There is a growing awareness among providers of the symptom burden experienced by cancer patients. Systematic symptom screening is difficult. Our plan was to evaluate a technology-based symptom screening process using touch-tone telephone and Internet in our rural outreach cancer program in Indiana. Would rural patients have adequate access to technologies for home-based symptom reporting?1) To determine access to touch-tone telephone service and Internet for patients in urban and rural clinics; 2) to determine barriers to access; 3) to determine willingness to use technology for home-based symptom reporting.Patients from representative clinics  in our network were surveyed. Inclusion criteria were age greater than 18, able to read, and diagnosis of malignancy.The response rate was 97%. Of 416 patients completing the survey , 95% had access to touch-tone telephone service, while 46% had Internet access . Higher rates of Internet access were related to younger patient age, current employment, and higher education and income. The primary barrier to Internet access was lack of interest. Use of the Internet for health related activities was less than 50%. The preferred means of symptom reporting in patients with internet access were the touch-tone telephone (70%), compared to reporting by the Internet (28%).Access to communication technologies appears adequate for home-based symptom reporting. The use of touch-tone telephone and Internet reporting, based upon patient preference, has the potential of enhancing symptom detection among cancer patients that is not dependent solely upon clinic visits and clinician inquiry. In recent years awareness of the symptom burden experienced by many cancer patients has grown ,2. At soWhile there is awareness among providers of symptom distress experienced by patients and there are effective symptom interventions, the problem in the day-to-day care of cancer patients is symptom identification . At a reCommunity Cancer Care (CCC) is an organization with home offices in Indianapolis, Indiana, that provides professional services and program development services to 23 hospitals throughout the state of Indiana. Professional oncology services are provided by 18 medical oncologists-hematologists who are employed by CCC or serve under contract. One psychiatrist, an advanced-practice nurse, and a certified nurse are dedicated to quality-of-life efforts. Each year, an average of 2500 new patients are seen in the network of clinics. At any given time, approximately 16,000 patients are receiving care in the CCC network. While the CCC has clinics in metropolitan Indianapolis, rural outreach and program development in rural hospitals have been a major focus of CCC since its inception in 1983. Twenty-one clinics are located in Indiana towns with populations less that 16,000. Twenty counties served by CCC have populations less than 45,000.Using paper and pencil scales we unsuccessfully tried to install a symptom screening process into the daily clinic workflow. The clinic process was slowed. Some patients could not complete the instruments. Patients' report of their symptoms could not be analyzed quickly and placed on the chart for the provider to use. Symptom screening was limited to the day of the clinic visit. We could not easily evaluate a patient's status between office visits. Trends in symptom occurrence were difficult to identify. With pencil and paper instruments it was a laborious and expensive process to establish a database for our patients' symptom reports, a necessary step in program evaluation.Because of these limitations, our goal is to develop a technology solution to gather, analyze, and present symptom reports to physicians and nurses. Several feasible options for reporting symptoms would include either a touch-tone telephone or an Internet connected computer. Because of well-documented differences between access to the telephone service and the Internet , we condThe study design and survey instrument were reviewed and approved by the Institutional Review Board of Community Medical Research Institute in Indianapolis.A convenience sample of cancer patients was gathered from the clinic network of CCC. Three urban clinics and seven rural clinics were conveniently selected for data collection. All of these sites had concentrated, busy clinic days during which patients could be recruited. Clinics were designated "urban" or "rural" based on their zip code being categorized urban or rural by the U.S. Department of Health and Human Services, Office of Rural Health Policy .Staff members at clinic sites were instructed to offer the survey instrument to all patients attending clinic during selected weeks of March and April 2003. All patients were volunteers. All patients had to be at least 18 years of age, be able to read, and have a diagnosis of malignancy (either solid tumor or blood). The number of patients who refused to complete the survey was recorded.The survey instrument included nine items about demographics and access to touch tone telephone service and