Araştırma Harry Winston Araştırma Vakfı tarafından desteklenmiştir.

1. Taze Çalışma Çözümler hazırlayın <ol><li> TBS: 0.05 M Tris Salin, pH 7.8 (TBS) Tamponlu. 60 ml TBS / mikroplak hazırlayın. </li><li> KKE: Domuz pankreatik elastaz, tip 1 (PPE) bir süspansiyon olarak sağlanmıştır. Iyice çalkalayın ve yaklaşık 0.01 U / ml TBS KKE sulandırın. 6 ml) solüsyonu / mikroplak çalışma hazırlayın. </li><li> SA 3 NA: Sukkinil-L-Ala-Ala-L-L-Ala-p-nitroanilid (SA 3 NA). Dimetil sülfoksit (DMSO) 0.1 M stok SA 3 NA çözeltisi hazırlayın ve -20 ° C&#39;de hemen kullanmak veya saklamak Stok SA 3 NA çözüm TBS içinde 1/100 sulandırarak 6ml çalışma çözüm / mikroplak hazırlayın. </li><li> Coomassie MAVİ: TBS% 10 Coomassie Brillant Blue R-250 (Coomassie Mavi) bir stok çözeltisi hazırlayın ve -20 ° C&#39;de hemen veya depoyu kullanın TBS içindeki stok solüsyonu 1/1000 seyreltilmesi ile 6 ml çalışma çözeltisi hazırlayın. </li></ol> 2. Tükürük hazırlayın <ol&gt; <li> Saliva KOLEKSİYON: duran konumda, örneğin ağız içinde sitrik asit veya sakızı gibi bir sialogogue yerleştirerek tükürük içine serum eksudasyon uyarır. Bir şekersiz damla limon sık dişsiz ve sakız çiğnemek edemiyoruz HIV-1 hastaları için uygundur. İlk 3 dakika boyunca gerektiği gibi yutmak ve sonraki 3 dakika için bir maskeli kabına tüm ağız tükürük toplamak. Bu, tükürük yaklaşık 2 ml sağlayacaktır. -80 Azından hemen kullanın veya saklamak ° C </li><li> Saliva TAŞIMA: Eğer 37 ° C ve buz üzerinde yerde donmuş, çözülme örneği. 2-4 taze veya çözülmüş örnek ° C&#39;nin 3 gün en fazla. Vorteks değil tükürük Sahibi ve bir serum eksuda içeren musin pipetleme kaçının. Tükürük işlerken Standart Önlemler kullanın <a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf</a> . </li></ol> 3. Mikroplaka Set-up <ol><li><strong&gt; TAMPON: Ölçümler üç nüsha halinde yapılır. , Bir mikrotitre plakası her bir kuyucuğa 130 uL TBS eklenir. </li><li> ÖRNEKLER: her bir test için de, 150 ul / kuyucuk bir son hacim 20 uL tükürük eklenir. Kabarcıklar tanıtma olmadan pipetleme ile karıştırın. </li><li> KONTROLLER: Pozitif kontrol KKD içerir, ancak tükürük. Negatif kontrol tükürük içerir, fakat herhangi bir PPE.To pozitif kontrol de 20 uL TBS eklenir. Negatif kontrol etmek de 20 uL tükürük eklenir. </li><li> KKE KULUÇKA: KKD çalışma çözümü iyice çalkalayın. De negatif kontrol kuyuları hariç her 50 uL KKE çalışma solüsyonu eklenir. Negatif kontrol kuyuları için, 50 uL TBS ekleyin. 23 ° C&#39;de 10 dakika inkübe </li><li> SUBSTRAT KULUÇKA: Her bir kuyuya 50 uL SA 3 NA çalışma çözeltisi ekleyin. 23 ° C&#39;de 45 dakika inkübe </li><li> Coomassie MAVİ:. 50 uL Coomassie Mavi çalışma çözüm Ekle &lt;/ Li&gt; <li> OKUMA: mikroplaka okuyucu absorbans, aktif α 1 PI ve protein içeriği temsil Coomassie Mavi algılamak için bir 595 nm temsil SA 3 NA bölünme algılamak için bir 405 nm okuyun. </li></ol> 4. Α 1 PI Endeksi hesaplayın <ol><li> ORTALAMA BİR 405 nm VE 595 nm normalleştirmek: Her üç nüsha için, ortalama hesaplamak. Numune ortalama Negatif Kontrol ortalama çıkarın. Plaka-to-plaka değişimi en aza indirmek için, aşağıdaki gibi Pozitif kontrol ortalama her Numune ortalama bölerek ortalama normalize: </li></ol><img alt="Denklem 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> Α 1 PI INEX HESAPLAYIN: normalize A 595 nm ile 405 nm bir normalize bölün: </li></ol> 5.. Temsilcisi Sonuçlar Endemik, kaynak sınırlı bölgelerde CD4 sayısı izlemek için bir nokta-bakım tarama testi olarak α-test kullanımı için uygun olup olmadıklarını belirlemek için, uyarılmış tükürük 20 kadın toplandı ve 11 erkek HIV-1 konular katılıyor kliniği için Kamerun rutin bakım. New York&#39;ta toplanan tükürük kullanarak α-testinin geliştirilmesi sırasında ön gözlemler ile tutarlı, tükürük α 1 PI Endeksi CD4 sayısı (r 2 = 0.91, p &lt;0.0001. N = 31) ile korele