We are grateful to Ms. Kaori Shimizu and Mr. Masaki Takasu of the Graduate School of Life Sciences, Toyo University, for their technical support. This project was supported by research grants from the Special Research Foundation and Bio-Nano Electronics Research Centre of Toyo University (to SK); by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to SK); by the New Project Fund for Risk Assessments, from the Ministry of Economy, Trade and Industry (to SK); by a Grant-in-Aid for Challenging Exploratory Research (award 23651028 to SK); by a Grant-in-Aid for Scientific Research (B) and (C) (award 23310026 and 26340030 to SK); and by a Grant-in-Aid for Strategic Research Base Project for Private Universities (award S1411016 to SK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Bu çalışmada kullanılan Japon Medaka sıkıntı ve rahatsızlık hafifletilmesi için göz önünde bulundurularak, insani Toyo Üniversitesi kurumsal kurallarına uygun olarak tedavi edildi. 1. Gümüş Nanocolloids (SNCS) <ol><li> Saflaştırılmış SNCS satın (20 mg / L-1,% 99.99 saflıkta, partikül damıtılmış su içinde süspansiyon haline getirilmiş 28.4 ± 8.5 nm ila ortalama çap). </li><li> Indüktif eşleşmiş plazma kütle spektrometresi ile gümüş saflık ve konsantrasyon doğrulamak (ICP-MS) kullanma kılavuzuna 8&#39;e göre analiz eder. ICP-MS için tedavi öncesi yöntemi Bölüm 7&#39;de açıklanan analizler. </li></ol> SNC Çözümleri Farklı tuzluluk ile (Gümüş Colloids ve Ag + Karışımlarının) 2. Hazırlık <ol><li> 60 g NaCl, 1.8 g KCI, 2.4 g CaCI2 · 2H 2 O ve 9.78 gr MgSO 4 · 7H 2 O Arıza sebebinin L 1 aşağıdakilerden oluşan 60 x ERM&#39;yi hazırlanmasırapure su; ultra saf su içinde NaHCO 3% 1.25 ile 7.0 arasında pH değeri ayarlanır. </li><li> gece boyunca 25 ° C&#39;de bir ERM çözeltisi karıştırılır. </li><li> seyreltilmiş ERM ile SNCS karıştırın. Her SNC-ERM karışık çözelti, 40 ml hazırlayın. Nihai konsantrasyon ERM farklı konsantrasyonlarda SNCS 10 mg / L -1 (1x, 5x, 10x, 15x, 20x, 30x veya). </li><li> Ultra saf su içinde% 0.625 ile 7.0 arasında SNC-ERM karışık çözelti NaHCO 3 pH ayarlayın. Ag + salma asidik koşullar 9 kolaylaştırılır, çünkü pH ayarlaması SNC çözeltisinin hazırlanması için çok önemlidir. </li><li> SNCS için bir referans bileşiği olarak iyodinin 3 kullanın. <ol><li> Sulandırılmış ERM ile Agno 3 karıştırın. 15.7 mg / L arasında bir iyodinin 3 konsantrasyonda iyodinin 3 -ERM karışık çözelti, 40 ml hazırlamak -1 ERM&#39;in farklı konsantrasyonlarda (10 mg / L-1 gümüş) (1 x, 5x, 10x, 15x, 20x veya 30x) . Not: AgNO 3 gümüş kolloid toksisitesini incelemek içinçözünür bir gümüş kaynağı solüsyonu, gümüş koloitler ve çözünür gümüş karışımı SNCS için bir referans bileşik olarak kullanılır. </li></ol></li></ol> 3. Medaka Kültür ve Yumurta Hasat <ol><li> Medaka (O. latipes) (turuncu-kırmızı suşu) (60 erkek ve 60 kadın) edinin. </li><li> gruplar olarak kültür Medaka (20 erkek ve bir grup olarak 20 kadın) bir Medaka akış kültür sistemi kullanılarak 3 L tanklarda 1x ERM. <ol><li> Aşağıdaki koşullarda Kültür: kültür