Bu çalışma, Avusturya Bilim Fonu&#39;nun (FWF P20619 ve BZ için P22988) tarafından desteklenmiştir.

Mikrodalga destekli numune hazırlama <ol><li> Numune hazırlama birinin başlangıç ​​elektron mikroskobu için otomatik mikrodalga doku işlemci (Leica EM AMW; Leica Microsystems, Viyana, Avusturya) programlamak gerekiyor önce TEM için mikrodalga destekli numune hazırlama için aşağıdaki protokolleri (Tablo 1): <img src="/files/ftp_upload/2950/2950table1.jpg" alt="Tablo 1" /> Tablo 1. Mikrodalga radyasyon kullanarak numune hazırlama için protokol. Farklı sütunlar (soldan sağa) gösterir: <ol><li> Vial numarası (Vial no.) Şişenin işlemcinin atlıkarınca içine yüklenir sırasını temsil eder. </li><li> Gerçek sürecinin Adım. </li><li> Flakonlarda Reaktifler. </li><li> Gerçek adım süresi. </li><li> Mikrodalga ışınlama kapatılmadan önce flakon içinde ulaşılır maksimum sıcaklık. </li><li> Mikrodalga radyasyon ayar: Sürekli = hızlı sıcaklık artışlarlaase, ayarlanan sıcaklık tutarak; Eğim = nazik sıcaklık artışı, sonunda ulaşılan nihai sıcaklığı; sıcaklığı 5 düştü kadar Atımlı = Hızlı sıcaklık artışı, güç kapatıldığında ° C, güç sıcaklığa ulaşmak için yeniden başladı. </li><li> Mikrodalga radyasyonun Maksimum güç. </li></ol></li><li> Taze numune hazırlama protokolü (Agar 100 epoksi reçine için 1.7 &#39;ye bakınız) açıklanan farklı adımları için çözümler hazırlamak, programlanan protokolü (Tablo 1) göre belirlenen şişeleri içine doldurun, atlıkarınca şişeleri yükleyin, ardından eklemek mikrodalga doku işlemci, ve içine carousel nihayet monomod odasına ilk flakon yükleyin. </li><li> 60mm Sørensen% 3 glutaraldehit (Agar Bilimsel Ltd, Stansted, İngiltere) bir damla bir modelleme mumu plaka üzerinde bir jilet ile Tütün Mozaik Virüsü (TMV) ile enfekte Nicotiana tabacum yapraklarını küçük bölümler (1mm 2) Kes Oda sıcaklığında fosfat tampon maddesi (pH 7.2)erature. </li><li> Hemen yaklaşık 200μm bir örgü genişliği ile belirlenen sepet içerisine ince cımbız ile bölümleri aktarın. Birbirlerinin üstüne sepetleri Yığın ve monomod odasının içine yerleştirin. Bakım örnekleri sürekli kurumasını yok ki yükleme ve sepetleri istifleme sırasında fiksatif solüsyonu ile kaplıdır dikkat edilmelidir. </li><li> Fiksasyon, dehidratasyon ve infiltrasyon için önceden programlanmış mikrodalga destekli numune hazırlama protokolü başlatın. </li><li> Numune hazırlama mikrodalga doku işlemci tarafından otomatik olarak gerçekleştirilir iken bölüm 3 (negatif boyanma) açıklandığı gibi kalan bitki materyali ile negatif boyanma ile devam edin. </li><li> Plastik bir kap içinde dolgu 24g Agar 100, 16g dodecenyl süksinik anhidrit ve 10 g metil nadic anhidrit (tüm bileşenler için Agar Bilimsel Ltd, Stansted, İngiltere bakınız), ısı: Taze aşağıdaki bileşenleri tarif gibi karıştırılarak Agar 100 epoksi reçine hazırlamak buna40 ° C ve iyice karıştırın. Benzil dimetilamin 1.2g ekleyin ve iyice karıştırın. Numune hazırlama protokolü sona (örneğin masa 1 adım 22 sırasında) gelir hemen önce belirlenen polimerizasyon içine Agar 100 epoksi reçine doldurun oluşturur. </li><li> Protokol tamamlandıktan sonra (tablo 1&#39;de basamak sonra 22) monovalent odasından gelen atlıkarınca son şişenin içine sızma numuneleri içeren yığın sepet bırakın. Mikrodalga cihazdan atlıkarınca çıkarın, sepetler Unstack ve belirlenen polimerizasyon