이 연구는 중앙 대학에 대한 기본 연구 기금에 의해 투자되었다, 부여 번호 2019GJZL04. 우리는 산림 재고 및 계획 아카데미에서 교수 Weisheng Zeng 교수 에게 감사, 국립 임업 및 초원 행정, 중국에 대한 액세스를 제공에 대한.

1. 데이터 준비
<ol><li>개별 트리 정보(1.3m의 유방 높이의 종 및 직경)와 플롯 정보(경사, 측면 및 고도)를 포함하는 모델링 데이터를 준비합니다. 이 연구에서는 중국 북서부 신장성에서 8일(2009년)과 9일(2014년) 중국 국유림 인벤토리에서 779개의 표본플롯을 관측한 21,898건의 관측을 통해 입수했다. 이 샘플 플롯은 1 Mu (0.067 ha에 해당하는 면적의 중국 단위)의 크기로 사각형 모양이며 4km x 8km의 격자 위에 체계적으로 배치됩니다. 참고: 모델링(기초 영역) 증분에 대한 데이터는 적어도 하나의 성장 기간(즉, 두 가지 관찰)이 필요합니다.</li>
	<li>623개의 샘플 플롯에서 17,145개의 관측값과 156개의 샘플 플롯에서 4,753개의 관측으로 구성된 모델 유효성 검사(모델 유효성 검사 데이터 집합)의 20%로 구성된 모델 피팅(모델 개발 데이터 집합)에 사용되는 샘플 플롯의 데이터의 80%와 데이터를 무작위로 두 개의 데이터 집합으로 나눕니다. 사용되는 주요 변수에 대한 설명 통계는 표 1에제공됩니다. 참고: 모델링 절차의 이 단계를 생략할 수 있으며 모든 데이터는 모델 개발에 사용됩니다.</li>
</ol><table border="1" fo:keep-together.within-page="1"><tbody>
<tr>
<td rowspan="2">변수</td>
			<td colspan="4">데이터 피팅</td>
			<td></td>
			<td colspan="4">유효성 검사 데이터</td>
		</tr>
<tr>
<td>분</td>
			<td>최대</td>
			<td>의미</td>
			<td>Sd.</td>
			<td></td>
			<td>분</td>
			<td>최대</td>
			<td>의미</td>
			<td>Sd.</td>
		</tr>
<tr>
<td>DBH 1(cm)</td>
			<td>5</td>
			<td>124.8</td>
			<td>19.9</td>
			<td>13.2</td>
			<td></td>
			<td>5</td>
			<td>101.5</td>
			<td>19.5</td>
			<td>13.4</td>
		</tr>
<tr>
<td>QMD(cm)</td>
			<td>6.7</td>
			<td>82.3</td>
			<td>22.5</td>
			<td>8.5</td>
			<td></td>
			<td>9.2</td>
			<td>73.3</td>
			<td>21.8</td>
			<td>9.2</td>
		</tr>
<tr>
<td>신분증(cm)</td>
			<td>0.1</td>
			<td>14.4</td>
			<td>1.1</td>
			<td>1</td>
			<td></td>
			<td>0.1</td>
			<td>16.9</td>
			<td>1</td>
			<td>1.1</td>
		</tr>
<tr>
<td>BAL (m3)</td>
			<td>0</td>
			<td>5.2</td>
			<td>1.7</td>
			<td>0.9</td>
			<td></td>
			<td>0</td>
			<td>5.4</td>
			<td>1.7</td>
			<td>1</td>
		</tr>
<tr>
<td>NT(나무/하)</td>
			<td>14.9</td>
			<td>3642</td>
			<td>1072</td>
			<td>673.7</td>
			<td></td>
			<td>14.9</td>
			<td>3418</td>
			<td>1205</td>
			<td>829.3</td>
		</tr>
<tr>
<td>BA (m2/ha)</td>
			<td>0.1</td>
			<td>77.5</td>
			<td>34.2</td>
			<td>13.9</td>
			<td></td>
			<td>0.1</td>
			<td>80.6</td>