the Internet. If patients indicated they did not have access to the Internet the survey instrument directed them to questions about reasons they did not have access. If patients indicated they did have access to the Internet, the survey instrument directed them to seven additional questions about how they use or might use the Internet.We used two-sample t-tests to test for mean differences and chi-square tests to test for differences in proportions of demographic characteristics across clinic setting and access to the Internet. Logistic regression models were used to evaluate access to the Internet as a function of clinic setting adjusting for demographic characteristics.Four hundred and sixteen patients completed the survey . The response rate was 97%. Thirteen patients refused to complete the survey stating they were too ill or too tired. Table Two-thirds (67%) of people cited lack of interest for not having Internet access. Other common reasons were unfamiliarity with the Internet (21%), cost (20%), and hesitation to use a computer (13%). There were no significant differences between the urban and rural patients regarding why they did not access the Internet.Fifty percent of patients with Internet access reported using it for health care purposes in both rural and urban clinics, and nearly 60% reported having used the Internet to seek information about their cancer. Among the 169 patients with Internet access who indicated their preferred method(s) for symptom reporting, the telephone was identified as the most popular method (70.4% of respondents) followed by Internet-based symptom reporting (28%) and touch-screen computer in the clinic waiting room (15%). Compared to urban patients, rural patients were somewhat more likely to prefer telephone symptom reporting (79% vs. 63%, P = .02) and less likely to prefer Internet bases reporting (20% vs. 35%, P = .02).Finally, 137 respondents indicated the different ways they might use the Internet for their health care. Requesting information from a physician or nurse was the most frequently cited potential use (77% of respondents). Other reasons included, submitting information about their own condition (59%), identifying and managing symptoms (54%), scheduling appointments (52%), and obtaining prescriptions (50%).The high rate 95%) of access to touch-tone telephone service among cancer patients in our network is comparable to data from other government surveys ,8. Inter5% of accBarriers to Internet use identified by patients and limited use of the Internet offer opportunities for better patient communication and education. Over half of the patients without Internet access reported they were not interested. Perhaps waiting room computers with links to cancer-related web sites with good educational and problem-solving content could spur interest. Educational programs for our cancer patients about the Internet and its use may also be helpful. Cost of Internet services did not seem to be a significant factor.These data suggest that a very significant proportion of cancer patients  were willing to use this modality to communicate with their cancer clinic for multiple tasks. While email may offer a convenient means of communication with a physician's office, there are many barriers to its use. Eysenbach has written a thorough review of the potential problems of liability and time pressures . While KThe findings of this survey must be interpreted with caution. While very few patients refused to complete the survey, the patient sample is a convenience sample not a total sample and not a random sample of our patients. With only 46% of our sample (191 patients) having access to the Internet, generalization should be cautious pending replication in a larger sample. The survey instrument did not include questions about readiness to use a touch tone phone for completing a symptom questionnaire by patients, and this is a limitation to the study.Our findings suggest that either touch-tone telephone or Internet-based computer methods might be used to collect home-based symptom ratings for cancer patients in both urban and rural centers. While access to technologies is adequate, acceptance and usability of such a system remains to be demonstrated. Patient preference for a telephone-based or Internet-based system can be definitively ascertained only after patients use both systems. With lack of interest being the most common barrier to Internet access, education and "get acquainted" programs for patients who do not have Internet access may be warranted. Alternatively, since many patients prefer touch tone telephone for symptom reporting, the use of IVR (Interactive Voice Recording) technology provides another way for symptom reporting, coupled with centralized nurse care management of cancer-related symptoms. Indeed, we are proceeding to test this in a study in which cancer patients will have an option of home-based symptom monitoring by either IVR or the internet coupled with centralized nurse care management of cancer-related symptoms. Patient resource centers