içeren.</li>
	<li>Dengelenmiş MCX kartuş başına bir örnek çözümü uygulayın.</li>
	<li>Kartuşları (v/v) % 1 Asetik asit içeren 1 mL su ile yıkayın.</li>
	<li>IAA 2 mL % 30 (v/v) Asetonitril (v/v) % 1 Asetik asit bir cam tüp içeren ile elute.</li>
	<li>Kartuşları (v/v) % 1 Asetik asit içeren 2 mL % 80'i (v/v) Asetonitril ile yıkayın.</li>
	<li>Kartuşları 2 mL su ve %5 sulu amonyak içeren 1 mL su ile yıkayın.</li>
	<li>Bir cam tüp %5 sulu amonyak içeren 2 mL % 60'ı (v/v) Asetonitril ile CKs elute.</li>
	<li>Vakum yoğunlaştırıcı kullanarak her hormon kesir solvent buharlaşır ve −30 ° C LC-MS/MS analizi kadar saklayın.</li>
</ol>3. IAA ve CKs LC-MS/MS analizi
<ol><li>Her hormon hulâsa bir tüp ile 600 µL metanol içinde dağıtılması ve çözüm için bir vida boyun toplam kurtarma şişe aktarmak. Vakum yoğunlaştırıcı kullanarak solvent buharlaşır.</li>
	<li>20 µL % 30 (v/v) Asetonitril IAA kesir ve CK kesirler vida boyun toplam kurtarma şişeleri %1 (v/v) Asetik asit içeren 20 µL suda çözülür.</li>
	<li>Pozitif iyon modunda bir HPLC sistemi ile donatılmış bir triple quadrupole MS sistemde örnekleri analiz. Not: Tablo 1' de listelenen gibi HPLC koşulları ayarlayın.<ol>
<li>IAA elüsyon %5 - %50 çözelti B üzerinde 7 dk, ikili bir degrade kullanmak sonra % 98 çözücü B tarafından artırmak ve 1 dk. için tutun ve sonra % 5 çözücü B, 2 min için yuva iğne ile equilibrate.</li>
		<li>CK elüsyon için % 2-% 40 çözücü B üzerinde 5 dk, % 40-% 70 çözücü B 7 min, ikili bir degrade kullanmak sonra % 95 çözücü B tarafından artırmak ve 1 dk. için tutun ve sonra 2 dk az %2 çözücü B tarafından için equilibrate enjeksiyon iç içe.</li>
		<li>Electrospray iyonlaşma (ESI) ayarla-MS parametreleri Tablo 2' de listelenen İyon kaynağı.</li>
	</ol>
</li>
	<li>(MRM) geçiş Tablo 3' te listelenen her analit miktar için izleme birden çok tepki kullanın.</li>
	<li>Döteryum etiketli standartlarına etiketlenmemiş oranı standart bir eğri karşı endojen IAA ve CK seviyeleri ölçmek.</li>
</ol>

