Carcinoma of the esophagus is an aggressive malignancy with an increasing incidence. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. The large and growing number of patients affected, the high mortality rates, the worldwide geographic variation in practice, and the large body of good quality research warrants a systematic review with meta-analysis.A systematic review and meta-analysis investigating the impact of neoadjuvant or adjuvant therapy on resectable thoracic esophageal cancer to inform evidence-based practice was produced.MEDLINE, CANCERLIT, Cochrane Library, EMBASE, and abstracts from the American Society of Clinical Oncology and the American Society for Therapeutic Radiology and Oncology were searched for trial reports.Included were randomized trials or meta-analyses of neoadjuvant or adjuvant treatments compared with surgery alone or other treatments in patients with resectable thoracic esophageal cancer. Outcomes of interest were survival, adverse effects, and quality of life. Either one- or three-year mortality data were pooled and reported as relative risk ratios.Thirty-four randomized controlled trials and six meta-analyses were obtained and grouped into 13 basic treatment approaches.Single randomized controlled trials detected no differences in mortality between treatments for the following comparisons:- Preoperative radiotherapy versus postoperative radiotherapy.- Preoperative and postoperative radiotherapy versus postoperative radiotherapy. Preoperative and postoperative radiotherapy was associated with a significantly higher mortality rate.- Postoperative chemotherapy versus postoperative radiotherapy.- Postoperative radiotherapy versus postoperative radiotherapy plus protein-bound polysaccharide versus chemoradiation versus chemoradiation plus protein-bound polysaccharide.Pooling one-year mortality detected no statistically significant differences in mortality between treatments for the following comparisons:- Preoperative radiotherapy compared with surgery alone .- Postoperative radiotherapy compared with surgery alone .- Preoperative chemotherapy versus surgery alone .- Preoperative and postoperative chemotherapy versus surgery alone .- Preoperative chemoradiation therapy versus surgery alone .Single randomized controlled trials detected differences in mortality between treatments for the following comparison:- Preoperative hyperthermia and chemoradiotherapy versus preoperative chemoradiotherapy in favour of hyperthermia.Pooling three-year mortality detected no statistically significant difference in mortality between treatments for the following comparison:- Postoperative chemotherapy compared with surgery alone .Pooling three-year mortality detected statistically significant differences between treatments for the following comparisons:- Preoperative chemoradiation therapy versus surgery alone  in favour of preoperative chemoradiation with surgery.- Preoperative chemotherapy compared with preoperative radiotherapy  in favour of preoperative radiotherapy.For adult patients with resectable thoracic esophageal cancer for whom surgery is considered appropriate, surgery alone  is recommended as the standard practice. Carcinoma of the esophagus is an aggressive malignancy that continues to kill more than 90% of people with the disease within five years . The inc2 = 0.02, p = 0.9) and a 52% relative increase in the risk of death at three years with radiotherapy compared with surgery alone .Surgical esophagectomy remains the preferred treatment for clinically localized thoracic esophageal carcinoma ,4-6. TwoThe failure of surgery alone is attributed to the systemic nature of the disease at the time of presentation ,8. EarlyThe large and growing number of patients affected, the high mortality rates, the geographic variation in practice, and the large body of good quality research evidence warrants a systematic review with meta-analysis.This systematic review was developed by the Practice Guidelines Initiative (PGI) of Cancer Care Ontario's Program in Evidence-based Care (PEBC). Evidence was selected and reviewed by two members of the PGI's Gastrointestinal Cancer Disease Site Group (DSG) and two methodologists. Members of the Gastrointestinal Cancer DSG disclosed potential conflict of interest information.This systematic review is a convenient and up-to-date source of the best available evidence on neoadjuvant or adjuvant therapy for resectable esophageal cancer. The body of evidence in this systematic review is primarily comprised of mature randomized controlled trial data; it forms the basis of a clinical practice guideline developed by the Gastrointestinal Cancer DSG published elsewhere (18). This systematic review and companion practice guideline are intended to promote evidence-based practice in Ontario, Canada. The PGI is editorially independent of Cancer Care Ontario and the Ontario Ministry of Health and Long-Term Care.The MEDLINE (1966 through October (week 2) 2003), CANCERLIT (1983 to October 2001), Cochrane Library , and EMBASE  databases were searched with no language restrictions. "Esophageal neoplasms" ) was combined with "chemotherapy, adjuvant" (MeSH), "radiotherapy, adjuvant" (MeSH), "immunotherapy, adjuvant" (MeSH), and each of the following phrases used as text words: "preoperative", "neoadjuvant", "chemotherapy", "radiotherapy", "radiation therapy", "irradiation", "immunotherapy", "chemoradiotherapy", "chemoradiation", and "hyperthermia". These terms were then combined with the search terms for the following study designs or publication types: practice guidelines, meta-analyses, and randomized controlled trials. Additionally, the conference proceedings of the 1997 to 2003 annual meetings of the American Society of Clinical Oncology (ASCO) and the 1999 to 2002 annual meetings of the American Society for Therapeutic Radiology and Oncology (ASTRO) were searched for reports of new or ongoing trials. Relevant articles and abstracts were reviewed, and the reference lists from these sources were searched for additional trials. This formal search was supplemented with published abstracts from thoracic surgery and oncology conferences, conversations with colleagues and experts in the field, and a review of textbooks related to esophageal oncology.Articles were included in this systematic review if they were fully published reports, abstracts, or meta-analyses of randomized controlled trials (RCT) of neoadjuvant or adjuvant treatments compared with surgery alone or surgery plus another treatment in patients with resectable and operable thoracic esophageal cancer. Data on survival had to be reported. Other outcomes of interest were adverse effects and quality of life. Reports of carcinomas located in the cervical esophagus were excluded.Because diverse treatment strategies were evaluated, the eligible studies were grouped into 13 basic treatment approaches (Table 2 (Chi-square) statistic using a planned cut-off for significance of p < 0.05. Potential sources of heterogeneity were postulated a priori and included study quality assessed with the Jadad scale [Heterogeneity of study results was assessed using a visual plot of the outcomes and by calculating the Xad scale  .Six randomized trials of preoperative radiotherapy and surgery versus surgery alone are presented in Table A published meta-analysis  using upFive randomized trials of surgery and postoperative radiotherapy compared with surgery alone are presented in Table Only Zieren et al. assessed quality of life. The results indicated more rapid recovery of quality of life with surgery alone compared with postoperative radiotherapy. Three trials ,29,47 de2 = 7.53, p = 0.11). The rate of local recurrence with radiotherapy was lower in three of the trials [The Gastrointestinal Cancer DSG pooled the five trials that reported one-year mortality data ,27-29,47e trials ,47, but e trials  noted thOne randomized trial evaluated preoperative radiotherapy versus postoperative radiotherapy with curative esophagectomy as part of a four-arm study . PatientIizuka et al.  reportedSeven randomized trials of preoperative chemotherapy and surgery versus surgery alone are presented in Table 2 = 8.26, p = 0.14).The Gastrointestinal Cancer DSG pooled the available data on preoperative chemotherapy with surgery versus surgery alone -34,37,48The first meta-analysis, by Bhansali et al. , pooled The second meta-analysis, by Urschel et al. , pooled The third meta-analysis  was a Co2 = 0.65, p = 0.42).Two randomized trials of preoperative and postoperative chemotherapy and surgery versus surgery alone ,36 Tabl were exaThree randomized trials of postoperative chemotherapy and surgery compared with surgery alone are presented in Table 2 = 0.08, p = 0.77).The Gastrointestinal Cancer DSG pooled the three-year mortality data for two trials ,40 Figu. The triEight randomized trials of combined modality neoadjuvant chemotherapy and radiotherapy are presented in Table 2 = 1.67, p = 0.89).The Gastrointestinal Cancer DSG pooled the one-year mortality data for the six trials with data available at one year ,42-46 .To compare the results between the two published meta-analyses ,55 with No randomized trials have evaluated postoperative chemotherapy combined with radiation versus surgery alone.One randomized trial evaluated postoperative chemotherapy versus postoperative radiotherapy following curative esophagectomy . PatientTwo randomized trials evaluating preoperative chemotherapy compared with preoperative radiotherapy were reviewed ,57. KelsOne randomized trial evaluated the role of preoperative bleomycin in addition to radiotherapy . SeventyOne Japanese trial evaluated protein-bound polysaccharide (PSK) as an adjunct to postoperative radiotherapy or chemoradiation in resected esophageal cancer . This trOne Japanese randomized trial, reported by Kitamura et al. , evaluatAdverse effects were inconsistently reported Tables ,4,5,6,7.Most trials excluded patients with cancers located in the cervical esophagus, and therefore the interpretation of this review is limited to tumours in the more distal two thirds.The options for neoadjuvant or adjuvant therapy for resectable thoracic esophageal cancer are many. On reviewing the results of randomized trials and meta-analyses, the Gastrointestinal Cancer DSG did not recommend neoadjuvant or adjuvant therapy based on the following:Preoperative radiotherapy does not improve survival compared with surgery alone. Postoperative radiotherapy may, in fact, be harmful .Preoperative chemoradiation does not appear to improve survival compared to surgery alone. Although the pooled analysis shows that all six studies are in the direction