연구는 해리 윈스턴 학술 진흥 재단에 의해 지원되었다.

1. 신선한 워킹 솔루션을 준비한다 <ol><li> TBS : 0.05 M 트리스는 식염, 산도에게 7.8 (TBS)을 버퍼. 60 ML TBS / microplate를 준비합니다. </li><li> PPE : 돼지의 췌장의 엘라스타제, 유형 1 (PPE) 정학로 제공됩니다. 잘 흔들 약 0.01 U / ML에 TBS에서 PPE를 희석. 6 ML)이 솔루션 / microplate 일을 준비합니다. </li><li> SA 3 NA : Succinyl, L 살라-L-살라-L-살라-P-nitroanilide (SA 세 없음). 디메틸 sulfoxide (DMSO)에 0.1 M 주식 SA 3 NA 솔루션을 준비하고 -20 ° C. 즉시 사용하거나 저장 주식 SA 3 NA 솔루션 TBS에서 100분의 1을 diluting하여 6ml 작업 솔루션 / microplate를 준비합니다. </li><li> COOMASSIE 블루 : TBS에서 10 % Coomassie 브릴리언트 블루 R-250 (Coomassie 파랑)의 주식 솔루션을 준비하고 -20 ° C. 즉시 또는 상점 사용 TBS 주식 솔루션 1천분의 1을 diluting하여 6 ML 작업 솔루션을 준비합니다. </li></ol> 2. 타액을 준비 <oL&gt; <li> 침이 컬렉션 서있는 위치에서, 같은 입안에 구연산 산성이나 껌 같은 sialogogue을 배치하여 타액으로 혈청 스며 나옴을 자극. 무설탕 레몬 드롭 자주 edentulous와 껌을 씹을 수없는 HIV-1 환자 편리합니다. 처음 3 분 동안 필요에 따라 삼키는하고, 다음 3 분위한 덮힌 컵에 전체 입을 타액을 수집합니다. 이것은 타액의 약 2 ML을 제공합니다. -80시 즉시 사용하거나 저장 ° C. </li><li> 타액 처리 :면 37 ° C와 얼음 장소에 냉동, 해동의 표본. 2-4에서 신선한 또는 해동 표본을 저장 ° C ~ 삼일 이상 더 이상은 아니야. 와동하지 타액 수행되며 혈청 exudate를 포함하지 mucin을 pipetting하지 마십시오. 침이 취급시주의 사항 표준을 사용 <a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf를</a> . </li></ol> 3. Microplate 설정까지 <ol><li><str긴 사연&gt; 버퍼 : 측정은 세중의로 진행됩니다. microtiter 접시의 각도에 130 μL TBS에 추가됩니다. </li><li> 표본 : 각 시험에 잘, 150 μl / 우물의 최종 부피 20 μL 타액이 추가됩니다. 거품을 도입하지 않고 pipetting하여 섞는다. </li><li> 제어 : 긍정적인 컨트롤 PPE를 포함하지만 아무 타액. 부정적인 컨트롤은 타액을 포함하지만 아무 PPE.To는 긍정적인 컨트롤이 그래요, 20 μL TBS를 추가하지 않습니다. 부정적인 컨트롤 그래요, 20 μL 타액에 추가됩니다. </li><li> PPE 보육 : PPE 작업 솔루션을 잘 흔들어. 물론 부정적인 제어 우물 제외한 각 50 μL PPE 작동 솔루션을 추가합니다. 부정적인 제어 우물로, 50 μL TBS를 추가합니다. 23 ° C.에 10 분 알을 품다 </li><li> 기판 보육 : 각 잘 ~ 50 μL SA 3 NA 작업 솔루션을 추가합니다. 23 ° C.에 45 분 품어 </li><li> COOMASSIE 블루 :. 50 μL Coomassie 파랑 작업 솔루션을 추가 &lt;/ 리&gt; <li> - 읽기 : microplate 리더의 흡광도, 활성 α 한 PI 및 단백질 함량을 나타내는 Coomassie 파랑을 감지하기 위해 595 nm의를 대표하는 SA 3 NA 절단을 감지하기 위해 405 nm의를 참조하십시오. </li></ol> 4. α 1 PI 지수를 계산 <ol><li> 평균 405 nm의 및 595 nm의을 정상화 : 각 세중의 경우, 평균을 계산합니다. 표본 평균에서 제외 제어 평균을 뺍니다. 플레이트 - 투 - 플레이트 편차를 최소화하려면 다음과 같이 긍정적인 제어 평균하여 각 표본 평균을 나누어 평균을 정상화 : </li></ol><img alt="등식 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> α 1 PI INEX를 계산 : 표준 595 nm의 의한 405 nm의 정규 나누기 : </li></ol> 5. 대표 결과 지방병, 리소스 제한된 지역에서 CD4 카운트를 모니터링하는 시점의 케어 선별 검사로 α-테스트 용도 적합 여부를 확인하려면 자극 타액 20 여성에서 수집되었다 11 남성의 HIV-1 과목 참석 클리닉에 대해 카메룬의 일상적인 관리. 