ortamının pH aralığı 6.2 ila 6.5 Işık: karanlık döngüsü: 16: 8 saat Kültür ortamının sıcaklığı: 24 ± 0.5 ° C kültür ortamının ozmotik basınç: 257 mOsm </li></ol></li><li> (Günde beş kez) (günde bir kez) saat 10:00 &#39;Artemia salina nauplii üzerinde Medaka Yem ve 09:00, 11:00, 13:00, 15:00 ve 17:00 yapay kuru balık diyet yem. <ol><li> A. elde salina nauplii. </li> &lt;li&gt; plastik kabı içinde bir% 3.0 tuz solüsyonu 5 L hazırlayın. </li><li> beher tuz çözeltisine tuzlu su karidesi yumurta 30 g ekleyin. </li><li> Bir havalandırma pompası kullanılarak (4 L / dk-1) geçirilerek, 48 saat boyunca 25 ° C&#39;de kuluçkaya. </li><li> 48 saat sonra, köpüren durdurun. </li><li> 5 ila 10 dk yumurtadan A. ayırmak için çözüm bekletin çıkmamış yumurta ve yumurta kabuğu arasından salina nauplii (çözelti alt kısmı) (çözelti üst kısmı). </li><li> durultma yoluyla çözeltinin üst tabakayı çıkarın. </li><li> 283 um gözenekli bir elekten çözelti alt kısmının filtre ve 198 um arasında açıklıkları olan bir net geçmesine nauplii toplar. </li><li> 6 saat içerisinde Medaka için nauplii besleyin. </li></ol></li><li> kadın Medaka olurken sonra, dişilerin organları hafifçe dış yumurta kümeleri kaldırmak veya sm kullanarak akvaryum dibinden yumurta toplamakTüm ağ (net büyüklüğü 5 cm x 5 cm, delik boyutu 0.2 mm x 0.2 mm). </li><li> 5 saniye boyunca musluk suyu akan yumurta küme durulayın. </li><li> 30x ERM çözüm durulanır yumurta kümelerinin tüm ekleyin. </li><li> 1 dakika sonra çözeltiden kümeleri kaldırın ve yavaşça kuru kağıt havlu ve silindir arasındaki yumurta kümeleri yerleştirin. </li><li> 30x ERM geri yumurta koymak. </li><li> bir mikroskop altında döllenmiş yumurta seçin. </li><li> forseps kullanarak altı-iyi plastik plakalar 1x ERM 810 yumurta, seçilen yerin. </li><li> 21. gelişim aşamasına kadar bir kuluçka 25 ± 0.1 ° C&#39;de kuluçkaya (Medaka embriyoların gelişim aşamaları Iwamatsu 10 çalışmalarından tanımlanmıştır.) </li><li> bir mikroskop altında gelişme aşamasında 21 inkübe yumurta dışarı seçin. </li><li> 1x ERM ile seçilen yumurta durulayın. </li><li> Pozlama deneyleri (bölüm 4) ile durulanır yumurta Konu. </li></ol> 4. Toksisite SNCS test veya AgNO 3 </sub&gt; Farklı ERM tuzluluk de <ol><li> Medaka yumurta yıkayın (aşama 21), test çözeltisi [SNCS (10 mg / L-1) ya da iyodinin 3 ile üç kez ERM&#39;in her bir konsantrasyonu (1 x, 5x (15.7 mg / L-1, 10 mg / L-1, gümüş gibi) , 10x, pH 7&#39;de 15x, 20x, 30x veya)]. Kontroller olarak, pH 7, 30 x ERM&#39;e 1 x yumurta kullanımı. </li><li> Altı oyuklu plastik plakalar her bir test çözeltisi, 5 ml 15 durulandı yumurta ekleyin. (SNC için poz deneyler üç kez yapın veya AgNO 3 toksisite her test solüsyonu kullanarak test.) </li><li> alüminyum folyo tabak sarın. </li><li> Enkübasyon kadar ya da 14 gün boyunca karanlıkta 25 ° C &#39;de sarılmış inkübe edin. </li><li> Biyolojik değişiklikler ve ölü yumurta maruz yumurta her 24 saat gözlemlemek (Şekil 1 ve 2). </li><li> Test çözümleri her 24 saat değişimi. </li><li> aşağıdaki gibi gözlemleri gerçekleştirin. <ol><li> maruz