favouring preoperative chemoradiation at one year, the pooled estimate did not achieve statistical significance. When examining the individual trial results, only the trial by Walsh et al.  detectedPreoperative cisplatin-based chemotherapy does not appear to improve survival. Four of the seven trials ,32-34 deThe available evidence from three randomized trials does not support the use of postoperative chemotherapy over surgery alone -41.Two novel approaches, immunotherapy and hyperthermia, were studied by two groups in Japan ,60. OgosExamination of the results of randomized trials, including the pooled analyses, fails to support the use of preoperative or postoperative adjuvant treatment of any type at this time for patients with resectable carcinoma of the thoracic esophagus. Overall, the evidence does not support the use of neoadjuvant or adjuvant therapy for patients with resectable cancer of the lower two-thirds of the esophagus. Surgical resection alone should remain the standard for localized thoracic esophageal cancer. Patient staging information can be found in Appendix 1  dose; PSK, protein-bound polysaccharide; MRC, Medical Research Council; PGCC, Practice Guidelines Coordinating Committee.Gy, Gray; OR, odds ratio; DSG, Disease Site Group; RR, (relative) risk ratio; PGI, Practice Guidelines Initiative; PEBC, Program in Evidence-based Care; MeSH, Medical subject heading; ASCO, American Society of Clinical Oncologists; ASTRO, American Society for Therapeutic Radiology and Oncology; RCT, randomized controlled trial; CI, confidence interval; XThe author(s) declare that they have no competing interests.RM, RW and LZ performed the initial literature search and created the initial drafts of the systematic review with input from other members of the Gastrointestinal Cancer DSG. RM, RW and BR created the final draft of this systematic review. All statistical analysis was performed by BR in consultation with RM and RW. Creation of the submitted manuscript was performed by BR and RM.The pre-publication history for this paper can be accessed here:Click here for file


Surprisingly little information on symptoms of paroxysmal atrial fibrillation is available in scientific literature. Using questionnaires, we have analyzed the symptoms associated with arrhythmia attacks.One hundred randomly-selected patients with idiopathic paroxysmal atrial fibrillation filled in a structured questionnaire.Psychic stress was the most common factor triggering arrhythmia (54%), followed by physical exertion (42%), tiredness (41%) coffee (25%) and infections (22%). Thirty-four patients cited alcohol, 26 in the form of red wine, 16 as white wine and 26 as spirits. Among these 34, red wine and spirits produced significantly more episodes of arrhythmia than white wine (p = 0.01 and 0.005 respectively).Symptoms during arrhythmia were palpitations while exerting (88%), reduced physical ability (87%), palpitations at rest (86%), shortage of breath during exertion (70%) and anxiety (59%). Significant differences between sexes were noted regarding swollen legs , nausea  and anxiety .Psychic stress was the commonest triggering factor in hospitalized patients with paroxysmal atrial fibrillation. Red wine and spirits were more proarrhythmic than white wine. Symptoms in women in connection with attacks of arrhythmia vary somewhat from those in men. Atrial fibrillation (AF) is nowadays divided into three different forms; paroxysmal, persistent and permanent . Even ifA group of one hundred patients suffering ECG-verified PAF and whose symptoms prompted hospital care were asked to fill in a structured questionnaire with 58 questions covering arrhythmia-triggering factors, time at which the attack starts and symptoms during attack. All patients completed the data by personal interviews by one of the authors. The vast majority of all patients were recruited during two periods of totally 14 months. In some questions, extra information could be supplied by the patients in their own words.  age of the entire group was 59.9 (22.4–79.2) years. Patients with previous histories of hypertension were significantly older than those without 65.0 (46.6–78.6) years as against 58.6 (22.4–79.2) years respectively. The women in the group were significantly older than the men 67.9 54.1–78.6) years and 58.2 (22.4–79.2) years respectively, p = 0.02 , attacks typically lasted less than one day, while a further 17% gave 1–7 days. No attack lasted more than one week.Thirty-five percent of the entire group woke up with episodes of fibrillation, 34% stated that attacks began with psychic stress, as defined by the patients. Thirty one percent of the episodes started during rest and 22% of the attacks followed physical exertion. Twenty-five percent could not think of any special factor initiating attacks. No differences in the onset of attacks between men and women could be established. Eighty-five percent of the group succeeded in identifying some sort of triggering-factor . There were no significant differences in the arrhythmia-provoking effects of spirits and red wine nor between men and women . Eight patients did not dare to travel. Sixty-six percent stated that their episodes of AF affected their relatives, while 32% answered this question in the negative. In their own words, they remarked that the main problem was their relatives' uneasiness.During attacks of AF, the most common symptoms were palpitations in connection with strain, reduced physical performance, palpitations when at rest, breathlessness during exertion and anxiety Table . FemalesAlthough PAF is one of the most common heart-disturbances causing patients to get in touch with medical care centres, surprisingly little information is available about the factors which trigger it and the symptoms associated with the onset of arrhythmia in larger groups of patients. This study has therefore been undertaken to determine which factors patients consider responsible for triggering arrhythmia and the symptoms that occur in connection with episodes of arrhythmia.From observations of heart-rate in sinus rhythm shortly before its onset, a separation of PAF into sympathetically-mediated and vagal forms has been suggested . EarlierIn our study, the 72 patients who thought that their attacks of arrhythmia usually occurred at about the same time of day gave this as the evening or at night. Hence a large fraction of those investigated should have vagal PAF since this often starts at night . DespiteAlcohol has long been considered to play an etiological role in PAF, a correlation underlined in the expression "holiday heart" . Since tIt is now generally accepted that most attacks of PAF are induced by ectopic impulses originating in the pulmonary veins ,20. BothAlthough a number of patients with PAF are without any symptoms , in geneThe limited amount of literature on the symptomology of PAF includes Quality of Life investigations, Case Reports and quantification of a few symptoms . InvestiThe most frequent symptoms in periods of AF reported by our group of patients included palpitations, reduced physical performance, palpitations when at rest, breathlessness when exerting oneself and anxiety. In an earlier report, the most pronounced symptoms were palpitations and anxiety as well as giddiness . That feSwollen legs can also be accounted for due to right-sided cardiac failure in some patients. That most patients experience definite symptoms following acute but transient attacks of AF can possibly depend on the increased activity of the sympathetic nervous system triggered by an attack of AF . It is pThis material was taken at a hospital and is thus not representative of all patients with PAF. The symptoms of our patients are so far advanced that hospitalization or a visit to a hospital was required. Ongoing treatment can have modified patients' recollections of anamnestic factors. The material was not taken consecutively, but randomness was favoured by lack of a systemic inclusion mechanism.Patients with hypertension are not excluded, even if subtle diastolic changes in the left ventricle and hence the left atrial performance could be caused by hypertension .Although symptoms associated with the onset of PAF may be age related, the present material is too limited to allow exploration of this relation.Most of the patients in a group being treated at a hospital for PAF consider psychic stress to be the factor triggering their arrhythmia.Red wine and spirits seems more prone to trigger attacks of AF than white wine.The symptoms of PAF are many and occur frequently.In women, PAF leads to significantly higher frequencies of swollen legs, indisposition and anxiety than in men.None.Author AH designed the investigation, collected all patient data, performed the statistical analysis and interpretation of the results, as well as the preparation of the manuscript.Author BMH assisted with the statistical analysis and the preparation of the manuscript.Author SBO supervised and designed the investigation as well as participated in the preparation of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:The complete questionnaire. The structured questionnaire with 58 questions covering arrhythmia-triggering factors, time at which the attack starts and symptoms during attack.Click here for file


