açıklanan Proje Alerji ve Enfeksiyon Hastalıkları Ulusal Enstitüsü Ödülü Numarası 5R01AI116895 tarafından desteklenmektedir. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Ulusal Alerji ve Bulaşıcı Hastalıklar Enstitüsü ve Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

1. Yaratma Örnekleri <ol><li> Isı C etkinleştirmek difficile buz üzerinde 30 dakika ve mağaza 65 ° C sıcaklıkta sporlar. Not: C Daha önce tarif edildiği gibi 7,9,10 difficile sporlarının üretilir ve saflaştırılabilir. </li><li> X, numune sayısı deneyi sırasında alınması gereken x + 1 ml çimlenme çözeltisi hazırlayın. Not: bakteri türlerinin çalışılan spor için çimlenme çözüm özgüdür. C içlin difficile spor çimlenmesi biz, 10 mM Tris (pH 7.5), 150 mM NaCI, deiyonize su içinde 100 mM glisin, 10 mM taurokolik asidin bir çözeltisi kullanılır. </li><li> tahlili başlamadan önce, korteks fragmanı tespiti için bir boş olarak hizmet etmek için sporların filizlenmeyi çözeltisi bir 1.0 mi bir numune alın. </li><li> Bir OD hızla çimlenme çözüm ve vorteks ~ 3,0 600 sporlar ekleyin. Not: B bizim çimlenme araştırmalar için subtilis aynı OD kullanılan 6C gibi 00 sporlar difficile. Not: sporların Bu miktar C sırasında izleme korteks fragmanı serbest bırakılması için çalıştı difficile spor çimlenmesi. Bu tahlilde kullanılan sporların miktarı, her bir bakteri ya da bir suş için deneysel olarak tespit edilmelidir. </li><li> Hemen 14,000 x g&#39;de 1 dakika için oda sıcaklığında sporlar ve santrifüj ilave edildikten sonra 1.2 ml lik bir numuneyi çıkarın. Bu sıfır zaman noktasıdır. </li><li> daha sonra korteks fragmanı analizleri için yeni bir tüpe için santrifüj numuneden süpernatan 1.0 mL aktarın. Not:, terbium floresan 9,18,19 dayanan bir deney kullanılarak çözelti içinde DPA tespiti için ayrı bir 100 ul numuneyi çıkar DPA izlenecek gerekirse. </li><li> -80 ° C&#39;de toplanan örnek dondurun. </li><li> Tüm zaman noktalarında tamamlanana kadar tekrarlayın düzenli aralıklarla 1.4 ve 1.5 adımları tekrarlayın. Not: Zaman santrifüj ve örnekleme, zaman noktaları daha az inci için gerekli olduğundanBir 2 dk dışında bizim işlem sırasında mümkün değildi, bu yüzden bizim örnekleme aralıkları kez 14 dakika boyunca her 2 dk. </li><li> 0, 12.5, 25, 50, 100, 250, 500 ve 5000: Şeker saptanması ve nicelenmesi azaltmak için pozitif bir kontrol olarak, N -acetylglucosamine (nmol) aşağıdaki miktarlarda bulunur. korteks örneklerinde olduğu gibi aynı zamanda bu örnekleri hazırlamak ve standart eğri sporlarını çimlenme tarafından oluşturulan verilere uygun olacak şekilde korteks örnekleri ile birlikte çalışacak. </li><li> her zaman nokta örnekleri toplandıktan sonra, numune liyofilize. Not: 2 ml vidalı kapaklı tüplerde bizim örnekleri dondurularak. </li></ol> 2. Cortex Fragments Ölçme <ol><li> % 1 fenol ve% 0.5 β-merkaptoetanol ile takviye edilmiş, 3 N HCI, 120 ul Liyofilize edilen numuneler, yeniden süspanse edin. </li><li> 2 ml&#39;lik vidalı kapaklı tüplere yeniden süspanse örnekleri aktarın. Not: tekrarlanabilirlik için, tüm liyofilize numune yeniden süspanse ve transfer edildiğinden emin olun. </li> &lt;li&gt; 4 saat 95 ° C de bir devri daim su banyosu içinde inkübe edin. </li><li> serin kadar inkübasyondan sonra, buz üzerinde örnekleri yerleştirin. </li><li> 3 M NaOH 120 ul örnekleri nötralize eder. </li><li> doymuş sodyum bikarbonat çözeltisi 80 ul her bir örnek ve vorteks bir% 5 asetik anhidrid çözeltisi 80 ul ekle. </li><li> 10 dakika oda sıcaklığında inkübe edin. </li><li> TAM 3 dakika boyunca geri 95 ° C su banyosunda örnekleri aktarın. Not: Bu adım hassas ve daha uzun süreler aşağı sinyal gelişimini etkileyebilir zamanıdır. </li><li> su banyosu örnekleri çıkarın ve buz üzerinde soğutun. </li><li> Her bir tüp ve vorteks 6.54% K 2 B 4 O 7 · 4H 2 O 400 ul ekleyin. </li><li> Tam olarak, 7 dakika boyunca 95 ° C de bir devri daim su banyosu içinde inkübe edin. Not: Bu adım hassas ve daha uzun süreler aşağı sinyal gelişimini etkileyebilir zamanıdır. </li><li> Kaldır5 dakika boyunca buz üzerinde örnekleri ve yer. </li></ol> 3. Renk reaktifi <ol><li> Numuneler buz (Adım 2.12) üzerinde iken, renk reaktifi hazırlamak. (; Ehrlich reaktifi DMAB) buzlu asetik asit, 1.9 ml p -dimethylaminobenzaldehyde 0.320 gr eritin. Not: reaktif zaman, sıcaklık ve ışığa karşı hassastır ve önceden yapılmamalıdır. </li><li> DMAB tamamen erir sonra, 10 N HCI ve vorteks 100 ul ekleyin. </li><li> buzlu asetik asit ve girdap 5 ml. </li></ol> 4. kolorimetrik reaksiyon <ol><li> Aktarım, her 100 ul yeni bir 1.5 ml mikrosantrifüj tüpüne korteks numunenin soğutuldu. </li><li> Her bir numune için taze hazırlanmış renk çözeltisi 700 ul ekleyin. </li><li> Derhal bir 37 ° C su banyosuna örnekleri transferi ve tam olarak 20 dakika süreyle inkübe edin. </li><li> berrak bir 96 oyuklu plakaya her bir numune 200 ul aktarın. </li><li> bir plaka üzerinde 585 nm&#39;de numune sayısal olarakokuyucu. Numuneler bir sarı renk başlamalıdır ve pozitif bir reaksiyon mor kayacak. Daha fazla korteks mevcut, derin mor renk. </li></ol>