formları içine ince cımbız kullanarak bunları yükleyebilirsiniz. Bakım örnekleri her zaman unstacking ve kurumasını yok ki yükleme sırasında Agar 100 epoksi reçine ile kaplıdır dikkat edilmelidir. </li><li> Birbirlerinin üstüne polimerizasyon formları yığını. Bakım örnekleri her zaman istifleme ve kurumasını yok ki yükleme sırasında Agar 100 epoksi reçine ile kaplıdır dikkat edilmelidir. </li><li> Mikrodalga tiss bir atlıkarınca daha önce kullanılmış şişeleri kaldırue işlemci, yığılmış sepetleri ile yükleyebilirsiniz ve mikrodalga doku işlemci içine carousel yerleştirin. </li><li> Önceden programlanmış polimerizasyon protokolü (tablo 1) başlatın. </li><li> Polimerizasyon mikrodalga doku işlemci tarafından otomatik olarak yapılır iken, bir transmisyon elektron mikroskobu ile negatif boyanmış parmaklıkları incelemek [örneğin Philips CM10 TEM, FEI (eski Philips), Eindhoven, Hollanda] ve bölüm 3 ve 4 (açıklandığı gibi görüntü analizleri Negatif boyama ve görüntü analizi). </li><li> Protokol Unstack, monovalent odasından gelen polimerizasyon biçimleri polimerizasyon biçimleri kaldırmak ve numuneler içeren polimerize bloklar kaldırmak tamamlandıktan sonra. Onlar şimdi bir mikrotom ile kesitli olmaya hazırız. </li></ol> 2. Kırpma ve kesit <ol><li> Ultra ince tutucu yaklaşık yapışma üst üzerinde numune ile 1cm üzerinden kesit için ayrı numune tutucular içine bir veya daha fazla blok yerleştirin. </li><li>TEM (örneğin Leica Reichert Ultratrim; Leica Microsystems) için bir örnek düzeltici ile blok Trim böylece maksimum blok yüzü. Uzunluğu ve (yüz boyutu bloke elmas bıçak boyutuna ayarlanması gerekebilir) elde edilir mümkün olduğu kadar yaprak malzeme içeren genişlikte 200μm 1mm. </li><li> 45 ° (örn. Diyatomelere Ultra 45, Gröpl, Tulln, Avusturya) bir bıçak açıda bir elmas bıçak kullanarak; bir ultramicrotome (Leica Microsystems örneğin Reichert Leica ULTRACUT S) ile Bölüm bloğu. Kısım kalınlığı yaklaşık 70 ila 90 nm olacak şekilde ayarlanmalıdır ve kesme hızı 1 mm / s civarında olmalıdır. Bir formvar (Agar Bilimsel Ltd) kaplı bakır veya nikel 200 kare mesh ızgara ile birden çok bölüm toplayınız. </li><li> Kısmen 2-ücretsiz CO oluşturmak için NaOH ile dolu bir petri 5 dakika; kurşun sitrat (42ml çift distile su ve 8ml 1N NaOH içinde çözülmüş 1.1 g kurşun sitrat Agar Bilimsel Ltd) ile ızgara üzerinde bölümler Post-leke çevre ve 15 mil için% 1 uranil asetat (Agar Scientific Ltd) ile nutes oda sıcaklığında damıtılmış su içinde çözülmüştür. Her post-boyama adım arasında 1 dakika için damıtılmış su ile ızgaraları yıkayın. Hava bir ızgara kutusuna ızgaraları kurutun. </li><li> Bir transmisyon elektron mikroskobu (örneğin Philips CM10 TEM, FEI, Eindhoven, Hollanda) ile bölümleri inceleyin. </li></ol> 3. Negatif boyama <ol><li> TMV ile enfekte yaprak malzeme 100mg hakkında ve 60mm Søfrensen fosfat tamponu (pH 7.2) 100μl bir mikroskop lamı üzerine bir jilet ile 2 dakika boyunca malzeme homojenize ham sap hazırlamak Hasat. </li><li> 4 ya da daha çok kuyucuk (alternatif olarak parafilm bir parça üzerinde homojenat aktarmak mümkündür) bir teflon kaplı mikroskop slaytı iyi ilk üzerine çıkan Homojenat 20uL aktarın. </li><li> Damla ve incu dönük formvar (Agar Bilimsel Ltd) kaplamalı yüzü Homojenat üstüne formvar kaplı ızgara yerleştirin5 dakika boyunca kesmek. </li><li> 200μl 60mm Sørensen fosfat tampon maddesi (pH 7.2) ve iki damla üzerinde ızgara yerleştirerek ızgara 2 dakika için 2 defa yıkanır. </li><li> Bir taze hazırlanmış% 2 fosfotungstik asit (Agar Scientific Ltd) 60mm Sørensen fosfat tampon maddesi (pH 6.5) içinde çözelti ile 1 dakika için ızgara inkübe edin. </li><li> Izgara çıkarın ve bir ızgara kutusuna kurumaya bırakın. </li><li> Bir transmisyon elektron mikroskobu (; FEI, Eindhoven, Hollanda örneğin Philips CM10 TEM) ile ızgara inceleyin. 