Ben yardım için deneysel tasarım kavramsallaştırma ve malzeme ile yardımcı bir tedavi rejimi ve otopsi kurulması yardım A. Pessier analizleri, MWH Chatfield, MJ Robak, K. kılıfları ve D. Lenger yardım için CL Richards-Zawacki teşekkür etmek istiyorum laboratuar ve çekimler sırasında kendi alanı kullanımı için destek ve Taronga Hayvanat Bahçesi&#39;nde M. McFadden ve P. Harlow. Proje kısmen LA Brannelly verilen Louisiana Çevre Eğitim Komisyonu hibe yoluyla ve Yaşam Teknolojileri tarafından Yaban Hayatı ve Balıkçılık Louisiana Bakanlığı tarafından finanse edildi.

Etik Onay: Bu çalışma ve yöntemleri Tulane Üniversitesi Hayvan Kullanım ve Bakım Komitesi (. Protokol no 0407) tarafından onaylanmıştır. 1.. Bd Enfeksiyon için test <ol><li> Bd için sürüntü <ol><li> Steril pamuklu çubukla sürüntü hayvanlar Ventrum beş kez, her uyluk, her tarafta, ve 45 darbeleri toplam her bacak üzerinde beş kez. Swab amfibi ciltte Bd için örnekleme noninvaziv bir yöntemdir. </li><li> Yavaşça DNA&#39;nın büyük miktar bez üzerinde toplanan sağlamak vuruşları sırasında ve arasında çubukla döndürün. </li><li> -20 ° C&#39;de 1.5 ml mikrotüplerde Mağazası bezlerden </li></ol></li><li> DNA Ekstraksiyon ve qPCR tahlil <ol><li> Hayvan dokusu için talimatlara uygun olarak, ticari olarak temin edilebilir DNA izolasyon kiti kullanılarak bezlerden alınan genomik DNA ekstrakte edin. </li><li> Boyle aşağıdaki kantitatif gerçek zamanlı PCR (qPCR) kullanılarak ekstre DNA analiz <em&gt; ve arkadaşları 17, aşağıdaki değişikliklerle birlikte yapılmıştır.: </li><li> Çift deiyonize su ile DNA ekstraksiyon numuneleri 01:10 sulandırmak. </li><li> PCR inhibisyonunu önlemek için, sığır serum albümini (BSA) içinde 0.7 ul ekleyin. </li><li> PCR inhibisyonu sonucu etkilemeyen kalmadığından emin olmak için, her numune içinde bir dahili pozitif kontrol içerir. </li><li> Pozitif ve negatif kontrol ve zoospore (enfeksiyon) yük tahmin etmek için seyreltme standartlar serisi ile örnekler. </li></ol></li></ol> 2. Aşılama <ol><li> Kültür Bd <ol><li> (40 dakika boyunca 121 ° C) otoklava ve soğumaya bırakılmıştır, 1 L ters ozmoz veya deiyonize edilmiş su, 10 g tripton eklenerek tripton suyu hazırlayın. Bir kez 23 ° C&#39;ye kadar soğutuldu, 1 ml penisilin-streptomisin ilave edin. </li><li> 23 ° C&#39;de yedi gün boyunca tripton suyu içinde (a Phyllomedusa maki ikinci Guabal, Panama izole J. Longcore ikinci JEL411) Bd suşu büyümek </li></ol></li><li> B aktarınRoth kültür Tripton ve agar plakaları için. <ol><li> Plakaları yapmak için, 1 L ters ozmoz veya deiyonize su ve otoklav (40 dakika boyunca 121 ° C) 10 g tripton ve 10 g agar ekleyin. </li><li> Otoklava karışım dokunmak için yeterince serin, ama congeals önce, bir laminer akış kaputu, Petri kutularına tam 1/4-1/3 dökün kez. </li><li> Agar tamamen pıhtılaşmış ve soğuduğunda, her plakaya 0.5 ml Bd suyu ekleyin ve plaka çevresinde yayılır. Kurumasını bekleyin. </li><li> Plakalar Bd zoospores, Parafilm ile mühür plakaları ile aşılanır kez. </li><li> Plakalar 5-7 gün boyunca 23 ° C&#39;de inkübe izin verin. Aşılamadan önce canlılığı sağlamak için hareket zoospore edin. </li></ol></li><li> Zoospore aşılama çözümü için Sel plakalar <ol><li> Bd inkübasyon ve büyüme 