تم دعم البحوث ومؤسسة هاري وينستون للبحوث.

1. إعداد الطازجة حلول العمل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التخزين المؤقت 0.05 متر تريس المالحة، ودرجة الحموضة 7.8 (TBS): TBS. تحضير 60 مل TBS / microplate. </li><li style=";text-align:right;direction:rtl"> معدات الوقاية الشخصية: خنزيري إيلاستاز البنكرياس، نوع 1 (PPE) وعلى النحو المنصوص عليه تعليق. يهز جيدا وتخفف من وسائل الحماية الشخصية في TBS إلى ما يقرب من 0.01 يو / مل. إعداد 6 مل العمل حل / microplate). </li><li style=";text-align:right;direction:rtl"> SA 3 NA: Succinyl-L-علاء-L-علاء-L-علاء-P-nitroanilide (SA 3 NA). تحضير 0.1 M الأسهم SA حل NA 3 في سلفوكسيد ميثيل (DMSO) واستخدامها على الفور أو تخزين عند درجة حرارة -20 درجة مئوية. إعداد 6ml حل الفريق / microplate بواسطة تمييع الأسهم SA حل NA 3 1/100 في TBS. </li><li style=";text-align:right;direction:rtl"> COOMASSIE BLUE: تحضير محلول المخزون بنسبة 10٪ الزرقاء الرائعة Coomassie R-250 (Coomassie الأزرق) في TBS واستخدامها على الفور أو متجر عند درجة حرارة -20 درجة مئوية. إعداد 6 مل حل الفريق من قبل تمييع الحل الأوراق المالية 1/1000 في TBS. </li></ol> 2. إعداد اللعاب <oL&gt; <li style=";text-align:right;direction:rtl"> جمع اللعاب: في وضع الوقوف، وتحفيز نضح المصل في اللعاب من خلال وضع مدر اللعاب مثل حمض الليمون أو العلكة في الفم. قطرة ليمون خالية من السكر غير مريحة للمرضى-1 فيروس نقص المناعة البشرية الذين هم أدرد في كثير من الأحيان وغير قادر على مضغ العلكة. ابتلاع حسب الحاجة ل3 دقيقة الأولى، وجمع كل لعاب الفم في كوب توج ل3 دقيقة المقبل. وهذا سيوفر حوالي 2 مل من اللعاب. استخدامها على الفور أو في تخزين -80 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> المناولة اللعاب: إذا المجمدة عينة ذوبان الجليد، على 37 درجة مئوية ومكان على الجليد. تخزين عينة طازجة أو مذاب في 2-4 درجة مئوية لا تزيد عن 3 أيام. لعاب لا تفعل وتجنب دوامة pipetting الميوسين الذي لا يحتوي على مصل الدم الافرازات. استخدام الاحتياطات العادية عند التعامل مع لعاب <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. Microplate مجموعة المتابعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"><strأونج&gt; BUFFER: يتم إجراء قياسات في ثلاث نسخ. إلى كل بئر من لوحة microtiter يضاف 130 TBS ميكرولتر. </li><li style=";text-align:right;direction:rtl"> عينات: إلى كل اختبار جيد، يضاف لعاب 20 ميكرولتر عن الحجم النهائي من 150 ميكروليتر / جيد. مزيج من قبل pipetting دون إدخال فقاعات. </li><li style=";text-align:right;direction:rtl"> الضوابط: التحكم إيجابي على معدات الوقاية الشخصية، ولكن لا لعاب. سيطرة سلبية يحتوي اللعاب، ولكن لا PPE.To سيطرة إيجابية جيدا يضاف 20 TBS ميكرولتر. لسيطرة سلبية تماما يضاف لعاب 20 ميكرولتر. </li><li