Kamerun toplanır. Ilişki bir doz-yanıt ilişkisi (Şekil 2A) ile tutarlı bir 3-parametreli sigmoidal eğri olarak tanımlandı. % 95 güven ve tahmin aralıkları sırasıyla mavi ve kırmızı çizgiler, (Şekil 2B) ile birlikte tasvir edilmektedir. Içindeönceki bir çalışmada, CD4 + T hücreleri dönemsellik 23 sinüs bisiklet ± HIV-1 dersten ve α 1 PI eksikliği (Pizz) 3 miras versiyonu olan olgularda 3.5 gün sergilediği belirlenmiştir. Tasvir burada 27 gün periyodisite (Şekil 3A) sergileyen olmayan bir HIV-1 Sağlıklı kontrol bisiklet CD4, bilgisayar tarafından üretilen sinüs eğrisi analizi temsili bir örnektir. CD4 Bilgisayar oluşturulan sinüs eğrisi analizi + 6 konularda T hücre değişiklikleri salınım 3 tepe-tepeye genlik ve eksen değerlerini vermiştir. Beklendiği üzere, salınım ekseni genlik (r 2 = 0.96, p = 0.009, n = 6) (Şekil 3B) ile korele idi. Hastalarda olacak düşük CD4 + T hücre sayısı, CD4 T hücre sayısı siklik değişiklikler küçüktü ve yüksek CD4 + T hücre sayısı olan hastalarda, siklik değişiklikler büyüktü. ÇünküŞekil 2 içinde gösterildiği regresyon hattı salınımı ve salınım ekseninin ekseni amplitüd bağlı olarak değişebilir etmek ve böylece regresyon hattı CD4 + T hücreleri beklenir ne kadar yukarıda ve aşağıda tahmin regresyon çizgisi için hesaplanan edilebilir, amplitüd ile ilişkilidir tahmin bisiklet (Şekil 3C). Şekil 3C ve Şekil 2B hesaplanan güven aralığı hesaplanan genlik overlaying, genlik (yeşil hat)% 95 içinde yer olduğu tespit edilmiştir güven aralığı (mavi çizgi) (Şekil 3D). <img alt="Şekil 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Şekil 1.. Prosedür Özet: ağızda Yeri sitrik asit ve tükürük toplamak. Üçlü bir mikroplaka kuyu için seyreltilmiş tükürük ekleyin. Her oyuğa PPE ekleyin ve 23 ° C&#39;de 15 dakika süreyle inkübe Her oyuğa PPE substrat ekleyin ve 23 ° C&#39;de 45 dakika süreyle inkübe. Coomassie Mavi ekleyin ve PPE aktivite tespit etmek ve 595 nm de proteini tespit etmek için 405 nm de okunur. <img alt="Şekil 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Şekil 2. Standart CD4 numaralandırma yöntemi ile α-testi karşılaştırılması. A) α-test uyarılan tüm ağız tükürük α 1 PI Endeksi miktarlarının tarafından yapıldı. CD4 sayısı standart akım sitometri kullanılarak bağımsız bir tıbbi laboratuvar tarafından yapıldı. Akım sitometri ve tükürük için kan aynı klinikte ziyaret toplandı. Α 1 PI Endeksi ve CD4 sayıları arasındaki ilişki, bir 3-parametreli sigmoidal eğrisi (r 2 = 0.91, p &lt;0.0001, n = 31). B)% 95 güven aralıkları ve tahmini mavi ve kırmızı olarak tasvir edilmektedir tanımlandı sırasıyla hatları,. <img alt="Şekil 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Şekil 3. CD4 c hesaplama Doğrulukα1PI Index.A kullanarak ounts) CD4 + T hücreleri olmayan bir HIV-1 sağlıklı kontrol 27 günlük periyodik sinüs bisiklet sergiledi. Salınım ekseni tasvir edilir (kırmızı noktalı çizgi). B) T hücre bisiklet 4 HIV-1 kişilerde salınım peak-tepe genliği ve eksen hesaplamak için kullanılan CD4, 1 sigara HIV Bilgisayar üretilen sinüs dalga analizi -1 Pizz konu ve parçası A. CD4 arasındaki ilişkiyi tasvir olmayan HIV-1 sağlıklı kontrol T + hücre genlik ve salınım ekseni 3-parametreli sigmoidal eğrisi (r 2 = 0.96, p = 0.009, n kabul edildi = 6). C) parçası B) &#39;de elde edilen salınım genlik ve ekseni arasındaki ilişkiyi sigmoidal Şekil 2 den regresyon hattı (siyah çizgi) boyunca nokta, beklenen amplitüd (yeşil hatlar) hesaplamak için kullanılmıştır. GE) beklenen amplitüd aralığı (yeşil hat)% 95 güven aralığı (belirgin olarak farklı bulunduolarak Mann Whitney Rank Sum Testi ve genlik hattı ve regresyon hattı (37 hücreleri arasındaki ortalama fark (% 95 güven aralığı ve regresyon hattı (63 hücre / ml arasındaki ortalama fark karşılaştırılmasına dayanarak) tarafından belirlenen mavi çizgiler) / ml), p = 0.001).