5-7 gün sonra, dokunun yaş 5 ml su ile her bir plak sel ve zoospor, 10 dakika boyunca ortam girmesine izin verir. </li><li> Zoospore suspe döküntaşınabilir bir kap içine nsion. </li><li> Bir hemasitometre ile zoospore konsantrasyonu tahmin. </li><li> Yaşlı musluk suyu ile 1 x 10 6 başına 3 ml &#39;lik bir konsantrasyona kadar seyreltin. </li></ol></li><li> Hayvanları aşılamak <ol><li> Tek tek 50 ml kaplarda, kendi Ventrum boyunca 3 ml aşılama karışımının dökülmesiyle her hayvanı aşılamak. İlave aşılama aşılama kabın tabanına damla izin verin. </li><li> Her bir hayvan, enfeksiyonun sağlama alınması için 24 st için aşı kapta kalır. </li><li> Aşılamadan 24 saat sonra, ayrı ayrı hayvanlar için dezenfekte terraryumlarda döndürür. </li><li> Enfeksiyon iki hafta boyunca inşa etmek için izin verin. Çubukla ve qPCR sonuçları ile enfeksiyon onaylayın ve tedaviye başlar. </li><li> BD-Negatif kontroller için 5 ml sel Bd içermeyen agar plakaları yukarıdaki gibi aynı yöntem kullanılarak musluk suyu ve mock aşılanması yaşlı hayvanlar. </li></ol></li></ol> 3. Tedavi Rejimi <ol><li> Prepamuamele banyosu re <ol><li> 1 L Amphibian Ringer Çözeltisi (- en etkili tedavi konsantrasyonu% 0.0025 itrakonazol) 2.50 ml sulu itrakonazol (% 1 Sporanox Oral çözelti) eklenir. <ol><li> Amphibian Ringer Solüsyonu tarifi: 1 L ters osmoz veya iyonu giderilmiş su, 6.6 g sodyum klorür, 0.15 g potasyum klorür, 0.15 g kalsiyum klorür ve 0.2 g sodyum bikarbonat. Otoklav çözeltisi (40 dakika boyunca 121 ° C) ve oda sıcaklığına soğumaya bırakın. </li><li> % 0.01 itrakonazol muamele konsantrasyonu için, 1 L Amphibian Ringer solüsyonuna 10,00 ml sulu itrakonazol (% 1 Sporanox Oral çözelti) ekleyin </li><li> % 0.005 itrakonazol tedavi konsantrasyonu için, 1 L Amphibian Ringer solüsyonuna 5.00 mL sulu itrakonazol (% 1 Sporanox: Oral solüsyon) ekleyin. </li></ol></li><li> Bd-pozitif ve Bd-negatif kontroller, hiç itrakonazol ile Amphibian Ringer Çözeltisi içine hayvanlar banyo. </li></ol></li><li> Tedavi &lt;ol&gt; <li> Muamele çözeltisinin 35 ml&#39;si ile dezenfekte 50 mi kaplarda tek tek amfibiler yerleştirin. </li><li> Emin hayvanlar tamamen hafifçe kabı dönen beş dakika boyunca kaplıdır. </li><li> Dezenfekte terraryumlarda hayvanları dönün. Altı gün boyunca tekrarlayın. </li></ol></li><li> Tedavi başarısını sağlamak <ol><li> Günlük tedavi Bd bulaşmasına Test hayvanları başlar. </li><li> Enfeksiyon sağlamak için dört hafta boyunca haftada temizlenir Bd enfeksiyon için test hayvanları bir hafta tedavi tamamlandıktan sonra, ve bir kez. </li></ol></li></ol> Bir Biohazard olarak 4. Bd <ol><li> Bireysel terraria haftalık dezenfekte edin. <ol><li> % 10 çamaşır suyu solüsyonu banyosunda terraria ve tedavi kaplar batırın. </li><li> Akan su ile iki kez, her ağartılmış kabı durulayın. </li><li> Kaplar tekrar kullanmadan önce 24 saat kurumasını bekleyin. </li></ol></li><li> Çapraz bulaşmayı önlemek için hayvanları tutarken temiz nitril eldiven giyinBd patojenin. Her hayvan taşıma arasındaki eldiven değiştirmek. </li><li> % 10 çamaşır suyu ile çözüm getirerek atmadan önce tüm sıvı atık dezenfekte edin. </li><li> Potansiyel Atmadan önce bd&#39;ye maruz kaldığı tüm katı atık otoklavlayın. </li></ol>