style=";text-align:right;direction:rtl"> PPE الحضانة: هزة PPE حل يعمل بشكل جيد. إضافة 50 عامل PPE ميكرولتر حل كل بئر باستثناء آبار مراقبة سلبية. إلى آبار مراقبة سلبية، إضافة 50 TBS ميكرولتر. احتضان 10 دقيقة في 23 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> التحضين الركيزة: أضف 50 ميكرولتر SA حل NA 3 العمل على كل بئر. احتضان 45 دقيقة في 23 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> COOMASSIE BLUE: أضف 50 ميكرولتر حل الأزرق Coomassie العمل &lt;<html> / لى&gt; <li style=";text-align:right;direction:rtl"> قراءة-OUT: اقرأ الامتصاصية في قارئ microplate، ونانومتر 405 للكشف عن انشقاق NA SA (3) الذي يمثل نشاطا α 1 PI وألف نانومتر 595 للكشف عن Coomassie الأزرق الذي يمثل نسبة البروتين. </li></ol> 4. α حساب مؤشر بي 1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطبيع المتوسط ​​نانومتر 405 و 595 ألف نانومتر: للحصول على كل ثلاث نسخ، وحساب متوسط. طرح متوسط ​​مراقبة سلبية من متوسط ​​العينة. للتقليل من لوحة إلى لوحة الاختلاف، وتطبيع المتوسطات بقسمة متوسط ​​كل نموذج من متوسط ​​مراقبة إيجابية على النحو التالي: </li></ol><img alt="المعادلة 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احسب 1 α PI INEX: اقسم تطبيع ألف نانومتر 405 نانومتر من قبل تطبيع 595 ألف: </li></ol>&quot;jove_content&quot;&gt; <img alt="المعادلة 2" src="/files/ftp_upload/3999/3999eq2.jpg" /> 5. ممثل النتائج لتحديد ما إذا كان α تجارب قد تكون مناسبة للاستخدام على انها اختبار فحص نقطة من الرعاية لرصد CD4 التهم المستوطنة في المناطق ذات الموارد المحدودة، وقد تم جمع لعاب حفزت من 20 أنثى و 11 ذكرا HIV-1 عيادة الموضوعات يحضرون لل الرعاية الروتينية في الكاميرون. بما يتفق مع الملاحظات الأولية خلال تطوير اختبار α باستخدام اللعاب التي جمعت في مدينة نيويورك، ومؤشر α بي 1 في اللعاب التي تم جمعها في الكاميرون ترتبط التهم CD4 (ص 2 = 0.91، P &lt;0.0001. ن = 31). تم تعريف العلاقة منحنى 3-المعلمة السيني وهو ما يتسق مع وجود علاقة بين الجرعة والاستجابة (الشكل 2A). وصفت ثقة 95٪، وفترات تنبؤ بخطوط زرقاء وحمراء، على التوالي (2B الشكل). فيدراسة سابقة، توصلنا إلى أن خلايا CD4 + T عرض الدراجات جيبية مع تواتر 23 ± 3.5 أيام في المواضيع-1 فيروس نقص المناعة البشرية والموضوعات مع إصدار ورثت من PI α نقص 1 (PIZZ) 3. هنا يصور مثال ممثل ولدت كمبيوتر الجيب تحليل منحنى CD4 ركوب الدراجات في غير HIV-1 مراقبة صحية دورية المعرض 27 يوما (الشكل 3A). الحاسوب ولدت منحنى جيب تحليل CD4 + الخلايا التائية أسفرت التغييرات في 6 موضوعات قيم الذروة إلى الذروة السعة ومحور التذبذب 3. كما هو متوقع، فقد ارتبط محور التذبذب مع السعة (ص 2 = 0.96، P = 0.009، ن = 6) (الشكل 3B). سوف CD4 في المرضى الذين يعانون انخفاض عدد خلايا تي +، والتغيرات الدورية في عدد الخلايا التائية CD4 كانت صغيرة، وعلى المرضى الذين يعانون من ارتفاع CD4 + عدد خلايا تي، التغيرات الدورية كانت كبيرة. لأنخط الانحدار صورت في الشكل 2 ويقدر يرتبط المحور من التذبذب ومحور التذبذب مع السعة، ويمكن حساب سعة للخط الانحدار وبالتالي تقدير مدى فوق وتحت ويتوقع أن CD4 خط الانحدار + الخلايا التائية أن تختلف نتيجة لل ركوب الدراجات (الشكل 3C). تتراكب مع سعة محسوب في 3C الشكل وفاصل الثقة محسوبة في الشكل 2B، وجد أن السعة (الخط الأخضر) يقع ضمن 95٪ فاصل الثقة (الخط الأزرق) (الشكل 3D). <img alt="الشكل 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> الشكل 1. ملخص الإجراء: حامض الستريك مكان في الفم وجمع اللعاب. إضافة إلى الآبار اللعاب المخفف من microplate في ثلاث نسخ. إضافة إلى كل وسائل الحماية الشخصية بشكل جيد واحتضان لمدة 15 دقيقة في 23 درجة مئوية. إضافة إلى معدات الوقاية الشخصية ركيزة كل جيدا واحتضان لمدة 45 دقيقة عند 23 درجة مئوية. إضافة Coomassie الأزرق وقراءة في 405 نانومتر للكشف عن نشاط وسائل الحماية الشخصية في 595 نيوتن متر للكشف عن البروتين. <img alt="الشكل 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> الشكل 2. مقارنة بين تجارب α مع أسلوب قياسي تعداد CD4. A) وقد أجريت تجارب بواسطة α quantitating مؤشر α بي 1 في تحفيز اللعاب الفم كله. وأجريت CD4 التهم من قبل مختبر طبي مستقل باستخدام معيار التدفق الخلوي. تم جمع الدم من أجل التدفق الخلوي واللعاب في زيارة عيادة نفسه. وقد تم تعريف العلاقة بين مؤشر α PI (1) والتهم CD4 بواسطة منحنى 3-المعلمة السيني (ص 2 = 0.91، P &lt;0.0001، ن = 31). ب) وصفت والثقة 95٪، وفترات تنبؤ في الأزرق والأحمر خطوط، على التوالي. <img alt="الشكل (3)" src="/files/ftp_upload/3999/3999fig3.jpg" /> الشكل 3. دقة حساب CD4 جounts باستخدام Index.A α1PI معارضها) خلايا CD4 + T الدراجات جيبية مع دورية يوم 27 في عنصر تحكم غير HIV-1 صحي. ويصور المحور من التذبذب (الخط المنقط باللون الأحمر). B) الحاسوب ولدت موجة جيبية تحليل CD4 T الدراجات استخدمت الخلية لحساب السعة الذروة إلى الذروة ومحور من التذبذب في 4 موضوعات-1 فيروس نقص المناعة البشرية (1)، غير فيروس نقص المناعة البشرية -1 PIzz الموضوع، وعدم HIV-1 مراقبة صحية صورت في ألف جزء العلاقة بين CD4 + T عرف سعة الخلية ومحور من التذبذب من قبل 3-المعلمة منحنى السيني (ص 2 = 0.96، P = 0.009، ن = 6). C) واستخدمت علاقة السيني بين السعة ومحور التذبذب المشتقة في جزء B) لحساب السعة المتوقعة (خطوط خضراء) من نقاط على طول خط الانحدار (الخط الأسود) من الشكل 2. D) والسعة المتوقعة وكان الفاصل الزمني (خطوط خضراء) تختلف اختلافا كبيرا من فترة الثقة 95٪ (الزرقاء خطوط) على النحو الذي يحدده اختبار مان ويتني مجموع الرتبة (على أساس المقارنة بين متوسط ​​الفرق بين فاصل الثقة 95٪ و خط الانحدار (63 خلية / ميكرولتر) والفرق بين متوسط ​​خط السعة وخط الانحدار (37 الخلايا / ميكرولتر)، P = 0.001).