with Internet access in outpatient clinics may be another mechanism to consider.The author(s) declare that they have no competing interests.MA: Assisted with data analysis and interpretation of results. Reviewed, corrected and submitted the final manuscript.DET: Designed the project including the questionnaire, obtained IRB approval, supervised completion of the survey, assisted with data analysis and writing the final manuscript.DB: Coordinated data collection from clinic sites and compiled survey results.KK: Assisted with data analysis, interpretation of results, in addition to review, preparation and submission of the final manuscript.AP: Carried out the data analysis and assisted with interpretation of results in addition to preparation of the methods and results sections of the manuscript.SE: Assisted in site recruitment and review of the manuscript.WMD: Assisted in site recruitment and review of the manuscript.The pre-publication history for this paper can be accessed here:


Light waves from an awaited signal—a white circle—arrive at the subject's eye; within a fraction of a second, the subject's thumb presses a button. Between eye and thumb lies the central nervous system, its feats of perception, integration, and response largely opaque to scientific scrutiny. Imaging techniques like magnetic resonance imaging can detail brain anatomy but can only broadly show changes in activity levels occurring over seconds—indirect echoes of brain function. Electrodes stuck to the scalp record coordinated neuronal symphonies, and wires inserted among neurons can capture the single-cell firing patterns of the individual instruments of the neural orchestra. But how these electrical signals map to information processing within and across neural circuits remains blurry. A new analysis sharpens the focus by separating individual brain wave patterns, measured from multiple sites across the scalp, into nine distinct process classes, each centered in an anatomically relevant brain area and producing predictable patterns as human subjects receive visual cues and produce responses.Scalp electroencephalograms (EEGs) are dominated by waves of synchronized neuronal activity at specific frequencies. Decades of research have associated wave patterns recorded at different scalp regions with different states of alertness—attending, drowsy, sleeping, or comatose; eyes open or closed—and gross abnormalities, such as seizure, brain damage, and tumor. In order to separate EEG responses to specific events from background, state-related activity, researchers repeat an experiment like the button-press exercise tens or hundreds of times and average the EEG across trials. By averaging out background activity, this technique reveals a characteristic waveform, called an event-related potential (ERP). It differs by electrode location, but often contains a large positive wave that peaks 300 milliseconds or more after an awaited visual cue.In the current paper, Scott Makeig et al. argue that ERP averaging removes important information about ongoing processes and their interactions with event-related responses. Instead of averaging multiple recordings from each of 31 electrode sites, the authors applied an algorithm that seeks independent signal sources contributing to the individual tracings. The researchers measured signal source activities by the frequency and phase of wave patterns and source locations by comparing signal strength and polarity at different electrodes. Altogether, the researchers identified nine classes of maximally independent sources, each having similar locations and activities across subjects. The results dovetail neatly with prior anatomical and functional observations.This analysis demonstrates that average waveforms identified in ERP studies probably sum multiple, separate processes from several brain regions. In particular, the large positive ERP seen 300 milliseconds or more after a visual cue reflects different waveforms from frontal, parietal, and occipital cortex—areas involved in task planning, spatial relationships and movement, and visual processing, respectively. In addition, this study showed a two-cycle burst of activity in the 4–8 (theta) frequency band after button presses—another common ERP feature. The theta activity was coordinated across several signal sources, and localized to areas associated with planning and motor control. Notably, the planning component seemed to lead the motor signal. Suppression or resynchronization of several EEG processes followed the visual cue or button press. The authors theorize that such coordination might influence the speed or impact of communication between brain areas and help retune attention after significant events.Using this approach in more subjects, and under differing conditions, could provide an unprecedented glimpse of how the brain translates perception and planning into action. The results suggest that EEG data contain an untapped richness of information that could give researchers and clinicians a new window into thought in action.