			<td>34.5</td>
			<td>15.3</td>
		</tr>
<tr>
<td>EL (m)</td>
			<td>2</td>
			<td>3302</td>
			<td>2189</td>
			<td>340.3</td>
			<td></td>
			<td>1441</td>
			<td>3380</td>
			<td>2256</td>
			<td>308.3</td>
		</tr>
</tbody></table>표 1. 피팅 및 유효성 검사 데이터에 대한 요약 통계입니다. DBH1: 유방 높이에서 초기 직경 1.3 m (DBH), DBH2: DBH는 5 년 의 성장 후 측정, QMD: 이차 평균 직경, ID: 5년 동안 직경 증분(DBH2 - DBH1),BAL: 피사체 트리보다 큰 나무의 기저면적(주제트리: 경쟁 지수를 계산한 나무), NT: 헥타르당 나무 수, BA: 헥타르당 나무 수, EL: 고도, S.D.: 표준 기량
2. 기본 모델 개발
<ol><li>개별 트리 기초 영역 증분에 영향을 주는 변수를 식별하려면 참조를 참조하십시오.</li>
	<li>데이터를 기반으로 변수를 선택하고 계산합니다. 일반적으로, 개별 나무 기저면적 증분은 트리 크기, 경쟁 및 사이트조건(27,28,29,30)의세 가지 변수 그룹에 의해 영향을 받습니다.<ol>
<li>DBH1,DBH 1(1/DBH1)의 <img alt="Equation 11" src="/files/ftp_upload/60827/60827eq11.jpg"> 역변환, DBH1(logDBH 1)또는 조합의 일반적인 로고릿헴과같은 나무 크기 효과를 고려한다.</li>
		<li>경쟁의 1면 지수와 같은 경쟁 효과를 고려하여 나무가 경험한 경쟁 수준을 보다 포괄적으로 정량화하고 스탠드 내의 사회적 위치를 보다 포괄적으로 정량화하십시오. 일방적 경쟁으로는 BAL 및 상대 밀도 지수(RD=DBH 1/QMD)가 포함됩니다. 양면 경쟁으로는 NT와 BA가 포함됩니다. 참고: 데이터를 사용할 수 있는 경우 거리 종속 경쟁 지수를 고려해야 합니다.</li>
		<li>위상(ASP), 경사(SL), EL. SL 및 ASP와 같은 사이트 효과를 스테이지 변환31을사용하여 포함해야 합니다.</li>
	</ol>
</li>
	<li>로그를 <img alt="Equation 12" src="/files/ftp_upload/60827/60827eq12.jpg"> 선택(- <img alt="Equation 11" src="/files/ftp_upload/60827/60827eq11.jpg"> +1) <img alt="Equation 12" src="/files/ftp_upload/60827/60827eq12.jpg"> (DBH2의정사각형)을 종속 변수로 나타낸다.</li>
	<li>단계별 회귀 방법을 사용하여 기본 모델을 개발합니다. 모델이 생물학적으로 합리적이고 독립적인 변수 간에 상당한 차이를 보이는지 확인합니다. 분산 인플레이션 계수(VIF)를 활용하여 다중 collinearity를 확인합니다.</li>
	<li>기본 모델에 는 독립적인 변수를 p&lt, 0.05 및 VIF & lt; 5로 둡니다.</li>
	<li>기본 모델 결과와 잔여 플롯을 출력합니다. 여기서 생산되는 기본 모델은 혼합 효과 모델의 추가 개발을 위한 토대가 됩니다.</li>
</ol>3. R 소프트웨어의 패키지 "nlme"와 선형 혼합 효과 모델 개발
<ol><li>모델 개발 데이터 집합을 읽고 패키지 "nlme"를 로드합니다. >model.development.dataset=read.csv("E:/DATA/JoVE/모델링 데이터.csv.csv 헤더=TRUE) >라이브러리(nlme)</li>
	<li>샘플 플롯을 임의 효과로 선택하여 혼합 효과 모델을 개발합니다.</li>
	<li>최대 가능성(ML) 메서드와 임의 효과의 가능한 모든 조합을 맞추고 결과를 출력합니다. >모델&lme(Y~1/DBH1+BAL+NT+EL, 데이터=model.development.dataset, 방법 ="ML", 무작위 =~1| 플롯) >요약(모델) 
	<ol>
