Biz yazının eleştirel okuma Dr Diane Baronas-Lowell teşekkür ederim. Bu çalışma, Fen Tohum Grant bir FAU Charles E. Schmidt Koleji ve KJ için Ellison Tıp Vakfı bir Yaşlanma Bursu tarafından desteklenen

1.. XLD (ksiloz Lizin desoksikolat) Agar Tabaklar XLD agar XLD agar plakaları üzerinde siyah koloniler görünür Salmonella, bir seçimli büyüme ortamı kullanılarak bir. Kontaminasyon endişeler vardır, ancak normal bir LB plakası ikame edilebilir. <ol><li> 5 ml iyonu giderilmiş su içinde 5.5 g XLD agar ve tekrar süspansiyon tartıldı. </li><li> Tüm ağar ıslak kadar iyice karıştırın. Topaklar gitti ve ortam tamamen yeniden süspansiyon haline gelinceye kadar deiyonize su 95 ml ilave edilir. </li><li> Tamamen (otoklavlamayın yok) eritmek için orta kaynatın. </li><li> 50 ° C oda sıcaklığında, ortam soğutun </li><li> Her biri 95 x 15 mm (çap x yükseklik) plaka (Parafilm ile sızdırmaz plakalar, 1 ay boyunca 4 ° C &#39;de muhafaza edilebilir) 25 ml agar dökün. </li></ol> 2.. Nematodu Büyüme Orta (NGM) RNAi Tabaklar Besleme C. hazırlanması elegans NGM plakalar, daha önce tarif edilmiştir 19. Burada bir prosedür kısaca RNAi besleme plakaları yapmak için NGM ortamı içine antibiyotik ampisilin ve RNAi kimyasal indüktör izopropil β-D-1-tiogalaktopiranosid (IPTG) ekleyin tarif edilir. <ol><li> 3 g NaCl ve 1 L iyonu giderilmiş su içinde 2.5 g Bacto pepton çözülür. </li><li> Ortam içine 17 gr Bacto ağar ekleyin. </li><li> 45 dakika boyunca ortam otoklav ve bir su banyosu içerisinde 50 ° C&#39;ye kadar ortam soğutun. </li><li> 1 ml kolesterol (% 95 etanol içinde 5 mg / ml), 1 ml 1 M CaCl2, 1 ml 1 M MgSO 4 ve 25 ml 1 M potasyum fosfat tamponu (pH 6.0): aşağıdaki çözümleri ekleyin. İyice karıştırın. </li><li> 1 ml 1 M IPTG ve 500 ul ampisilin (steril su içinde 100 mg / ml) eklenir. </li><li> Iyi çözüm karıştırın ve Pipet yardımcısı kullanarak ve steril prosedürleri takip 25 mi serolojik pipet 60 x 15 mm (çap x yükseklik) petri tabak içine dökün. 12 ml agar ile her bir plak doldurun. Plakalar kadar 1 ay boyunca 4 ° C&#39;de saklanabilir. </li></ol> Temel autophagy geni bec-1 Salmonella enfeksiyonuna karşı savunma bir konak genin işlevini incelemek için bir örnek olarak kullanılmıştır. Deney prosedürleri, Şekil 1 ve Tablo 1 &#39;de gösterilmiştir. Bulaşmasına RNAi ile muamele edilmiş hayvanların hazırlanması için protokol, parantez içinde verilmektedir, her deney aşamasının gün izler. <ol><li> Bec-1 RNAi besleme inoküle ve 100 mg / ml ampisilin (Gün 1) ile desteklenmiş 2 ml LB ortam içine -80 ° C donmuş bakteri pul yerleştirilerek boş vektör L4440 RNAi bakteri besleme kontrolü. Taze RNAi bakteri olması tüm deney boyunca haftada bir kez bu adımı tekrarlayın. Kullanılmadığı zaman, 4 ° C buzdolabı içinde kültür saklayın. </li><li> RNAi tabakalarına RNAi bakteri kültürü gece boyunca 100 ml tohum. Üç bec-1 RNAi ve üç kontrol boş v hazırlayınRNAi plakaları ector. 