<ol>
	<li>Haberlandt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kulturversuche+mit+isolierten+Pflanzenzellen.">Kulturversuche mit isolierten Pflanzenzellen.</a> Sitzungsber. Math.-Naturwiss. Kl. Akad. Wiss. Wien. 111, 69-92 (1902).</li><li>Skoog, F., Miller, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+regulation+of+growth+and+organ+formation+in+plant+tissues+cultured+in+vitro.">Chemical regulation of growth and organ formation in plant tissues cultured in vitro.</a> Symp Soc Exp Biol. 11, 118-130 (1957).</li><li>Ganeshan, S., Caswell, K. L., Kartha, K. K., Chibbar, R. N., Khachatourians, G. G., McHughen, A., Scorza, R., Nip, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transgenic plants and crops. , 69-84 (2002).</li><li>Teshima, D., Ikeda, K., Satake, M., Aoyama, T., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+emetic+alkaloid+by+in+vitro+culture+of+Cephaelis+ipecacuanha.">Production of emetic alkaloid by in vitro culture of Cephaelis ipecacuanha.</a> Plant Cell Rep. 7 (4), 278-280 (1988).</li><li>Chatterjee, S. K., Nandi, R. P., Ghosh, N. C., Atal, C. K., Kapur, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+and+utilixzation+of+medicinal+plants.">Cultivation and utilixzation of medicinal plants.</a> Regional Research Laboratory, Council of Scientific and Industrial Research. , 295-301 (1982).</li><li>Yoshimatsu, K., Shimomura, K., Bajaj, Y. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biotechnology in Agriculture and Forestry 21, Medicinal and Aromatic Plants IV. , 87-103 (1993).</li><li>Ideda, K., Teshima, D., Aoyama, T., Satake, M., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+propagation+of+Cephaelis+ipecacuanha.">Clonal propagation of Cephaelis ipecacuanha.</a> Plant Cell Rep. 7 (4), 288-291 (1988).</li><li>Yoshimatsu, K., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+shoot+formation+on+internodal+segments+and+alkaloid+formation+in+the+regenerates+of+Cephaelis+ipecacuanha+A.+Richard.">Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard.</a> Plant Cell Rep. 9 (10), 567-570 (1991).</li><li>Koike, I., Taniguchi, K., Shimomura, K., Umehara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+endogenous+indole-3-acetic+acid+and+cytokinins+during+adventitious+shoot+formation+in+ipecac.">Dynamics of endogenous indole-3-acetic acid and cytokinins during adventitious shoot formation in ipecac.</a> J. Plant Growth Regul. , (2017).</li><li>Gamborg, O. L., Miller, R. A., Ojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+requirements+of+suspension+cultures+of+soybean+root+cells.">Nutrient requirements of suspension cultures of soybean root cells.</a> Exp. Cell Res. 50 (1), 151-158 (1968).</li><li>Watad, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adventitious+shoot+formation+from+carnation+stem+segments:+a+comparison+of+different+culture+procedures.">Adventitious shoot formation from carnation stem segments: a comparison of different culture procedures.</a> Scientia Hortic. 65 (4), 313-320 (1996).</li><li>Ajithkumar, D., Seeni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+clonal+multiplication+through+in+vitro+axillary+shoot+proliferation+of+Aegle+marmelos+(L.)+Corr.,+a+medicinal+tree.">Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree.</a> Plant Cell Rep. 17 (5), 422-426 (1998).</li><li>Tiwari, V., Tiwari, K. N., Singh, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+of+cytokinins+on+in+vitro+propagation+of+Bacopa+monniera.">Comparative studies of cytokinins on in vitro propagation of Bacopa monniera.</a> Plant Cell Tiss. Org. Cult. 66 (1), 9-16 (2001).</li><li>Rao, M. S., Purohit, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+shoot+bud+differentiation+and+plantlet+regeneration+in+Celastrus+paniculatus+Willd.">In vitro shoot bud differentiation and plantlet regeneration in Celastrus paniculatus Willd.</a> Biol. Plant. 50 (4), 501-506 (2006).</li><li>Sanikhani, M., Frello, S., Serek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDZ+induces+shoot+regeneration+in+various+Kalancho&#235;+blossfeldiana+Poelln+cultivars+in+the+absence+of+auxin.">TDZ induces shoot regeneration in various Kalancho&#235; blossfeldiana Poelln cultivars in the absence of auxin.</a> Plant Cell Tiss. Org. Cult. 85 (1), 75-82 (2006).</li><li>Yoshimoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+negatively+regulates+cell+death+by+controlling+NPR1-dependent+salicylic+acid+signaling+during+senescence+and+the+innate+immune+response+in+Arabidopsis.">Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.</a> Plant Cell. 21 (9), 2914-2927 (2009).</li><li>Schaller, G. E., Bishopp, A., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin-yang+of+hormones:+cytokinin+and+auxin+interactions+in+plant+development.">The yin-yang of hormones: cytokinin and auxin interactions in plant development.</a> Plant Cell. 27 (1), 44-63 (2015).</li></ol>

Yazarlar onlar hiçbir çıkar çatışması var bildirin.