뉴욕시에서 수집된 타액을 사용하는 α-시험 개발하는 동안 예비 관찰과 일치, 타액의 α 1 PI 지수는 CD4 카운트 (R 2 = 0.91, P &lt;0.0001. N = 31)와 상호 카메룬에서 수집했습니다. 관계는 선량 - 반응 관계 (그림 2A)과 일치 3 - 매개 변수 sigmoidal 곡선에 의해 정의되었다. 95 %의 신뢰와 예측 간격이 각각 파란색과 빨간색 선, (그림 2B)로 표시됩니다. 에이전의 연구, 우리는 CD4 + T 세포는 주기성 23 sinusoidal 사이클링 ± HIV-1 과목과 α 1 PI 결핍증 (PIZZ) 3 상속된 버전 과목 3.5 일 전시 사실을 확인했습니다. 묘사 본 27 일간의 주기성 (그림 3A)를 전시가 아닌 HIV-1 건강한 통제 사이클링 CD4의 컴퓨터가 만든 사인 곡선 분석의 대표적인 예입니다. CD4의 컴퓨터가 만든 사인 곡선 분석 + 6 과목 T 세포 변화는 진동 3의 피크 - 투 - 피크 진폭 및 축에 대한 가치관이 나왔고. 예상되어지는대로 진동 축이 진폭 (R 2 = 0.96, P = 0.009, N = 6) (그림 3B)와 상호되었습니다. 환자 것입 낮은 CD4 T + 셀 카운트는 CD4 T 세포 카운트의 주기적 변화가 작았, 높은 CD4 + T 세포 카운트 환자에서 주기적 변화가 큰 있었다. 때문에그림 2에 묘사된 회귀 선 진동과 진동 축 축이 진폭이에 의해 변경할함으로써 회귀 선 CD4 + T 세포가 예상되어지는 얼마만큼 위 아래 추정 회귀 선에 대해 계산할 수, 진폭과 서로 관련되어 추정 사이클링 (그림 3C). 그림 3C와 그림 2B에서 계산된 신뢰 구간 계산 진폭 오버레이, 그것은 진폭이 (녹색 라인) 95% 내에 놓여 것으로 나타났다 신뢰 구간 (파란색 선) (그림 3D). <img alt="그림 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> 1 그림. 절차 요약 : 입안에 장소 구연산 산성과 타액을 수집합니다. 세중의에 microplate의 우물로 희석 타액을 추가합니다. 각도에 PPE를 추가하고 23 ° C.에 15 분 동안 품어 각도에 PPE 기판을 추가하고 23 ° C에서 45 분 동안 품어. Coomassie 블루를 추가하고 PPE 활동을 감지하고 595 nm의에서 단백질을 감지하는 405 nm의에서 읽어보세요. <img alt="그림 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> 그림 2. 표준 CD4 열거 방식으로 α-테스트의 비교. ) α-시험이 자극된 전체 입을 타액에서 α 한 PI 지수를 quantitating에 의해 수행되었다. CD4 카운트는 표준 유동세포계측법를 사용하는 독립 의학 연구소에 의해 수행되었다. 유동세포계측법과 타액을위한 혈액은 같은 병원 방문에서 수집된되었다. α 1 PI 지수와 CD4 카운트 사이의 관계는 3-파라미터 sigmoidal 곡선 (R 2 = 0.91, P &lt;0.0001, N = 31). B) 95 %의 신뢰와 예측 간격은 파란색과 빨간색으로 그려져있다 정의한 각각 선,. <img alt="그림 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> 그림 3. CD4에게 C를 계산의 정확성α1PI Index.A를 사용 ounts)은 CD4 + T 세포가 아닌 HIV-1 건강 관리 27 일간의 주기성으로 sinusoidal 자전거를 전시. 진동 축이 그려져있다 (빨간 점선). B) T 세포 사이클이 4 HIV-1 과목 진동 피크 - 투 - 피크 진폭과 축을 계산하는 데 사용되었다 CD4, 1 이외의 HIV의 컴퓨터 생성된 사인 웨이브 분석 -1 PIzz 제목과 부품 A에서 CD4 간의 관계를 묘사되지 않은 HIV-1 건강 관리 T + 세포 진폭과 진동 축 3 - 매개 변수 sigmoidal 곡선 (R 2 = 0.96, P = 0.009, N에 의해 정의되었다 = 6). C) 부분 B)에서 파생되는 진동의 진폭과 축 사이의 sigmoidal 관계는 그림 2에서 회귀 선 (검은 선)을 따라 포인트의 예상 진폭 (녹색 라인)을 계산하는 데 사용되었다. D) 예상 진폭 간격 (녹색 라인)는 95 % 신뢰 구간 (에서 크게 달랐다로 맨 휘트니 순위 합 시험과 진폭 선과 회귀 선 (37 세포 사이의 평균 차이 (95 % 신뢰 구간과 회귀 선 (63 셀 / μl 사이의 평균 차이 비교 기준)에 따라 결정 파란색 라인) / μl), P = 0.001).

