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Functional Evolution of a Cold-induced Gene, CBF3, in Arabidopsis thaliana Ecotypes
No full text我們以28個生態型的阿拉伯芥研究受冷誘導基因，CBF3的功能性演化。 CBF3是受冷調控的訊息傳導路徑上重要的“開關”。我們以不同的軟體分析了1297-bp長的啟動子區域序列以及909-bp轉錄區域序列。經過4oC，1.5小時冷處理後，我們以real-rime PCR測量不同生態型之CBF3表現差異，並將此表現差異和以啟動子序列建構之親緣樹互相對照，試圖找出決定該基因表現之重要序列。大部分啟動子及轉錄區域的序列變異符合中性演化，然而藉由sliding window method of Tajima’s D test，我們偵測到不同形式的天擇存在。雖然啟動子上一段區域可能受到balancing selection，然而此一結果之生物上的意義仍未知。我們也在transcriptional activation domain的C端偵測到以低頻率存在之alleles，此一現象也可能由天擇決定。在蛋白質序列的演化上，CBF3大致受到purifying selection作用。然而跟高度保守的AP2 domain相較，Ka/Ks的比值在transcriptional activation domain有劇烈的波動。我們在此區域找到Ka/Ks比值超過1的區段。經由codeml程式(PAML package)的檢測，我們在此區段中偵測到ㄧ個可能是受到positive selection作用的胺基酸(151E)。此胺基酸在7個生態型中被置換為alanine。根據前人對於CBF1之transcriptional activation domain的研究，此一胺基酸(151E)若被取代為alanine，則其活化下游基因表現之能力會增強。我們因此推斷positive selection選擇此一突變，以增加阿拉伯芥植株對於冷的耐受性。經由研究對偶基因特異性表現(allele-specific expression)的策略，我們在啟動子上找到2個核苷酸序列與CBF3低表現量相關。我們也找到ㄧ個天然的CBF3 knockout line，Kas-2。此一發現可供我們進一步釐清CBF3在植物抗冷中扮演的角色。We are interested in functional evolution of CBF3, one of the “master switches” in cold-regulated pathways, in 28 ecotypes of Arabidopsis thaliana. We analyzed sequence polymorphism of 1297-bp promoter and 909-bp transcriptional unit (TU) regions with the aids of different software. After 1.5h chilling treatment (4oC), the CBF3 expression levels were measured by real-time PCR, and the expression variations were mapped to the phylogeny tree constructed according to promoter sequences polymorphisms. The promoter and TU evolution are compatible with neutral evolution while heterogeneity of natural selection was also detected by the sliding window method of Tajima’s D test. A region in promoter was possibly subject to balancing selection, however, the meaning of this is not clear at present. We also found selection against low frequency alleles in C-terminal transcriptional activation domain. In protein sequence evolution, the CBF3 is under strong purifying selection which is against deleterious substitution. However, in contrast to highly conserved AP2 domain, the Ka/Ks ratio fluctuated in transcriptional activation domain in paired comparisons among CBF paralogs. A region with ω more than one was found. The amino acid residue within this region, 151E, was identified by codeml (PAML package) as positively selected site. 151E was substituted by 151A in seven ecotypes we have studied. According to a previous report of trans-activation assay in the transcriptional activation domain, this substitution led to stronger trans-activity. We believe this substitution was favored by positive selection to acquire better cold-tolerance for Arabidopsis in the wild. Using the allele-specific expression approach, we identified two nucleotide sites that are related to low expression of CBF3 after chilling treatment. Kas-2, a CBF3 natural knockout line, was found in this study and will be useful to evaluate the roles of CBF3 in freezing tolerance in Arabidopsis.Abstract 1
中文摘要 2
INTRODUCTION 3
Molecular evolution and statistics to detect evolutionary process 3
Advantages of using natural variation to predict functional sequences 4
The necessity of searching the link of genotype-phenotype 5
In protein evolution 5
Allele-specific expression approach 6
CBF gene family in Arabidopsis thaliana 7
Divergence of CBF paralogs 8
Strategies and goals of CBF functional evolution research 10
Objectives that have been obtained in this report: 10
MATERIALS AND METHODS 12
Arabidopsis thaliana ecotypes 12
DNA extraction, PCR, and Sequencing 12
Molecular evolution and population genetic analysis 13
Cold-treatment and sampling procedure 14
RNA extraction and cDNA synthesis 14