Arabidopsis core promoters.Core promoters are the gene regulatory regions most proximal to the transcription start site (TSS), central to the formation of pre-initiation complexes and for combinatorial gene regulation. The DNA elements required for core promoter function in plants are poorly understood. To establish the sequence motifs that characterize plant core promoters and to compare them to the corresponding sequences in animals, we took advantage of available full-length cDNAs (FL-cDNAs) and predicted upstream regulatory sequences to carry out the analysis of 12,749 Arabidopsis core promoters. One of them corresponded to the TATA element, for which an in-depth analysis resulted in the generation of robust TATA Nucleotide Frequency Matrices (NFMs) capable of predicting Arabidopsis TATA elements with a high degree of confidence. We established that approximately 29% of all Arabidopsis promoters contain TATA motifs, clustered around position -32 with respect to the TSS. The presence of TATA elements was associated with genes represented more frequently in EST collections and with shorter 5' UTRs. No cis-elements were found over-represented in TATA-less, compared to TATA-containing promoters.Using a combination of expectation maximization and Gibbs sampling methods, we identified several motifs overrepresented in Arabidopsis promoters. The percentage of TATA-containing promoters is much lower than commonly recognized, yet comparable to the number of Drosophila promoters containing a TATA element. Although several other DNA elements were identified as over-represented in Arabidopsis promoters, they are present in only a small fraction of the genes and they represent elements not previously described in animals, suggesting a distinct architecture of the core promoters of plant and animal genes.Our studies provide a first genome-wide illustration of the composition and structure of core  To obtain the  regions, we first checked the length of the 5' UTRs, which was shorter than 50 bp for 2,649 genes and which was interrupted by introns in 2,179 genes. To include into our analyses these cases, three different strategies were followed. If the 5' UTR was longer than 50 bp, and no introns were present in the corresponding  region , a direct retrieval of the  region was performed from the TAIR 5' UTR dataset. If the 5' UTR was shorter than 50 bp and no intron interrupted this region , we extended the 5' UTR to 50 nt with a fragment of the immediately adjacent downstream coding sequence using the TIGR cDNA dataset. Finally, if the 5' UTR was shorter than 50 nt and an intron interrupted this region (32 genes), we manually retrieved the  region from the genomic sequence using TAIR's SeqViewer. After these analyzes, we were able to generate datasets corresponding to the ,  and  regions from a total of 12,749 genes. These datasets were used for all the subsequent analyzes in this study.Arabidopsis core promoters, we first searched for DNA elements conserved in the  and  regions of the 12,749 Arabidopsis genes. The search was carried using both MEME and AlignACE (see Methods). Motifs correspond to short sequences (6–10 bp), often recognized by a DNA-binding protein, and which can be represented by a consensus sequence. While the total number of motifs retrieved per region with these algorithms was 16 and 32 respectively, only motifs detected in at least 50 sequences with either MEME or AlignACE are shown  for each one of these motifs from the results of the MEME search (see Methods). The NFMs for each of these motifs, provided as Arabidopsis genes  interval. Motif 3 has all the characteristics of a TATA box n microsatellites have been extensively researched in humans  region. Motif 10 resembles the (GAA), +50 regn humans , but notMotif 13, with the consensus T/A CCGGCGA , was found to be only slightly overrepresented in the  region, compared to the  background model   region and establish where each of them localized a TATA with the highest probability. As shown in Figure Arabidopsis genes is 129 nt , the 12,749 sequences utilized here are represented in average by 13.02 ESTs, suggesting that the available FL-cDNAs are likely to correspond to genes expressed at a higher level than the average Arabidopsis gene. Interestingly, however, TATA-containing genes were represented in average by 17.6 ESTs  were manually parsed out independent of the number of occurrences or positional preferences. The obtained motifs were plotted according to their positions within the regions and their consensus sequences were graphed using WebLogo version 2.7  and  regions, we used the higher order probabilistic model MotifScanner from MotifSampler version 3.0 . The searomoters . For theArabidopsis  and  regions are ~65% A/T to ~35% C/G and ~61% A/T to ~39% C/G, respectively, a pseudo-random set of 50 bp sequences was generated for each region to be tested with the matrices as a way of determining the chances of finding the motifs candidates in a stochastic environment. This information was then used together with the search results obtained from the real data to support the confidence of the findings.After establishing that the distribution of nucleotides in the For the analysis of the TATA motif, the TATA NFM previously described  was usedArabidopsis genes with the ones found in the motif clusters. Under the assumption that the contribution of a gene to transcription activity is related to the number of its detected ESTs, we downloaded a dataset from TAIR that accounts for the number of ESTs submissions per locus  and  regions of 12,749  and [+1,Click here for fileNucleotide Frequency Matrices for all the motifs shown in Figure Click here for file


Africa in the twenty-first century is faced with a heavy burden of disease, combined with ill-equipped medical systems and underdeveloped technological capacity. A major challenge for the international community is to bring scientific and technological advances like genomics to bear on the health priorities of poorer countries. The New Partnership for Africa's Development has identified science and technology as a key platform for Africa's renewal. Recognizing the timeliness of this issue, the African Centre for Technology Studies and the University of Toronto Joint Centre for Bioethics co-organized a course on Genomics and Public Health Policy in Nairobi, Kenya, the first of a series of similar courses to take place in the developing world. This article presents the findings and recommendations that emerged from this process, recommendations which suggest that a regional approach to developing sound science and technology policies is the key to harnessing genome-related biotechnology to improve health and contribute to human development in Africa.The objectives of the course were to familiarize participants with the current status and implications of genomics for health in Africa; to provide frameworks for analyzing and debating the policy and ethical questions; and to begin developing a network across different sectors by sharing perspectives and building relationships. To achieve these goals the course brought together a diverse group of stakeholders from academic research centres, the media, non-governmental, voluntary and legal organizations to stimulate multi-sectoral debate around issues of policy. Topics included scientific advances in genomics innovation systems and business models, international regulatory frameworks, as well as ethical and legal issues.Seven main recommendations emerged: establish a network for sustained dialogue among participants; identify champions among politicians; use the New Plan for African Development (NEPAD) as entry point onto political agenda; commission an African capacity survey in genomics-related R&D to determine areas of strength; undertake a detailed study of R&D models with demonstrated success in the developing world, i.e. China, India, Cuba, Brazil; establish seven regional research centres of excellence; and, create sustainable financing mechanisms. A concrete outcome of this intensive five-day course was the establishment of the African Genome Policy Forum, a multi-stakeholder forum to foster further discussion on policy.With African leaders engaged in the New Partnership for Africa's Development, science and technology is well poised to play a valuable role in Africa's renewal, by contributing to economic development and to improved health. Africa's first course on Genomics and Public Health Policy aspired to contribute to the effort to bring this issue to the forefront of the policy debate, focusing on genomics through the lens of public health. The process that has led to this course has served as a model for three subsequent courses , and the establishment of similar regional networks on genomics and policy, which could form the basis for inter-regional dialogue in the future. Inequities in global health continue to be among the major challenges facing the international community . Despite, and the Institute for Molecular and Cell Biology-Africa (IMCB-A), founded in 1999 to study the molecular mechanisms of tropical infections. A further example is the new Biosciences Facility for Eastern and Central Africa that was recently launched as part of a NEPAD initiative . Par. Pare.g.e.g. . GenomicThe most efficient means of garnering political support is often to go directly to the politicians themselves – those who have been supportive or outspoken of the issues in question – to put the subject before their colleagues. The course itself represented an important step in this direction, as it brought together a spectrum of stakeholders, including academics, civil society, and government officials. The course, and the subsequently established network, therefore furnished an opportunity for direct communication and dialogue among individuals with a shared vision, including policymakers in a position to "champion" the issues and proposals that emerged from the course to their colleagues and others.NEPAD offers a possible forum to bring the subject of genomics-related biotechnology onto the political agenda, and provides a means of informing African leaders of genomics and its relevance to improving health and development in Africa. In particular, the AGPF recommends the establishment by NEPAD of an 'African Genomics Committee', which would provide a plan for utilizing genomics and other new technologies to enhance health in Africa, advocate for increased investment in S & T, target other relevant stakeholders in individual countries, educate policy makers about the need for a strong R&D base established through partnerships across Africa, and organize steering committees to identify gaps and implement strategies for improvement.Participants agreed on the need to consider emerging technologies like genomics in light of Africa's specific health challenges, and consequently on the importance of prioritizing these and identifying strategic entry points. Infectious  diseases, genetic and other non-communicable disorders, sanitation, nutrition, environmental pollution and loss of biodiversity were all proposed as areas requiring concerted attention, with a special emphasis on the potential for using genomics-related biotechnology to target the three biggest killers in Africa: malaria, HIV/AIDS and tuberculosis. There are already well-known African-led initiatives to apply scientific innovation to combat important health concerns, such as the Multilateral Initiative on Malaria, and the African Malaria Vaccine Testing Network (AMVTN). It will be important to build on existing success stories, and to identify gaps in terms of priority health areas receiving inadequate attention. This will help to focus efforts and to more efficiently channel limited resource, both financial and human. A regional approach, which has since been adopted by NEPAD, was proposed as a promising mechanism for harnessing existing competence to address local needs.This survey would identify strategic areas of strength, such