This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).

NOT: Önceki tükürük örnekleri sağlayarak tüm konular Nationwide Çocuk Hastanesi, insan deneklerin tedavisi için yönergelere uygun bilgilendirilmiş onam formu. 1. Tükürük Toplama ve Saklama <ol><li> Tükürük toplama önce, öznenin ağız konu su ile ağzınızı çalkalayın sahip ve yeme kaçınarak veya numune toplama önce 30 dakika boyunca içerek gıda veya diğer yabancı maddelerden arındırılmış olduğundan emin olun. </li><li> Başlığın veya borunun iç dokunmamak için emin bir DNA sabitleme tamponu 2, 2.5 ml bir 15 ml santrifüj tüpü açın ve söz konusu tampon çözeltisi içine, tükürük 2,5 mi tükürük vardır. Not: tükürük fazla 2,5 ml Toplama DNA sabitleştirme tamponu, numune oranı yetersiz bir ikinci örnek bozulmasına yol açabilir. Çok az tükürük Toplama protokol beklenen verimi azaltacaktır. Toplama hacimleri değerlendirmek için, t tarafında sayılı geçişlerini kullanmakdiye tüp. </li><li> Kapağı yerine yerleştirin ve bu karışım homojen hale getirene kadar inversiyon yolu ile karıştırılır. Çalkalama gerekli değildir. Kısa süreli depolama veya uzun süreli depolama boyunca 4 ° C (&gt; 3 ay), oda sıcaklığında örnekleri saklayın. </li></ol> 2. İlk Hazırlıklar ve Hücre Lizis <ol><li> Önceki ekstraksiyon başlangıç, 37 ° C bir su banyosu ısı ve bir buz kovası hazırlar. Üç 15 ml konik santrifüj tüpleri her numune için ekstre edilmiştir gerekli olacaktır. Üç tüp, hücre ve protein topağı, son ekstre gDNA ve izopropanol ve etanol süpernatantlar tutmak için kullanılacaktır. </li><li> 15 saniye sonra orta hızda vorteks depolama örnekleri ve ters örnekleri birkaç kez almak. </li><li> Temiz bir 15 ml&#39;lik santrifüj tüpüne numune 2.5 ml koyun ve Celi Lysis Çözüm 5 ml ilave ediniz. Ters, numune 50 kez karıştırılır ve 30 dakika oda sıcaklığında inkübe edin. </li></ol> 3. RNA Kaldırma <ol><li> Rnaz A Sol 40 ul ekleyin100 mg / ml Katkı ve 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> 3 dakika boyunca buz üzerinde 37 ° C su banyosu numuneyi çıkarın ve soğuk. <ol><li> RNaz bir inkübasyondan sonra, protokol DNA rehidrasyon aşama boyunca 65 ° C&#39;ye kadar su banyosu sıcaklığını arttırabilir. </li></ol></li></ol> 4. Protein ve Lipid Kaldırma <ol><li> Ml / 20 mg Proteinaz K çözeltisi 50 ul ters çevirme ile birkaç kez karıştırın ve 30 dakika en az oda sıcaklığında inkübe edilir. Not: Bu protokolü için olası bir duraklama noktasıdır. Ekstraksiyon tamamlanabilir kadar Proteinaz K solüsyonu ilave edildikten sonra, numune, 4 ° C&#39;de saklanabilir. En fazla 24 saat boyunca 4 ° C &#39;de depolama, ekstraksiyon verimi veya DNA kalitesi üzerinde önemli bir etkiye sahip olduğu gösterilmemiştir. Bu aşamada uzun süreli depolama henüz değerlendirilmemiştir. </li><li> 10 dakika boyunca buz üzerinde yüksek hızda 20 saniye, ve yeri boyunca kuvvetli bir şekilde, girdap protein çökeltme çözeltisinin 1.7 ml ilave edilir. </li&gt; <li> Örnekler 3000 x g&#39;de 10 dakika ve 4 ° C&#39;de 10 dakika santrifüj boyunca buz üzerinde soğutuldu sonra. Çöken proteinler, sıkı bir topak oluşturmak devam etmek gerekir. Topak sıkı değildir ya da çözelti hala puslu olması halinde, numuneler daha 5 dakika boyunca buz üzerinde soğutulabilir ve santrifüj işlemi tekrarlanır. Numuneler, bir sıkı bir pelet sağlamak için buz üzerinde tutulmalıdır. </li></ol> 5. izolasyonu ve saflaştırılması gDNA <ol><li> Temiz bir 15 ml&#39;lik bir santrifüj tüpü içine, bir pipet 5 ml izopropanol ve 20 mg / ml saf Glikojen Çözüm 8 ul. </li><li> Çöken protein, pelet geride bırakarak, izopropanol ve glikojen çözeltisi içeren bir tüp içine adım 4,3 gDNA ihtiva eden üst sıvıyı dökün. Üst fazı ilave edildikten sonra yavaşça 3.000 x g ve 4 ° C&#39;de 30 dakika boyunca ters Numune ve santrifüj ile 50 kez karıştırılır. </li><li> Temiz bir 15 ml tüp içine yavaşça süpernatant dökün. Üstte yüzen maddenin çıkarılmasından sonra,% 70 etanol, 1 ml eklebirkaç kez yavaşça sallanarak hafifçe çökelen pelet üzerine etanol taşıyarak pelet yıkayın. Tüp içinde etanol saklayın. </li><li> İlk yıkama, 2,000 xg&#39;de 1 dakika ve 20 ° C için örnek santrifüj. Bu santrifüj işlemi, 4 ° C ya da 20 ° C &#39;de yapılabilir. Sıcaklıkta hiçbir belirgin etkisi, bu aşama için gösterilmiştir. </li><li> Pelet İlk yıkama ve santrifüj ardından yavaş yavaş tüpten etanol yıkama dökün ve atın, sonra adımları 5.3 ve 5.4 tekrarlayarak bir ikinci yıkama yapar. </li><li> İkinci yıkamadan gelen süpernatan ayrılmasından sonra, 15 dakika boyunca hava ile kuruması için pelet sağlar. <ol><li> Numune tamamen kurumuş olduğunda, başka bir 15 dakika boyunca hava ile kurumaya. </li></ol></li></ol> GDNA&#39;sından 6. Rehidrasyon <ol><li> Örnek kuruduktan sonra, kurutulan gDNA pelet yeniden sulanması Tris-EDTA içinde 300 ul ilave edin. </li><li> Bir orta hızda ve yerde 5 saniye vorteks örnek1 saat boyunca 65 ° C sıcak su banyosu. </li><li> Su banyosundan çıkarın ve numune, oda sıcaklığında O / N inkübe edin. Not: Tüm ürünler ve reaktifler Malzeme, Tablo l&#39;de listelenen ikinci, hem de Tablo 4. </li></ol>