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الإعلان عن أي تضارب في المصالح.

المصل غير مخفف يحتوي على متوسط ​​36 ميكرومتر α 1 PI و 3.6 ميكرومتر α 2 macroglobulin (α 2 م) فارق بمقدار 10 أضعاف 8. كلا البروتينات تنافس للربط إلى معدات الوقاية الشخصية. وقد تم استغلال هذه الخصائص اثنين والتركيز والانجذاب معدات الوقاية الشخصية، لوضع طريقة ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> مدر اللعاب، وحامض الستريك 0.05M </td><td> جمعية الإمارات للغوص والحلويات خال من السكر، 4900 خلفي N.20th شارع، فيلادلفيا، بنسلفانيا 19144 </td><td> قطرات الليمون الخالي من السكر </td></tr><tr><td> الخنزيري إيلاستاز البنكرياس، نوع 1 (EC 3.4.21.36) </td><td> سيغما الدريخ، وسانت لويس، MO 63103 </td><td> E1250 </td></tr><tr><td> Succinyl-L-علاء-L-علاء-L-علاء-P-nitroanilide </td><td> سيغما الدريخ </td><td> S4760 </td></tr><tr><td> Coomassie بريليانت الأزرق R-250 </td><td> سيغما الدريخ </td><td> B7920 </td></tr><tr><td> زراعة الأنسجة المعاملة، و 96 microplates جيد </td><td> بيكتون ديكنسون، فرانكلين البحيرات، NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Microplate القارئ مع المرشحات لل405 نانومتر و 595 نانومتر </td><td> MTX مختبر أنظمة، 8456 تايكو الطريق، بويlding D، فيينا، فيرجينيا 22182 </td><td> Dynex P97277 </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

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

ووصف طريقة تعداد CD4، وα اختبار، والذي يستخدم لعاب كامل لتوفير سريعة ودقيقة CD4 التهم. وα اختبار تكاليف البنسات ويلغي الحاجة لتقنية الكواشف، والتدريب مكلفة مثل الاجسام المضادة، والأجهزة، والتبريد، ونقل العينات، فضلا عن جمع ومعالجة الدم.

وα الاختبار: رابيد تعداد CD4 خالية من الخلايا عن طريق اللعاب الجامعة

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

13.8K Views.  Weill Cornell Medical College. ووصف طريقة تعداد CD4، وα اختبار، والذي يستخدم لعاب كامل لتوفير سريعة ودقيقة CD4 التهم. وα اختبار تكاليف البنسات ويلغي الحاجة لتقنية الكواشف، والتدريب مكلفة مثل الاجسام المضادة، والأجهزة، والتبريد، ونقل العينات، فضلا عن جمع ومعالجة الدم.

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.

دقيقة وبسيطة قياس الموالية للالتهابات سايتوكاين 1β ايل باستخدام تحفيز الدم الجامعة الفحص

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

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.

السريع في نقاط الفحص من كفاءة الرعاية تخثر الدم الكامل في Enoxaparin

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.

أخذ العينات الإنسان Peptidome اللعاب الأصلية باستخدام مسبار الترشيح الفائق المصاصة، مثل: تبسيط وتعزيز الكشف الببتيد لقياس الطيف الكتلي السريرية

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.

جمع واستخراج الحمض النووي من اللعاب لتسلسل الجيل القادم

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.

التقطير السريع وعزل مكونات الدم الكامل في عينات تم الحصول عليها من إعداد المجتمعية

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.

تقييم خصائص مضادة للالتهابات والتخثر غشائي الخلايا باستخدام الثقافة خلية ثلاثية الأبعاد والدم كله غير أنتيكواجولاتيد

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.

تقدير عدد النيفرون في الكلي الكامل باستخدام طريقه النقع الحمضي

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.

التقييم السريع لسمية المركبات الكيميائية باستخدام أجنة سمك الحمار الوحشي

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.

توصيف سريع للمرونة اللزجة لمخاط مجرى الهواء باستخدام مقياس الريومتر على الطاولة

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.