<ol>
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Çıkar çatışması ilan etti.

Seyreltilmemiş Serum median 36 mM içeren α 1 PI ve 3.6 mM α 2 makroglobulin (α 2 M) 10 kat farkı 8. Her iki protein KKD bağlanmak için yarışırlar. Bu iki özellik, konsantrasyon ve PPE yakınlığı, özellikle α 2 M 8 rakip varlığında, normal serum α 1 PI ölçebilen bir yöntem geliştirmek için istismar edilmiştir. Bu yöntem,% 0.3 serum az 50 nM μ 1 PI algılayabilir. % 2,5 serum dilüsyonunda, KKD&#39;ler içi...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sialogogue, 0.05M sitrik asit </td><td> Eda en tatlandırılmamış Şekerleri, 4900 Arka N.20th Street, Philadelphia, PA 19144 </td><td> Şeker-Free limon damlatarak </td></tr><tr><td> Domuz pankreatik elastaz, tip 1 (AK 3.4.21.36) </td><td> Sigma-Aldrich, St. Louis, MO 63.103 </td><td> E1250 </td></tr><tr><td> Sukkinil-L-Ala-Ala-L-L-Ala-p-nitroanilid </td><td> Sigma-Aldrich </td><td> S4760 </td></tr><tr><td> Coomassie Brillant Blue R-250 </td><td> Sigma-Aldrich </td><td> B7920 </td></tr><tr><td> Doku kültürü ile tedavi edilen, 96 mikroplaklar </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> 405 nm ve 595 nm filtreleri ile Mikroplaka Okuyucu </td><td> MTX Lab Sistemleri, 8456 Tyco Road, Building D, Viyana, Virginia 22.182 </td><td> Dynex P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