21000X veya daha yüksek bir primer büyütmede transmisyon elektron mikroskobu ile rastgele seçilen olumsuz lekeli virionlar en az 10 görüntü (en az 10 veya daha fazla virionlar her görüntü üzerinde görünür olmalıdır) atın. Bakım tüm görüntüler de aynı büyütme olduğunu alınmalıdır. </li></ol> 4. Görüntü analizi <ol><li> Alınan mikrografları, üzerinde en azından 100 rasgele seçilmiş tek bir virüs partiküllerinin uzunluğu ve genişliği ölçülürherhangi bir görüntü analizi bilgisayar yazılımı kullanarak olumsuz lekeli örnekleri partikül analizi aracı veya Optimas ile [örneğin Hücre D (Olympus, Yaşam ve Malzeme Bilimleri Europe GmbH, Hamburg, Almanya) 6.5.1-(Medya Sibernetik Inc, Bethesda, Maryland, USA)]. </li><li> Negatif boyama yöntemleri ve TEM ile görselleştirilmiş virüs partiküllerinin bir ortalama uzunluk ve genişlik elde etmek için ortalama ve standart sapma hesaplanır. </li><li> Açıkça virüs hastalıkları belirlemek için, literatürde bilinen virüs hastalıkları tarafından uyarılan virüsler ve yapısal değişikliklerin bir uzunluk boyutlarına sahip ve yapısal özellikleri (2.5 de elde edilmiştir) karşılaştırın. </li></ol> 5. Temsilcisi sonuçları: Gibi mikrodalga destekli numune hazırlama tipik TMV indüklenen ultrastrüktürel değişiklikler sonra paralel biçimde hizalanmış virionlar içeren geniş alanlar enfekte Nicotian arasında sitoplazmada transmisyon elektron mikroskobu ile gözlenebilirbir tabacum hücreleri (Şekil 1A). Ayrıca TMV ile enfekte olan ham sap TMV parçacıklar negatif boyanma sonra kıvrımlı, çubuk şeklindeki yapılar (Şekil 1B) olarak gözlenebilir bırakır. 100 virüs partiküllerinin Görüntü analizi uzunluk ve genişlik olarak 17nm olarak 280 nm (Şekil 2) TMV bir ortalama boyutu ortaya çıkardı. TMV ile enfekte olmuş hücrelerine Bu çalışmada gözlenen virionlar ebadında bir ince yapı TMV parçacıkların tütün ve boyut aralığı içinde TMV bağlı ince yapı özelliklerine uygun olarak, daha önce literatürde bildirilen 9-15 olduğu tespit edilmiştir. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Şekil 1" /> TMV ile enfekte yaprak hücreleri ve virionlar Şekil 1. Transmisyon elektron mikroskop. A) Görüntü Nicotiana tabacum mikrodalga destekli bitki numune hazırlama sonrası TMV ile enfekte mezofil yaprak hücrelerinin ince yapısı gösterir. Sitoplazmada biriken paralel hizalanmış virionlar geniş alana dikkat.C nişasta = kloroplast (St), M = Mitokondri, N = nükleus, V = vakuol, Bar = 2μm. B) Görüntü enfekte yaprakların sap negatif boyanma tespit edildi virionlar gösterir. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Şekil 2," /> Şekil 2. Virionlar nispi büyüklüğü. Uzunluğu ve negatif boyanma onlar elektron mikroskobu göründüğü gibi enfekte yaprakların sap sonra TMV-parçacıkların genişliği Göreli dağılımı. (Uzunluk / genişlik ortalama ± standart sapma) değerleri ortalamaları 100 virionlar hesaplandı.