Real-time PCR analysis 15
RESULTS 17
Sequence polymorphism in CBF3 (AT4G25480) 17
The evolutionary distances among CBF paralogs 18
CBF3 nucleotide diversity is compatible with neutral evolution while heterogeneity of selection strength was also detected 19
A probable selective sweep occurred in both promoter and coding sequence but no significant heterogeneity of polymorphism to divergence ratios was identified 19
The CBF3 protein is under strong purifying selection but significant differences of selection constraints were detected in different functional domains 20
A positively selected site was identified within transcriptional activation domain 21
CBF3 expressions in ecotypes after chilling treatment 22
Real-time PCR data calibration 22
Variation of CBF3 expression levels in different ecotypes 23
DISCUSSION 25
The technical bias in real-time PCR and sampling 25
The sequence polymorphism in CBF3 gene 25
The link of coding sequence diversity to previous functional assay 27
Positive selection in CBF3 protein sequence 28
Matching the expression levels with phylogeny constructed according to promoter sequences 29
FIGURES and TABLES 31
REFERENCES 46
Appendix 5
Dynamic Multistate SROC and HSROC with Bayesian Directed Acyclic Graphic Model in Population-based Colorectal Cancer Screening with Fecal Immunochemical Test
No full text研究背景 接受者作業特徵(ROC)曲線已廣泛使用於評估診斷工具的表現，然而其用於評估大規模社區癌症篩檢工具卻有其限制。主於問題根源於無症狀臨床症前期癌症的偵測會受到不完全確診偏誤的影響。而且這個影響又和追蹤時間和癌症平均滯留期有關。所謂平均滯留期代表由臨床症前期進入臨床期之速率。 本研究動機來自於對於評估台灣地區社區及全國癌症篩檢工具表現的需求，會受到上述二個問題的影響。本研究欲以統合分析的角度來對接受者作業特徵曲線著手，不論是來自多個研究或對有不同追蹤時間的單一研究為對象，以解決上述這二個問題。而欲對接受者作業特徵曲線進行平均滯留期的調整，則必須將多階段隨機過程整合考量納入既有的接受者作業特徵曲線總合分析方法。 研究目標 1． 利用總合接受者作業特徵曲線及多階層總合接受特作業特徵曲線來評估以族群為基礎之大腸直腸癌症篩檢工具表現。此類工具受到上述不完全確診偏誤及追蹤時間影響。並使用不同方式進行參數估計，包括蒙地卡羅-馬可夫鏈法、廣義線式模式下之最大概似估計法、非線性混合模式下之動差法。 2． 發展貝氏有向非循環圖形模型。其目的為連結用於處理多研究或多切點間正確性和閥值選擇的總合接受者作業特徵曲線，以及單一研究或單一切點內之隨機真陽或偽陽性結果。 3． 以總合接受者作業特徵曲線的型式，以貝氏有向非循環圖形模型之方法來得到以平均滯留期調整後的接受者作業特徵曲線。 材料與方法 本研究使用三個不同的免疫糞便潛血法大腸直腸癌症篩檢資料做為資料分析來源。第一個是基隆地區整合式篩檢資料，第二個是台灣全國大腸直腸癌篩檢資料，第三個則是公開發表的資料，涵蓋以糞便為基礎之篩檢，包含化學糞便潛血、免疫糞便潛血法，以及腸鏡篩檢。前二個初級資料我們可得到所有受檢個案的量化數字。所有個案包含無病個案、篩檢偵案癌症個案、以及間期癌症個案。每種類型資料我們因此都有有多個不同的敏感度特異度資料。 我們先對基隆免疫糞便潛血資料建構接受者作業特徵曲線。方法是以20、40、60、80、100、150、250、450 ng/ml等值區分不同切點。篩檢陰性個案，若其在二十四個月內產生間隔癌症，則視為偽陰性。並使用約登指數(Youden index)用於決定最適切點。並使用參數化方法，包接總合接受者作業特徵曲線及多階層總合接受者特徵作業特徵曲線，來評估其總合曲線之情形，以得到包含診斷勝算比、閥值、其曲線下面積等整體指標。 接下來我們使用自行發展之貝氏有向非循環圖形模型，來處理總合接受者作業特徵曲線及多階層總合接受特作業特徵曲線裡的問題。傳統方法裡這兩個模型內得到的診斷勝算比、閥值是確定性的；而且當違反異質性假設時，要得到曲線下面積也很困難。 在貝氏架構下，我們接著結合考量大腸直腸癌自然史之多階段模型，來處理設定切點下偽陰性的問題。經過求得的需要挍正的個案數量，我們接著即可建構出動態多階段模式總合接受者作業特徵曲線(MSROC)。 我們將這些方法運用於台灣國家大腸直腸癌篩檢資料。最後我們亦納入公開出版的一些資料納入作後設分析，以得到目前最佳的的證據。 主要結果及結論 從實務的角度來看大腸直腸癌症篩檢免疫糞便潛血法，上述方法得到幾個有趣的發現。 1． 在不考量追蹤時間以及臨床滯留期調整的情形下，以傳統接受者作業特徵曲線來分析免疫糞便潛血法於以族群為基礎之大腸直腸癌症篩檢，會得到太過樂觀的結果：87%的曲線下面積，以及80%對稱點值(Q*)。 2． 以多階段模式總合接受者作業特徵曲線及多階層總合接受特徵者作業曲線，來分析免疫糞便潛血法於以族群為基礎之大腸直腸癌症篩檢，則會得到相當分歧的結果。 3． 假定所有的間隔癌症都是偽陰性所挍正的接受者作業特徵曲線，會得到太保守的結果：71%的曲線下面積，以及66%對稱點值(Q*)。 4． 以三階段馬克夫過程考量間期癌症可能來自偽陰性及新發生的情況下，所挍正的接受者作業特徵曲線，可以得到最佳的點估計：79%的曲線下面積，以及73%對稱點值(Q*)。 從方法學的觀點來看 本論文首先提出以總合接受者作業特徵曲線及多階層總合接受者作業特徵曲線，結合以間隔癌為基礎之追蹤研究設計，應用於考量時間因素及後續追蹤下，以族群為基礎之癌症篩檢工具評估。我們接著發展出貝氏有向非循環圖形模型，在以總合接受者作業特徵曲線及多階層總合接受者作業特徵曲線的架構下來作參數估計。如此更具彈性來處理可觀察與不可觀察的異質性，包括涵蓋固定模式下的共變量，以及納入變動模式分析。第三步我們發展出創新的動態多階段模式總合接受者作業特徵曲線及多階層總合接受者特徵作業特徵曲線模型。此模型結合貝氏有向非循環圖形模型建構，可在三階段馬克夫模型下，估計出癌症發生率及平均滯留期，用以挍正相關曲線以相關估數。應用此貝氏DAG MSROC模型於以族群為基礎之癌症篩檢，可得到不偏的總合接受者作業特徵曲線及多階層總合接受者特徵作業，用以評估免疫糞便潛血法用於以族群為基礎之大腸直腸癌症篩檢之工具表現。