<ol>
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관심의 어떠한 충돌 선언 없습니다.

Undiluted 혈청은 평균 36 μm의를 포함 α 한 PI 및 3.6 μm의 α 2 macroglobulin (α 2 M) 10 배 차이 8. 두 단백질은 PPE에 바인딩을 위해 경쟁합니다. 이러한 두 가지 특성, 농도 및 PPE 친화력은 특별히 α 2 M 8을 경쟁의 존재에 정상 혈청에서 α 한 PI를 측정할 수있는 방법을 개발하기 위해 악용되었습니다. 이 방법은 0.3 % 혈청에서와 같은 작은 50 나노미터...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Sialogogue, 0.05M 시트르산 </td><td> 에다의 Sugarfree 캔디, 4900 후면 N.20th 거리, 필라델피아, PA 19144 </td><td> 무설탕 레몬 물방울 </td></tr><tr><td> 돼지의 췌장의 엘라스타제, 유형 1 (EC 3.4.21.36) </td><td> 시그마 - 올드 리치, 세인트 루이스, 미주리 63,103 </td><td> E1250 </td></tr><tr><td> Succinyl-L-살라-L-살라-L-살라-P-nitroanilide </td><td> 시그마 - 올드 리치 </td><td> S4760 </td></tr><tr><td> Coomassie 브릴리언트 블루 R-250 </td><td> 시그마 - 올드 리치 </td><td> B7920 </td></tr><tr><td> 조직 문화 - 대우, 96 잘 microplates </td><td> Becton 디킨슨, 프랭클린 호수, 뉴저지 07417-1886 </td><td> 35-3072 </td></tr><tr><td> 405 nm의 및 595 nm의 용 필터 Microplate Reader를 </td><td> MTX 랩 시스템, 8456 Tyco 번지 부이lding D, 비엔나, 버지니아 22182 </td><td> Dynex P97277 </td></tr></tbody></table>