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Çıkar çatışması ilan etti.

TEM için Mikrodalga destekli bitki numune hazırlama birkaç saat 3,4,16 içinde hızlı ve güvenilir ultrastrüktürel verilerini sağlamak için kanıtlanmıştır. Bu çalışmada kullanılan yöntem ile elde organelleri ve membranlar ince yapısal korunması örnek fiksasyon muazzam bir azalma avantajı ile 5,15 konvansiyonel ve cryofixed örnekleri benzer ve yaklaşık 2 saat 3 gün veya daha uzun zaman almasi. Bu, literatürde mevcut TEM için hızlı numune hazırlama protokol temsil eder. Bu çalışmada açıklanan yöntemi TMV bağlı ultrastrüktürel değişiklikler ve viral ajan kendisini açık ve hızlı kimlik izin negatif boyama yöntemleri ile TEM için mikrodalga destekli bitki numune hazırlama birleştirdi. TMV indüklenen ultrastrüktürel değişiklikler transmisyon elektrik fiksasyon başlangıcından sonra yaklaşık 4 saat içinde, kırpma kesit ve post-boyama sonra incelenmiştir olabilirtron mikroskop. Tam otomatik numune hazırlama modunun kullanılması viral ajan boyutu ve genişliğini belirlemek için geçici olarak negatif boyama yapmak için araştırmacı bırakılmış. Böylece, bu yöntem bitki fitopatolojinin tarım ve bilimsel deneyler ileride kullanmak için büyük önem taşımaktadır yaklaşık yarım gün bitki virüs hastalıklarının net ve hızlı tanı sağlar sonucuna varabiliriz. Bu yöntem, aynı zamanda, insan ve hayvan hastalıklarının hızlı teşhis için kullanılabilir gibi tıp ve veteriner patoloji, gelecekteki kullanım için büyük bir potansiyele sahiptir.

<table border="1"><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Glutaraldehit </td><td> Agar Bilimsel Ltd </td><td> R1312 </td><td></td></tr><tr><td> Osmiyum tetroksit </td><td> Agar Bilimsel Ltd </td><td> R1022 </td><td></td></tr><tr><td> Agar 100 reçine </td><td> Agar Bilimsel Ltd </td><td> R1043 </td><td></td></tr><tr><td> Dodecenyl süksinik anhidrit </td><td> Agar Bilimsel Ltd </td><td> R1051 </td><td></td></tr><tr><td> Metil nadic anhidrit </td><td> Agar Bilimsel Ltd </td><td> R1081 </td><td></td></tr><tr><td> Benzil dimetilamin </td><td> Agar Bilimsel Ltd </td><td> R1060 </td><td></td></tr><tr><td> Sitrat Kurşun </td><td> Agar Bilimsel Ltd </td><td> R1210 </td><td></td></tr><tr><td> Uranil asetat </td><td> Agar Bilimsel Ltd </td><td> R1260A </td><td></td></tr><tr><td> Fosfotungstik asit </td><td> Agar Bilimsel Ltd </td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td> Agar Bilimsel Ltd </td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Leica EM AMW </td><td> Leica Microsystems </td><td></td><td></td></tr><tr><td> Leica (Reichert) Ultratrim </td><td> Leica Microsystems </td><td></td><td> Yeni modeli mevcuttur </td></tr><tr><td> Leica (Reichert) ULTRACUT S </td><td> Leica Microsystems </td><td></td><td> Yeni modeli mevcuttur </td></tr><tr><td> Diyatomelere Ultra 45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td> Philips CM10 TEM </td><td> FEI (eski Philips) </td><td> &lt;/ Td&gt; <td> Yeni modeli mevcuttur </td></tr><tr><td> Hücre D </td><td> Olympus A.Ş. </td><td></td><td></td></tr><tr><td> Optimas 6.5.1 </td><td> Medya Sibernetik A.Ş. </td><td></td><td> Yeni sürümü mevcut </td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Virüsler tarafından oluşturulan ultrastrüktürel değişikliklerin incelenmesi genellikle açıkça Bitkilerde viral hastalıkların saptanması için gereklidir. Transmisyon elektron mikroskobu (TEM) için geleneksel numune hazırlama ile bu soruşturmaların birkaç gün 1,2 alabilir ve bu nedenle bitki virüs hastalıklarının hızlı tanı için uygun değildir. Mikrodalga tespit ölçüde geleneksel numune hazırlama 3-5 sonra gözlemlenen ile benzer sonuçlar ile ince yapı TEM incelemeler için numune hazırlama süresini azaltmak için kullanılabilir. Birçok farklı ölçüye mikrodalga cihazlar başarılı tespit ve TEM araştırmalar 5-8 için biyolojik numunelerin gömülmesi için kullanılabilecek şu anda mevcuttur. Bu çalışmada Tütün Mozaik Virüsü göstermektedirler (TMV) o T mikrodalga destekli numune hazırlama ile yaklaşık yarım gün içinde yapraklarda ultrastrüktürel değişiklikler teşhis etmek mümkün olduğunu Nicotiana tabacum bitkileri enfekteEM. Birçok adımlar yine elle 6-8 yapılmak zorundadır ve bu nedenle daha fazla zaman ve emek alıcı olan diğer mevcut cihazlar aksine neredeyse tamamen otomatik olarak 5 numune hazırlama yapar gibi biz piyasada satılan bir mikrodalga cihazı ile bu çalışma gerçekleştirmek için seçtiniz. Numune hazırlama kalan TMV-enfekte yaprakları ve ince yapısı ve boyutu, aşağıdaki inceleme sap içinde viral parçacıkların tam otomatik olarak negatif boyama gerçekleştirilir olarak tespit ve gömme sırasında gerçekleştirilebilir.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Bu çalışma, transmisyon elektron mikroskobu ve negatif boyama yöntemleri için mikrodalga destekli bitki numune hazırlama kombinasyonu kullanarak yaklaşık yarım gün bitki virüs hastalıklarının hızlı ve net bir teşhis sağlayan bir yöntem açıklanır.