Background While receiver operating characteristics (ROC) curve has been widely used to evaluate the performance of diagnostic tool its application to evaluate the performance of screening tool used in population-based cancer screening is not straightforward because the identification of asymptomatic cancer, staying in pre-clinical screen-detectable phase (PCDP), is often faced with incomplete ascertainment that is highly dependent on follow-up time and mean sojourn time (MST) of cancer in question, representing the rate of disease progression from PCDP to CP (clinical phase). We are motivated by evaluating the performance of one community-based and the expanded nationwide population-based screening program in Taiwan. Two issues indicated above are encountered. To solve these two, different studies or single study with different follow-up times can be handled in the context of meta-analysis of ROC. The ROC curve adjusting for MST is solved by the combination of multistate stochastic process with the developed meta-analytic ROC method. Aims 1. We used summary ROC (SROC) and hierarchical ROC (HROC) curve with different estimation methods, including Monte Carlo Markov Chan (MCMC), generalized linear model with maximum likelihood estimation (MLE), and non-linear mixed model with moment method (ME) to evaluate the performance of population-based colorectal cancer screening to deal with incomplete ascertainment due to different follow-up times; 2. We then developed Bayesian directed graphic model to link both diagnostic accuracy and threshold encoded in the context of SROC underpinning at study level with the random outcome of true positive and false positive at individual level; 3. We then combined Bayesian DAG model under the context of SROC with multistate Markov underpinning to derived mean sojourn time(MST)-adjusted ROC. Material and Methods Three sources of dataset were used for the analysis of ROC for FIT in the CRC screening. First, primary data on FIT screening embedded in the Keelung Community-based Integrated Screening (KCIS) were used. The second data are also primary data but derived from the nationwide FIT screening in Taiwan. The third source for meta-analysis was derived from published data, of which faecal-based CRC screening, including both guaic-feacal occult blood test and FIT, and scopy-based CRC screening programs were enrolled. For the two primary dataset, we kept the quantitative value of FIT for all subjects, including disease-free subjects, screen-detected and interval cancers. Therefore, a series of sensitivity and specificity with varying cut-off for positive FIT were derived for the further ROC analysis. We firstly depicted the crude ROC curve in the KCIS data with FIT cut-off levels at 20, 40, 60, 80, 100, 150, 250, 450 ng/ml, whereas interval cancers occurring within 24 months after previous negative screen was treated as false negative. The Youden index was used to assess the optimal cut-off level. The parametric methods of summary ROC (SROC) and hierarchical SROC (HSROC) were applied to assess the behavior of SROC. Summary measures of diagnostic odds ratio, threshold, and AUC given homogeneity between studies were obtained. A Bayesian directed acyclic graphic (DAG) model for SROC was developed to tackle the problems of SROC and HSROC, in which the diagnostic log-odds ratio and diagnostic threshold were deterministic, and the difficulty of obtain AUC when the assumption of heterogeneous studies was violated. A further model which takes into account the possible false negative cases for those with FIT level lower than the cut-off levels by combing a multi-state model for the disease natural history for CRC was developed under the context of Bayesian DAG model. With the corrected number of false negative cases, we can obtain the multi-state summary ROC (MSROC) . All the methods were also applied to the Taiwanese nationwide CRC screening data. A final meta-analysis incorporating selected studies from literature gives the pooled results of ROC for FIT screening given the current state-of-art evidence. Main Results and Conclusions From the practical aspect of population-based colorectal cancer screening with FIT, evaluating performance of FIT test with the developed methodologies mentioned above can throw light on several interesting findings. 