Etki dağıtım tanımlamak için fiş boşaltmaya karışan organogenesis, bu ne zaman etki exogenously explants inducing için uygulanan organogenesis phytohormone ücretsiz ortamda, gözlenen bitki malzemeler kullanmak önemlidir sürgünler veya kökleri, onlar hücre farklılaşma ve organogenesis fabrikaları doğasında plastisite değerlendirmek üzere bütün explant etkiler. Kültür medyada phytohormone ücretsiz Dianthus caryophyllus L.11, Aegle marmelos (L.) Corrêa12, Bacopa monnieri (L.) Pennell13, gibi diğer bitki türü üzerinde adventif sürgünler indüklenen Celastrus paniculatus Willd. 14ve Kalanchoë blossfeldiana Poelln. 15. bu bitki türü protokolünde uygulamak mümkün olacaktır.
Biz IAA, tZ, tZR, IP ve Asetonitril IPR ve katı faz çıkarma tarafından arıtılmış. Çünkü tüm etki saflaştırılmış (ve dahil olmak üzere gibberellinler, absisik asit, jasmonic asit, salisilik asit)16kartuş sütunları (HLB, MCX ve zayıf anyon değişim (balmumu)) üç tür orijinal yöntemi kullanır. HLB sütun bir polimer ters fazlı jelleştirici, MCX sütun aynı katyon değişim gruplarıyla kullanır, ve balmumu sütun aynı zayıf anyon-Satım gruplarıyla kullanır. Orijinal yöntemi ile MCX sütunda İkinci adımda sulu amonyak içeren % 60 Asetonitril (temel) CKs ve IAA (asidik) içeren bir balmumu sütunundaki son adımda % 1 Asetik asit % 80 Asetonitril ile elutes. Auxin ve antagonistically bitki büyüme17düzenleyen etkileşim, CKs bizim odak olduğu gibi Basitleştirilmiş Protokolü sadece HLB ve MCX sütunları kullanır; IAA % 1 Asetik asit ilk adım HLB sütunu içeren % 30 Asetonitril ile eluted. LC-MS/MS analiz için numune hazırlama üzerinden iki gün alır.
Asetonitril solvent phytohormone arıtma kartuş sütunlardaki sırasında kurumasına izin verilmemelidir. Eğer işe yararsa, cam tüpe sıkışmış ve örnek kayıp örnek Asetonitril etki olmasını önlemek için içinde resuspend. Bu protokol için algılama sınırıdır iyon kapanı MS sistemi ile ~ 10 pg IAA ve CKs üzerinden 10-30 mg taze dokular için. Küçük miktarlarda analiz etmek için daha fazla örnek toplamak için veya MS daha yüksek hassasiyet ile kullanmak için gerekli olacaktır.
Phytohormone analiz bitki büyüme ve gelişmesi değerlendirilmesi için önemli bir tekniktir. Bu yöntemi kullanarak, biz en iyi kültür durumu hala bilinmeyen nerede auxin ve bitki türleri oluşumunda adventif ateş tedavisinde CK zamanlamasını belirlemek mümkün olabilir. Phytohormone miktar giderek daha önemli hale geldikçe, burada açıklanan LC-MS/MS Protokolü yüksek hassasiyet ve çözünürlük ile analiz küçük örnekleri sağlayacaktır. Bir önceki yöntem bizim basitleştirilmesi arıtma ve analiz kolaylaştırmak ve yüksek çok yönlülük ve tekrarlanabilirlik getir. Gelecekte, bu yöntem diğer bitki türü organogenesis sırasında endojen auxin ve CK dinamiklerini araştırmak için uygulanabilir.