It is an absolute necessity to continually assess the quality of health information on the Internet. Quality-controlled subject gateways are Internet services which apply a selected set of targeted measures to support systematic resource discovery.The CISMeF health gateway became a contributor to the MedCIRCLE project to evaluate 270 health information providers. The transparency heritage consists of using the evaluation performed on providers that are referenced in the CISMeF catalogue for evaluating the documents they publish, thus passing on the transparency label from the publishers to their documents.Each site rated in CISMeF has a record in the CISMeF database that generates an RDF into HTML file. The search tool Doc'CISMeF displays information originating from every publisher evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository. Starting with 270 websites, this trust heritage has led to 6,480 evaluated resources in CISMeF (49.8% of the 13,012 resources included in CISMeF).With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party. The availability of Internet health tools and services has been increasing at a phenomenal rate in recent years making the Internet a major source of knowledge for healthcare professionals, medical students and also patients and the general public. This increase has made it an absolute necessity to continually assess the quality of health information on the Internet. Indeed, creating a Web site is relatively easy, therefore uncontrolled health information can be launched by virtually anyone with access to the Internet. Peer review is often absent throughout this media as opposed to scientific journals. There have been numerous debates about the variable quality of health information on the Internet and its impact on public health . There iIn the past five years, a lot of authors have scrutinized the quality of the health content available on the Internet. These studies, assessing the quality of health information, have been extensively reviewed by Eysenbach et al. . In the As of today, access to accurate and trustworthy health information on the Internet is not an easy task; there are a great number of directories and search engines available in this new media . But, thThe objective of CISMeF  ,7 is to ) [CISMeF is a quality-controlled subject gateway initiated by the Rouen University Hospital (RUH). Each of the following phases proposed by Koch , which c) , (b) col The CISMeF Web site opened in February 1995. In December 2003, 13,012 resources had been indexed, with an average of 55 new resources indexed each week. CISMeF is considered by most professionals as the reference health institutional Web site in France with as many as 25,000 unique machines visiting this Web site by working day. Doc'CISMeF is the search tool of the CISMeF gateway [CISMeF is manually maintained. In CISMeF, a resource is defined as 1) a Web site or 2) high-quality documents from this Web site. CISMeF describes and indexes the most important sources of institutional health information in French, in order to allow them to be searched quickly and precisely. A great variety of resources are indexed, in terms of resource type , and resource format (html, pdf, etc.). Its Universal Resource Locator (URL) is  gateway .In 1997, because the quality of health information became an important issue for the building and maintenance of a trustworthy health gateway, the CISMeF team participated in the development of a user guide named Net Scoring . The goa) [In 2002, CISMeF became a contributor to the MedCIRCLE project (URL: ) . The aim) . MedCIRCThe main deliverable of this project for the CISMeF team was the evaluation and rating of 270 health information providers (or publishers) which who release health resources in French on a regular basis. Because CISMeF catalogues and indexes not only Web sites but also and mainly quality-controlled documents from those health publishers, we introduced the concept of "transparency transitivity" or " transparency heritage". It consists in applying to these documents the evaluation performed for their publishers, thus passing on the transparency label from the publishers to their documents.Health professionals have begun to realize that it is their responsibility to guide consumers and patients to the best available medical information on the web. Many national governments and medical societies have acknowledged that it is their responsibility to help users to identify "good quality" information sources and have begun to develop national health gateways , portal sites and other forms of "infomediaries" such as seals of approval  or certiHowever, current approaches do not harness any of the advantages of the Web as a decentralized, distributed information system. There is a need for "next generation" tools, including intelligent knowledge-based tools, allowing consumers to positively and actively identify reliable health information that suits their needs.) for laypersons, listing consumer health information sites. Before MedCIRCLE, documents had been evaluated using the DISCERN instrument [) represents the medical profession of Barcelona. To this date, in the project "Web Medica Acreditada", COMB has accredited more than 300 Spanish health websites from Spain and Latin America [The three application partners of MedCIRCLE, besides CISMeF in France, were the Agency for Quality in Medicine (AQuMed) in Germany and the Official Medical College of