37 ° C gece boyunca (Gün 2) de inkübe edin. </li><li> 37 ° C inkübatör RNAi plakaları kaldırmak ve onları bankta oda sıcaklığına kadar soğumasını bekleyin. Besili L4 vahşi tip N2 Hermafroditler Pick up ve onları RNAi-1 bec ve boş vektör RNAi plakaları kontrol transfer. , Üçlü plakalar üzerinde, plaka başına üç solucanlar yerleştirin. Aşama 3.2 (Gün 3) &#39;de anlatılan ile aynı günde, RNAi plakaları hazırlar. </li><li> Adım 3.3 &#39;de hazırlanan taze gelen RNAi plakaları 36-40 saat ve transfer solucanlar için 20 ° C inkübatör solucanlar ile RNAi inkübe edin. Solucanlar aktarılır sonra 64 saat (Gün 4) için 20 ° C inkübatör içinde inkübe edin. </li></ol> 4. Enfeksiyon için Salmonella hazırlayın <ol><li> Streak Salmonella -80 ° C dondurulmuş 1 XLD agar plaka üzerinde stok ve gece (5 Gün 37 ° C plaka inkübe </stRong&gt;). </li><li> Bir iyi izole edilmiş siyah Salmonella koloni almak ve (Gün 6) bir karışımı çalkalanarak 37 ° C&#39;de 2 ml LB ortamı içinde inoküle. </li><li> 1 C. Tohum 80 ml ​​Salmonella gecede kültür elegans 60 x 15 mm (çap x yükseklik) NGM agar plaka ve toplam 6 plakaları hazırlamak. 6 saat boyunca oda sıcaklığında inkübe edin. Bakteri kültürü plakasının (Gün 7) üzerine, bir çim kuru ve oluşturmalıdır. </li></ol> Salmonella, 5. Infect RNAi ile muamele Worms <ol><li> Transferi bec-1 RNAi tedavi ve Salmonella plakalarına boş vektör RNAi tedavi L4 N2 çift cinsiyetli (Adım 3 kurmak solucanlar döl) kontrol eder. Her bir grup için 3 levhaları üzerinde plaka başına 40 solucanlar yerleştirin. 48 saat (Gün 7) 20 ° C&#39;de sonsuz inkübe edin. </li><li> Adım 3.1 ve 3.2 (Gün 7&#39;de tarif edildiği gibi, taze RNAi plakaları bir takım hazırlanması ve <strong&gt; Gün 8). </li><li> 48 saatlik enfeksiyondan sonra, aşama 5.2 &#39;de hazırlanan karşılık gelen RNAi plakalara Salmonella ile enfekte olmuş solucan aktarın ve 20 ° C (gün 9) de inkübe edilir. </li></ol> 6.. Kurtuluş Testi <ol><li> Günlük solucanlar hayatta Puan ve yumurtlama zamanında taze gelen RNAi plakaları solucanlar transfer. Adım 3.1 ve 3.2 &#39;de tarif edildiği gibi önceki her sarmal transfer taze RNAi plakaları bir dizi hazırlayın. Yavaşça bir uçtan basık platin tel ile solucan vücudu (baş, orta kısım ve kuyruk) dokunun. Solucan gövdesinin hiçbir hareket görülmediği takdirde bir solucan ölü olarak puanlanır. </li><li> Günlük solucan veya her gün hayatta kalma Puan ve solucanlar yumurta döşeme durdurmak sonra iki kez bir hafta taze gelen RNAi plakaları solucanlar transfer. </li><li> Tüm solucanlar Öldükten sonra, bir veri kümesi olarak üçlü plakaları sağkalım verileri havuzu. GraphPad P olarak uygun istatistiksel yazılım içine her grubun girdi sağkalım verilerisağkalım eğrileri oluşturmak ve Kaplan-Meier sağkalım analizi yapmak Rism. Tüm deney sonucu teyit etmek için en az bir kez tekrarlanır. </li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

