この研究は、中央大学のための基礎研究資金によって資金提供されました, 助成金番号 2019GJZL04.中国国立林業・草原局森林インベントリー・アンド・プランニングアカデミーのヴァイシェン・ゼン教授に、データへのアクセスを提供してくれたことに感謝します。

1. データ準備
<ol><li>個々の樹木情報(1.3 mの胸の高さで種と直径)とプロット情報(傾斜、傾斜、傾斜、標高)を含むモデリングデータを準備します。この研究では、中国北西部の新疆省の8番目(2009年)と9日(2014年)の中国国有林在庫からデータが得られ、779のサンプルプロットの21,898の観測が含まれています。これらのサンプルプロットは、1Μ(中国の面積は0.067ヘクタールに相当)の大きさの正方形で、4km x 8kmのグリッド上に体系的に配置されています。 注: モデリング (基底領域) 増分のデータには、少なくとも 1 つの成長期間 (つまり、2 つの観測値) が必要です。</li>
	<li>モデルフィッティングに使用されるサンプルプロットのデータの80%をランダムに2つのデータセットに分割し、623のサンプルプロットから17,145個の観測値と、156のサンプルプロットから4,753個の観測値から構成されるモデル検証(モデル検証データセット)の20%で構成されます。使用する主要変数の記述統計は 、表 1に示されています。 注: モデリング手順のこの手順は省略でき、すべてのデータはモデル開発に使用されます。</li>
</ol><table border="1" fo:keep-together.within-page="1"><tbody>
<tr>
<td rowspan="2">変数</td>
			<td colspan="4">フィッティングデータ</td>
			<td></td>
			<td colspan="4">検証データ</td>
		</tr>
<tr>
<td>分</td>
			<td>最大</td>
			<td>意味</td>
			<td>S.D.</td>
			<td></td>
			<td>分</td>
			<td>最大</td>
			<td>意味</td>
			<td>S.D.</td>
		</tr>
<tr>
<td>DBH1 (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>ID (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>バル (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 (木/ハ)</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>表 1.適合データと検証データの要約統計量 :DBH1: 胸の高さ1.3 m (DBH) での初期直径、 DBH2: 成長5年後に測定された DBH、 QMD:二次平均直径、ID:直径の増分5年間(DBH2 - DBH 1)、BAL:対象木(被験者ツリー:競争指数を計算した木)よりも大きい木の基礎面積、NT:ヘクタールあたりの木の数、BA:ヘクタール当たりの基底面積、EL:標高、S.D.:標準偏差。
2. 基本的なモデル開発
<ol><li>参照を参照して、個々のツリーの基礎領域の増分に影響する変数を識別します。</li>
	<li>データに基づいて変数を選択し、計算します。一般に、個々のツリーの基底領域の増分は、ツリー サイズ、競合、およびサイト条件 27、28、29、30 の 3 つの変数グループによって影響を受けます。<ol>
<li>DBH1、DBH1の二乗 <img alt="Equation 11" src="/files/ftp_upload/60827/60827eq11.jpg"> ()、DBH 1 の逆変換 (1/DBH1)、および DBH1 の一般的な対数 (logDBH1)またはそれらの組み合わせなどのツリー サイズの効果を考慮します。</li>
		<li>競争の一面と両側の両方のインデックスのような競争効果を考慮して、ツリーが経験する競争のレベルとスタンド内の社会的地位をより包括的に定量化します。一方的な競合には、BAL と相対密度指数 (RD=DBH1/QMD) が含まれます。二面競争にはNTとBAが含まれます。 注: データが利用可能な場合は、距離依存の競合指数を考慮する必要があります。</li>
		<li>ステージの変換31を使用して、アスペクト (ASP)、傾斜角 (SL)、EL SL、および EL. SL および ASP などのサイト効果を含める必要があることを考慮します。</li>
	</ol>
</li>
	<li><img alt="Equation 12" src="/files/ftp_upload/60827/60827eq12.jpg"> <img alt="Equation 11" src="/files/ftp_upload/60827/60827eq11.jpg"> 従属変数として log( - +1) ( <img alt="Equation 12" src="/files/ftp_upload/60827/60827eq12.jpg"> DBH2の二乗を示す) を選択します。</li>
	<li>ステップワイズ回帰法を使用して基本モデルを開発します。モデルが生物学的に妥当であり、独立変数間で有意な差が生じられるようにしてください。分散膨張係数 (VIF) を利用して多重共線性をチェックします。</li>
	<li>基本モデルでは、独立変数は p < 0.05 および VIF < 5 のままにしておきます。</li>
	<li>基本モデルの結果と残差プロットを出力します。ここで作り出された基本モデルは、ミックスエフェクトモデルのさらなる発展の基盤となる。</li>
</ol>Rソフトウェアのパッケージ"nlme"を用いて線形混合効果モデル開発
<ol><li>モデル開発データセットを読み取り、パッケージ "nlme" を読み込みます。 >model.development.dataset=read.csv("E:/DATA/JoVE/モデリングデータ.csv" ヘッダー=真) >ライブラリ(nlme)</li>
	<li>混合効果モデルを作成するには、ランダム効果としてサンプルプロットを選択します。</li>
	<li>ランダム効果のすべての可能な組み合わせを最尤法(ML)法に適合し、結果を出力します。 >モデル<-lme(Y~1/DBH1+BAL+NT+EL,データ=モデル.開発.データセット、 メソッド="ML"、ランダム=~1|プロット) >概要(モデル) 
	<ol>
<li>ランダム=~1を設定すると、ランダムパラメータに対する切片が指定されます。すべての組み合わせが適合するまで、ランダムステートメントを変更します。例えば、1/DBH1および BAL をランダム・パラメーターとして設定する場合、コードは次のようになります。さらに、適合の過程で、適合モデルの非収束によるエラーをコードが報告する場合があります。</li>
	</ol></li>
	<li>アカイケの情報基準(AIC)、ベイズ情報基準(BIC)、対数尤度(Loglik)、尤度比検定(LRT)で最適なモデルを選択します。 >anova(モデル.1、モデル.6) >anova(モデル.6、モデル.23) >anova(モデル.23、モデル.30)</li>
	<li>Riの構造を確認します。Ri32の不均一性と自己相関に対処する。Riは次のように記述されます。 <img alt="Equation 1" src="/files/ftp_upload/60827/60827eq1.jpg">(1) ここで、σ 2はモデルの残差分散に等しい未知のスケーリング係数であり、Giは異種関係を表す対角行列であり、Γ iは自己相関を記述する行列である。<ol>