<li>랜덤 =~1을 설정하는 것은 임의의 매개 변수에 대한 가로채기입니다. 모든 조합이 장착될 때까지 임의문을 변경합니다. 예를 들어 1/DBH1 및 BAL을 임의 매개 변수로 설정하려면 코드는 다음과 같습니다: 임의 =~1/DBH1+BAL-1. 또한 피팅 과정에서 코드는 장착된 모델의 수렴되지 않아 오류를 보고할 수 있습니다.</li>
	</ol></li>
	<li>아카이케의 정보 기준(AIC), 베이지안 정보 기준(BIC), 로가릿가능성(Loglik), 가능성 비율 테스트(LRT)로 최고의 모델을 선택합니다. >아노바 (모델.1, 모델.6) >아노바 (모델.6, 모델.23) >아노바 (모델.23, 모델.30)</li>
	<li>R i의 구조를 결정합니다. Ri32의이종과 자기 상관 관계를 해결하십시오. Ri는 다음과 같이 작성됩니다. <img alt="Equation 1" src="/files/ftp_upload/60827/60827eq1.jpg">(1) σ2가 모델 잔여 분산과 동일한 알 수 없는 스케일링 계수인 경우 Gi는 이종성을 설명하는 대각선 행렬이며 Γ나는 자기 상관 관계를 설명하는 매트릭스입니다.<ol>
<li>잔류물잔류가 잔류 음모로부터 이종성이 있는지 관찰한다. 이종성(잔류에는 명확한 패턴 또는 추세가 있음)이 있는 경우, 오류 분산 구조를 모델링하기 위해 상수 플러스 전원 기능, 전원 함수 및 지수 함수와 같은 자주 사용되는 세 가지 분산 함수를 소개합니다. >Model.30.1&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.개발.데이터셋, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯 가중치=varConstPower(폼=~ 장착(.)) >요약(모델.30.1) >Model.30.2&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.개발.데이터셋, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯 가중치=varPower(폼=~ 장착(.)) >요약(모델.30.2) >Model.30.3&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.개발.데이터셋, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯 가중치=varExp(폼=~ 장착(.)) >요약(모델.30.3)
</li>
		<li>AIC, BIC, Loglik 및 LRT에 따라 모델에 가장 적합한 분산 함수를 결정합니다. >아노바(모델.30, 모델.30.1) >아노바(모델.30, 모델.30.2) >아노바 (모델.30, 모델.30.3)
</li>
		<li>일반적으로 사용되는 세 가지 자동 상관 구조(CS), 일차 자동 회귀 구조 [AR(1)], 선차 자동 회귀 및 이동 평균 구조 [ARMA(1,1]]의 조합 --자기 상관 관계를 고려합니다. >Model.30.3.1&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.development.dataset, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯, 가중치=varExp(폼=~장착(.)), 코르= 코르콤심()) >요약(모델.30.3.1) >Model.30.3.2&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.development.dataset, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯, 가중치=바렉스(폼=~ 장착(.)), 코르=코라1()) >요약(모델.30.3.2) >Model.30.3.3&lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.development.dataset, 메서드="ML", 랜덤=~1/DBH1+BAL+NT| 플롯, 가중치=varExp(폼=~ 장착(.)), 코르=코르아르마(q=1,p=1)) >요약(모델.30.3.3)
</li>