Gottlieb Haberlandt (1854-1945) kavramı, "totipotency" hangi bitki tarafından hücreleri bölmek, ayırmak ve düz-den sonra onların önceki farklılaşma belirli hücre tipleri olgun bitkiler1içine tüm bitkiler yeniden, evlenme teklif etti. Doku kültürü içinde olup bitki rejenerasyonu veya değil bağlı olmak kombinasyonu ve exogenously uygulanan etki büyüme orta yoğunlukta tarafından belirlenir. Skoog ve Miller adventif kökleri bir düşük oran2içeren ortamda indüklenen Oysa adventif sürgünler tütün Kallus kültürü CKs yüksek bir oranı için auxins, içeren ortamda gelen indüklenen bulundu. Bu bulgu beri doku kültürü yaygın transgenik bitkiler3yenilenme ve ekonomik açıdan önemli bitkileri yayılması için kullanılmıştır. Adventif çekimleri dışında yapraklar, kökler ve internodes gibi sürgün apikal meristem doku üzerinden bağlı olmak. Phytohormone tedavi indüksiyon adventif sürgünlerin birçok bitki türü için gereklidir. Ancak, optimum konsantrasyonları ve kombinasyonları türler tarafından ve explants kullanılan dokular arasında farklı. Böylece, çok çaba optimum konsantrasyonları ve deneyler için etki kombinasyonları belirlenmesinde gider.
Carapichea ipecacuanha (Brot.) L. Andersson (Ipecac) emetine ve cephaeline, başta olmak üzere4kökleri gibi alkaloidler içeren tıbbi bir bitkidir. Kök özleri bir balgam söktürücü, kusturucu ve bir amoebicide5kullanılır. Ipecac doğal olarak Brezilya tropikal yağmur ormanları içinde büyür rağmen tohum kültüründe ayarlamak isteksiz ve Japonya, onun soğuk iklim6ile tohum depolama sırasında çimlenme oranı azalır. Bunun yerine, doku kültürü tarafından yayılır, hangi adventif ateş internodes oluşumu en verimli yöntem7,8. İlginçtir, adventif çekimleri bu tür phytohormone tedavi8olmadan bağlı olmak.
Epidermis callusing olmadan internodal ventrikül apikal bölgede, ancak Bazal Bölge9üzerinde adventif sürgünler oluşur. Bu fark, muhtemelen phytohormonal yönetmelik altında doku polarite internodal segmentleri, ın gösterir. Ipecac kültür sistemi değişiklikleri endojen phytohormone düzeylerinde adventif ateş oluşumu sırasında analiz etmek eşsiz bir fırsat sağlar. Burada bizim yöntem ve endojen düzeyde bir auxin (indol-3-Asetik asit (IAA)) ve dört CKs analizi için tanıtmak (isopentenyl adenin (IP), isopentenyl adenin riboside (IPR), trans-zeatin (tZ) ve trans-zeatin riboside (tZR)) LC-MS/MS kullanımı ile internodal segmentlerinde.

<table border="0" cellpadding="0" cellspacing="0" style="width:677px;" width="678">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]indole-3-acetic acid</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">031 1531</td>
			<td style="width:167px;">Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]trans-zeatin</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0301</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[2H5]trans-zeatin riboside</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0311</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H6]N6-isopentenyl adenine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0161</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">[2H6]N6-isopentenyl adenosine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0171</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">indole-3-acetic acid</td>
			<td style="width:176px;">Wako</td>
			<td>098 00181</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trans-zeatin</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">Z0876 5MG</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-zeatin riboside</td>
			<td style="width:176px;">Wako</td>
			<td>262 01081</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenine</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">D7674 1G</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenosine</td>
			<td style="width:176px;">ACROS ORGANICS</td>
			<td style="width:119px;">22648 1000</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">acetonitrile hypergrade for LC-MS LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.00029.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Water for chromatography LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.15333.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:176px;">SHIMADZU</td>
			<td style="width:119px;">Prominence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS</td>
			<td style="width:176px;">Sciex</td>
			<td style="width:119px;">3200QTRAP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis HLB 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">WAT094225</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">screw neck total recovery vial</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002805</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000274</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Acquity UPLC BEH C18, 2.1x100 mm</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002350</td>
			<td>UPLC column</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Proshell 120 EC-C18, 2.1x50 mm</td>
			<td style="width:176px;">Agilent</td>
			<td style="width:119px;">699775-902</td>
			<td>UPLC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">DHS1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser II</td>
			<td style="width:176px;">QIAGEN</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 4</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Savant SpeedVac/Refregerated vapor trap</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SPD111/RVT4104</td>
			<td>vacuum concentrartor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable glass tobe (13x100 mm)</td>
			<td style="width:176px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile petri dish</td>
			<td style="width:176px;">INA OPTICA</td>
			<td style="width:119px;">I-90-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

Adventif ateş oluşumu, transgenik bitkiler yenilenme ve ekonomik açıdan önemli bitkileri yayılması için önemli bir tekniktir. Phytohormone tedavi çoğu türler adventif sürgünler indüksiyon için gereklidir. Olup olmadığı sürgünler adventif bağlı olmak auxin ve cytokinin (CK) arasındaki dengeyi belirlenir düzeyleri. Çok çaba optimum konsantrasyonları ve etki explants kullanılan her doku ve her bitki türü birleşimlerini belirlenmesinde gider. Ipecac içinde ancak, adventif sürgünler kültür orta phytohormone tedavi olmadan internodal kesimlerindeki indüklenen. Bu Ipecac değerlendirilmek üzere hücre farklılaşması için doğal plastisite sağlar. Ipecac adventif sürgünler ikna etmek için phytohormone ücretsiz B5 Orta % 0,2 gellan sakız ile 5 hafta boyunca katılaşmış bir 14-h ışık/10-h karanlık döngüsü 15 µmol m−2 s– 1 ışık altında 24 ° C'de internodal kesimleri kültürlü. Phytohormone dynamics adventif ateş oluşumu sırasında araştırmak için endojen indol-3-Asetik asit ve CKs segmentlerinde sıvı Kromatografi-tandem kütle spektrometresi tarafından LC-MS/MS şiddetindeydi. Bu yöntem basit bir şekilde analiz endojen indol-3-Asetik asit ve CKs düzeyleri sağlar. Organogenesis diğer bitki türleri içinde sırasında endojen auxin ve CK dinamiklerini araştırmak için uygulanabilir.