<li>残差が残差プロットからの不均一性であるかどうかを観察します。不均一性がある場合(残差には明確なパターンまたはトレンドがある)、誤差分散構造をモデル化するために、3つの頻繁に使用される分散関数(定数プラスパワー関数、指数関数)を導入します。 >モデル.30.1<-lme(Y~1/DBH1+BAL +NT+EL,データ=モデル.開発.データセット,メソッド=ML",ランダム=〜1/DBH1+BAL+NT|プロット 重み=varConstPower(フォーム=〜フィット(.)) >概要(モデル.30.1) >モデル.30.2<-lme(Y~1/DBH1+BAL +NT+EL,データ=モデル.開発.データセット,メソッド="ML",ランダム=〜1/DBH1+BAL+NT|プロット 重み=varPower(フォーム=〜フィット(.)) >概要(モデル.30.2) >モデル.30.3<-lme(Y~1/DBH1+BAL +NT+EL,データ=モデル.開発.データセット,メソッド=ML",ランダム=〜1/DBH1+BAL+NT|プロット 重み=varExp(フォーム=〜フィット(.)) >概要(モデル.30.3)
</li>
		<li>AIC、BIC、Loglik、および LRT に従って、モデルの最適分散関数を決定します。 >アノバ(モデル.30、モデル.30.1) >アノバ(モデル.30、モデル.30.2) >アノバ(モデル.30、モデル.30.3)
</li>
		<li>一般的に使用される自己相関構造(複合対称構造(CS)、一次自己回帰構造[AR(1)]、および第1次自己回帰構造と移動平均構造[ARMA(1,1)]の組み合わせ(自己相関を考慮する)を導入します。 >Model.30.3.1<-lme(Y~1/DBH1+BAL+NT+EL,データ=モデル.開発.データセット,メソッド="ML", ランダム=~1/DBH1+BAL+NT|プロット、ウェイト=varExp(フォーム=〜フィット(.))、コル=コル=コープシム()) >概要(モデル.30.3.1) >Model.30.3.2<-lme(Y~1/DBH1+BAL+NT+EL,データ=モデル.開発.データセット,メソッド="ML", ランダム=~1/DBH1+BAL+NT|プロット、ウェイト=varExp(フォーム=〜フィット(.))、コル=corAR1()) >概要(モデル.30.3.2) >Model.30.3.3<-lme(Y~1/DBH1+BAL+NT+EL,データ=モデル.開発.データセット,メソッド="ML", ランダム=~1/DBH1+BAL+NT|プロット、ウェイト=varExp(フォーム=〜フィット(.))、コル=コルアルマ(q=1,p=1)) >概要(モデル.30.3.3)
</li>
		<li>AIC、BIC、Loglik、および LRT に従って最適な自己相関構造を決定します。 >anova(モデル.30.3,モデル.30.3.2) 注意: 不均一性と自己相関がない場合 、Gi と Γi は定義できません。</li>
		<li>制限された最尤法(REML)法を使用して、混合効果モデルの最終結果を出力します。 >混合.モデル<-lme(Y~1/DBH1+BAL +NT+EL,データ=モデル.開発.データセット,メソッド="REML",ランダム=1/DBH1+BAL+NT|プロット ウェイト=varExp(フォーム=〜フィット(.))、コル=corAR1()) >サマリー(混合モデル)
</li>
	</ol></li>
</ol>4. バイアス補正
<ol><li>対数スケールの最終モデルを使用して、基底領域増分の予測値を元のスケールに変換します。ただし、このような対数変換モデルからの予測値の線形逆変換は、関連するログ変換バイアスを生成します。対数偏差に対処するために、補正係数が得られ、予測方程式に統合され、特定のツリーの実際の予測基底面積増分が推定されます[式(2)]: <img alt="Equation 2" src="/files/ftp_upload/60827/60827eq2.jpg">(2) ここで <img alt="Equation 13" src="/files/ftp_upload/60827/60827eq13.jpg"> 、モデルから基底面積増分の対数値が予測 <img alt="Equation 14" src="/files/ftp_upload/60827/60827eq14.jpg"> され、対数変換バイアスを補正した後の5年間の基底領域増分の予測後方変換値 <img alt="Equation 15" src="/files/ftp_upload/60827/60827eq15.jpg"> である。プロットにおけるランダム効果からの差異σ 2は残差分散である。</li>
	<li>基底領域の増分 <img alt="Equation 14" src="/files/ftp_upload/60827/60827eq14.jpg"> () を直径増分に変換します。</li>
</ol>5. モデル予測と評価
<ol><li>セクション 1.2 で作成されたモデル検証データセットを予測用に準備します。</li>
	<li>線形混合効果モデルを使用して、個々のツリーの基底領域の増分を予測します。ランダム成分は、次の最善の線形偏りのない予測変数を使用して計算されました。 <img alt="Equation 3" src="/files/ftp_upload/60827/60827eq3.jpg">(3) ここで <img alt="Equation 16" src="/files/ftp_upload/60827/60827eq16.jpg"> 、ランダム成分のベクトルであり <img alt="Equation 17" src="/files/ftp_upload/60827/60827eq17.jpg"> 、プロット間変動の分散共分散行列 <img alt="Equation 18" src="/files/ftp_upload/60827/60827eq18.jpg"> であり、相補的な観測値で作用するランダム成分の設計行列 <img alt="Equation 19" src="/files/ftp_upload/60827/60827eq19.jpg"> であり、その成分は基底面積増分と固定効果モデルを使用した予測増分の差によって与えられる残留ベクトルです。</li>
	<li>以下の3つの統計指標23,33を用いて、基本モデルと線形混合効果モデルの予測能力を評価し、比較する。 <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) ここで obji は基礎面積の増分であり 、esti は予測された基底領域の増分であり、 <img alt="Equation 20" src="/files/ftp_upload/60827/60827eq20.jpg"> 観測値の平均値、N は観測値の数です。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed-model+pairwise+multiple+comparisons+of+repeated+measures+means.">Mixed-model pairwise multiple comparisons of repeated measures means.</a> Psychological Methods. 6 (3), 282-296 (2001).</li><li>Hayes, A. F., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+heteroskedasticity-consistent+standard+error+estimators+in+OLS+regression:+An+introduction+and+software+implementation.">Using heteroskedasticity-consistent standard error estimators in OLS regression: An introduction and software implementation.</a> Behavior Research Methods. 