C. elegans onun besin kaynağı olarak bakteri yiyor basit bir genetik model organizmadır. Böylece, C arasındaki etkileşimleri araştırmak için bir bağırsak patojen ile normal bakteriyel gıda yerine kolaydır elegans ve seçilen patojen. Burada bir protokol Salmonella enfeksiyonu ve C birleştirmek için açıklanan elegans RNAi Salmonella enfeksiyona karşı savunma konak genlerin rolünü incelemek için tedavi besleme. Önceki enfeksiyon protokol...

Serbest yaşayan toprak nematod Caenorhabditis elegans birçok biyolojik soruları incelemek için kullanılan basit ve genetik uysal bir model organizmadır. C. elegans egemen kendini gübreleme çift cinsiyetli olarak var. Erkekler kendiliğinden gametogenezi 1,2 sırasında X kromozomu olmayan disjunction tarafından oluşturulur. Bol yiyecek, C varlığında elegans sürekli yetişkin Dört larva aşamalardan geçerek gelişir. Sıcaklık da C etkilemektedir elegans gelişimi; Daha hızlı gelişimi, daha yüksek sıcaklıklarda görülmektedir. Laboratuvarda, C. elegans gıda 1,2 olarak tohumlanmış bakteri Escherichia coli (suş OP50) ile agar plakaları üzerinde 20 ° C&#39;lik bir sabit bir sıcaklıkta kültürlenir. Son on yıl içinde, C. elegans konak-patojen 3-5 incelemek için bir omurgasız organizma olarak ortaya çıkmıştır. Doğada, C. elegans onun nutrien olarak bakteri yiyort kaynak 1,2. Bu normal bakteri laboratuar gıda, OP50, kolayca C arasındaki etkileşimi incelemek için diğer patojenler ile ikame edilebilir elegans ve seçilen herhangi bir patojen. Bu koşullar altında, bağırsak enfeksiyonun birincil sitesidir. Gerçekten de, bakteriyel patojenlerin geniş bir yelpazede ölümcül C enfekte ettiği gösterilmiştir elegans 3-5. Gram-negatif bakteri Salmonella çapında 6,7 insan gıda kaynaklı hastalığa neden olan bir gastrointestinal patojendir. C. Bu bakteri çoğaltır ve kalıcı bağırsak enfeksiyonları 8-10 sergiler gibi elegans Salmonella typhimurium için iyi bir model ev sahipliği yapmaktadır. C. elegans roman hem tanımlamak için kullanılmıştır, ve daha önceden bilinen Salmonella hastalık oluşturma faktörleri 11. İlginç bir şekilde, C. elegans bağışıklık sistemi başarıyla Salmonella çoğaltma sınırlar. O Inhib önce rapor edilmiştirautophagy genlerin enfraruj C&#39;de artan Salmonella çoğaltma işler elegans, enfekte solucan 10 erken ölüm ile sonuçlanır. (Burada Otofaji olarak anılacaktır) Macroautophagy bozulması için 12 lizozom teslimat için sitoplazma ve ayırmak için hücre içi organellere zarlarının yeniden düzenlenmesini içeren bir dinamik bir süreçtir. Autophagy C. Salmonella çoğaltma sınırlamak için bildirilmiştir elegans ve memeliler de 10,13. C. elegans genomu ilk çok hücreli ökaryot genom olduğu; Bu RNAi tedavi 14-16 yanıt verici olmaktadır. Ayrıca, RNAi RNAi 16,17 besleme olarak bilinen hedef genin, bir iki-şeritli RNA içeren bakteri yemek için solucan tabi tutulması ile etkili bir şekilde idare edilebilir. Tüm genom RNAi besleme kütüphaneler genom RNAi 16,18 taranması için oluşturulan edilmiştir. Burada, bir Salmonella enfeksiyonu protocol RNAi test C&#39;ye izin beslenme ile birleştiğinde Salmonella enfeksiyona karşı koruma kabiliyetleri için ilgi çekici elegans genler.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">BP9723-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XLD agar</td>
			<td>EMD Chemicals</td>
			<td>1.05287.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Fisher</td>
			<td>DF0140-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Fisher</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td>Gold Biotechnology</td>
			<td>12481C50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Fisher&#160;</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 95 x 15mm</td>
			<td>Fisher</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 60 x 15mm&#160;</td>
			<td>Fisher</td>
			<td>08-757-13A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Serological pipet</td>
			<td>Fisher</td>
			<td>13-668-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Express Pipet-Aid</td>
			<td>Fisher</td>
			<td>13-675-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ6000 shaking incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival</td>
			<td>I-36DL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