1st hafta hiçbir adventif sürgünler oluşmuş. 2nd hafta küçük sürgünler ortaya çıktı. 3rd ve 4th hafta, sayısı çoğunlukla apikal bölgede (ı ve II) artan vurur (Şekil 2A). 5inci hafta, sayı sürgünler yapıldı yaklaşık 7 içinde bölge ı ve II (Şekil 2B) bölgesinde 5. Buna ek olarak, sadece birkaç sürgünler bölgelerde III ve IV kuruldu.
Kültür önce IAA düzeyi bölgede biraz daha yüksek ben (4,1 pg/mg, taze ağırlık (FW)) daha bölgelerde II-IV (~ 2,5 pg/mg FW; Şekil 3). 1st hafta IAA seviyesi artar büyük ölçüde Bölge IV (11,4 pg/mg FW) ve bölgeler ı-III (1.5-2.2 pg/mg FW) biraz azalmış. 2nd hafta için Bölge IV IAA düzeyi azalmış ~ 4,4 pg/mg FW, Bazal Bölge IAA birikimi geçici olduğunu belirten. Kültür 5 hafta tarafından bir IAA konsantrasyon gradyanı, artan bölgesinden ben Bölge IV düzeyleri ile ortaya çıktı.
Kültür daha önce birçok CKs (şekil 3) yalnızca izleme düzeylerini vardı. 1st hafta 13,8 pg/mg FW bölgesinde II ve 18.1 pg/mg FW bölgesi III tZR seviyesi artar. Düzeyleri sonra yavaş yavaş üzerinden 5 hafta kültür azalmıştır. Öte yandan, tZ, IP ve IPR düzeyde değişti sadece biraz kültür sırasında.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56902/56902fig1.jpg"> Resim 1 : İpeka hazırlanması için adventif ateş oluşumu. (A)internodal bir kesimi (8 mm uzun) temiz bir bankta rejenere Ipecac kesilir. (B) ilk Internode adventif ateş oluşumu için kullanıldı. (C) kesimleri 25 mL phytohormone-Alerjik B5 orta adventif sürgünler ikna etmek için Petri yemeklerinde kültürlü. Ölçek çubuğu 1 cm. = <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56902/56902fig2.jpg"> Resim 2 : Adventif dağılımı internodal bir kesiminde kurulan vuruyor. (A)adventif sürgünler 0-5 hafta sonra kültür kurdu. Ölçek çubuğu 5 mm. (B) = kesimleri dört bölgeye (ı-IV) bölümlenmiş ve her bölgedeki adventif sürgün sayısı hafta 5 sayılır. Veri anlamına gelir ± SEM vardır (n = 3). On segmentleri her deneyde kullanılmıştır. NF = bulunamadı. Bu rakam Koike vd değiştirildi 9 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56902/56902fig3.jpg"> Şekil 3 : Zaman-ders analiz internodal kesimleri phytohormone düzeylerinin. Segmentleri dört bölgeye (ı-IV) ayrı kaldık. Endojen IAA ve her bölgedeki CKs (tZ, tZR, IP ve IPR) LC-MS/MS tarafından sayısal. Çünkü IP ve IPR düzeyleri çok düşük, yakınlaştırılmış bir grafik içinde aynı grafik eklenir. Veri anlamına gelir ± SEM vardır (n = 3). Sekiz segmentleri her deneyde kullanılmıştır. Bu rakam Koike vd değiştirildi 9 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Sütun</td>
			<td>IAA: ACQUITY BEH C18 φ2.1 × 100 1.7 µm</td>
		</tr>
<tr>
<td></td>
			<td>CKs: Poroshell EC-C18 φ2.1 × 50 2.7 µm</td>
		</tr>
<tr>
<td>Sıcaklık</td>
			<td>40ºC</td>
		</tr>
<tr>
<td>Mobil faz</td>
			<td>Çözelti c: distile su + % 0.05 Asetik asit</td>
		</tr>
<tr>
<td></td>
			<td>Solvent B: Asetonitril + % 0.05 Asetik asit</td>
		</tr>
<tr>
<td>Akış hızı</td>
			<td>0,35 ml/dk</td>
		</tr>
<tr>
<td>Enjeksiyon hacmi</td>
			<td>18 µl</td>
		</tr>
</tbody></table>Tablo 1: HPLC analiz IAA ve CK durumda.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Perde gaz (a.u.)*</td>
			<td>10 / 40 **</td>
		</tr>
<tr>
<td>Çarpışma gaz (a.u.)*</td>
			<td>5 / 3 **</td>
		</tr>
<tr>
<td>İyon sprey voltaj (V)</td>
			<td>5500</td>
		</tr>
<tr>
<td>Sıcaklık (ºC)</td>
			<td>600</td>
		</tr>
<tr>
<td>İyon kaynağı gaz 1 (a.u.)*</td>
			<td>30</td>
		</tr>
<tr>
<td>İyon kaynağı gaz 2 (a.u.)*</td>
			<td>40 / 80 **</td>
		</tr>
<tr>
<td>Çözünürlük</td>
			<td>Birim</td>
		</tr>
</tbody></table>Tablo 2: İyon kaynağı parametrelerinin. * rasgele birimleri. ** IAA analiz/CK analizi. Bu tablo Koike vd değiştirildi 9
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>Q1 (m/z)</td>
			<td>Q3 (m/z)</td>
			<td>Declustering potansiyel (V)</td>
			<td>Giriş potansiyel (V)</td>
			<td>Çarpışma enerji (V)</td>
		</tr>
<tr>
<td>IAA</td>
			<td>176</td>
			<td>130</td>
			<td>26</td>
			<td>6.5</td>
			<td>21</td>
		</tr>
<tr>
<td>d5-IAA</td>
			<td>181</td>
			<td>134</td>
			<td>36</td>
			<td>4.0</td>
			<td>23</td>
		</tr>
<tr>
<td>tZ</td>
			<td>220</td>
			<td>136</td>
			<td>36</td>
			<td>5.0</td>
			<td>23</td>
		</tr>
<tr>
<td>d5- tZ</td>
			<td>225</td>
			<td>137</td>
			<td>31</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
<tr>
<td>tZR</td>
			<td>352</td>
			<td>220</td>
			<td>41</td>
			<td>5.5</td>
			<td>23</td>
		</tr>
<tr>
<td>d5- tZR</td>
			<td>357</td>
			<td>225</td>
			<td>51</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
<tr>
<td>IP</td>
			<td>204</td>
			<td>136</td>
			<td>31</td>
			<td>9.0</td>
			<td>19</td>
		</tr>
<tr>
<td>d6- IP</td>
			<td>210</td>
			<td>137</td>
			<td>31</td>
			<td>5.5</td>
			<td>21</td>
		</tr>
<tr>
<td>IPR</td>
			<td>336</td>
			<td>204</td>
			<td>31</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
<tr>
<td>d6- IPR</td>
			<td>342</td>
			<td>210</td>
			<td>36</td>
			<td>5.5</td>
			<td>21</td>
		</tr>
</tbody></table>Tablo 3: LC-MS/MS analizi IAA ve CKs MRM geçişler. Bu tablo Koike vd değiştirildi 9