kalma 6. günde, o (15 saniyede) kalp hızını saymakbir kronometre (Şekil 3a) ile bir mikroskop altında F Medaka embriyolar. </li><li> Maruziyet 6. günde, bir mikrometre (Şekil 3b) kullanılarak bir mikroskop altında Medaka embriyoların göz boyutu (çap) ölçün. </li><li> Kuluçka gününde, bir mikrometre (Şekil 3c) kullanarak bir mikroskop altında larva tam vücut uzunluklarının. </li><li> 14 gün (Şekil 3d) üzerinde yumurtadan maruz yumurta sayısını saymak. </li></ol></li></ol> SNC Çözüm Çözünür Gümüş ve Gümüş Analizi 5. İzolasyon <ol><li> 14,000 x bir 3 kDa&#39;lık bir zar filtre ile filtre her SNC çözeltisi (gümüş kolloidler ve çözünür gümüş bir karışımı) çözünür gümüş izole g ve 10 dakika süre ile 4 ° C. 1x ERM&#39;in agrege SNCS bildirilen ortalama çapı 67.8 mil 11, çünkü SNCS çözünür gümüş izole etmek için 3 kDa&#39;lık bir zar filtresi kullanınnd Ag + o 0.162 nm 12&#39;dir; 3 kDa&#39;lık bir membran 2 nm 13 ya da daha fazla bir çapa sahip olan parçacıklar hariç tutar. </li><li> ICP-MS yapan kılavuzu 8&#39;e göre, ICP-MS analizi (Şekil 3E) tarafından filtre edilmiş çözelti (= çözünebilen gümüş konsantrasyonu), 50 ul gümüş konsantrasyonu ölçümü. ICP-MS için tedavi öncesi yöntemi Bölüm 7&#39;de açıklanan analizler. </li></ol> Medaka Embriyolar Gümüş Biyoakümülasyon 6. Ölçüm <ol><li> Bölüm 4&#39;de anlatıldığı gibi Medaka SNCS yumurta (evre 21) ya da iyodinin 3 Açığa. </li><li> Maruziyet 6. günde, Medaka Kitap 14&#39;te tarif edilen protokole göre enzim kuluçka Medaka&#39;nın kullanılarak yumurta (yani, dechorion) koryon çıkarın. </li><li> ICP-MS işletim manuel 8 (Şekil 3f) &#39;ye göre ICP-MS analizi ile dechorioned yumurta gümüş konsantrasyonu ölçün. önICP-MS yöntemi, Bölüm 7&#39;de açıklanan analizler. </li></ol> ICP-MS Analizi ile Gümüş Konsantrasyon 7. ölçümü <ol><li> (; Bölüm 1 gümüş konsantrasyonu doğrulama için) örnekleri [gümüş çözeltisi 50 ul ekleyin; Üç dechorionated embriyolar (bölüm 5); ya da bir 50 mL teflon çanağa filtre edilmiş çözelti (bölüm 5)], 50 ul. </li><li> 50 ml&#39;lik bir cam kap, aşırı saf, nitrik asit 2.0 ml ilave edilir. </li><li> (yaklaşık 3 saat) kurur hemen öncesine kadar 110 ° C&#39;de sıcak bir plaka üzerinde karışımı ısıtın. </li><li> Tamamen organik madde çözülür 2.0 ultra saf nitrik asit ilave edildi ve beher için hidrojen peroksit, 0.5 ml ekleyin. </li><li> (yaklaşık 3 saat) kurur hemen öncesine kadar sıcak plaka üzerinde yine karışımı ısıtın. </li><li> % 1.0, aşırı saf, nitrik asit çözeltisinin 4 ml artığı içinde çözündürülür. </li><li> Transfer santrifüj tüpüne 4 ml. </li><li> (Üç kez olmak üzere toplam) iki kez 7.7 7.6 tekrarlayın. Nihai hacim 12.0;mi. </li><li> Kullanma kılavuzuna 8&#39;e göre ICP-MS analizi ile (% 1.0 ultra saf nitrik asit içinde eritilmiş) numune gümüş konsantrasyonu ölçümü. <ol><li> Bir iç ve bir dış standart solüsyonu kullanın gümüş konsantrasyonu ölçmek için (Malzeme Listesi). iç ve dış standart bir çözüm Laboratuvar Akreditasyon Amerikan Derneği (A2LA) tarafından akredite edilmiştir. Gümüş tespit limitleri 0.0018 ng / ml -1 (çözüm) ve 0.016 ng mg ağırlık -1 (embriyo gövdesini) idi. </li></ol></li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD Guidelines for the Testing of Chemicals, Section 2 Effects on Biotic Systems. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> National Coastal Condition Report. , (2001).</li><li>Sakaizumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+inorganic+salts+on+mercury-compound+toxicity+to+the+embryos+of+the+Medaka,+Oryzias+latipes.">Effect of inorganic salts on mercury-compound toxicity to the embryos of the Medaka, Oryzias latipes.</a> J. Fac. Sci. Univ. Tokyo. 14 (4), 369-384 (1980).</li><li>Sumitani, K., Kashiwada, S., Osaki, K., Yamada, M., Mohri, S., Yasumasu, S., 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=Medaka+(Oryzias+latipes)+Embryo+toxicity+of+treated+leachate+from+waste-landfill+sites.">Medaka (Oryzias latipes) Embryo toxicity of treated leachate from waste-landfill sites.</a> J. Jpn. Soc. Waste Manage. Exp. 15 (6), 472-479 (2004).</li><li>Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Nanoparticles+in+the+See-through+Medaka+(Oryzias+latipes).">Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes).</a> EHP. 114 (11), 1697-1702 (2006).</li><li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Integrated Book for the Biology of the Medaka. , (2006).</li><li>Miyamoto, T., Machida, T., Kawashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environmental+salinity+on+the+development+of+chloride+cells+of+freshwater+and+brackish-water+medaka,+Oryzias+latipes.">Influence of environmental salinity on the development of chloride cells of freshwater and brackish-water medaka, Oryzias latipes.</a> Zoo. Sci. 3 (5), 859-865 (1986).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> XSERIES 2 ICP-MS Getting Started Guide Revision B - 121 9590. , (2007).</li><li>Kashiwada, S., Ariza, M. E., Kawaguchi, T., Nakagame, Y., Jayasinghe, B. S., Gartner, 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=Silver+nanocolloids+disrupt+medaka+embryogenesis+through+vital+gene+expressions.">Silver nanocolloids disrupt medaka embryogenesis through vital gene expressions.</a> ES &amp; T. 46 (11), 6278-6287 (2012).</li><li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Mech. Dev. 121, 605-618 (2004).</li><li>Kataoka, C., Ariyoshi, T., Kawaguchi, H., Nagasaka, S., Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity+increases+the+toxicity+of+silver+nanocolloids+to+Japanese+medaka+embryos.">Salinity increases the toxicity of silver nanocolloids to Japanese medaka embryos.</a> Environ. Sci.: Nano. 2, 94-103 (2014).</li><li>Shannon, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+effective+ionic+radii+and+systematic+studies+of+interatomic+distances+in+halides+and+chalcogenides.">Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides.</a> Acta Cryst. 32, 751-767 (1976).</li><li>Wakamatsu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Medaka Book, 6.1: Preparation of hatching enzyme. , (2015).</li></ol>

The authors declare that they have no competing financial interests.

Medaka deniz suyu son derece hoşgörülü bir tatlı su balığıdır; o da bu balığın orijinal doğal yaşam Japon sahil 6 kapalı tuzlu su olduğu bilinmemektedir. Bu nedenle, Medaka balık klorid hücreleri 7 iyi gelişmiştir. Bu benzersiz özellik balık sadece tek bir tür kullanarak (tatlı su deniz suyu için) tuzluluk bir fonksiyonu olarak çevre kimyasalların toksisite test etmek için yeni bir yol ile bilim adamları sağlar. aşamada 21, Medaka yumurta ...

1979 yılında test kimyasallar için Ekonomik İşbirliği ve Kalkınma Örgütü (OECD) test kriterlerine Teşkilatı&#39;nın kuruluşundan bu yana, 38 deney kılavuzları kurallar, Biyotik Sistemleri 1 üzerine etkileri Bölüm 2&#39;de yayınlanmıştır. tatlı su habitatlarının, yani tatlı su bitkilerden olmuştur test Sudaki organizmalar Tüm; yosun; Böyle piresi ve chironomids olarak omurgasızlar; Bu tür Medaka, Zebra balığı ve gökkuşağı alabalığı gibi balıklar. tuzlu su ortamlarına kıyasla, tatlı su ortamları daha doğrudan insan ekonomik ve endüstriyel faaliyetler etkilenir. Onlar kirlilikten daha yüksek risk altındadır, çünkü bu nedenle, tatlı su ortamları test için ön plana çıkmışlardır. Haliç dahil kıyı bölgelerde, tuzluluk acı su ve deniz suyu koşulları arasında değişir ve bu alanlar genellikle endüstriyel aktivite 2 tarafından kirletilmektedir. Kıyı alanları ve bunların ilişkili sulak h ile karakterizedirüksek ekolojik biyolojik çeşitlilik ve verimlilik. Kıyı ekosistemleri nedenle kimyasal kirlenmeye karşı korunmalıdır. Ancak, acı su ve deniz suyu ortamlarında ekotoksikolojik araştırma var sınırlı kalmıştır. Sakaizumi 3 Japon Medaka yumurta metil civa ve tuzluluk arasındaki toksik etkileşimler okudu ve test çözeltisi ozmotik basıncı artırarak metil civa toksisitesini arttırdı bulundu. . Sumitani ark 4 sızıntı suyu toksisitesini araştırmak için Medaka yumurta kullanıldı; Onlar yumurtalara sızıntı suyunun osmotik denklik embriyogenez sırasında anormallikleri uyaran anahtarı olduğunu ortaya koymuştur. Buna ek olarak, Kashiwada 5 plastik nanopartiküller (çapı 39,4 nm) kolay tuzlu koşullar altında Medaka yumurta koryon (15x embriyo yetiştirme ortamı (ERM)) üzerinden nüfuz bildirmiştir. Tipik bir küçük balık modeli, Japon Medaka (Oryzias latipes </em&gt;) Temel biyoloji ve ekotoksikolojisine 6 kullanılmaktadır. Japon Medaka deniz suyuna tatlı gelen dolayı son derece gelişmiş klorür hücrelerinin 7 arasında değişen koşullarda yaşayabilir. Bunlar tuzluluk geniş bir yelpazede olan koşullarda test etmek için yararlı olduğu umulmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Silver nanocolloids</td>
			<td style="width:188px;">Utopia Silver Supplements</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">NaCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31319-45</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">KCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">28513-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#183;2H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">06730-15</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">21002-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3&#160;</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31212-25</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgNO3</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31018-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>HORIBA, Ltd.</td>
			<td style="width:119px;">F-51S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balance</td>
			<td>Mettler-Toledo International Inc.</td>
			<td style="width:119px;">MS204S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka (Oryzias latipes) orange-red strain&#160;</td>
			<td colspan="2">National Institute for Environmental Studies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka flow-through culturing system</td>
			<td>Meito Suien Co.</td>
			<td>MEITOsystem</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Artemia salina nauplii eggs</td>
			<td>Japan pet design Co. Ltd</td>
			<td>4975677033759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aeration pomp</td>
			<td>Japan pet design Co. Ltd</td>
			<td>non-noise w300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otohime larval &#946;-1</td>
			<td style="width:188px;">Marubeni Nissin Feed Co. Ltd</td>
			<td style="width:119px;">Otohime larval &#946;-1</td>
			<td>Artificial dry fish diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td style="width:188px;">Leica microsystems</td>
			<td style="width:119px;">M165FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micrometer</td>
			<td>Fujikogaku, Ltd.</td>
			<td>10450023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Nksystem</td>
			<td>TG-180-5LB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">shaker</td>
			<td style="width:188px;">ELMI Ltd.</td>
			<td style="width:119px;">Aizkraukles 21-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plastic plates</td>
			<td>Greiner CELLSTAR</td>
			<td style="width:119px;">M8562-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum foil</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">6-713-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stopwatch</td>
			<td>DRETEC Co. Ltd.</td>
			<td style="width:119px;">SW-111YE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-kDa membrane filter</td>
			<td>EMD Millipore Corporation</td>
			<td colspan="2">0.5-mL centrifugal-type filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-mL Teflon beaker</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">33431097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-538</td>
			<td>For internal standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-622</td>
			<td>For external standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ultrapure nitric acid</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">28163-5B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">hydrogen peroxide&#160;</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">18084-1B</td>
			<td>for atomic absorption spectrometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ICP-MS</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Thermo Scientific X Series 2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hot plate</td>
			<td>Tiger Co.</td>
			<td>CRC-A300</td>
			<td></td>
		</tr>
	</tbody>
</table>