Son on yıl içinde, C. elegans gram-negatif bakteri Salmonella typhimurium karşı ev sahibi savunma da dahil olmak üzere, ana ve patojen arasındaki etkileşimleri incelemek için bir omurgasız organizma olarak ortaya çıkmıştır. Salmonella C bağırsakta inatçı enfeksiyon oluşturur elegans ve enfekte olmuş hayvanlar arasında erken ölüm ile sonuçlanır. Bağışıklık bir takım mekanizmalar C de tespit edilmiştir Salmonella enfeksiyonlarına karşı savunmak için elegans. Autophagy, evrimsel olarak muhafaza edilmiş lizozomal bozunma yolağı, C. Salmonella çoğaltma sınırlamak için gösterilmiştir elegans ve memelilerde. Burada, protokol C bulaştırmak için açıklanan Solucanlar Salmonella, insanlarda enfeksiyona benzer bir süre için, Salmonella maruz edildiği Salmonella typhimurium ile elegans. Salmonella enfeksiyonu önemli C ömrü kısaltır elegans &lt;/ Em&gt;. Bir örnek olarak temel autophagy geni bec-1 kullanarak, C ile bu enfeksiyon yöntemi Birleştirilen elegans RNAi yaklaşım besleme ve bu protokol C işlevini incelemek için kullanılabilir gösterdi Salmonella enfeksiyonu karşı savunmada elegans konak genler. Yana C. elegans tüm genom RNAi kütüphaneler kullanılabilir, bu protokol kapsamlı C taranması için mümkün kılan genom RNAi kütüphaneleri kullanılarak Salmonella ve diğer bağırsak patojenlere karşı korumak elegans genler.

20 ° C&#39;de, vahşi tip N2 solucanlar medyan ömrü. Salmonella enfeksiyonu belirgin 10.5 gün N2 solucanlar medyan ömrünü azaltır 17 gün (Şekil 2A ve Tablo 2) (p = 0.0002, log-rank testi) (Şekil 2A .) Bir C. elegans geninin Salmonella hastalıklara karşı önemli bir rol oynar, kendi inhibisyon Salmonella enfeksiyona karşı yatkınlığın vermek olacağı tahmin edilmektedir. Aslında, Sa...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans konak-patojen etkileşimleri çalışmak için yeni bir genetik model olarak ortaya çıkmıştır. Burada C bulaştırmak için bir protokol açıklar Salmonella enfeksiyona karşı savunma konak genlerin rolünü incelemek için çift iplikli RNAi girişim tekniği ile birleştiğinde Salmonella typhimurium ile elegans.

Bulaştırmak için bir protokol<em&gt; Caenorhabditis elegans</em&gt; Ile<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K Görüntüleme.  Florida Atlantic University.  C. elegans konak-patojen etkileşimleri çalışmak için yeni bir genetik model olarak ortaya çıkmıştır. Burada C bulaştırmak için bir protokol açıklar Salmonella enfeksiyona karşı savunma konak genlerin rolünü incelemek için çift iplikli RNAi girişim tekniği ile birleştiğinde Salmonella typhimurium ile elegans. 

Video: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Allelotyping belirlenmesi için bir PCR Salmonella enterica Serovars Enteritidis, Hadar, Heidelberg ve Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

İmmünoloji ve Enfeksiyon

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Fenotipinin yüksek verim Deneyi<em&gt; Salmonella enterica</em&gt; Typhimurium Derneği, Invasion, ve Makrofagların Çoğaltma

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol<em&gt; Clostridium difficile</em&gt; Antibiyotik Tedavisine Yanıt Vermek

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Virülans ve Aeromonas enfeksiyon Caenorhabditis elegans modelindeki patogenezinde değerlendirilmesi

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Fare Buzağılarına Tutarlı Masaj Benzeri Pertürbasyonların Uygulanması ve Bunun Sonucu Kas içi Basınç Değişikliklerinin İzlenmesi

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Insan kaynaklı pluripotent kök hücresini kullanma- Salmonella ve diğer patojenlere karşı epitelyal hücre korumasını Incelemek ve değiştirmek Için bağırsak organoidleri türetilmiştir

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Kronik Salmonella EnfeksiyonuNa Bağlı Bağırsak Fibrozisi

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Methods Article

Bulaştırmak için bir protokol