1. The performance of FIT in population-based CRC screening evaluated with empirical ROC curve yields too optimistic results, 87% AUChom and 80% Q* without considering follow-up time and adjusting for mean sojourn time. 2. The performance of FIT in population-based CRC screening evaluated with summary and hierarchical summary ROC curve varies with follow-up time and MST. 3. Assuming all interval cancers arising from false negative cases give too conservative results, 71% AUChom and 66% Q*. 4. The corrected ROC curve with refined parameters from three-state Markov process considering interval cancer composed of false negative cases and newly incident cases gives base-case estimates, 79%AUChom and 73% Q*. From the viewpoint of methodology This thesis first proposed summary ROC and hierarchical SROC in combination with interval-cancer-based follow-up study design to evaluate performance of screening tool used in population-based cancer screening with the consideration of time dimension of follow-up time. We then develop Bayesian directed acyclic graphic (DAG) model to estimate the parameters defined under the framework of SROC and HSROC with great flexibility of considering observed and unobserved heterogeneity corresponding to covariates (fixed effects) and random-effect (the incorporation of random-effect. The third step was to develop a novel dynamic multistate SROC and HSROC method with Bayesian DAG underpinning to correct both curves and relevant parameters with the estimated preclinical incidence rate and mean sojourn time (MST) obtained from a three-state Markov process. Applications of the proposed Bayesian DAG MSROC model to population-based colorectal cancer screening can be helpful for calibrating SROC and HSROC curves for unbiased evaluation of performance of FIT test in population-based screenin
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Molecular population genetics and gene expression analysis of duplicated <it>CBF </it>genes of <it>Arabidopsis thaliana</it>
Get PDFAbstract Background CBF/DREB duplicate genes are widely distributed in higher plants and encode transcriptional factors, or CBFs, which bind a DNA regulatory element and impart responsiveness to low temperatures and dehydration. Results We explored patterns of genetic variations of CBF1, -2, and -3 from 34 accessions of Arabidopsis thaliana. Molecular population genetic analyses of these genes indicated that CBF2 has much reduced nucleotide diversity in the transcriptional unit and promoter, suggesting that CBF2 has been subjected to a recent adaptive sweep, which agrees with reports of a regulatory protein of CBF2. Investigating the ratios of Ka/Ks between all paired CBF paralogus genes, high conservation of the AP2 domain was observed, and the major divergence of proteins was the result of relaxation in two regions within the transcriptional activation domain which was under positive selection after CBF duplication. With respect to the level of CBF gene expression, several mutated nucleotides in the promoters of CBF3 and -1 of specific ecotypes might be responsible for its consistently low expression. Conclusion We concluded from our data that important evolutionary changes in CBF1, -2, and -3 may have primarily occurred at the level of gene regulation as well as in protein function.</p
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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