salinity-dependent toxicity assay of silver nanocolloids using medaka eggs

Tuzluluk su ortamında önemli bir özelliğidir. Sudaki organizmalar için tatlı su, tuzlu su ve deniz suyunun yaşam alanları tanımlar. kimyasallar ve Sucul organizmalar için ekolojik riskler değerlendirmelerinin toksisite testleri sıklıkla tatlı yapılmaktadır, ancak Sucul organizmalar için kimyasalların toksisite pH, sıcaklık, tuzluluk ve bağlıdır. Hiçbir yöntem Sudaki organizmalar için toksisite tuzluluk bağımlılığını test etmek için, ancak var. Onlar tatlı su, tuzlu su ve deniz suyuna adapte çünkü Burada, Medaka (Oryzias latipes) kullanılır. embriyo yetiştirme ortamı (ERM) (1x, 5x, 10x, 15x, 20x, 30x ve) yumurta (1x ERM ve 30x ERM Medaka gümüş nanocolloidal parçacıklar (SNCS) toksisitesini test etmek için kullanılmıştır sahip ozmotik basıncı eşdeğer farklı konsantrasyonlarda sırasıyla tatlı ve deniz suyu, için). Altı oyuklu plastik tabak olarak, üç kopya halinde 15 Medaka yumurta 10 mg / L ve en SNCS maruz bırakıldı# 8722, 1, karanlıkta, pH 7 ve 25 ° C &#39;de ERM&#39;in farklı konsantrasyonlarda. Gün 6 ve gündüz (bölüm 4) kuluçkalık üzerine larva tam vücut uzunluğu 15 sn ve göz çapı başına kalp hızını ölçmek için bir diseksiyon mikroskobu ve bir mikrometre kullanılır. embriyolar kuluçka veya gün 14 kadar gözlenmiştir; Daha sonra 14 gün (bölüm 4) her gün çıkma oranı sayıldı. embriyolarda gümüş birikimi görmek için tümevarımsal gümüş testi çözümleri konsantrasyonu (Bölüm 5) ve dechorionated embriyolar (bölüm 6) açıkça tuzluluk arttıkça artan Medaka embriyolar SNCS KAG toksisitesini ölçmek için plazma kütle spektrometresi birleştiğinde kullandık. Bu yeni yöntem bizi farklı tuzluluk kimyasalların toksisite test etmenizi sağlar.

SNC toksisite tuzluluk etkisi çok açıktı: deformite veya ölüm indüksiyon tuzluluk bağımlı (Şekil 1 ve 2). Biz (10 mg / L -1) -exposed embriyolar SNC fenotipik biyobelirteçleri (kalp atım hızı, göz boyutu, tam vücut uzunluğu ve çıkma oranı) ölçüldü. Bu fenotipik biyomarkırlar tuzluluk bağımlı SNC toksisite ortaya koydu. Kalp hızı 29.6 32.2 atım / kontr...

Watch this Scientific Journal Video about Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs at JoVE.com

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Medaka Yumurta kullanma Gümüş Nanocolloids tuzluluk bağımlı zehirlilik Deneyi

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

salinity-dependent-toxicity-assay-silver-nanocolloids-using-medaka

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

7.9K Görüntüleme.  Toyo University. Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Video: Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Kimyasal Güvenlik Tarama ve Sistem Biyolojisi Veri Üretimi Gelişim Toksisite Tahliller Tabanlı İnsan Pluripotent Kök Hücre

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Gelişim Biyolojisi

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Nematod kullanma Germline Kimyasal zehirlilik Kapsamlı Değerlendirme<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Statik Organ Kültürü kullanarak Palatal Fusion incelenmesi yöntemi

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epikardiyal dışsal gelişimi Kültürü Deneyi ve<em&gt; Ex Vivo</em&gt; Epikardiyal türetilmiş Hücre Göç Değerlendirilmesi

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

İlaç Tedavi ve<em&gt; İn Vivo</em&gt; Bir Medaka Balık Osteoporoz Modelinde Osteoblast-Osteoklast Etkileşimler Görüntüleme

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Perisitlerden tahlil filizlenen bir endotel hücre boncuk birleşmeyle

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC tüp-formasyon assay doğal ürünlerin anjiyogenezi üzerinde etkisini değerlendirmek için

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Enjekte Gryllus bimaculatus Yumurta

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Endotel Kolonisi Oluşturan Hücreler ve Mezenkimal Kök Hücreler tarafından Oluşturulan Eş Kültür Sfaoidler Kullanılarak Üç Boyutlu Anjiyogenez Tsay Sistemi

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Yakınlık Ligasyon Tayini kullanılarak C. elegans Germline Ribonükleoprotein Kompleksi Montaj Yerinde Algılama

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...