<ol>
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Yazar, hiçbir rakip mali çıkarlarını bildirir.

Bu protokolü kullanarak biz güvenle ve etkili amfibialarda Bd için itrakonazol tedavisi tedavi dozunu ve süresini azaltmak için başardık. Düşük tedavi dozu ve süresi (altı gün boyunca 0.0025%) subklinik enfekte I. başarılı bir tedavi oldu nebulifer. Daha düşük bir doz ve tedavi süresi hem de etkinliği Amfibilerin bütün türlerin ve yaş sınıfları için kullanılacak hiçbir güvenli ve etkili tedaviler şu anda olduğu gibi, Bd enfeksiyon tedavileri için kli...

Amfibiler şu anda tüm omurgalı takson 1 biyoçeşitliliğin büyük düşüş yaşıyoruz. . Chytridiomycosis, chytrid fungal patojen Batrachochytrium dendrobatidis (BD) neden olduğu bir deri hastalığıdır, Bd biyoçeşitlilik kaybına neden rekor kötü patojen olan bu dramatik düşüşler 2 önemli nedenlerinden biridir: amfibiyenlere 600 tür üzerinde bozar ve var 200 kadar türü kritik tehlike altında veya 2,3 soyu tükenmiş olan 300&#39;den fazla türün düşüşler neden oldu. Amfibi chytrid mantar özgü ev sahibi değil ve ciddi hastalığa neden ilerleme Bd enfeksiyon için izin konağın hangi nitelikleri belli değildir. Chytridiomycosis elektrolit kaybı ve kalp yetmezliği ile sonuçlanan 4, deride iyon kanallarını bozarak ölüme neden olur. Aynı zamanda, (herhangi bir Bd zoosporlar ihtiva) Bd yüzer tetarlar bir bağışıklık tepkisi 5 Leadin neden olurg araştırmacılar Bd ürün 6 toksik üretir inanıyorum. Birçok türün hızla azalmaktadır Çünkü, esir güvence koloniler ve ıslah programlarında önemli koruma önlemleri ve bazı türler için tek can simidi olabilir. Kurulması ve hastalıksız koloni bakımı amfibi yönetimi tesisleri için bir sorundur. Bd enfeksiyonların tedavisi için mevcut kemoterapötik yöntemler, etkili olmasına rağmen, tedavi edilen hayvanlar için zararlı olabilir ve tek bir tedavi yöntemi türleri ve amfibi 7 yaş sınıflar arasında başarılı olmuştur. Bd enfeksiyonu için tedaviler birkaç klinik denemeleri 7 gerçekleştirilmiştir. Tedavinin üç en sık kullanılan yöntemler, ısı terapisi, kloramfenikol ve itrakonazol vardır. Isı terapisi az yan etkileri, çoklu hayat aşamalarında kullanılabilir olması için görünen bir nonchemotherapeutic tedavi seçeneği olduğunu ve homo olduğunumüttefiki etkili. Bir tedavi denemede, Chatfield ve Richards-Zawacki 8 ile muamele edilen hayvanların% 96, 10 gün boyunca 30 ° C &#39;de muhafaza edildikten sonra enfeksiyonların temizlenir olduğunu buldu. Bu deneme% 100 başarılı olmasa da, başkalarının, kendi türleri için ısı terapisi değerlendirmek için bir temel sağlar. Isı tedavisi, birçok türün için etkili olmalarına rağmen, uzun bir süre boyunca yüksek sıcaklıklarda kalamaz birçok dağ türler için pratik değildir. Kloramfenikol, bir bakteri öldürücü, bir çok türde Bd tedavisinde başarılı bir şekilde kullanılmıştır, ve in vitro olarak mantar gelişimini inhibe edilmiştir. Bir antibakteriyel olarak, kloramfenikol, özellikle mantar zarar vermez, ancak ökaryotik hücrelerde apoptosisi 9 neden olduğu bilinmektedir. Bu