HIV hastalığı izlemek için uygun fiyatlı CD4 sayımı için acil bir ihtiyaç vardır. CD4 sayımı kan hücreleri, sadece şu anda kabul edilebilir bir yöntem üzerinde CD4 ölçmek için özellikle monoklonal antikorlar zaman ve sıcaklık sınırlamaları, teknik gelişmişliği ve reaktiflerin maliyeti nedeniyle kaynak sınırlı bölgelerde, ulaşamayacağı. Yaygın olarak kullanılan bir maliyet tasarrufu ve zaman tasarrufu sağlayan laboratuvar stratejisi yerine tedbir bazı kan değerlerinin daha hesaplamaktır. Örneğin, LDL düzeyleri total kolesterol, HDL, trigliseritler ve 1 ölçülen seviyeleri kullanılarak hesaplanır. Bu nedenle, doğrudan CD4 + T hücre sayısı CD4 hesaplanması için doğru bir yöntem sağlayabilir regüle hücre içermeyen ögelerinden tanımlama ögelerinden fizyolojik ilgisi nedeniyle sayılır. Kan girin ve CD4 + T hücreleri dolaşımdaki olmaya yazgılı olan kök hücrelerin sayısı bir kemokin CXCL12 tarafından belirlenirnd reseptörü lokomosyon 2 üzerindeki etkisi nedeniyle CXCR4. Kana kök hücre hareketin süreci ayrıca hücre yüzeyinde insan lökosit elastaz (HLE CS) ve HLE CS-reaktif aktif α 1 proteinaz inhibitörü (α 1 PI, α 1 antitripsin, SerpinA1) 3 ile düzenlenir. HIV-1 hastalığı, α 1 PI hastalığı süreçler 4 gerçekleşmemesinden dolayı inaktive edilir. HIV-1 hastalığının erken asemptomatik kategoride, aktif α 1 PI arıtılmamış, HIV-1 hasta (medyan = 12 uM ve semptomatik kategoriye 4, 5 sırasında normal düzeyinin elde edilmesi için% 100 olarak normalin altında olduğu bulunmuştur. Bu desen değil yetersiz sentezi, proteolitik inaktivasyon veya oksijenasyonu, bağışıklık inaktivasyon atfedilmiştir. Biz&gt; 220 CD4 hücre / ul HIV-1 kişilerde, CD4 sayısı (r 2 aktif α 1 PI serum düzeyleri ile ilişkili olduğu görüldü = 0.93, p &amp;lt; 0.0001, n = 26) ve inaktif α 1 PI (r 2 = 0.91, p &lt;0.0001, n = 26) 5. HIV-1 enfekte olan ve olmayan konulara α 1 PI İdaresi α 1 PI CD4 sayısının düzenleyen katılır + T hücreleri kan 3 düşündüren CD4 sayısı dramatik artış ile sonuçlanmıştır. Stimülasyon ile, tüm tükürük aktif α 1 PI ve bu ölçüm korelasyon ölçümü bunu CD4 sayıları hesaplamak doğru bir yöntem olduğunu kanıtlayan izin vermek için yeterli seröz eksuda (plazma tükürük içine kan damarı duvarlarının geçer proteinli madde içeren) içerir. Kısaca, bu sakız veya sitrik asit gibi sialogogues tüm ağız tükürük içine serum eksudasyon uyarır. Serum eksudasyon uyararak sonra, tükürük serum α 1 PI aktivitesi elastaz aktivitesini inhibe etme kapasitesi ile ölçülür. Domuz pankreatik elastaz (KKE)elastaz bir hazır ucuz bir kaynağıdır. PPE kendine özgü substratlar, kolorimetrik peptid bir tanesi, sukinil-L-Ala-Ala-L-L-Ala-p-nitroanilid (SA yarma gelen PPE önleyen bir bire-bir kompleks oluşturarak α 1 PI bağlanır 3 NA). Oda sıcaklığında 10 dakika boyunca PPE bir satüre konsantrasyonu ile kuluçkaya tükürük tükürük tüm aktif α 1 PI için KKD&#39;nin bağlayıcı sağlar. Aktif α 1 PI KKD&#39;nin edilen inhibisyon PPE substrat SA 3 NA. (Şekil 1) eklenmesi ile ölçülebilir. CD4 sayımları kan hacmi (CD4 hücre / ul) cinsinden ölçülür rağmen, tükürük α 1 Pİ konsantrasyonu hacmi gün ve kişi süresince önemli ölçüde değişebilir değil tükürük hacmi beri, tükrük serum konsantrasyonu ile ilgilidir 6 kişi için. Ancak, tükürük neredeyse tüm protein serum içeriği nedeniyle ve tükürük protein içeriği mea olduğunu7 surable. Böylece, tükürük aktif α 1 PI tükürük protein içeriği bir oranı olarak hesaplanır ve α 1 PI Endeksi denir. Sonuçlar burada α 1 PI Endeksi CD4 sayıları hesaplamak için doğru ve kesin fizyolojik bir yöntem sağladığını göstermiştir sundu.