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The authors have nothing to disclose.

Bu prosedür, büyük ölçüde DNA, kaliteden ödün vermeden, standart yöntemlere göre daha yüksek molekül ağırlıklı DNA verimini geliştirilmiş bir optimize edilmiş DNA ekstraksiyon protokolüdür. Verim çoğunda büyük etkisi ile kritik adım, yaygın olarak 11 dağıtılan değildi biri hariç, burada gözden yayınlanmış herhangi bir protokol daha etanol yağış sırasında uzun bir santrifüj aşamasını kapsamaktadır adım 5.2 oldu. Bu daha santrifüj ile bağlantılı DNA kalitesinde...

İnsan genetik çalışmalar için yüksek kalitede DNA temininde hastalık geni bulma işlemi esastır. Kan, tükürük toplama daha pahalı olması da invaziv işlemler gerektiren ve rağmen, fonksiyonel çalışmalar için sonsuz bir DNA kaynağı veya iPSCs olarak ölümsüzleşti hücre hatları oluşturmak için tercih edilir ve hücre hatları mevcut olmadığında bazen kan DNA kullanılır. Bununla birlikte, kan elde eğitimli bir flebotomist gerektirir ve kan tükürük 1 daha kısa bir yarı-ömrü vardır. O toplanan ve böylece iyi hastane ve laboratuvarlardan 2 su toplama alanı dışında potansiyel denek havuzu artan flebotomist için gerek kalmadan posta yoluyla gönderilen olabilir beri tükürük DNA elde etmek daha az pahalı ve daha kolaydır. Konular yerine kan 3, 4 bir tükürük örneği vererek seçeneği varken Çalışması okullaşma gelişmiş olabilir. Miktar ve tükürük DNA kalitesi ile ilgili endişeler bizi yaygın sınırlı olabilirtükürük 2, 3, 4, 5 önemli miktarda elde etmedi eski yanak bezlerden yöntemleri üzerinde DNA testi için mililitre başına 4.3 x 10 5 hücre ortalama ile, bütün tükürük uygunluğunu gösteren çok sayıda çalışma son çalışmalarda rağmen e, 6. mütevazı bir edebiyat microarray tabanlı yöntemler 8, dahil genotiplendirme uygulamalar için bütün tükürük edilen DNA uygunluğunu gösteren var iken 9, 10, herhangi bir çalışma inceleyen gelecek nesil dizileme (NGS). Bütün bu tükürük DNA ekstraksiyon protokolü optimize etmek için hedefe kolaylıkla ortak reaktifler ve sarf malzemeleri ile laboratuarlarda uygulanan maliyet etkin bir şekilde genetik uygulamalar için miktar ve kalitesini en üst düzeye çıkarmak oldu. Tükürük DNA ekstraksiyon çeşitli prosedürler gerektirir: 1) toplama ve depolama, 2) hücre parçalama, 3) RNaz tedavisi, 4) protein çökelmesi, 5) etanol yağış, 6) DNA rehidrasyon. Tarif edilen DNA sabitleştirme Tampon çözelti,Daha önce 2, yeterince değişiklik olmadan fonksiyonları. RNaz tedavi ve DNA rehidrasyon adımları optimize için hiçbir girişimde bulunuldu. Kalan her adım için, verimi etkileyebilecek çeşitli değişkenler tespit edilmiştir. Her bir değişken ayrı ayrı manipüle edilmiş ve verim ve kalitesinde iyileşme istatistiksel olarak değerlendirildi. Veriminin ve / veya DNA kalitesini geliştirdiği gösterildi değişkenler için, en uygun değerleri final protokolüne dahil edilmiştir.