eylem mekanizması tam olarak bilinmemektedir, ancak kloramfenikol tedavisi, hücre içi sporangia öldürme, deri apoptoza neden olduğu iddia edilmiştir. Herhangi bir klinik deneme, bu nedenle yapılmıştır ve olmasına rağmendoz ve tedavi süresi, değerlendirilmemiştir güncel tedavi protokolleri genellikle karasal amfibiler için bu tedavi pratik yapma, 2-4 hafta 10,11 için suya batmaya gerektirir. İnsan maruz aplastik anemi 12 nadir bir formu ile ilişkili olduğu için kloramfenikol de veteriner kullanımı için bazı ülkelerde sınırlı olmuştur. İtrakonazol, bir azol antifungal, en yaygın olarak tutsak amfibiler Bd enfeksiyonunun tedavisi için kullanılmaktadır. En yaygın olarak kullanılan protokol beş dakika 11 gün boyunca günde bir% 0.01 itrakonazol çözeltisi içinde banyo hayvan. Bu protokol, amfibiler için etkili iken, amfibiler 11,13 klinik çalışmalarda dayanmamaktadır, memeli uygulama için kullanılan standart elde edilmiş ve. Bu protokol, özellikle genus Rana ve tetarlar ve son Metamorphs 11, bazı türlerde olumsuz yan etkilere neden olabilir.Tadpole tedavi itrakonazol çok daha düşük bir konsantrasyonda (% 0.0005) kullanılarak başarılı olmuştur, ama depigmentasyon 14 ile ilişkilendirilmiştir. Bu aynı konsantrasyon son Metamorphs üzerinde trialed ve enfeksiyonu 15 tedavi de başarısız olduğu bulunmuştur. Itrakonazol tedavisi ile olumsuz yan etkilerin riski olduğundan, bazı hekimler 11 gün boyunca bir yarım olağan konsantrasyon (% 0.005) kullanılan ve bu Bd ENFEKSĠYONLARI 16 kür başarılı bulduk. Bu çalışma Bd enfeksiyonunu tedavi etmek için 15 itraconazol&#39;den kullanarak bir klinik tedavi çalışmasının bir özetidir. Protokol itrakonazol ile Bd enfekte hayvanların tedavisinde güvenli ve etkili hem de en düşük doz ve tedavi süresini bulmak için bir girişimde açıklanır. Bu çalışmada kullanılan hayvanların körfez sahili toads son Metamorphs, Incilius nebulifer idi.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Amfibiler herhangi bir omurgalı sınıfının büyük düşüş yaşandığı ve bu düşüşler önde gelen nedeni hastalık chytridiomycosis neden olan bir mantar patojen, Batrachochytrium dendrobatidis (BD), olduğunu. Esir güvence kolonileri tehdit amfibi türlerinin için dünya çapında önemli olan ve nesli kritik tehlike olanlar için tek can simidi olabilir. Hastalıksız koloni bakımı esir yöneticileri bir önceliktir, tüm türler için ve yaşam evreleri arasında henüz güvenli ve etkili tedaviler tespit edilmemiştir. Yaygın olarak kullanılan bir dozaj, bazı bireyler ve türler için zararlı olabilir, ancak en yaygın olarak kullanılan kemoterapötik tedavi, itrakonazol olduğu. Biz itrakonazol daha düşük ve daha güvenli ama etkili doz Bd enfeksiyonları tedavi bulunamadı olmadığını değerlendirmek için klinik tedavi deneme gerçekleştirildi. Bulduğumuz bu 0.01-0.0025 &#39;den% tedavi konsantrasyonu giderek düşürülür ve 11 ila tedavi süresini azaltarak5 dk hamam -6 gün, kurbağalar daha az yan etkileri ve daha az tedavi ile ilişkili mortalite ile Bd enfeksiyon tedavi edilebilir.