Barcelona (COMB). AQuMed was founded in March 1995 as a joint institution of the German Medical Association and the National Association of Statutory Health Insurance Physicians. AQuMed established a health gateway  thesaurus from the US National Library of Medicine, and several metadata element sets : (a) 11 ) [HIDDEL is a metadata language and an ontology, which enables the expression of descriptive and evaluative annotations in XML/RDF. The first version of HIDDEL was initially developed during the MedCERTAIN project  . HIDDEL ) , a basic) . HIDDEL evaluate health information providers. It presents three levels of evaluation: (a) self-disclosure (b) evaluation by non-medical experts, and (c) evaluation by medical experts. As a quality-controlled subject gateway, CISMeF uses HIDDEL only as a third-party.HIDDEL is based on existing quality criteria such as the Health On the Net (HON) Code of Conduct . It was The HIDDEL vocabulary can be downloaded freely from the MedCIRCLE Web site, as long as the sources are acknowledged and requests for changes or expansions are fed back to the community. At present HIDDEL is available in four languages: English, German, French and Spanish. The use of this controlled vocabulary enables automatic translation (except for free text). The heritage process was made possible because of HIDDEL's dual structure: on the one hand, Infoprovider metadata, describing to the health information provider , and on the other hand, Sitespecific metadata devoted to one Web site evaluation (e.g., language). In CISMeF, we have applied Sitespecific metadata to each resource  from a publisher already included in the CISMeF database. The name of the person responsible for the quality of the Web site, which is one of the Infoprovider metadata, is the same for every document of the Web site. On the contrary, the language of the document, which is one of the Sitespecific metadata, may vary from one document to another. The CISMeF team implemented the whole HIDDEL structure in the CISMeF database, which involved the creation of triggers, thus ensuring automated transfer from CISMeF to HIDDEL metadata, and the creation of new forms (interface recasting) to deal with non-CISMeF metadata. Because the HIDDEL elements are optional and repeatable, CISMeF has selected a number of 70 metadata among the 305. Most of the metadata previously used in CISMeF and in particular the Dublin Core are also included in the HIDDEL language. These metadata were automatically triggered in the HIDDEL language.The interoperability process consists of an exchange of RDF files, containing experts' annotations "written" in HIDDEL. The semantic-based Archer Annotation System deals with RDF annotations reception. Archer is a Web application that allows annotating health information Web sites using the HIDDEL vocabulary. It is a technical platform and an organizational infrastructure that can be used by consumers, health information providers, and third party rating services. The first version of Archer was implemented as a part of MedCERTAIN, and further enhanced in the course of the successor project MedCIRCLE to allow the exchange of metadata between third party rating organizations.) into the generated HTML pages, making them not only machine-readable (as every HTML page is) but also machine-processable. Therefore, one of the main goals of this metadata element set was fulfilled easily: it became interoperable with other Internet services. Moreover, an RDF Scheme describing CISMeF specific metadata was created (URL: ).On another ground, through its search engine Doc'CISMeF, CISMeF provides external links to Archer backend servlets, and internal links to rated sites disclosure  included in the CISMeF database: national agencies, medical societies, universities and hospitals. CISMeF first checks the publishers' information without asking the health information provider to self-declare any metadata as described in the MedCERTAIN quality management process.CISMeF used HIDDEL to select and evaluate the 270 health publishers most represented in CISMeF and made the results of their evaluations explicit and accessible using RDF metadata. These were exported into the searchable MedCIRCLE Open Directory.) displays the information originating from each of the publishers that were evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository where HIDDEL metadata elements are displayed  has been previously accredited a third party.Nonetheless, every resource included in CISMeF and those evaluated by the MedCIRCLE process are quality-controlled. The MedCIRCLE consortium takes a very neutral approach and does not impose but strongly recommends certain procedures or minimum metadata, taking into account that collaborating gateways, accreditors, certifiers, raters may approach from very different angles.CISMeF has applied full heritage from the evaluated publishers: each document from a MedCIRCLE rated publisher, indexed in CISMeF, will also receive the MedCIRCLE button of the publisher with the same link to MedCIRCLE central repository. The idea is to keep the common Infoprovider elements for every document, and to use CISMeF metadata to disclose Sitespecific elements specific to each document, in particular the indexing with the MeSH thesaurus. At the end of the project in December 2003, starting from 270 websites, the translation from CISMeF metadata to HIDDEL, led to 6,480 evaluated resources in CISMeF in September 2003 (49.8% of the 13,012 resources included in CISMeF). All CISMeF selected HIDDEL metadata (70 out of 305) are displayed in the Doc'CISMeF record in RDF into HTML. The top five publishers indexed in CISMeF, which produced trustworthy documents in French are: Grenoble Medical School (N = 435), Health Canada (n = 275), Strasbourg Medical School (N = 263), and French Ministry of Health (N = 248).Every new document  included in CISMeF from one of the 270 main publishers evaluated through the MedCIRCLE process inherits automatically: (a) a MedCIRCLE button linking to the repository and (b) HIDDEL metadata included in CISMeF database and displayed in the Doc'CISMeF record in RDF into HTML. On the other hand, a document that comes from one of the 270 publishers evaluated within the MedCIRCLE project but not included in the CISMeF database will not receive a MedCIRCLE button in the CISMeF gateway. However, this more global transitivity could be applied in a more generic search tool such as Google.Since February 2003, when the MedCIRCLE button became operational in the CISMeF gateway, the CISMeF team has decided to go on (keep) applying the MedCIRCLE transparency process after the end of the project with the following rules: (a) check every month if there is a new publisher with five documents already included in CISMeF and (b) if so, begin the MedCIRCLE process for these publishers and apply transparency heritage for their respective documents. We applied these rules after the end of the EU-funding project. In March 2004, the CISMeF database contained a complete evaluation through the MedCIRCLE process for 346 publishers  and 7,053 documents from these publishers (53.3% of the 13,227 resources included in CISMeF).In the MedCIRCLE repository, end-users can access an aggregate view of what people say about a certain Web site by clicking: The CISMeF gateway is one of many possible producers of trustworthy metadata regarding a health information provider. Metadata from the Open Directory can also be fed into search engines and other gateways.) consortium (URL: ): this HON tool bar is indicating if the site is accredited directly by HON or indirectly by one specific accredited Web sites as health gateways such as CISMeF or MedlinePlus. In this context, HON is creating a seal of quality trust heritage where MedCIRCLE is creating a seal of transparency heritage. A formal evaluation of these two examples of heritage  is mandatory to check their respective hypothetical added value.As the number of health related Web sites worldwide has been estimated as being around 100,000, complete coverage by a single third party evaluation body is impossible. Instead, a collaborative approach as shown in this paper has to be promoted, whereby different rating services or organizations use comparable standards and a common metadata language. More recently, the Health on the Net Foundation has developed a HON tool bar in the course of the EU-funded Active Health , which partially granted the MedCIRCLE consortium.One of the main findings of the MedCIRCLE consortium in the course of the project has been that there is no absolute objective quality of a Web site. Quality is to a certain degree subjective, may vary in time and also according to the eye of the beholder. A Web site that a consumer looking for health information finds acceptable one day may be unacceptable another day. For example, a consumer may search general information for one drug (e.g. after a TV show) and finds advice on a patient information Web site. Later, the same consumer searches for the same drug for his/her child. But this time, he/she checks if the respective Web site is sponsored by a pharmaceutical company. He/she comes to the same Web site as before, but this time this Web site will be unacceptable. The context of the search changed, making a general advice acceptable and a specific advice unacceptable. By providing metadata about health related websites, MedCIRCLE allows the health information consumers to decide themselves if a website is of good quality. Trust is improved by enhancing transparency. More globally, the MedCIRCLE consortium will lead to a safer Internet by providing a seal of transparency to over 7,000 resources on the Internet via the CISMeF search engine. The precaution principle is now widely accepted in the European Union, which develops Action Plans, such as the Action Plan for Safer Use of the Internet  by providing a seal of transparency. The Netizen (citizen on the Internet) has the active role to evaluate the reliability when reading the HIDDEL metadata after clicking the MedCIRCLE button. With a seal of trust, the role of the Netizen is more passive but leads to a faster trust information access. Nevertheless, a drawback of the MedCIRCLE button is a possible misunderstanding by end-users, who might confuse it with a quality seal and therefore may forget to actively press the MedCIRCLE button, which may hinder their valid judgement. Here again, a formal evaluation of theses two approaches (transparency vs. trust) is necessary.implicit third party. One of the main results of the MedCIRCLE project, from the CISMeF team's point of view, is that CISMeF proceeded from being an implicit to being an explicit third party thanks to the creation of the MedCIRCLE button now used for 346 publishers (2.6% of the resources included in CISMeF) and 7,053 resources (53.3% of the resources included in CISMeF), which multiplies by 20 the number of resources