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Ipecac düğümler arası kültür sırasında endojen Auxin ve Cytokinin miktar

Adventif sürgünler Ipecac phytohormone tedavi olmadan internodal kesimlerindeki bağlı olmak. Phytohormone dynamics adventif ateş oluşumu sırasında değerlendirmek için endojen auxin ve cytokinin LC-MS/MS tarafından internodal kesimleri içinde ölçülür.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

Nanyang Technological University

Research

JoVE Journal

Environment

8.9K Görüntüleme.  Toyo University. Adventif sürgünler Ipecac phytohormone tedavi olmadan internodal kesimlerindeki bağlı olmak. Phytohormone dynamics adventif ateş oluşumu sırasında değerlendirmek için endojen auxin ve cytokinin LC-MS/MS tarafından internodal kesimleri içinde ölçülür.

Video: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Enterovirus Kültür ve RT-qPCR Su Norovirüs Oluşum EPA Metot 1615 Ölçüm. Virüs Örneklerinin I. Koleksiyonu

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Çevre

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Ağır Metal ve Mikroalglerden Verimlilik Diğer İnorganik Bulaşanlar Kantitasyonu

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Gerçek zamanlı PCR ile Kıyı tortulları ve Niceleme 16S rRNA Genler ve Transkriptler eşzamanlı DNA-RNA Ekstraksiyon

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

Su akışı ağırlıklı ve tarım kanallar üzerinden askıya alınan parçacıklar drenaj olaylar sırasında yakalama

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Kekik esans uygulama etkisi et kurutma sırasında mikrobiyal yükü

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

Kayalar ve Mineral toplamları x-ışını kırınım sinkrotron tabanlı kullanarak soğuk sıkıştırma sırasında stres dağıtım

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

Kimyasal yalıtım, miktar ve sürüngenler cilt lipidler, ayrılık

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

Termal-Optik Organik/Elemental Karbon Ölçümlerinin Split Nokta Analizi ve Belirsizlik Niceliği

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Taramalı Elektron Mikroskobu için Embriyo, Yumurta Kabuğu ve Mantar Kültürünün İşlenmesi

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Korozif Baca Gazlı Tezgah Ölçekli Fotobiyoreaktörde Mikroalg Ekimi ve Biyokütle Kantifikasyonu

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...