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

A CD4 numaralandırma yöntemi, α-testi, hızlı ve doğru CD4 sayımları sağlamak üzere tam tükürük kullanan tarif edilmektedir. Α-test maliyetleri pennies ve bu monoklonal antikorlar, enstrümantasyon, soğutma, numunelerin taşınması gibi toplama ve kan işleme gibi teknik eğitim, pahalı reaktifler için gereksinimini ortadan kaldırır.

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 ...

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

13.7K Görüntüleme.  Weill Cornell Medical College. A CD4 numaralandırma yöntemi, α-testi, hızlı ve doğru CD4 sayımları sağlamak üzere tam tükürük kullanan tarif edilmektedir. Α-test maliyetleri pennies ve bu monoklonal antikorlar, enstrümantasyon, soğutma, numunelerin taşınması gibi toplama ve kan işleme gibi teknik eğitim, pahalı reaktifler için gereksinimini ortadan kaldırır.

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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Tam Kan Stimülasyon Testi kullanarak Pro-inflamatuar sitokin IL-1β Doğru ve Basit Ölçme

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

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Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Tüm Kan enoksaparin Antikoagülan Etkinliğinin Rapid Point-of-Care Assay

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Bir Lollipop-Like Ultrafiltrasyon Probe Kullanımı İnsan Yerli Tükürük Peptidome Örnekleme: Klinik Kütle Spektrometresi için Peptid Algılama basitleştirin ve geliştirin

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

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 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 ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Topluluk tabanlı Ayar Elde Edilen Örneklerin hızlı Fraksiyonlama ve Tam Kan Bileşenlerinin İzolasyonu

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Endotel hücreleri kullanarak 3D hücre kültürü ve sigara anticoagulated tam kan antikoagülan ve anti-enflamatuar özellikleri değerlendirilmesi

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Asit maserasyon yöntemini kullanarak tüm böbrek netron numarasının tahmini

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Zebrabalığı Embriyoları Kullanılarak Kimyasal Bileşiklerin Toksisitesinin Hızlı Değerlendirilmesi

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

Masaüstü Reometre Kullanarak Hava Yolu Mukusunun Hızlı Viskoelastik Karakterizasyonu

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.