with a seal of trust.While using Net Scoring, CISMeF was acting as an The HIDDEL language is totally embedded in the CISMeF metadata element sets. This allows a very easy interoperability with MedCIRCLE tools, and more specifically Archer. Semantic Web approaches already used in the MedCIRCLE project could open up new ways for educating health professionals and consumers and reaching less savvy health professionals and consumers, because part of the intelligence and knowledge currently required to critically appraise information on the health professional or consumer Web site could be built into the search tools. The feasibility of this approach has been already demonstrated by CISMeF but also by the German (AQuMed) and Spanish partners (COMB). The impact on health professionals and consumers is subject to ongoing investigation within the MedCIRCLE project. The Semantic Web may provide the health professional or the consumer with a greater capacity to determine the reliability of a given health information provider or service than the Web in its current form.With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party.None declared.SJD had the original idea of seal transparency and drafted the manuscript. DH and TRRB developed respectively in France and Germany the programs to implement the seal transparency in CISMeF & MedCIRCLE Web sites.The pre-publication history for this paper can be accessed here:


The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection.We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background) mutation rate, the selection coefficient, and the effective population size.The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed. Due to transcription factors that bind to specific sites on DNA. These binding sites contain about 10 – 15 base pairs relevant for binding and are mostly located in the cis-regulatory promoter region of a gene. A cis-regulatory region in E. coli is about 300 base pairs long and contains a few transcription factor binding sites [fuzzy, that is, different sites for the same factor differ by about 20 – 30 percent of the bases relevant for binding [binding cooperativity can be related to various functions. In a genetic switch such as the famous phage lambda switch in Escherichia coli [signal integration, leading to the expression of the downstream gene only when both kinds of factors are present simultaneously [modules interspersed between regions containing no sites. In many known cases, the expression of a gene depends on the simultaneous presence of several factors. Well-studied examples of regulatory networks in eukaryotes include the sea urchin Strongylocentrotus purpuratussea [Drosophila [To a large extent, gene regulation is the control of transcription. It is accomplished by a number of regulatory proteins called ng sites . There mhia coli , it prodaneously . A promoratussea  and the osophila .fitness landscape for binding site sequences is constructed (see the discussion in the next section). The resulting mutation-selection equilibrium is analysed using a mean-field quasispecies approach [mutational entropy as a possible reason: the single or few sequence states with optimal binding of the transcription factor can be outweighed by the vastly higher number of sub-optimal states at some mutational distance from the optimal binding sequence. This effect is similar to the fuzziness of amino acid sequences in proteins discussed in [The sequence statistics of binding sites has been addressed in two recent theoretical studies ,11. Baseapproach . This apapproach ,11, fuzzussed in .losses of function in both copies [new functions of genes as a response to specific demands. This task must be accomplished mainly by sequence evolution of regulatory DNA. There are examples of highly conserved regulatory sequences with a conserved function but binding sites can also appear, disappear, or alter their sequence even between relatively closely related species; see, e.g., refs. [Drosophila [From an evolutionary perspective, explaining the molecular programming of regulatory networks presents a striking problem. The diversification of higher eukaryotes, in particular, requires the efficient generation and alteration of regulatory binding interactions. One likely mode of evolution is gene duplications with subsequent complementary h copies ,17. Howe., refs. -22. Thisosophila . The traquantitative analysis of binding site evolution. We study a theoretical model that takes into account point mutations, selection, and genetic drift. The form of selection is inferred from the biophysics of the binding interactions between transcription factors and DNA. We derive the stationary distribution of binding sites under selection, which shows specific correlations between nucleotide frequencies at different positions in a binding site. The non-stationary solutions of the model describe efficient adaptive pathways for the molecular evolution of regulatory networks by point mutations. This efficiency can be quantified in terms of the length of the binding motif, and the length of the promoter region, and the fitness landscape for factor binding, which is amenable to quite explicit modeling.The modes of regulatory sequence evolution and their relative importance remain largely to be explored. Contributions may arise from point mutations, slippage processes , and larnewly arising selection pressure is estimated in terms of the characteristic time scales of