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The authors have nothing to disclose.

stimülasyon üzerine çimlenme süreci geçiren sporları makromoleküler sentez için gerek kalmadan kendi direnç özelliklerini kaybederler. Spor çimlenmesi tetiklendiğinde, spor çekirdek su 2 karşılığında DPA serbest bırakır. Nedeniyle atıl sporun yüksek DPA içeriğine, spor çekirdek genişleme 2 önünde bir engel olarak, muhtemelen, hareket ederek şişme çekirdek engeller yoğun olarak ozmotik basınç ile özel bir peptidoglikan, korteks altındadır. <p class="jove_cont...

Endosporlar metabolik bakteriler olumsuz ortamlarda devam izin bakterilerin uyuyan biçimleridir. Birçok spor oluşturan Bakterilerde Spor Oluşumu besin maddesinde yoksun kalma ile indüklenen, ancak bu işlem, oksijen ve diğer streslere 1 pH, maruz değişiklikleri ile kontrol edilebilir. Onların metabolik dormant spor formunda iken, bakteri, UV radyasyonu, kuruma, yüksek sıcaklıklarda ve 2 dondurma karşı. Sporlanma sürecine bilginin çoğu model organizma çalışmalarda, Bacillus geliyor. Sporlanma süreci DNA replikasyonu ve eksenel flamanlı 3,4 oluşumu ile başlar. Asimetrik septum daha sonra iki eşitsiz boyutlu bölmeye hücreyi böler. Daha büyük bölme, ana hücresi, küçük bölmesini, forespore engulfs. anne hücresi ve Dorman bırakmadan, sporu ve sonunda lyses anne hücreyi olgun gen ifadesini koordine forespore Hemçevreye 1 içine t spor. spor yapısı ve bileşimi birçok bakteri türleri boyunca muhafaza edilir. Spor çekirdek vejetatif hücrenin daha düşük bir su içeriğine sahiptir ve dipikolinik asit (DPA) 1,2 ile zengindir. spor oluşumu sırasında, DPA su karşılığında spor iç kısım içine pompalanır. Çekirdeği çevreleyen en sporlu bakterilerin, çimlenme (germinants) uyaran küçük moleküller tanıyan reseptörleri 2 bulunduğu, bir iç spor bir zardır. Sadece spor iç zarının dışında bulunan hücre duvarı peptidoglikan bir tabakadır. Bir uzman peptidoglikan tabakanın (korteks) hücre duvarı peptidoglikan çevreleyen ve [N-asetilglukosamin (NAG) ve N-asetilmuramik asit (NAM) alternatif] hücre duvarı peptidoglikan aynı bileşenlerin çoğu oluşmaktadır. Bununla birlikte, korteks NAM tortularının yaklaşık olarak% 50 muramic-δ-laktam 5,6 dönüştürülmüş </s&gt; Yukarı. spor çimlenme sırasında bu muramic-δ-laktam artıkları böylece korteks değil hücre duvarı (çimlenme tamamlamak için gerekli bir süreç) bozulmuş sağlayan, spor korteks parçalayıcı enzimlerin (SCLEs) tarafından kabul edilmektedir. Korteksi çevreleyen bir dış zar ve kaplama proteinlerinin 2 birkaç kat olduğunu. çimlenme sürecini kontrol sporlu bakteriler için büyük önem taşımaktadır. Filizlenen reseptörler kendi germinants 2 etkileşimde bulunduğunuzda Çimlenme başlatılır. Birçok spor oluşturan bakteri, bu germinants 2 olan amino asitler, şekerler, nükleotid, iyon ya da bunların kombinasyonları bulunmaktadır. C difficile spor çimlenmesi bazı safra asitleri [örneğin, taurokolik asit (TA)] ve amino asitler (örneğin, glisin) 7-10 kombinasyonları ile başlatılır. C arasında farklılıklar olmasına rağmen difficile çimlenme yolu ve okudu yolların diğer sporluörneğin B. bakteriler, subtilis, tüm ortak bir vejetatif hücre çimlenmiş sporun 2,8 büyümeye izin spor korteksi düşmesine mutlak bir gerekliliktir. Ya SLEC (B. subtilis içinde bulunduğu gibi) (birçok Clostridia bulunduğu gibi) Korteks bozunma SCLEs CwlJ / SleB ile gerçekleştirilebilir. Korteks hidroliz hücre duvarı ve sporun üzerinde kısıtlama azaltır. Bu, tam çekirdek rehidrasyon, hücre metabolizması 2 için gerekli olan proteinlerin birçok yeniden aktive gerekli bir adım için izin verir. Sporların filiz olduğunda, bir faz-karanlık durumu ve bu işlem için bir faz parlak durumundan atıl spor değişiklikleri 600 optik yoğunluk (OD) bir değişiklik ile ölçülebilir nm 11. Bir önceki raporda OD bu değişimin çok sporla 12 DPA salınımına bağlı olduğunu göstermektedir. Bizim son çalışma sırasında, biz C. zamanlamasını karşılaştırmak için çalıştı difficile spor çimlenmesi ve izlenenDPA ve korteks parçalarının 9 sürümü. Onlar sporlar çimlenme tarafından piyasaya sürülecek başladı bu çalışma için korteks parçalarının serbest bırakılmasını izlemek için önemliydi. Burada kullanılan kolorimetrik deney indirgeyici uçları olan şekerleri tespit etmek için bir yöntem dayanmaktaydı Ghuysen ve ark., 13 geliştirilmiştir. Diğerleri indirgen şekerlerin 14 tespiti için protokoller tarif etmişlerdir ya da bu protokolleri 15 modifiye Çünkü, bu konuda literatür kafa karıştırıcı olabilir. Burada, C çimlenme kurtuldu indirgeyici şekerlerin kolorimetrik tespiti için detay bir adım-adım yöntemi biz difficile sporlarının. Bu çalışmada C kullanır rağmen difficile sporları, diğer sporlu bakterilerden sporların çimlenmesi sırasında salınan indirgeyici şekerler bu protokol 9,16,17 ile tespit edebiliyoruz.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Tablo 1 NAG standartlarını kullanarak tipik sonuçları göstermektedir. Veriler, standart bir eğri oluşturmak için kullanılmaktadır. Tablo 2, 100 mM glisin, 10 mM TA (çimlenme arttıran koşullarda) ya da 100 mM glisin, sadece (non-çimlenen koşullar) ile takviye edilmiş tampon çimlenme bir çimlenme deneyinde tipik sonuçlan göstermektedir. Ta, C. yokluğunda difficile sporlar çimlenir yoktur ve çözeltide korteks parça...

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Bakteriyel Spor Filizlenmesinin sırasında Cortex Fragments algılama

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.3K Görüntüleme.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Video: Detecting Cortex Fragments During Bacterial Spore Germination

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Çoğaltım ve Yeşil floresan proteini ifade Maedi-visna Virüs bir Enfeksiyöz Moleküler Clone algılama

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

İmmünoloji ve Enfeksiyon

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periyodik Asit Schiff Boyama ile periferik kan mononükleer hücreleri glikojen algılama

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Memeli Hücreleri içinde fonksiyonel Bakteriyel Efektör elektroporasyonu

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Sıtma enfeksiyon sırasında hücre dışı vezikül ile değerlendirilmesi

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Yöntemleri için algılama sitotoksik Amyloids aşağıdaki enfeksiyon pulmoner endotel hücre Pseudomonas aeruginosa tarafından

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

Spore adsorpsiyon enzimler ve antijenleri için Nonrecombinant görüntü sistemi olarak

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Bir yüksek-den geçerek Shigella-belirli bakterisidal tahlil

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Bakteriyel Spor Filizlenmesinin sırasında Cortex Fragments algılama

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