		<li>AIC, BIC, Loglik 및 LRT에 따라 최고의 자기 상관 구조를 결정합니다. >아노바 (모델.30.3, 모델.30.3.2) 참고: 이종성 및 자기 상관관계가 없는 경우 Gi와 Γi를 정의할 수 없습니다.</li>
		<li>제한된 최대 가능성(REML) 방법을 사용하여 혼합 효과 모델의 최종 결과를 출력합니다. >Mixed.model&lt;lme(Y~1/DBH1+BAL+NT+EL, 데이터=모델.개발.데이터셋, 메서드="REML", 랜덤=~1/DBH1+BAL+NT| 플롯 가중치=varExp(폼=~ 장착(.)), 코르=코르아1()) >요약(혼합.모델)
</li>
	</ol></li>
</ol>4. 바이어스 수정
<ol><li>로그리스믹 스케일의 최종 모델을 사용하여 기초 영역 증분의 예측값을 원래 축척으로 변환합니다. 그러나 로그 변환 모델에서 예측값의 선형 백 변환은 연결된 로그 변환 편향을 생성합니다. 로그 바이어스를 처리하기 위해 보정 계수가 파생되고 예측 방정식에 통합되어 지정된 트리 [방정식(2)]에 대한 실제 예측 기저 영역 증분을 추정합니다. <img alt="Equation 2" src="/files/ftp_upload/60827/60827eq2.jpg">(2) <img alt="Equation 13" src="/files/ftp_upload/60827/60827eq13.jpg">여기서 모델으로부터 기저면적증의 로그와스믹값이 예측되는 반면, <img alt="Equation 14" src="/files/ftp_upload/60827/60827eq14.jpg"> 로그 변환 편향을 보정한 후 5년 동안 기저면적증의 예측된 역변화값은 <img alt="Equation 15" src="/files/ftp_upload/60827/60827eq15.jpg"> 플롯및 σ2의 무작위 효과로부터의 분산이다.</li>
	<li>기저 면적 <img alt="Equation 14" src="/files/ftp_upload/60827/60827eq14.jpg"> 증분()을 직경 증분으로 변환합니다.</li>
</ol>5. 모델 예측 및 평가
<ol><li>예측을 위해 섹션 1.2에서 생성된 모델 유효성 검사 데이터 집합을 준비합니다.</li>
	<li>선형 혼합 효과 모델을 사용하여 개별 트리 기저 영역 증분을 예측합니다. 임의 구성 요소는 다음과 같은 최고의 선형 편향되지 않은 예측 변수를 사용하여 계산되었습니다. <img alt="Equation 3" src="/files/ftp_upload/60827/60827eq3.jpg">(3) <img alt="Equation 16" src="/files/ftp_upload/60827/60827eq16.jpg">임의 구성 요소에 대한 벡터는 어디; 플롯 간 <img alt="Equation 17" src="/files/ftp_upload/60827/60827eq17.jpg"> 가변성에 대한 분산-코바변 <img alt="Equation 18" src="/files/ftp_upload/60827/60827eq18.jpg"> 매트릭스; 상호 보완적인 관측에서 작용하는 임의구성요소에 대한 설계 매트릭스; <img alt="Equation 19" src="/files/ftp_upload/60827/60827eq19.jpg"> 고정효과 모델을 이용한 기저 면적 증분과 예측된 증분의 차이에 의해 구성요소가 주어지는 잔류 벡터이다.</li>
	<li>다음 세 가지 통계지표(23,33)를사용하여 기본 모델 및 선형 혼합 효과 모델의 예측 능력을 평가하고 비교한다. <img alt="Equation 4" src="/files/ftp_upload/60827/60827eq4.jpg">(4) <img alt="Equation 5" src="/files/ftp_upload/60827/60827eq5.jpg">(5) <img alt="Equation 6" src="/files/ftp_upload/60827/60827eq6.jpg">(6) obji가 기저 면적 증분인 경우, esti는 예측된 기저 면적 증분이며, <img alt="Equation 20" src="/files/ftp_upload/60827/60827eq20.jpg"> 관측의 평균이며, N은 관측수의 수입니다.</li>
</ol>