39 (4), 709-722 (2007).</li><li>Gutzwiller, K. J., Riffell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using Statistical Models to Study Temporal Dynamics of Animal-Landscape Relations. , (2007).</li><li>Calama, R., Montero, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Multilevel linear mixed model for tree diameter increment in stone pine (Pinus pinea): a calibrating approach. 39, (2005).</li><li>Vonesh, E. F., Chinchilli, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+and+nonlinear+models+for+the+analysis+of+repeated+measurements.">Linear and nonlinear models for the analysis of repeated measurements.</a> Journal of Biopharmaceutical Statistics. 18 (4), 595-610 (1996).</li><li>Zobel, J. M., Ek, A. R., Burk, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Forest+Inventory+and+Analysis+surveys,+basal+area+models,+and+fitting+methods+for+the+aspen+forest+type+in+Minnesota.">Comparison of Forest Inventory and Analysis surveys, basal area models, and fitting methods for the aspen forest type in Minnesota.</a> Forest Ecology &amp; Management. 262 (2), 188-194 (2011).</li><li>Sharma, M., Parton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Height-diameter+equations+for+boreal+tree+species+in+Ontario+using+a+mixed-effects+modeling+approach.">Height-diameter equations for boreal tree species in Ontario using a mixed-effects modeling approach.</a> Forest Ecology &amp; Management. 249 (3), 187-198 (2007).</li><li>Crecente-Campo, F., Tom&#233;, M., Soares, P., Di&#233;guez-Aranda, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalized+nonlinear+mixed-effects+height&#8211;diameter+model+for+Eucalyptus+globulus+L.+in+northwestern+Spain.">A generalized nonlinear mixed-effects height&#8211;diameter model for Eucalyptus globulus L. in northwestern Spain.</a> Forest Ecology &amp; Management. 259 (5), 943-952 (2010).</li><li>Fu, L., Sharma, R. P., Hao, K., Tang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalized+interregional+nonlinear+mixed-effects+crown+width+model+for+Prince+Rupprecht+larch+in+northern+China.">A generalized interregional nonlinear mixed-effects crown width model for Prince Rupprecht larch in northern China.</a> Forest Ecology &amp; Management. 389 (2017), 364-373 (2017).</li><li>Hao, X., Yujun, S., Xinjie, W., Jin, W., Yao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+mixed-effects+models+to+describe+individual+tree+crown+width+for+China-fir+in+Fujian+Province,+southeast+China.">Linear mixed-effects models to describe individual tree crown width for China-fir in Fujian Province, southeast China.</a> Plos One. 10 (4), 0122257 (2015).</li><li>Vanderschaaf, C. L., Burkhart, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+methods+to+estimate+Reineke's+Maximum+Size-Density+Relationship+species+boundary+line+slope.">Comparing methods to estimate Reineke's Maximum Size-Density Relationship species boundary line slope.</a> Forest Science. 53 (3), 435-442 (2007).</li><li>Zhang, L., Bi, H., Gove, J. H., Heath, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+alternative+methods+for+estimating+the+self-thinning+boundary+line.">A comparison of alternative methods for estimating the self-thinning boundary line.</a> Canadian Journal of Forest Research. 35 (6), 1507-1514 (2005).</li><li>Hart, D. R., Chute, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+von+Bertalanffy+growth+parameters+from+growth+increment+data+using+a+linear+mixed-effects+model,+with+an+application+to+the+sea+scallop+Placopecten+magellanicus.">Estimating von Bertalanffy growth parameters from growth increment data using a linear mixed-effects model, with an application to the sea scallop Placopecten magellanicus.