as existing centres of excellence, potential areas of improvement, and health priorities receive inadequate attention. It would also serve to identify local and national innovators, and to inform the structuring of Regional Centres of Excellence described below.For several years, genomics has been linked with a number of high-profile, intensely controversial issues like human cloning and genetically modified organisms. While emerging technologies like genomics raise a number of important ethical and social issues that deserve careful consideration , a nuancPublic engagement was seen to form part of a long-term strategy for capacity building, and raising the overall profile of science and technology in Africa. The discussions reflected a conception of capacity strengthening as intimately linked with quality education – at all levels, and across disciplines. Core to this debate among course participants was the belief that endogenous capacity must be developed in order that Africa can begin to be self-sufficient, and itself become an innovator. Participants identified the following categories as needing attention:There is a need to introduce innovative techniques to teach science and technology in the classroom, in order to generate interest and aptitude in the subject matter from an early stage in the educational process. Besides contemporary scientific approaches, indigenous knowledge and its applications to health could also be a relevant component to include in the curriculum.Those in a position to shape policy should be familiarized with codes of ethics pertaining to their field; moreover, they should be educated about how best to capitalize on international frameworks  in order to ensure that their countries benefit from such arrangements, and are not exploited. Policy makers should develop strategies for negotiating their interests collectively in international forums, when appropriate, given shared needs and values.There is a general need to strengthen capacity in the area of communication, in particular on increasing the level of science literacy among the media. This might include integrating journalism and science programs at the college and university levels. There is a corresponding need to improve the ability of scientists to communicate the relevance of their work to the public, and to policy-makers.There is a great need to build capacity in Africa with regard to the ethical, legal and social issues (ELSI) which inevitably accompany the emergence of new technologies. Strategies would in many cases involve sensitizing the public to issues of relevance, such as their rights as patients and participants in research , encouraging dialogue about the social consequences of introducing new technologies into traditional settings, and putting frameworks in place (e.g. ethics review boards) to ensure that ethical, quality and safety standards before research is undertaken.Networks provide a means of generating new ideas, pooling the creative energies of individuals, and exchanging advice and expertise around a particular area of focus, in this case genomics and health policy. Such networks could play an advocacy role, combining the voices and the influence of key players from diverse disciplines and sectors, to advance a common aim. Collaborations, at the level of institutions – both within and between countries and regions – would facilitate the transfer of both knowledge and technology. During the course, it was pointed out that there is a particular need to encourage linkages between universities and industry to, among other things, facilitate the move from research and development to product generation and commercialization. This could include mechanisms to facilitate relationships between universities undertaking research in biotechnology and local industries. Institutional partnerships and collaborations at all levels, including internationally, can mean the channelling of resources to common areas of focus, and pooling the relative strengths and resources of partner institutions [Along with the need to strengthen the R&D base in science and technology, participants of the course identified a related need to increase the emphasis on commercialization – not only as a tool for sparking innovation but also to permit the generation of capital necessary to sustain the industry. An important step in the process of moving toward commercialization is the forming of alliances within countries, between universities and industry, sometimes known as "cross-linking". The fruitfulness of the Africa course, where people from across sectors and sub-regions came together with a common mission, re-enforced the value and the importance of establishing cross-sectoral networks and collaborations. itutions . Such coEnsuring that the benefits of science and technology, including emerging fields like genomics, requires a long-term strategy for sustained investment.Three models were suggestedThe establishment of an African Science and Technology Fund, dedicated to supporting research and development in the area of health-related biotechnology, would rely upon the contribution of African governments.The establishment of an Investment Fund for genome-related biotechnologies for improving health would represent an innovative approach to obtaining capital, providing a further incentive for investors to put money into development by creating a fund that provides a return on investment, as well as furnishing funds for advancement. Such a fund might be dedicated to providing capital for the development of mature, or future, health-related technologies.Capitalizing on existing funds allocated for research related to diseases afflicting Africa, such as the WHO's Global Fund to Fight   AIDS, Tuberculosis and Malaria. Genomics and biotechnology represents a powerful set of tools for health improvement, and the World Health Organization through its Genomics and World Health (2002) report has raised it as an important issue deserving international attention. It is important to use this positive emphasis to give weight to the case for the relevance of biotechnology to health in developing countries, particularly for policy makers.With respect to R&D, there are already areas of strength on the continent; it is crucial to identify localized expertise, and to establish linkages with centres elsewhere in the region, as well as abroad, to ensure the transfer of knowledge and of technology, and to facilitate human resource development. Infrastructure must be developed to attract qualified African researchers to remain in or to return to Africa – both to support them, technically, intellectually, and socially and to provide them with similar opportunities for creativity and growth as may be found in other locales. The Biosciences Facility, established in 2003 by NEPAD, takes up this challenge, promoting "scientific excellence by bringing together a critical mass of scientists drawn from national, regional and international institutions in state-of-the-art facilities where they can undertake cutting-edge research to help solve the most important development constraints faced by the poor in Africa" . While tDeveloping countries in various parts of the world have proven that they too can have strong technology sectors, and make important contributions in terms of science and innovation. Their successes represent an opportunity to bring to the attention of politicians that there are countries succeeding in genomics. A detailed study of these models can provide important insights into how Africa can capitalize on the promise of genomics and biotechnology, particularly as it relates to health. In 2003, the Joint Centre for Bioethics completed a qualitative study of R&D in biotechnology in South Africa; similar studies are underway in Cuba, Egypt and China. Research of this kind could feed into more systematic efforts in the region to better understand how some developing countries, including those in Africa, have managed to develop S&T research and manufacturing capacity in the health sector.Africa. These regional centres of excellence need not preclude the existence of national centres of excellence. The Biosciences Facility is modelled on such an approach.The proposed centres would be distributed across Northern, Southern, Eastern, Western and Central African sub-regions. Each centre would have its own area of focus, in terms of targeted health problems, depending on regional expertise. The Centres would not be the sole preserve of each region, but would in fact use the strengths and specializations of each region to achieve the goal of harnessing genomics to improve health in The course on Genomics and Public Health Policy in Africa was carefully designed, with inputs from both its Canadian and African co-organizers, to have a programme and participant profile reflecting the inter-disciplinarity of the issue being considered. Genomics cuts across S&T, environmental, development, industrial, education and health policy and generates important ethical, legal and social issues. It therefore requires a genuinely participatory and multi-stakeholder approach, as well as frank discussions about both the potential promise and perils of a relatively new science.The strength of the course, as reflected in the evaluations submitted by participants, was the rare opportunity for discussion and networking among opinion leaders from different sectors. Both during and between sessions, participants exchanged perspectives and experiences with others from different regions of the content, and from different disciplines. Senior political officials, journalists, academics, and civil society representatives worked together in Study Teams to create proposals. Discussions were lively and open, with broad participation from those in attendance. However, a weakness of the course was the absence of industry representatives, who would certainly have contributed an important and valuable point of view. The small number of women participants was also a notable disadvantage. Later courses modelled on the Nairobi offering  had greater success in drawing participants from industry and obtaining a better gender balance. Notably, however, the recommendations that emerged from these courses, while reflecting differences due to regional priorities and context, did not vary considerably despite the broader contribution, particularly from the private sector ., as well as a web-based discussion board. While there was some initial activity on the discussion board, this eventually subsided, and was soon evident that this approach had failed. In an effort to revive the momentum and to solicit ideas from AGPF members about how to best move forward with the network, a short survey was sent to members asking what their needs were, both in terms of the network as well as in terms of the technical facilities at their disposal. The response rate was extremely low; however, those who provided feedback confirmed what the participation level suggested: namely, that information technology facilities in Africa are such that very few individuals, outside of some well-equipped academic or private institutions, have regular access to the internet. The web-based discussion board was, therefore, in practice a highly unsustainable option for the majority of participants. The point was also raised that it was not enough to be connected electronically; there was also a need to share a more tangible goal or project, and to have a more visible leader from within the group, to galvanize efforts and motivate continued interaction. One respondent explained that finding the time to contribute to such networks is extraordinarily difficult for many Africans, who often "wear many hats". As a result, a general interest was insufficient to justify diverting time from other tasks; a concrete, realizable goal was essential for engaging individuals who already feel over-stretched. As a consequence of these inputs, an email-based forum was established, since most AGPF members have better access to email than to the internet, and a moderator was temporarily appointed over the group. Activity on the forum improved and continues today, more than two years later, though interventions are irregular and generally extend to the sharing of information or material of interest, rather than discussions about issues.A major outcome of the Nairobi course, and one which had strong support from participants, was the creation of a virtual network to facilitate ongoing interaction and discussion. Within two weeks of its completion, a website was created for the course The India course on Genomics and Public Health Policy was held in January 2003, less than one year after the inaugural Nairobi effort. Based on feedback from the previous course, the questionnaire requesting feedback about participants' technical and substantive needs in relation to the creation of a network was distributed during the course, to permit the creation of a network that was much more responsive to the needs of the participants. Moderators from among the participants were nominated before the course' end and their roles clarified, to facilitate the sustainability and autonomy of the network.Later in 2003, two further courses were held in Oman and in Venezuela, both of which added a further element demonstrating the learning from the first two courses. On both occasions, the Joint Centre for Bioethics collaborated with the Regional Offices of the World Health Organization; in the first instance, with the Eastern Mediterranean office (EMRO) and in the second, with the Pan-American Health Organization (PAHO). This collaboration ensured that the recommendations of each course had an institutional structure through which they could be channelled, to reach the ear of decision-makers. EMRO and PAHO have extensive links with ministries of health within their regions, as well as with representatives from civil society and industry. This provided an opportunity for the results of the course to have a much wider impact. By contract, the impact of the Nairobi course is very much linked to the efforts of individual participants to engage with their constituencies and with the NEPAD initiative, of which one of their members is now a senior actor. The Forum developed following the Nairobi course has not provided a framework to drive action the way it was initially intended; however, it continues to provide a portal for information-sharing and dialogue.The executive course on Genomics and Public Health Policy in Africa was the first of its kind to be held on the continent. The response of participants indicated a tremendous enthusiasm for and interest in discussing the emerging technology of genomics and its applications for addressing the health woes of Africans. The sessions covered a spectrum of topics, from basic science, to ethics, business models and international frameworks – exemplifying the range of intersecting issues relevant to informed discussions about genomics and related policy. The course also was a demonstration of the fruitfulness of a multi-stakeholder approach. An important aim of the course was to encourage network-building and the development of meaningful interactions, as a foundation for sustained dialogue among opinion leaders. Participants were encouraged to develop independent proposals in a collaborative environment, rather than to be passive recipients of "expertise" from the session leaders. The result was a series of concrete proposals for action, and the establishment of an e-network to provide a forum for ongoing communication, discussion and elaboration of the issues and proposals raised during the course. Several participants agreed to raise the proposals and themes articulated to their colleagues; the course also generated some publicity, as journalists invited to attend and to participate actively in the meeting reported on the key issues in various media ,17; see ; see 17;across regions in the developing world. Each of the three executive course held to-date has addressed similar themes in relation to genomics and health; but each has also been adapted to the particular context and interests of the host country or region. This has partly been achieved through active collaboration between the Joint Centre for Bioethics and the host institutions. The electronic networks provide a means of generating a long-term impact, driven by participants who are empowered, in their particular capacities, to take forward the ideas shared and the proposals developed through their interaction. The Nairobi course also highlighted the importance of being proactive in soliciting suggestions from participants about creative means of virtual networking that realistically address the poor information technology infrastructure in most parts of Africa. It also was instructive in demonstrating that a network is not itself self-sustaining; it must be driven by a clear, shared vision among participants, and possibly even a concrete and realizable project. Moreover, ideally a moderator from within the group should take leadership in feeding the forum, and motivating ongoing participation.Since the completion of this course, three more offerings have taken place, one in India in collaboration with the Indian Council for Medical Research (ICMR) in January 2003, another in Oman in August 2003, and a third in Venezuela in 2004. A fourth course is being planned for a venue in South-east Asia. The Nairobi offering demonstrated clearly the receptiveness of African researchers and policy makers to such an initiative, and captured the vision of a cross-section of stakeholders around how to ensure that the new wave of scientific promise does not pass them by, or crush them in its wake, but instead is harnessed for better health and to further economic development in their region . The couconcrete proposals to inform policymaking.The New Partnership for Africa's Development (NEPAD) has made science and technology (including genomics and biotechnology) a key platform in its plan for economic renewal ,9. IndeeThe author(s) declare that they have no competing interests.All authors participated in and contributed to the course. ACS drafted the manuscript. PAS and ASD conceived of the course, refined the manuscript for critical content and approved final version; and with JM, participated the course design and its coordination. AGFP members provided intellectual input, through their lively discussions and proposals during the Course on Genomics & Public Health Policy in Africa, held 4–8 March 2002.. This course was funded primarily by Genome Canada and the International Development Research Centre (Canada). PAS holds a Distinguished Investigator Award from the Canadian Institutes for Health Research. ASD is supported by the McLaughlin Centre for Molecular Medicine. The African Centre for Technology Studies, which hosted the course, was supported by the Norwegian Agency for Development Co-operation.The Canadian Program from Genomics and Global Health is funded by several sources listed at


Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of  In plants, RNA-mediated silencing pathways have diversified in unique ways. This study elucidates the specific functions of some of the key regulators in development, chromatin structure, and pathogen defense Arabidopsis), which catalyzes accurate excision of the mature miRNA  , only one of which (DCL1) has been assigned a definitive function in small RNA biogenesis  . RDR6 istructure . In manytructure , althougtructure . Arabidoogenesis . Biochemogenesis . Endogenogenesis . Exogenoogenesis .Both siRNAs and miRNAs function post-transcriptionally to suppress or inactivate target RNAs. siRNAs guide sequence-specific nucleolytic activity of the RNA-induced silencing complex (RISC) to complementary target sequences . Among oSchizosaccharomyces pombe and Arabidopsis, endogenous siRNAs from repeated sequences corresponding to centromeres, transposons, and retroelements are relatively abundant   . And, se] locus) . The RNA] locus) .Arabidopsis. Plants with point mutations or insertions in several members of the DCL and RDR gene families were examined. The data indicate that plants genetically diversified several factors involved in formation of functionally distinct small RNAs.Using a genetic approach, we show here the existence of three small RNA-generating pathways with unique requirements in Arabidopsis miRNA formation. As shown for miR-171, miR-159  and DCL3 (At3g43920) genes, respectively  and RDR2 (At4g11130), respectively -treated leaves, as shown previously  was analyzed in parallel with the rdr1 and rdr2 mutants. This rdr6-1 mutant displayed a weak virus-susceptibility phenotype that was consistent with previously reported sde1 and sgs2 mutants (RDR6 transcript levels were detected between wild-type and rdr6-1 mutant plants (data not shown).At least two factors, DCL1 and HEN1 (HUA ENHANCER1), are involved in  miR-159 A, and se miR-159 , mutantsulations B. Plantsleotides  . The siRNA sequences were identified at 5,299 genomic loci , 5S rDNA repeats, and nonrepetitive sequence was calculated C. siRNAsAtSN1) was selected for detailed analysis. AtSN1-derived siRNA formation requires AGO4 . The endogenous siRNA requirement for RDR2 contrasted