<ol>
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저자는 공개 할 것이 없습니다.

혼합 효과 모델의 개발에 대 한 중요 한 문제는 어떤 매개 변수 임의 효과로 처리 될 수 있습니다 결정 하 고 고정 효과 간주 되어야 한다34,35. 두 가지 방법이 제안되었습니다. 가장 일반적인 방법은 모든 매개 변수를 임의의 효과로 처리한 다음 AIC, BIC, Loglik 및 LRT에서 선택한 최상의 모델을 사용하는 것입니다. 이것은 우리의 연구35에?...

짝수 숙성 된 단일 문화와 비교하여, 여러 목표를 가진 고르지 못한 숙성 혼합 종 산림 관리는 최근1,2,3의관심을 증가시켰습니다. 특히 복잡한 고르지 못한 혼합종 숲4에대해 강력한 산림 관리 전략을 수립하기 위해서는 다양한 관리 대안이 필요하다. 산림 의 성장과 수율 모델은 다양한 관리 계획에 따라 나무 또는 서 개발 및 수확을 예측하기 위해 광범위하게 사용되어왔다 5,6,7. 숲의 성장과 수율 모델은 개별 트리 모델, 크기 클래스 모델 및 전스탠드 성장 모델6,7,8로분류됩니다. 안타깝게도 크기 급 모델과 전스탠드 모델은 고르지 못한 숙성 된 혼합 종 숲에 적합하지 않으며, 산림 관리 의사 결정 과정을 지원하기 위해 보다 자세한 설명이 필요합니다. 이러한 이유로, 개별 나무 의 성장 및 수율 모델은 다양한 종 조성, 구조 및 관리 전략9,10,11로산림 스탠드에 대한 예측을 할 수있는 능력으로 인해 지난 수십 년 동안 증가 된 관심을 받고 있습니다.
일반 최소 제곱(OLS)회귀는 개별 트리 성장모델(12,13,14,15)의개발을 위해 가장 일반적으로 사용되는 방법입니다. 동일한 샘플링 단위(즉, 샘플 플롯 또는 트리)에서 고정된 시간 동안 반복적으로 수집된 개별 트리 성장 모델에 대한 데이터 집합은관측수(10,16)의독립성 및 높은 공간 적 및 시간적 상관관계가 없는 계층적 적재 구조를 갖는다. 계층적 적 재계 구조는 OLS 회귀의 기본 가정을 위반합니다: 즉 독립적 인 잔류물 및 일반적으로 동일한 분산 데이터를 분산합니다. 따라서 OLS 회귀의 사용은 필연적으로 이러한데이터(13,14)에대한 파라미터 추정치의 표준 오차에 대한 편향된추정치를생성한다.
혼합 효과 모델은 반복측정 데이터, 세로 데이터 및 다단계 데이터와 같은 복잡한 구조로 데이터를 분석할 수 있는 강력한 도구를 제공합니다. 혼합 효과 모델은 고정 된 구성 요소, 전체 채우기에 공통, 그리고 각 샘플링 수준에 특정 하는 임의구성 요소로 구성 됩니다. 또한, 혼합효과 모델은 비대각선 분산-공변구조행렬(17,18,19)을정의함으로써 공간과 시간의 이종성 및 자기상관성을 고려한다. 이러한 이유로, 혼합 효과 모델은 직경 높이모델(20, 21,크라운 모델22,23,자가 감산 모델24,25,및 성장 모델26,27)과같은 임업에 광범위하게 사용되어 왔다.
여기서 주요 목적은 선형 혼합 효과 접근 방식을 사용하여 개별 트리 기저 영역 증분 모델을 개발하는 것이었습니다. 우리는 혼합 효과 접근 방식이 광범위하게 적용될 수 있기를 바랍니다.