</a> Ices Journal of Marine Science. 66 (9), 2165-2175 (2009).</li><li>Uzoh, F. C. C., Oliver, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+tree+diameter+increment+model+for+managed+even-aged+stands+of+ponderosa+pine+throughout+the+western+United+States+using+a+multilevel+linear+mixed+effects+model.">Individual tree diameter increment model for managed even-aged stands of ponderosa pine throughout the western United States using a multilevel linear mixed effects model.</a> Forest Ecology &amp; Management. 256 (3), 438-445 (2008).</li><li>Cond&#233;s, S., Sterba, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+an+individual+tree+growth+model+for+Pinus+halepensis+Mill.+in+the+Spanish+region+of+Murcia+with+yield+tables+gained+from+the+same+area.">Comparing an individual tree growth model for Pinus halepensis Mill. in the Spanish region of Murcia with yield tables gained from the same area.</a> European Journal of Forest Research. 127 (3), 253-261 (2008).</li><li>Pokharel, B., Dech, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed-effects+basal+area+increment+models+for+tree+species+in+the+boreal+forest+of+Ontario,+Canada+using+an+ecological+land+classification+approach+to+incorporate+site+effects.">Mixed-effects basal area increment models for tree species in the boreal forest of Ontario, Canada using an ecological land classification approach to incorporate site effects.</a> Forestry. 85 (2), 255-270 (2012).</li><li>Wykoff, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+basal+area+increment+model+for+individual+conifers+in+the+northern+Rocky+Mountains.">A basal area increment model for individual conifers in the northern Rocky Mountains.</a> Forest Science. 36 (4), 1077-1104 (1990).</li><li>Stage, A. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Error+Structure+for+Forestry+Yield+Models.">Generalized Error Structure for Forestry Yield Models.</a> Forest Science. 33 (2), 423-444 (1987).</li><li>Zhao, L., Li, C., Tang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual-tree+diameter+growth+model+for+fir+plantations+based+on+multi-level+linear+mixed+effects+models+across+southeast+China.">Individual-tree diameter growth model for fir plantations based on multi-level linear mixed effects models across southeast China.</a> Journal of Forest Research. 18 (4), 305-315 (2013).</li><li>Hall, D. B., Bailey, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+Prediction+of+Forest+Growth+Variables+Based+on+Multilevel+Nonlinear+Mixed+Models.">Modeling and Prediction of Forest Growth Variables Based on Multilevel Nonlinear Mixed Models.</a> Forest Science. 47 (3), 311-321 (2001).</li><li>Yang, Y., Huang, S., Meng, S. X., Trincado, G., Vanderschaaf, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multilevel+individual+tree+basal+area+increment+model+for+aspen+in+boreal+mixedwood+stands+:+Journal+canadien+de+la+recherche+forestie&#768;re.">A multilevel individual tree basal area increment model for aspen in boreal mixedwood stands : Journal canadien de la recherche forestie&#768;re.</a> Revue Canadienne De Recherche Foresti&#232;re. 39 (39), 2203-2214 (2009).</li><li>Pinheiro, J. C., Bates, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed-effects+models+in+S+and+S-Plus.">Mixed-effects models in S and S-Plus.</a> Publications of the American Statistical Association. 96 (455), 1135-1136 (2000).</li></ol>