<table border="0" cellpadding="0" cellspacing="0" width="260">
	<tbody>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">15ml Centrifuge Tubes</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">12-565-268</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Cell Lysis Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158908</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Proteinase K</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:84px;">P6556</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Protein Precipitation Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158912</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Isopropanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">A416-4</td>
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			<td height="33" style="height:33px;width:88px;">Glycogen</td>
			<td style="width:88px;">EZ-BioResearch</td>
			<td style="width:84px;">S1003</td>
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			<td height="22" style="height:22px;width:88px;">70% Ethanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">04-355-305</td>
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		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Tris-EDTA (TE)</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP2473-1</td>
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		<tr height="33">
			<td height="33" style="height:33px;width:88px;">NaCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">AC194090010</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Tris HCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP1757-100</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">EDTA(0.5M) Solution</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">03-500-506</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Sodium Dodecyl Sulfate</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP166-100&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
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			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
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			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Name of Equipment</td>
			<td style="width:88px;">Distributor</td>
			<td style="width:84px;">Catalog#</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:88px;">Analog Vortex Mixer</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">02-215-365</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Centrifuge 5810R</td>
			<td style="width:88px;">Eppendorf</td>
			<td style="width:84px;">5811 000.010</td>
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	</tbody>
</table>

collection and extraction of saliva dna for next generation sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA, bir DNA ekstraksiyon eşleştirilmiş bir dizi gerçekleştirilmiştir ekstraksiyon için optimal parametreleri belirlemek. Tek bir tükürük örnek bölünmüş ve her bölümü belirli bir değişken için iki muhtemel değere biri ile test edilmiştir. Eşleştirilmiş her testin en az sekiz çoğaltır (örneğin, tek bir tükürük örneği ve ilk 50 ° C inkübasyon olmadan hem ekstraksiyon test etmek için bölündü) yapıldı. Toplam DNA verimi, 260/280 değeri, 260/230 değeri ve parçalanma de?...

Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

Collection and Extraction of Saliva DNA for Next Generation Sequencing

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Yeni Nesil dizinleri için Toplama ve Tükrük DNA çıkarımı

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

collection-and-extraction-of-saliva-dna-for-next-generation-sequencing

Research

JoVE Journal

Medicine

39.0K Görüntüleme.  The Research Institute at Nationwide Children's Hospital. DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Video: Collection and Extraction of Saliva DNA for Next Generation Sequencing

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Biyomedikal Uygulamalar için Aljinatta Mikrokürelerin Üretimi

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

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The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

Α-testi: Tüm Tükürük Kullanarak Hızlı Hücre içermeyen CD4 sayımı

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

İnsan Pankreas için Toplama Protokolü

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

Biyomarker Çalışmaları için bir sonraki nesil Doku Mikrodizi (ngTMA) Protokol

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

Düşük kaliteli ve düşük miktar Tümör Biyopsilerinde ve Islak ve kuru Bench Süreçleri Entegrasyonu Hedefli optimize eder nesil Sıralama

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

Macaques Karaciğer ve Mezenterik lenf nodu Seri Koleksiyonu için Laparoskopik Tekniği

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.
Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing.&#160;Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.
In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

Tüm genom sıralama Candida glabrata algılama, işaretçileri, Antifungal ilaç direnci için

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Metabolomik ve Lipidomik Analizler için Beyin Tümörlerinin Yerinde Örnekleme sivesi ve ekstraksiyonu

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke survivors. The diagnosis and characterization of these symptoms in individual patients can be challenging and often requires skilled clinical staff. Virtual reality (VR) presents an opportunity to develop novel assessment tools for patients with USN.
We aimed to design and build a VR tool to detect and characterize subtle USN symptoms, and to test the tool on subjects treated with inhibitory repetitive transcranial magnetic stimulation (TMS) of cortical regions associated with USN.
We created three experimental conditions by applying TMS to two distinct regions of cortex associated with visuospatial processing- the superior temporal gyrus (STG) and the supramarginal gyrus (SMG) - and applied sham TMS as a control. We then placed subjects in a virtual reality environment in which they were asked to identify the flowers with lateral asymmetries of flowers distributed across bushes in both hemispaces, with dynamic difficulty adjustment based on each subject's performance. 
We found significant differences in average head yaw between subjects stimulated at the STG and those stimulated at the SMG and marginally significant effects in the average visual axis.
VR technology is becoming more accessible, affordable, and robust, presenting an exciting opportunity to create useful and novel game-like tools. In conjunction with TMS, these tools could be used to study specific, isolated, artificial neurological deficits in healthy subjects, informing the creation of VR-based diagnostic tools for patients with deficits due to acquired brain injury. This study is the first to our knowledge in which artificially generated USN symptoms have been evaluated with a VR task.

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

Tek Taraflı Mekansal İhmalleri Değerlendirmek için Sanal Gerçeklik Araçları: Veri Toplama için Yeni Bir Fırsat

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke ...