Jean-François Papon, Bruno Louis, Estelle Escudier ve Paris-Est PCD tanı merkezinin tüm ekip üyelerine, PCD tanı merkezlerini ziyaret ettikleri ve sayısız değişim sırasında müsaitlikleri ve içten karşılamaları için teşekkür ederiz. Ayrıca Robert Hirst'e ve Leicester'ın PCD merkezindeki tüm ekip üyelerine karşılamaları, zamanları, tavsiyeleri ve uzmanlıkları için teşekkür ederiz.

Bu yazarların açıklayacak hiçbir şeyleri yok.

Bu makale, COVID-19 pandemisi sırasında uygun enfeksiyon kontrolü hususları için yapılan ayarlamalarla birlikte, burun fırçalama örneklerini kullanarak CFA için standart bir çalışma prosedürü sağlamayı amaçlamaktadır. PCD tanısı zordur ve şu anda nazal nitrik oksit ölçümü, DHSV kullanarak CFA, iletim elektron mikroskobu (TEM) kullanarak siliyer ultrayapısal analiz, immünofloresan kullanarak siliyer proteinlerin etiketlenmesi ve PCD'ye neden olan genler için genetik test dahil olmak üzere ulu...

Primer siliyer diskinezi (PKH), solunum kirpiklerinin durağan, yavaş veya diskinetik olduğu, mukosiliyer klirens bozukluğuna ve kronik oto-sino-pulmoner hastalığa yol açan kalıtsal heterojen hareketli siliopatidir 1,2,3,4. PKH'nin klinik bulguları erken bebeklik döneminde başlayan kronik ıslak öksürük ve kronik burun tıkanıklığı, bronşektaziye yol açan tekrarlayan veya kronik üst ve alt solunum yolu enfeksiyonları, tekrarlayan veya kronik otitis media ve sinüzittir 5,6,7. PKH hastalarının yaklaşık yarısı situs inversus veya situs ambiguus gibi organ lateralite defektleri ile başvurmaktadır. Bazı hastalar ayrıca erkeklerde hareketsiz sperm ve kadınlarda Fallop tüplerinde hareketsiz kirpikler nedeniyle infertilite sorunları ile başvurmaktadır 1,2,8. PCD nadirdir, ancak prevalansı tanımlamak zordur ve 1: 10.000 ila 1: 20.000 9,10 arasında değişmektedir. Ancak PKH'nin gerçek prevalansının tanıdaki güçlükler ve klinik şüphe eksikliği nedeniyle daha yüksek olduğu düşünülmektedir. PCD semptomları, diğer akut veya kronik solunum yollarının ortak solunum bulgularını taklit eder ve tanıyı doğrulamanın tanısal zorlukları iyi bilinmektedir, bu da yetersiz tedavi ve takibe yol açmaktadır 2,5,9,11.
Dijital Yüksek Hızlı Videomikroskopi (DHSV) kullanan siliyer videomikroskopi, PCD 4,8,12,13 için tanı araçlarından biridir. DHSV, siliyer vuruş frekansı (CBF) ve vuruş paterni(CBP) analizi 2,14,15,16'dan oluşan siliyer fonksiyonel analizi (CFA) gerçekleştirmek için en uygun yöntem olarak kabul edilir. DHSV, genellikle burun fırçalamadan elde edilen canlı solunum epiteli kullanır13.
Mevcut COVID-19 salgını göz önüne alındığında, kanıtlar altta yatan solunum yolu hastalığının COVID-19 enfeksiyonundan sonra daha kötü sonuçlara yol açabileceğini gösterdiğinden, PCD tanısının doğrulanması artık daha da önemlidir17,18. Mevcut pandemi sırasında güvenli ve etkili bir PCD tanı hizmeti, doğrulanmış PCD hastalarının genel popülasyona kıyasla ek koruyucu önlemlerden yararlanmasına da izin verecektir19.
COVID-19'un bulaşması öncelikle damlacık yayılımıyoluyla gerçekleşir 20. Asemptomatik (veya minimal semptomatik) hastalardan yüksek bulaşma potansiyeli, burun örneği20'deki yüksek viral yük ile önerilmektedir. Ek olarak, viral partiküller aerosol haline gelirse, havada en az 3 saat kalırlar21. Bu nedenle, solunum sağlığı çalışanları, tanı teknikleri için klinik bakım ve örnek toplama işlemi gerçekleştirirken yüksek bir viral yük rezervuarına maruz kalmaktadır22. Ayrıca, canlı solunum örneklerinin manipülasyonu, teknisyeni COVID-19 kontaminasyonuna maruz bırakır. COVID-19 hastalarına bakan solunum hekimleri ve KBB cerrahları için en iyi uygulama önerileri23 uygulanırken, COVID-19 pandemisi sırasında DHSV'nin uygulanmasına yönelik önerilerin eksikliği vardır.
Bir PCD teşhis hizmeti sunmaya devam etmek için, sağlık çalışanının (numune toplama gerçekleştiren) ve teknisyenin (numune işleme gerçekleştiren) güvenliğini sağlarken, siliyer videomikroskopi protokolünün COVID-19 pandemisi sırasında uyarlanması gerekiyordu. Siliyer videomikroskopi tekniği şu anda araştırma hizmeti ve uzmanlaşmış tanı merkezleri ile sınırlıdır, çünkü CFA kapsamlı eğitim ve deneyim gerektirir. Ayrıca, şu anda, DHSV 4,13 kullanarak numunelerin işlenmesi ve analiz edilmesi için standardizasyon ve hassas çalışma prosedürü eksikliği vardır.
Bu makalenin amacı, DHSV için standart çalışma prosedürlerini, özellikle canlı burun epitelini örneklendirirken ve işlerken enfeksiyon kontrol önlemlerine ve güvenliğe atıfta bulunarak tanımlamaktır. Bu, mevcut COVID-19 salgınına rağmen yüksek kaliteli PCD tanı ve bakımının devam etmesini sağlayacaktır.

<table>
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>FisherScientific</td>
			<td>352096</td>
			<td>15 ml High-Clarity Polypropylene Conical Tube with lid</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>LONZA</td>
			<td>17-836E</td>
			<td>Antifungal solution</td>
		</tr>
		<tr>
			<td>Blakesley-weil nasal forceps</td>
			<td>NOVO SURGICAL</td>
			<td>E7739-12</td>
			<td>Used to hold the brush to perform the nasal brushing</td>
		</tr>
		<tr>
			<td>Bronchial cytology brush</td>
			<td>CONMED</td>
			<td>129</td>
			<td>Used for nasal brushing</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>NUOVA APTACA</td>
			<td>2150/SG</td>
			<td>Used for COVID-19 testing</td>
		</tr>
		<tr>
			<td>Digitial high-speed videomicroscopy camera</td>
			<td>IDTeu Innovation in motion</td>
			<td>CrashCam Mini 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>ThermoScientific</td>
			<td>12372098</td>
			<td>Microscope slides used to create the visualization chamber</td>
		</tr>
		<tr>
			<td>Heated Box</td>
			<td>IBIDI cells in focus</td>
			<td>10918</td>
			<td>Used to heat the sample</td>
		</tr>
		<tr>
			<td>Inverted Light microscope</td>
			<td>Zeiss</td>
			<td>AXIO Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Heater</td>
			<td>TOKAI HIT</td>
			<td>TPiE-LH</td>
			<td>Used to heat the oil immersion lens</td>
		</tr>
		<tr>
			<td>Medium 199 (M199), HEPES</td>
			<td>TermoFisher Scientific</td>
			<td>12340030</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Motion Studio X64</td>
			<td>IDT Motion</td>
			<td>version 2.14.01</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Oil</td>
			<td>FischerScientific, Carl Zeiss</td>
			<td>11825153</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular cover slip</td>
			<td>VWR</td>
			<td>631-0145</td>
			<td>Used to cover the visualization chamber</td>
		</tr>
		<tr>
			<td>Spacer (Ispacer) 0.25 mm</td>
			<td>Sunjinlab</td>
			<td>IS203</td>
			<td>Used for the creation of the hermetic closed visualization chamber</td>
		</tr>
		<tr>
			<td>Square cover slip</td>
			<td>VWR</td>
			<td>631-0122</td>
			<td>Used for the creation of lab-built open visualization chamber</td>
		</tr>
		<tr>
			<td>Streptomycin/Penicillin</td>
			<td>FisherScientific, Gibco</td>
			<td>11548876</td>
			<td>Antiobiotics solution</td>
		</tr>
	</tbody>
</table>

nasal brushing sampling and processing using digital high speed ciliary videomicroscopy &ndash; adaptation for the covid-19 pandemic

Primer Siliyer Diskinezi (PKH), önemli otosinopulmoner hastalığa yol açan genetik motil bir siliopatidir. PCD tanısı, farklı tanı yöntemlerindeki zorluklar nedeniyle sıklıkla kaçırılır veya gecikir. PCD için tanı araçlarından biri olan Dijital Yüksek Hızlı Videomikroskopi (DHSV) kullanan siliyer videomikroskopi, siliyer vuruş frekansı (CBF) ve vuruş paterni (CBP) analizinden oluşan siliyer fonksiyonel analiz (CFA) yapmak için en uygun yöntem olarak kabul edilir. Bununla birlikte, DHSV, numunelerin işlenmesi ve analiz edilmesi için standartlaştırılmış, yayınlanmış çalışma prosedüründen yoksundur. Ayrıca, COVID-19 pandemisi sırasında önemli bir enfeksiyon kontrol sorunu olan canlı solunum yolu epitelini kullanır. Bu sağlık krizi sırasında tanısal bir hizmet sunmaya devam etmek için, siliyer videomikroskopi protokolü yeterli enfeksiyon kontrol önlemlerini içerecek şekilde uyarlanmıştır.
Burada, siliyer solunum numunelerinin numune alınması ve laboratuvarda işlenmesi için gözden geçirilmiş bir protokolü açıklıyoruz ve COVID-19 enfeksiyon kontrol önlemlerine uymak için yapılan uyarlamaları vurguluyoruz. Bu protokole göre işlenen ve analiz edilen 16 sağlıklı denekten alınan burun fırçalama örneklerinden elde edilen CFA'nın temsili sonuçları açıklanmıştır. Ayrıca, kalite seçim kriterlerini karşılamayan numuneler artık CFA'ya izin verdiğinden, optimum kalitede epitel siliye şeritlerin elde edilmesinin ve işlenmesinin önemini de gösteriyoruz, bu da bu tekniğin tanısal güvenilirliğini ve verimliliğini potansiyel olarak azaltıyor.

Tekniğin etkinliğini göstermek için, CFA'nın sonuçlarını 16 sağlıklı yetişkin gönüllüden oluşan bir seride sunuyoruz (5 erkek, yaş aralığı 22-54 yaş).
Toplam 16 gönüllüden 14'ünden (4 erkek, yaş aralığı 24-54 yaş) alınan burun fırçalama örnekleri, CFA'yı gerçekleştirmek için gereken seçim kriterlerini karşılayan yeterli uygun epitel kenarları sağlamıştır. Bu 14 burun fırçalama örneğinden toplam 242 siliyer kenar kaydedildi ve 212 kenar tanımla...

Watch this Scientific Journal Video about Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy Ã¢â‚¬â€œ Adaptation for the COVID-19 Pandemic at JoVE.com

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

PCD tanısı için başarılı ve yüksek kaliteli siliyer fonksiyonel analizi garanti etmek için, solunum epitel örneklemesi ve işlenmesi için hassas ve dikkatli bir yöntem şarttır. COVID-19 pandemisi sırasında PCD tanı hizmeti vermeye devam etmek için, siliyer videomikroskopi protokolü uygun enfeksiyon kontrol önlemlerini içerecek şekilde güncellenmiştir.

Dijital Yüksek Hızlı Siliyer Videomikroskopi Kullanarak Burun Fırçalama Örneklemesi ve İşleme – COVID-19 Pandemisine Adaptasyon

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

nasal-brushing-sampling-processing-using-digital-high-speed-ciliary

Research

JoVE Journal

Immunology and Infection

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

4.5K Görüntüleme.  University of Liège. PCD tanısı için başarılı ve yüksek kaliteli siliyer fonksiyonel analizi garanti etmek için, solunum epitel örneklemesi ve işlenmesi için hassas ve dikkatli bir yöntem şarttır. COVID-19 pandemisi sırasında PCD tanı hizmeti vermeye devam etmek için, siliyer videomikroskopi protokolü uygun enfeksiyon kontrol önlemlerini içerecek şekilde güncellenmiştir.

Video: Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy – Adaptation for the COVID-19 Pandemic

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Egzersizle indüklenen Lenfosit Apoptoz belirlenmesi için parmak sopa Kan Örnekleme Metodolojisi

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

İmmünoloji ve Enfeksiyon

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Cryo-Elektron Tomografi ve Otomatik Sub-tomogram Ortalaması kullanarak HIV Zarf Glikoproteinler Moleküler Yapılarının Belirlenmesi

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

Granülosit-bağımlı otoantikor indüklenen Cilt Kabarcıklanma

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Yüksek Verimli/Yüksek İçerikli Taramaya Uyarlanmış Hücre İçi Mikobakterilerin Nicelleştirilmesi için Mikroskobik Fenotipik Bir Tahlil

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Sigara litik Ekzositoz görselleştirme<em&gt; Cryptococcus neoformans</em&gt; Dijital Işık mikroskopi ile Makrofagların gelen

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Domuz influenza A virüs Algılama ve İzolasyonu için Burun Mendil

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

Dijital Çizgi-kodlama Platform&#39;u kullanarak bulaşmasını Mikroplar Gen İfadesi profil

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Kan işleme için hızlı bir yöntem insan kanı plazma peptid düzeylerinin veriminin arttırılması için

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Yüzeyler İnsan Norovirus Tespiti sürüntü Örnekleme Yöntemi

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Electrochemiluminescence deneyleri için insan adacık otoantikorlar

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...