著者らは開示するものは何もない。

混合効果モデルの開発のための重要な問題は、どのパラメータがランダム効果として扱うことができるか、どのパラメータが固定効果34、35と考えるべきかを決定することです。2つの方法が提案されている。最も一般的なアプローチは、すべてのパラメータをランダムエフェクトとして扱い、AIC、BIC、Loglik、およびLRTによって選択された最良のモデ...

偶数年の単一培養と比較して、複数の目的を持つ不均一な熟成混合種森林管理は、最近1、2、3の注目を集めています。堅牢な森林管理戦略を策定するためには、異なる管理の選択肢の予測が必要であり、特に複雑な不均一な混合種森林4に対して必要である。森林の成長と収穫モデルは、樹木を予測したり、様々な管理スキーム5、6、7の下で開発と収穫を立ち上げるために広く使用されています。森林の成長と収量モデルは、個別ツリーモデル、サイズクラスモデル、および全スタンド成長モデル6、7、8に分類されます。残念ながら、サイズクラスのモデルと全スタンドモデルは、森林管理の意思決定プロセスをサポートするためにより詳細な説明を必要とする不均一な老朽化した混合種の森林には適していません。このため、樹木の成長と収量モデルは、森林の予測を行う能力が、さまざまな種の組成、構造、および管理戦略9、10、11を持つため、ここ数十年で注目を集めています。
通常の最小二乗(OLS)回帰は、個々の木の成長モデル12、13、14、15の開発に最も一般的に使用される方法です。同じサンプリング単位 (サンプルプロットまたはツリー) 上で一定の時間にわたって繰り返し収集された個々のツリー成長モデルのデータセットは、階層的確率構造を持ち、観測値 10,16の間に独立性の欠如と高い空間的および時間的相関があります。階層的確率構造は、OLS回帰の基本的な仮定、すなわち独立した残差と等分散を伴う正規分布データに違反します。したがって、OLS回帰を使用すると、必然的にこれらのデータ13,14に対するパラメータ推定値の標準誤差の偏った推定値が生成されます。
混合効果モデルは、反復測定データ、縦方向データ、マルチレベルデータなど、複雑な構造を持つデータを分析するための強力なツールです。混合効果モデルは、完全な母集団に共通の固定コンポーネントと、各サンプリングレベルに固有のランダムコンポーネントの両方で構成されます。さらに、混合効果モデルは、非対角分散共分散構造行列17,18,19を定義することにより、空間と時間における不均一性と自己相関性を考慮に入れる。このため、混合効果モデルは、直径高さモデル20、21、クラウンモデル22、23、自己薄化モデル24、25、および成長モデル26、27など、林業で広く使用されています。
ここでの主な目的は、線形混合効果アプローチを用いて、個々の木の基底領域増分モデルを開発することであった。混合効果アプローチが広く適用されることを願っています。