with the miRNAs, which exhibited complete insensitivity to each of the rdr mutations tested .The HEN1 protein was implicated in post-transcriptional silencing of sense-, but not hairpin-forming, transgenes . We testants see B. The siAtSN1 siRNA accumulation and methylation of cytosine positions at the AtSN1 locus , CpNpG (43.1%), and asymmetric CpHpH (16.3%) methylation in Col-0 wild-type plants  was unaffected , whereas sensitivity to MspI indicates lack of methylation at only CpNpG sites. In wild-type Col-0 and La-ing MspI B. In rdrAtSN1 in rdr2-1 and dcl3-1 mutant lines. Loci containing genes encoding a retrotransposon reverse transcriptase and phosphofructokinase β subunit (PFK) were used as positive controls for sequences associated primarily with K9- and K4-methylated histone H3, respectively . The changes in H3 methylation shown here are similar to those at several heterochromatic or silenced loci in ago4 mutant plants (Chromatin immunoprecipitation (ChIP) assays were used to detect changes in H3K4 and H3K9 methylation at ectively . At AtSNants see C. This wants see C. The ext plants .AtSN1-derived transcripts was measured in rdr2-1 and dcl3-1 mutant plants and compared against the level of PFK transcript using semiquantitative RT-PCR. As shown in AtSN1 transcripts were detected in wild-type Col-0 plants. However, the normalized level of AtSN1 transcripts was over 8- and 3-fold higher in rdr2-1 and dcl3-1 mutant plants, respectively, compared to wild-type plants. Therefore, loss of siRNA-forming capability correlated with loss of heterochromatic marks and elevated transcript levels at an endogenous locus that is normally silenced at the chromatin level.The level of Nicotiana benthamiana, using an Agrobacterium infiltration assay. Subcellular accumulation sites for these proteins were compared to those of β-glucurodinase (GUS)–GFP (cytosolic control) and nuclear inclusion a protein (NIa)–GFP (nuclear control). The DCL3–GFP, HEN1–GFP, and GFP–AGO4 fusion proteins were detected exclusively in the nucleus  fusions in a heterologous plant,  nucleus , indicatdcl mutant series. Two of the viruses, a GFP-tagged version of turnip mosaic virus (TuMV–GFP) and turnip crinkle virus (TCV), infect Arabidopsis systemically and cause moderate to severe disease symptoms. The third virus, cucumber mosaic virus strain Y (CMV-Y), infects plants systemically, but causes only mild symptoms. Wild-type (Col-0 and La-er) and mutant plants were inoculated on rosette leaves, and upper, noninoculated tissue (cauline leaves and inflorescences) was analyzed for virus-specific siRNAs at 7 and 14 d post-inoculation (dpi).The involvement of DCL1, DCL2, and DCL3 in siRNA formation in response to infection by three dissimilar RNA viruses was tested using the dcl1-7, dcl2-1, and dcl3-1 mutant plants, siRNAs accumulated to levels that were similar to those in infected wild-type plants at 7 and 14 dpi  or significantly higher than  the titers in wild-type plants  lower compared to infected wild-type plants  class. While DCL3 undoubtedly functions as the ribonuclease to process dsRNA precursors, RDR2 presumably functions as a polymerase to form dsRNA molecules de novo using templates resulting from transcription of DNA. At some loci, however, RDR2 may be unnecessary as a catalytic subunit, but rather contribute to the formation or stability of a complex that contains active DCL3. This could be the case at some sites, such as the siRNA02 locus, that contain inverted duplications and that may form transcripts with extensive dsRNA structure. Interestingly, accumulation of siRNAs specific to a hairpin construct was shown to be RdRp dependent in fission yeast . At somedcl2-1 plants, which exhibited delayed viral siRNA accumulation and increased susceptibility and sensitivity. However, there are several reasons to suspect that multiple antiviral, siRNA-generating systems exist. siRNAs triggered by TCV were not eliminated in dcl2-1 plants, but rather siRNA accumulation was delayed. Although this could be due to incomplete loss of DCL2 function in the mutant, it could also reflect the existence of secondary or redundant DCL activities. Among the three viruses tested, two were unaffected by the dcl2-1 mutation. This strongly implies the existence of one or more other siRNA-generating activities with unique or redundant antiviral specificity. Further, the DCL2-dependent system may have functions in addition to those associated with antiviral defense. The DCL2–GFP fusion protein was detected primarily in the nucleus, whereas TCV replicates and accumulates outside of the nucleus. Experiments to determine the genetic requirements for RDR1 and RDR2 during antiviral silencing against the three viruses were inconclusive, again possibly the result of functional redundancies or the presence of confounding viral RdRp activities (rdr6 (sde1/sgs2) mutants were deficient in CMV-induced silencing. Additionally, RDR1 contributed to defense against tobamoviruses.The third system functions in antiviral defense and involves DCL2. Loss of this system was specifically detected in TCV-infected tivities . Mourraitivities , on the DCL gene family, the members do not correlate one-for-one with those in Arabidopsis . Further study is required to correlate the DCL activities from wheat germ with those in Arabidopsis.Caenorhabditis elegans and human, for example, contain only one DICER  accumulate to such low levels in ago4 and hen1 mutants? One possibility is that heterochromatic marks (DNA and H3K9 methylation) and associated factors serve to recruit RDR2, DCL3, or both to specific sites on chromatin, thus establishing a reinforcement loop. Loss of heterochromatin in an ago4 mutant, for example, would result in failure to recruit the siRNA-generating enzymes to transcripts originating from a target locus and, therefore, the absence of siRNAs. This hypothesis, however, does not hold for some other siRNA-generating sites, such as those that yield cluster2 siRNAs and siRNA02. Accumulation of siRNAs from these sites is unaffected or even enhanced in ago4 and hen1 mutants. In wild-type plants, these loci are both hypomethylated at CpG and non-CpG sites and are associated with histone H3 that largely lacks K9 methylation (data not shown). The siRNAs formed from these loci clearly require RDR2 and DCL3, but they appear not to affect chromatin structure. These siRNAs may be sequestered elsewhere in the cell and unable to interact with chromatin or chromatin-associated factors.Accumulation of siRNA from endogenous loci and transgenes does not necessarily require AGO4 , suggesting that AGO4 acts downstream of siRNA formation to direct DNA methylation. Losses of AGO4 and HEN1 have nearly identical effects on all siRNAs tested, possibly because HEN1 and AGO4 affect a similar point in the pathway. If AGO4 and HEN1 function downstream of siRNA formation, why do siRNAs derived from some sites  of DCL2 (At3g03300). dcl3-1 has a T-DNA insertion within predicted exon 7 of DCL3 (At3g43920) at a point 2,136 nucleotides beyond the ATG in genomic DNA. This introduces four codons after the serine 288 codon, followed by a premature stop codon. rdr1-1 has a T-DNA insertion within predicted exon 1 after nucleotide 2,366 beyond the ATG of RDR1 (At1g14790). rdr2-1 has a T-DNA insertion within predicted exon 1 (in front of nucleotide 316 from the ATG) of RDR2 (At4g11130). rdr6-1 has a T-DNA insertion within predicted exon 2  of RDR6 . Each insertion line was backcrossed twice to Col-0 and brought to homozygosity. Additional information about the insertion lines are provided in the supplemental online materials.All plants were grown under standard greenhouse conditions. The eviously . Other mdcl1-7, hen1-1, ago4-1, and cmt3-7 mutants, La-er was the wild-type control.For analysis of each insertion mutant, Col-0 was the wild-type control plant. For Arabidopsis inflo-rescence tissue was used for miRNA and endogenous siRNA analysis. Probes for miR-171 and AtSN1-siRNA analysis were described previously  was treated with sodium bisulfite and amplified using primers AtSN1-BS1 (5′-GTTGTATAAGTTTAGTTTTAATTTTAYGGATYAGTATTAATTT-3′) and AtSN1-BS2 (5′-CAATATACRATCCAAAAAACARTTATTAAAATAATATCTTAA-3′). At least 18 independent clones were sequenced for each genotype.Cytosine methylation was analyzed by bisulfite sequencing of genomic DNA or by DNA blot assay following digestion with methylation-sensitive restriction endonucleases, as described elsewhere . The regAtSN1 in wild-type Col-0 and rdr2-1 and dcl3-1 mutants was measured relative to that at internal control loci, At4g04040 and At4g03800. The data were then normalized against the values measured in Col-0.ChIP assays were done using antibodies specific for dimethyl-histone H3K4  or dimethyl-histone H3K9  as described elsewhere . Methyla35S:DCL3–GFP construct contained the DCL3 coding region fused to GFP coding sequence, flanked by the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The expression cassette was cloned in pSLJ755I5. All other GFP fusion constructs were made by cloning the coding sequence into pGWB5  or pGWB6 , a set of gateway-compatible binary vectors designed for 35S promoter-driven expression of GFP fusion proteins . Cloning using gateway vectors was done using reagents and protocols from Invitrogen . Constructs were introduced into Agrobacterium tumefaciens strain GV2260 and expressed in N. benthamiana leaves as described previously  were infected with TuMV–GFP, CMV-Y, and TCV as described previously (Wild-type and mutant eviously . At 7 aneviously .Arabidopsis genome was done using RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html) and Repbase (http://www.girinst.org/index.html).Computational identification of repeat sequences, including transposons and retroelements, in the Arabidopsis siRNAs and miRNAs, including those that were analyzed in this work, can be found in the Arabidopsis Small RNA Project database .Further information about Figure S1Arabidopsis DCL and RDR genes and location of T-DNA insertion sites in mutant lines.(A) Exon (bars)/intron (lines) organization of the DCL2 and DCL3 mRNA in Col-0 and the respective mutants. DNA fragments corresponding to nucleotides 2,652–3,292 of the DCL2 open reading frame and nucleotides 2,805–3,571 of the DCL3 open reading frame were used as hybridization probes. As a control, the blots were stripped and hybridized with a β-tubulin-specific probe ((B) RNA blot analysis  for ic probe .RDR1 and RDR2 mRNA in Col-0 and the respective mutants. DNA fragments corresponding to nucleotides 2,900–3,300 of the RDR1 open reading frame and nucleotides 10–271 of the RDR2 open reading frame were used as gene-specific probes. RNA samples from SA-treated leaf tissues were also included in the analysis.(C) RNA blot analysis  for (5.9 MB EPS).Click here for additional data file.Figure S2N. benthamiana using an Agrobacterium-injection procedure. Leaf tissue from injected zones was excised at 2 dpi for immunoblot assay using a monoclonal antibody against GFP and confocal microscopy (see The 35S promoter-driven GFP fusion constructs were transiently expressed in copy see . An arro(10.8 MB EPS).Click here for additional data file.Table S1(25 KB DOC).Click here for additional data file.Table S2(24 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the entities discussed in this paper are At1g14790 (NM_101348), At3g03300 (NM_111200), At3g43920 (NM_114260), At3g49500 (NM_114810), At4g11130 (NM_117183), chromosome I (NC_003070.3), chromosome III (NC_003074.4), and siRNA02 (AF501743).The GenBank  accession numbers for the T-DNA insertion lines discussed in this paper are rdr1-1 (SAIL_672F11), rdr2-1 (SAIL_1277H08), and rdr6-1 (SAIL_388H03).The SAIL (formerly Garlic)  accession numbers for the T-DNA insertion lines discussed in the paper are dcl2-1 (SALK_064627) and dcl3-1 (SALK_005512).The SIGnAL database (


Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS)–based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS) outbreak in Singapore.The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV) sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host.This study has further demonstrated the importance of improving clinical and epidemiological studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order to allow the most effective control. Mass spectrometry-based sequence analysis provides a sensitive and accurate method to characterize genetic variation of the SARS coronavirus in clinical samples During infectious disease outbreaks, due to either new or established agents, extensive information gathering is required to enable identification of the source, transmission routes, and the effect of containment policies. It is becoming increasingly clear that traditional approaches based on travel and contact tracing are not sufficient for tracking an outbreak. New sequence-based techniques for pathogen detection and identification have the potential to become perhaps the most important component of these investigations, as demonstrated in the recent worldwide effort in fighting the epidemic of severe acute respiratory syndrome (SARS). The discovery of the SARS coronavirus (SARS-CoV) as the etiological agent for SARS was a major breakthrough , which wDirect sequence analysis of a pathogen in a large number of clinical samples, particularly at a population level, is generally cumbersome, technically challenging, and expensive. Therefore, a rapid, sensitive, high-throughput, and cost-effective screening method would greatly facilitate large-scale characterization of genetic variation of pathogens at a population level. A mass spectrometry (MS)–based method for detecting single nucleotide polymorphisms has been routinely used as a high-throughput method for genotyping human samples, and, thus, we sought to extend this methodology to detect pathogen sequence variations. Here, we demonstrate the high sensitivity of the MS-based analysis in detecting SARS-CoV sequence variations and apply it to analyzing both cultured viral isolates and uncultured tissue samples of SARS-CoV.The SARS-CoV samples used in the sensitivity study were previously described . BrieflyVero cell cultured viral isolates were obtained from 13 Singapore patients: the presumed index case (patient Sin2500) of the Singapore SARS outbreak, whose date of illness onset was 25 February 2004; three primary contacts , whose dates of onset were 9 March 2004 (Sin2677 and Sin2774) and 14 March 2004 (Sin2748); one secondary contact (patient Sin2679), who was believed to have contracted SARS from index patient Sin2500 through another primary contact not included in this study and whose date of illness onset was 15 March 2004; and another eight patients , who were believed to be the fifth- or six-generation contacts of index patient Sin2500 (based on contact tracing records) and whose dates of illness onset ranged from 2 April to 14 April 2004. All the patients fitted the World Health Organization case definition for probable SARS [For the spiked human blood samples, RNA was extracted from 200 μl of blood into 50 μl of water using a HighPure RNA kit . For the clinical samples, RNA was extracted into 30 μl of water using a QiAmp viral RNA mini kit . RNA samples were reverse transcribed into cDNA using 2 μl of RNA as template, a SuperScript kit , and 13 sequence-specific primers . All cDNReal-time quantitative PCR analyses of the RNA samples from spiked human blood were performed using a Lightcycler Sars-CoV quantification kit (Roche). Each analysis was done using 1 μl of RNA and in accordance with the manufacturers' instructions.Twenty-one single nucleotide variations (SNVs) of SARS-CoV were analyzed in this study. Eight of these were identified from our previous sequence analysis of five Singapore SARS-CoV viral isolates and represent variations , and 13 A primer extension genotyping assay was designed for each SARS-CoV SNV using SpectroDesigner software  and analyzed using the MassARRAY system (Sequenom) and the recommended protocol for the MADLI-TOF (matrix-assisted laser desorption ionization/time-of-flight) MS-based genotyping analysis . One micThe analytical detection limit of the combined RNA preparation/RT-PCR/MALDI-TOF MS system was determined by probit analysis  using th6 (SB1), 3.84 × 103 (SB2), 2.17 × 103 (SB3), 6.21 × 102 (SB4), and 1.20 × 102 (SB5) copies per microliter , 12 of the 15 analyses of the SB4 sample , and six of the 15 analyses of the SB5 sample . According to probit analysis, this corresponds to a 95% probability of detection when at least 75 copies of viral RNA  are present per reaction, and still a 50% detection chance at 38 copies of virus RNA per reaction . All 64 sham spiked samples gave negative genotype calls.Having demonstrated the high sensitivity of the MS-based analysis, we typed 18 SARS-CoV SNVs in the cultured viral isolates from nine Singapore patients, including five early Singapore cases  and four later Singapore cases .Of the 18 SNVs analyzed, nine were detected in at least two SARS isolates . AssuminIn addition, the sequence variant sharing pattern at SNVs 28,008, 548, 1,727, and 13,347 can further differentiate the four fifth- and six-generation isolates into three different sub-lineages  defined The molecular relationship among the viral isolates derived from MS-based viral sequence analysis is consistent with the one derived from the whole-genome sequence analysis of the same isolates , clearlyWe also typed the 18 SNVs in four uncultured lung tissue samples from patients Sin842, Sin848, Sin849, and Sin852, and compared their sequence variations with their matched cultured isolates .Of the 72 direct sequence variation comparisons (18 SNVs in four sample pairs), nine differences were identified. Six of these were due to heterogeneous sequences in primary tissue samples. For example, the cultured isolate from patient Sin849 showed the sequence variant T:T:C at SNV positions 548, 1,727, and 13,347, a subset of the heterogeneous T/C:T/C:T/C variant observed in the matched lung tissue sample from the same patient. Direct comparison of the MS spectrums of the three different sequence variants  at SNV position 1,727 in In addition, another three sequence variant differences were observed between the paired cultured and tissue samples from patient Sin848 at SNV positions14,807, 26,205, and 26,509, where the tissue sample showed the sequence variant C:C:T, but the matched culture isolate showed the variant T:T:C . In all The application of tagging and thus tracking of SARS-CoV strains using viral lineage- and/or strain-specific sequence variants was further demonstrated in our investigation of a clinical sample from a German patient. This patient stayed in Hanoi, Vietnam, before sharing an airplane flight with an early Singapore SARS patient on his way to New York via Frankfurt . German We genotyped the virus directly from a brochoalveolar lavage specimen from the German patient at four SNV positions  that were distinctive for the early Singapore SARS-CoV isolates see . The seqThrough the application of this MS mini-sequencing approach to the SARS outbreak in Singapore we have demonstrated the precision that pathogen sequence data can add to an epidemiological investigation. We analyzed 18 SARS-CoV SNVs and determined the molecular relationship among the viral isolates from 13 Singapore SARS patients see  from difA further application of MS-based viral sequence variation analysis in tracking the virus strain and thus the transmission of SARS-CoV was demonstrated by our confirmation of the Singapore origin of a SARS-CoV isolate from a German patient. Travel and contact tracing records for this German patient indicated more than one potential exposure to SARS-CoV, and because virus could not be cultured from the patient, it was difficult to pinpoint the origin of his infection by classical sequencing methods that require virus enrichment by culture. By genotyping the four SNV positions that showed unique variants in the early Singapore SARS-CoV isolates, we confirmed that this German patient was indeed infected by an early Singapore virus strain, most likely in a hitherto unnoticed aircraft transmission event from an early Singaporean patient who was later hospitalized with SARS in Germany . TherefoDirect viral sequence variation analysis of uncultured lung tissue samples identified cases of heterogeneous viral sequences in single patient samples. As the SARS-CoV virus is a single-strand RNA virus, the discovery of different sequences in a single tissue sample suggests the presence of multiple viral sequence variants, or quasispsecies, within the host when the sample was retrieved. Our result has further confirmed a recent observation of SARS-CoV quasispecies in individual patients  and is c3–104 RNA copies per milliliter), which is close to the detection limit of real-time RT-PCR based diagnostic tests  [Our study has clearly demonstrated the advantages of MS-based genetic analysis as a method for large-scale viral genetic characterization in clinical samples. Firstly, MS-based analysis has high sensitivity, providing successful detection of virus more than 95% of the time at virus concentrations as low as 75 RNA copies per reaction  ,21,22 aneaction) ,22,23,24MS-based sequence variation analysis is complementary to the