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development of an individual-tree basal area increment model using a linear mixed-effects approach

여기서 중국 북서부 신장성에 위치한 779개의 샘플 플롯에서 21898 피케아 아스페라타 나무를 포함한 데이터 세트를 기반으로 5년 기저면적 증분의 개별 트리 모델을 개발했습니다. 동일한 샘플링 단위의 관측 값 간의 높은 상관관계를 방지하기 위해 난사 적 가변성을 고려하여 임의의 플롯 효과가 있는 선형 혼합 효과 접근 방식을 사용하여 모델을 개발했습니다. 나무 크기, 경쟁 및 사이트 조건에 대한 인덱스와 같은 다양한 트리 및 스탠드 레벨 변수가 잔여 가변성을 설명하기 위한 고정 효과로 포함되었습니다. 또한, 이종분증 및 자기상관성은 분산 기능 및 자기 상관 구조를 도입하여 기술되었다. 최적의 선형 혼합 효과 모델은 아카이케의 정보 기준, 베이지안 정보 기준, 로가릿암 가능성 및 가능성 비율 테스트와 같은 여러 가지 적합성 통계에 의해 결정되었습니다. 그 결과 개별 나무 기저 면적 증분의 중요한 변수는 유방 높이에서 직경의 역 변환, 피사체 트리보다 큰 나무의 기저 면적, 헥타르당 나무 수 및 고도가 있음을 나타냈다. 또한 분산 구조의 오차는 지수 함수에 의해 가장 성공적으로 모델링되었으며, 1차 자동 회귀 구조(AR(1)에 의해 자가 상관관계가 현저히 수정되었다. 리니어 혼합 효과 모델의 성능은 일반 최소 제곱 회귀를 사용하여 모델에 비해 크게 향상되었습니다.

P. 아스페라타에 대한 기본 기저영역 증분 모델은 방정식(7)으로 표현되었다. 매개 변수 추정, 해당 표준 오류 및 적합성 부족 통계가 표 2에표시됩니다. 잔여 플롯은 그림 1에표시됩니다. 잔류물의 발음이 관찰되었다. <img alt="Equation 7" src="/files/ftp_upload/60827/60827eq7.jpg">(7)
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Watch this Scientific Journal Video about Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach at JoVE.com

Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

혼합 효과 모델은 임업의 계층적 스토세스 구조로 데이터를 분석하기 위한 유연하고 유용한 도구이며 산림 성장 모델의 성능을 크게 향상시키는 데 사용할 수도 있습니다. 여기서선형 혼합 효과 모델과 관련된 정보를 합성하는 프로토콜이 제시됩니다.

선형 혼합 효과 접근 방식을 사용하여 개별 트리 기초 영역 증분 모델 개발

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots ...

development-an-individual-tree-basal-area-increment-model-using

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Biology

3.2K 조회수.  Beijing Forestry University. 혼합 효과 모델은 임업의 계층적 스토세스 구조로 데이터를 분석하기 위한 유연하고 유용한 도구이며 산림 성장 모델의 성능을 크게 향상시키는 데 사용할 수도 있습니다. 여기서선형 혼합 효과 모델과 관련된 정보를 합성하는 프로토콜이 제시됩니다.

비디오: Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabricated 포스트 - 배열 감지기 (mPADs) : 기계적 힘을 분리하는 접근

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

연구

생물학

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.,5,12,20) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7 with a multiobjective evolutionary algorithm SPEA226, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.

This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.

SWAT 모델을 사용하여 농업 보호 관행의 공간 Multiobjective 최적화 및 진화 알고리즘

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

주름 식민지 개발을 사용하여 Biofilm 형성을 평가하기 위해 세미 양적 접근

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

멀티 플렉스 항체 검사 방법을 사용하여 Luminex xMAP 분석에 캡처 엘리사의 전환

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Zebrafish 개발하는 동안 Photoconvertible 단백질을 사용하여 셀 추적

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

메추라기 오리 키메라를 사용하여 두개 안면 개발에 종 - 특정 포스트 평가

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

담관의 보행을 연구하기위한 Whole-mount Immunohistochemical method의 사용<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7 세포 단층층의 세포 기판 접착 영역 및 세포 모양 분포의 정량화

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...