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			<td height="21" style="height:21px;width:216px;">Computer</td>
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development of an individual-tree basal area increment model using a linear mixed-effects approach

ここでは、中国北西部の新疆省にある779のサンプルプロットから21898 ピセアアスペラタ の木を含むデータセットに基づいて、5年間の基底面積増分の個々の木モデルを開発しました。同じサンプリングユニットからの観測値間の高い相関を防ぐために、確率的変動性を考慮してランダムプロット効果を用いた線形混合効果アプローチを用いてモデルを開発しました。樹木サイズ、競合、サイト条件のインデックスなど、さまざまなツリーレベルおよびスタンドレベル変数が、残留変動性を説明するための固定効果として含まれていました。また、不均一性と自己相関性は、分散関数と自己相関構造を導入して説明した。最適な線形混合効果モデルは、アカイケの情報基準、ベイズ情報基準、対数尤度、尤度比検定など、いくつかの適合統計によって決定されました。その結果、個々の木の基礎面積の増分の有意な変数は、乳房の高さでの直径の逆変換、被験者の木よりも大きい木の基礎面積、ヘクタール当たりの木の数、および標高であることを示した。さらに、分散構造の誤差は指数関数によって最もうまくモデル化され、自己相関は一次自己回帰構造(AR(1)によって有意に補正された。線形混合効果モデルのパフォーマンスは、通常の最小二乗回帰を使用してモデルに対して大幅に改善されました。

P.アスペラータの基礎面積増分モデルの基本は、式(7)で表した。パラメータ推定値、対応する標準誤差、および適合しない統計量を表 2に示します。残差プロットは図1に示されています。残差の顕著な不均一性が観察された。 <img alt="Equation 7" src="/files/ftp_upload/60827/60827eq7.jpg">(7)
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			<td>推定...

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

混合効果モデルは、林業における階層的確率構造を持つデータを分析するための柔軟で有用なツールであり、森林成長モデルのパフォーマンスを大幅に改善するためにも使用できます。ここでは、線形混合効果モデルに関する情報を合成するプロトコルを提示する。

線形混合効果アプローチを用いた個別木基底領域増分モデルの開発

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

Research

JoVE Journal

Biology

3.3K ビュー.  Beijing Forestry University. 混合効果モデルは、林業における階層的確率構造を持つデータを分析するための柔軟で有用なツールであり、森林成長モデルのパフォーマンスを大幅に改善するためにも使用できます。ここでは、線形混合効果モデルに関する情報を合成するプロトコルを提示する。

ビデオ: 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.

微細加工後のアレイ検出器（mPADs）：機械的な力を単離するためのアプローチ

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

生物学

Development of New Therapeutic Applications Using Microfluidics

マイクロ流体を使用した新規治療用途の開発

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モデルを用いた農業保全の実践の空間的な多目的最適化と進化的アルゴリズム

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.

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

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.

多重抗体スクリーニング法を用いたLuminex XMAPアッセイへの捕捉ELISAの変換

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.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

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.

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

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.

内部陽性対照との定量的リコンビナーゼポリメラーゼ増幅アッセイの開発

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.

全身免疫組織化学的方法を用いて胆道の内腔を調べる<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.