identification of new sequence variation by direct sequence analysis and is particularly suitable for investigating agents for which there is already extensive sequence information. Direct sequence analysis is still the “gold standard” for identifying new sequence variations, but it is inefficient and is not necessary for characterizing known sequence variations in a large number of samples. A combination of initial characterization of genome sequence by direct sequence analysis in a subset of samples and the subsequent analysis of informative genetic variations via a MS-based approach is more efficient and suitable for large-scale population investigations. The genome sequences of a wide variety of pathogens and strains are being rapidly accumulated. In bacterial pathogens, strain sequence information is frequently limited to a relatively small number of genes , whereas in viruses such as influenza, extensive genomic knowledge is accumulating. Such accumulation of both partial- and whole-genome sequence information for pathogens will further extend the usefulness of this approach.In summary, our reassessment of the SARS-CoV transmission route in Singapore using MS-based viral sequence variation analysis highlighted the limitation of conventional epidemiological analysis based on travel and contact tracing, and the importance of informing clinical and epidemiological investigation of pathogen transmission by genetic analysis. With its demonstrated high throughput , sensitiFigure S16 copies per reaction (line a), 1.01 × 105 copies per reaction (line b), 9.4 × 103 copies per reaction (line c), 8.9 × 102 copies per reaction (line d), and 1.07 × 102 copies per reaction (line e). The virus loads determined in the five spiked human blood samples were as follows: SB1, 1.64 × 106 copies per reaction; SB2, 3.84 × 103 copies per reaction; SB3, 2.17 × 103 copies per reaction; SB4, 6.21 × 102 copies per reaction; and SB5, 1.20 × 102 copies per reaction. NTC, non-template control.The x-axis denotes the cycle number of the quantitative PCR assay, and the y-axis denotes fluorescence intensity (F2) over the background level. RNA standards were as follows: 1.05 × 10(244 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession number for the Frankfurt-1 isolate is AY291315 and for the Urbani isolate is AY278741.The GenBank (Molecular biology (studying the makeup and function of molecules) is increasingly being used to track outbreaks of infectious diseases. For example, molecular biology can help to identify the cause of a disease  and understand how it spreads.These researchers had previously used molecular biology techniques to study different strains of the SARS virus, which causes the often fatal disease called severe acute respiratory syndrome. They had found that different strains could be distinguished from each other on the basis of specific genetic “fingerprints.” They now wanted to find quick and easy ways to determine the identity of particular viral strains found in sick patients.They used a molecular biology technique called mass spectrometry. They took samples from patients  and determined whether they could detect the SARS virus, identify specific strains, and distinguish between them.They found that mass spectrometry is a useful tool for detecting the SARS virus and for distinguishing between different strains. They also found that they could use this tool to help understand how the SARS virus had been transmitted between specific patients.For this technique to work, the researchers needed pre-existing information about genetic differences between strains. This means that detailed DNA sequencing is necessary to find these differences in the first place and to discover new ones as the virus evolves.The authors suggest that combining initial genetic sequencing of the different strains with the mass spectrometry technique to analyze subsequently large numbers of samples is the most efficient and cost-effective approach.Science Magazine:http://www.sciencemag.org/feature/data/sars/Public access Web pages on SARS from http://www.genomenewsnetwork.org/articles/05_03/sars_3.shtmlNews article on the SARS genome on the Genome Network News Web site: http://www.gis.a-star.edu.sg/homepage/default.jspGenome Institute of Singapore (GIS): http://www.gis.a-star.edu.sg/homepage/gismediapress.jsp?pid=19GIS's press release on differences between SARS strains:


The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer. Wnt binding to its receptor causes the stabilization and nuclear localization of β-catenin. Nuclear β-catenin then functions to activate transcription in conjunction with the transcription factor TCF. A recent report has challenged this basic precept of the Wnt signaling field, arguing that the nuclear localization of β-catenin may be unrelated to its function and that β-catenin functions at the plasma membrane to activate this signaling pathway. Here we present evidence that the pathway in fact does depend on the nuclear localization of β-catenin. We reexamine the functionality of various truncations of β-catenin and find that only the most severe truncations are true signaling-null mutations. Further, we define a signaling-null condition and use it to show that membrane-tethered β-catenin is insufficient to activate transcription. We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability. These alleles allow us to show that the membrane-bound form of activated β-catenin does indeed depend on the endogenous protein. Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby. Our data clearly show that nuclear localization of β-catenin is in fact necessary for Wnt pathway activation. An uncertainty about Wnt signalling -- and in particular about the role of beta-catenin -- is laid to rest Drosophila ortholog wingless (wg) is a segment polarity gene that defines posterior cell fates in each of the larval segments . This complex is responsible for the retention of Armadillo (Arm) in the cytoplasm, for its phosphorylation, and thus for its targeting for ubiquitination and destruction. When the complex is inactivated by Dsh, the intracellular levels of Arm increase, and Arm enters the nucleus, where in combination with the transcription factor TCF/Pangolin, it activates the transcription of genes such as cyclin D and c-myc  causes activation of the Wnt pathway without entering the nucleus. However, this is not the first time that the controversy about the location of Arm/β-catenin function has arisen. Previously, a group working with amphibian embryos had found that membrane-tethered plakoglobin, a close relative of β-catenin, can activate Wnt signaling  is required for patterning.In this study, we reexamine Arm function using three classes of previously described arm gene were classified into three groups based upon their phenotypes and the position of stop codons that result in truncated proteins. The “weak” class has the smallest truncations and is represented by XM19arm. In germline clones  does not allow proper progression through oogenesis and germline clones do not make eggs (S18)  . We haveypes see G and 1H.8 . We haS18 has no effect on the cuticle of wild-type embryos  might reveal some signaling capacity of those alleles as well. Experiments of this kind have been difficult with ArmΔArm, given that it lacks the α-catenin-binding site and fails to rescue the junctional defect in “medium” and “strong” endogenous arm allele backgrounds. We have found that by expressing both ArmΔArm and ArmS18, we can recover intact embryos in all backgrounds tested. We find that “medium” and “weak” alleles can be induced to activate transcription, but the “strong” arm allele cannot .When Armignaling . Chan anignaling  suggest radation . Both muradation . Both muradation I and 5J.radation K and 5L.radation A and 5B.ΔArm is expressed in wild-type epidermis. If ArmΔArm functions independently of the endogenous protein, then all cells should assume the naked cell fate. However, this does not occur . Under these conditions, all cells in the cuticle take on the naked cell fate . Therefore, the activity of C-terminally truncated arm alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby  to reduce Cby levels in F1aarm embryos with and without ArmΔArm. In the absence of ArmΔArm, i.e., in embryos where most ArmF1a protein is cytoplasmic, Cby RNAi has no effect  weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by Arm compare C and 5D.y of Cby , a nucleo effect D. Howeve compare B to 6C. ΔArm cannot activate transcription on its own. Based on these findings, we propose that membrane-tethered Arm, whether wild-type or activated, cannot activate transcription on its own. It does, however, have a profound effect on the endogenous form, forcing both “weak” and “medium” alleles to translocate to the nucleus and activate transcription. Our findings extend and build upon the original nuclear localization of Arm model  does not signal in the presence of uniform ArmΔArm , which we expressed in O43A01arm mutant embryos , consistent with the “medium” alleles retaining the TCF-binding region  for details on mutants used. Hypomorphic mutants of arm are as follows: LM134arm TCC to TTC at nucleotide 2776, F1aarm CGC to CAC at nucleotide 2990, XM19arm stop codon at nucleotide 3850, O43A01arm stop codon at nucleotide 3404, XP33arm stop codon at nucleotide 3466, XK22arm stop codon at nucleotide 3013. Other alleles used were S044230axin , M11–1zw3, V26dsh, Q8apc1, d40apc2, and IG22wg.The wild-type strain used was Oregon R. See Flybase /Y (zygotic)LM134 zw3M11–1arm /Y (zygotic)F1aarm /Y (zygotic)LM134arm /Y (zygotic)F1aarm /Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)F1a zw3M11–1arm /Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)LM134arm /Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)LM134 zw3M11–1arm /Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)O43A01arm /Y (zygotic)O43A01arm /Y (zygotic); ArmS18 (zygotic)F1aarm /Y (zygotic); Arm–GAL4/UAS–WIZ–Cby, UAS–ΔArm (zygotic)F1aarm /Y (zygotic); Arm–GAL4/UAS–WIZ–Cby (zygotic)XK22arm /Y (zygotic); ArmS18 XK22arm /Y (zygotic); MAT–GAL4/UAS–ΔArm (zygotic); ArmS18 S18, a mirystoylated, membrane-tethered full-length Arm . The antibodies used were anti-Arm , rabbit anti-Arm .Embryos were lysed in extract buffer , and the extracts were separated by 7.5% SDS-PAGE and were blotted as described elsewhere a. MaternAccession Numbershttp://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes and alleles discussed in this paper are Q8apc1 (U77947), d40apc2 (AF091430), arm (X54468), S044230axin (AF086811), V26dsh (U02491), IG22wg (NM 164746), and M11–1zw3 (X54005).The GenBank (