Tüm hayvanlar dört hafta sona tedavi sonrasında BD-negatif olsaydı Itrakonazol tedavi başarılı kabul edildi. Tedavi başlamadan önce, tedavisi bittiği, dört hafta sonra, Bd pozitif kontrol hayvanları 16,765.12 bir ortalama zoospore yük varken, aşılamadan iki hafta sonra, her Bd ile aşılanmış hayvanları, ((17,34 ±) 1,121.7 bir zoospore ortalama bir yük ile, enfekte edilmiştir ) 14643,08 ±. Incilius nebulifer tedavisi (n = 15), altı g...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Amfibiler Batrachochytrium dendrobatidis enfeksiyon, azaltılmış konsantrasyonu (% 0.0025) ve genel olarak kullanılan daha itrakonazol daha kısa süre (altı gün boyunca beş dakika banyolar) kullanılarak ortadan kaldırılabilir. Daha az mortalite ve daha az olumsuz yan etkileri bu modifiye tedavi protokolü kullanılarak gözlenmiştir.

İndirimli Itraconazole Konsantrasyon ve Süreleri Tedavisi Başarılı mısınız<em&gt; Batrachochytrium dendrobatidis</em&gt; Amfibi Enfeksiyon

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K Görüntüleme.  James Cook University.  Amfibiler Batrachochytrium dendrobatidis enfeksiyon, azaltılmış konsantrasyonu (% 0.0025) ve genel olarak kullanılan daha itrakonazol daha kısa süre (altı gün boyunca beş dakika banyolar) kullanılarak ortadan kaldırılabilir. Daha az mortalite ve daha az olumsuz yan etkileri bu modifiye tedavi protokolü kullanılarak gözlenmiştir. 

Video: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Alexa Fluor Etiketleme Dang Virüs görselleştirme

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

İmmünoloji ve Enfeksiyon

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Karşı Antibiyotik Etkinlik Test Yapay Balgam Orta Kullanımı<em&gt; Pseudomonas aeruginosa</em&gt; Kistik Fibrozis Akciğer Daha İlgili Koşullar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Toplam Protein Ekstraksiyon ve 2-D Jel Elektroforez Yöntemleri<em&gt; Burkholderia</em&gt; Türler

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Küçük Hint firavunları (Herpestes Auropunctatus) sera bir oral kuduz aşılama biyolojik Marker olarak kullanım Için Iophenoxic asit analogları Analizi

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Farelerde Gardnerella vaginalis Mesane Maruziyeti Tarafından Tetiklenen Tekrarlayan Escherichia coli İdrar Yolu Enfeksiyonu

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Enfeksiyöz Mononükleozlu Çocuklardan Periferik Kan Örneklerinde İmmün Hücre Alt Popülasyonlarının Ayrılması

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

Japon Ensefaliti Aşılı Çocuklarda Lenfositlerin Tespiti için Akış Sitometreleri Arasında Laboratuvarlar Arasında Transferin Standardizasyonu

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...