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

HIV 질환을 모니터링하기 위해 저렴한 CD4 열거를위한 긴급 필요가있다. CD4 열거는 혈액 세포, 오직 현재 결제 방법에 대한 CD4를 측정하기위한 특정 단클론 항체의 시간 및 온도 제한, 기술 세련미와 시약 비용으로 인해 자원이 제한된 지역에 범위 밖입니다. 일반적으로 사용되는 비용 절감 및 시간 절약 실험실 전략은 오히려 측정 특정 혈액 값보다 계산하는 것입니다. 예를 들어, LDL 수치는 총 콜레스테롤, HDL 및 트리 글리세 리드가 1 측정된 수준을 사용하여 계산됩니다. 따라서 직접 CD4의 개수 + T 세포는 CD4을 계산하는 정확한 방법을 제공할 수를 조절 세포가없는 상호의 신분은 상호의 생리 관련으로 인해 계산됩니다. 혈액을 입력하고 CD4에게 + T 세포를 순환이되도록 운명되는 줄기 세포의 수는 케모카인 CXCL12에 의해 결정됩니다ND는 수용체는 운동 2 자신의 영향력으로 인해 CXCR4. 혈액으로 줄기 세포 운동의 과정은 또한 세포 표면에 인간 백혈구 엘라스타제 (HLE CS) 및 HLE CS-반응성 활성 α 1 proteinase 억제제 (α 한 PI, α 1 antitrypsin, SerpinA1) 3에 의해 규제됩니다. HIV-1 질환에서 α 한 PI는 질병 프로세스 4 ~ 인해 inactivated있다. HIV-1 질병의 조기 asymptomatic 카테고리에서 활성 α 한 PI는 치료 HIV-1 환자 (평균 = 12 μm의, 그리고 증상 범주 4, 5 중 정상적인 수준을 달성하는 100 % 아래 정상적인 것으로 발견되었다.이 패턴이 아닌 부족한 합성, proteolytic 불활 성화 또는 산소로, 면역 불활 성화에 기인되었습니다. 우리는&gt; 220 CD4 세포 / μl와 HIV-1 과목에서 CD4 카운트가 (R 2 개 활성 α 한 PI의 혈청 수준과 상호 것으로 관찰 = 0.93, P &amp;그건, 0.0001, N = 26) 및 비활성 α 한 PI (R 2 = 0.91, P &lt;0.0001, N = 26) 5. HIV-1 감염과 감염되지 않은 과목에 대한 α 1 PI의 행정은 α 한 PI는 CD4의 수를 규제에 참여 + T 세포가 혈액 3에서 제안 CD4 카운트 극적으로 증가 결과. 자극을 통해 전체 타액 활성 α 한 사설 탐정이 측정의 상관 관계의 측정 그것이 CD4 카운트를 계산하는 정확한 방법이라는 증거입니다 있도록 충분한 묽은 exudate을 (플라즈마가 타액으로 혈관 벽을 통과 proteinaceous 재료를 포함)가 포함되어 있습니다. 간단히 같은 껌이나 구연산 등의 산성 sialogogues은 전체 입을 타액으로 혈청 스며 나옴을 자극. 혈청 스며 나옴을 자극 후 타액의 혈청 α 한 PI의 활동 엘라스타제 활동을 억제하기위한 능력에 의해 측정됩니다. 돼지의 췌장 엘라스타제 (PPE)엘라스타제의 즉시 사용할 수 저렴한 소스입니다. PPE는 특정 기판, colorimetric 펩타이드입니다 중 하나, succinyl, L 살라-L-살라-L-살라-P-nitroanilide (SA를 cleaving에서 PPE을 방지 일대일 복잡한을 형성 α 한 PI에 바인딩 3 NA). 상온에서 10 분 용 보호구의 포화 농도와 타액을 잠복기 것은 타액에있는 모든 활성 α 한 PI에 PPE의 바인딩이 가능합니다. 활성 α 한 PI에 의해 PPE의 결과 억제는 PPE 기판 SA 3 NA합니다. (그림 1)를 추가하여 측정할 수 있습니다. CD4 카운트는 혈액 부피 (CD4 세포 / μl)의 측면에서 측정하고 있지만, 타액의 α 1 PI의 농도는 볼륨이 날짜와 사람 중에 상당히 달라질 수없는 타액의 볼륨 때문에, 타액의 혈청 농도와 관련되어 사람 6. 그러나, 타액의 거의 모든 단백질은 혈청 컨텐츠로 인해이며, 타액의 단백질 함량은 내 잘못이다7 surable. 따라서 타액에서 활동중인 α 한 PI는 타액 단백질 콘텐츠에 대한 비율로 계산되며, α 1 PI 인덱스 되나있다. 결과는 본 α 한 PI 지수는 CD4 카운트를 계산하기위한 정확하고 정밀한 physiologic 방법을 제공하는 입증 제시.

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

CD4 열거 방법, α-테스트는 신속하고 정확한 CD4 카운트를 제공하기 위해 전체 타액을 사용하는 설명되어 있습니다. α-테스트 비용 동전과 같은 단클론 항체, 계측, 냉동, 샘플 운송뿐만 아니라 수집 및 혈액의 처리와 같은 기술 교육, 값비싼 시약의 필요성을 없애줍니다.

α-테스트 : 전체 타액을 사용하여 빠른 셀 프리 CD4의 열거

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 조회수.  Weill Cornell Medical College. CD4 열거 방법, α-테스트는 신속하고 정확한 CD4 카운트를 제공하기 위해 전체 타액을 사용하는 설명되어 있습니다. α-테스트 비용 동전과 같은 단클론 항체, 계측, 냉동, 샘플 운송뿐만 아니라 수집 및 혈액의 처리와 같은 기술 교육, 값비싼 시약의 필요성을 없애줍니다.

비디오: 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.

전체 혈액 자극 분석을 사용하여 프로 염증성 시토킨 IL - 1β의 정확하고 간단한 측정

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.

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.

롤리팝 - 같은 Ultrafiltration 프로브를 사용하여 인간의 원주민 타액 Peptidome를 샘플링 : 임상 질량 분광법을위한 펩타이드 감지를 단순화하고 향상

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.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

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.

지역 사회 기반의 설정에서 획득 샘플의 신속한 분별 및 전체 혈액 성분의 분리

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.

3D 세포 배양 및 비 anticoagulated 전 혈을 사용 하 여 내 피 세포의 항 응 혈 약 그리고 항 염증 제 속성에 대 한 평가

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.

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.

Zebrafish 배아를 이용한 화학 화합물의 독성에 대한 신속한 평가

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.

벤치탑 레오미터를 이용한 기도 점액의 신속한 점탄성 특성화

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.