Mikrodalga Transmisyon Elektron Mikroskobu ile Bitki Virüs Hastalıkları Hızlı Tanı Destekli

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K Görüntüleme.  University of Graz. Bu çalışma, transmisyon elektron mikroskobu ve negatif boyama yöntemleri için mikrodalga destekli bitki numune hazırlama kombinasyonu kullanarak yaklaşık yarım gün bitki virüs hastalıklarının hızlı ve net bir teşhis sağlayan bir yöntem açıklanır.

Video: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Yabani Kuşlar Kuş Gribi Virüsü Hızlı Teşhis: Alanında Taşınabilir RRT-PCR ve Dondurulmuş kuru Reaktifler Kullanımı

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

İmmünoloji ve Enfeksiyon

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero Hücre Kültür Testi ile Alan toplanan Sivrisinekler Enfeksiyon Virüs Algılama

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-Elektron Mikroskopi tarafından HIV-1 kapsid Meclisleri Yapısı ve iteratif Helisel Gerçek uzay İmar

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Sistein Kalıntılarının Solvent Erişilebilirlik Analizi<em&gt; Mısır rayado fino virüsü</emÜretilen&gt; Virüs gibi parçacıklar<em&gt; Nicotiana benthamiana</emVLP&#39;ler için Peptidlerin&gt; Bitkiler ve Çapraz bağlama

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Yüksek çözünürlüklü elektron mikroskobu<em&gt; Helikobakter pilori</emDemir Kullanılabilirlik Değişen Koşullarda Üretilen&gt; Çağ Tip IV Salgı Sistemi Pili

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronic minigenomlar İçeren virüs benzeri partiküller ile Biyogüvenlik Seviyesi 2 Koşullarında Ebola virüsü Ömrü Modelleme

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Pamuk Yeşilkurt bir densovirüs Host Fitness Host doku Dağılımı, İletim-modunu ve Etkisi incelenmesi için Protokoller

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Tam kan örneklerinde virüs tespiti için hızlı, güvenli ve basit el ile başucu nükleik asit çıkarma

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

Antimikrobiyal sinerji mürekkep püskürtmeli yazıcı yoluyla tarafından test otomatik Dama Tahtası dizi ve el ile zaman-kill yöntem

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...