mutation, selection, and drift. For Drosophila, it may be as short as 105 years even for moderate selection pressures. However, this pathway is found to depend crucially on the presence of selection. It would be too slow under neutral evolution, in contrast to the results of [With the parameters found in natural systems, our model predicts that a new binding site for a given transcription factor can be generated by a fast series of adaptive substitutions, even if the expression of the corresponding gene bears even a modest fitness advantage. The evolutionary time required for site formation in response to a sults of , see alssults of . CooperakBT) between a transcription factor and its binding site is, to a good approximation, the sum of independent contributions from a small number of important positions of the binding site sequence, , with  ≈ 10 - 15 [εi depend on the position i and on the nucleotide ai at that position. There is typically one particular nucleotide  preferred for binding; the sequence  is called the target sequence. The target sequence can be inferred as the consensus sequence of a sufficiently large number of equivalent sites. The so-called energy matrix εi(a) has been determined experimentally for some factors from in vitro measurements of the binding affinity for each single-nucleotide mutant of the target sequence. Typical values for the loss in binding energy are 1–3 kBT per single-nucleotide mismatch away from the target sequence. In this paper, we use the further approximation εi = ε if ai =  and ε = 0 otherwise, the so-called two-state model [ is then, up to an irrelevant constant, simply given by its Hamming distance r to the target sequence: E/kBT = εr. (The Hamming distance is defined as the number of positions with a mismatch ai ≠ .)The binding energy (measured in units of  10 - 15 -30. The te model . The binεi(a) tend to reduce the number of crucial steps in the adaptive process and thereby to further increase its speed.It is important to note the status of this "minimal model" of binding energies for the discussion in this paper. Both approximations underlying the model can be violated. Even though typical mismatch energies are of the same order of magnitude, there can be considerable differences between different substitutions at one position and between different nucleotide positions. Moreover, deviations from the approximate additivity of binding energies for the single nucleotide positions have also been observed. However, these complications do not affect the order-of-magnitude estimates for adaptive sequence evolution. As it will become clear, the efficiency of binding site formation depends only on the qualitative shape of the fitness landscapes derived below. In these landscapes, the regime of weakly-binding sequences and of strongly-binding sequences are separated by only a few single nucleotide substitutions. The relative magnitude of the fitness increase of these substitutions does not matter in first approximation. Indeed, inhomogeneities in the values of the Within the two-state model, the binding probability of the factor in thermodynamic equilibrium isε is the binding energy per nucleotide mismatch and ερ is the chemical potential measuring the factor concentration. Both parameters are expressed in units of kBT and hence dimensionless. Appropriate values for typical binding sites have been discussed extensively in refs. [ε should take values around 2, which is consistent with the measurements for known transcription factors mentioned above [background sites elsewhere in the genome (which have a sequence similar to the target sequence by chance), and on the functional sites in the in the genome other than the binding site in question that may compete for the same protein. Binding to background sites does not significantly reduce the binding to a specific functional site [ρ ≈ (log nf)/ε ≈ 2 - 4, given observed factor numbers nf of about 50 – 5000 [Here in refs. ,13. It ied above -30. The fitness landscape quantifies the fitness F of each sequence state at the binding site. Fitness differences arise due to different expression levels of the regulated gene, and these in turn depend on the binding of the transcription factors. It is only these fitness differences that enter the population dynamics   of binding site sequences in the next section. Following the conceptual framework of ref. [cellular states (labelled by the index α) with different transcription factor concentrations, i.e., with different chemical potentials ρα. These cellular states can be thought of as different stages within a cell cycle. In each state, the fitness depends on the expression level of the regulated gene in a specific way. This expression level is determined by the binding probability pα of the transcription factor. Assuming that both dependencies are linear  and that the cellular states contribute additively to the overall fitness F, we obtainA  of ref. , we assuselection coefficient sα is defined as the fitness difference (due to different expression of the downstream gene) between the cases of complete factor binding and no binding in the state α. Such fitness differences can now be measured directly in viral systems [r only. We note that the fitness F is measured relative to that of a phenotype with zero binding probability in any state α.Here the  systems . Insertion state favours expression of the gene, the off state disfavours it. It is then natural to assume selection coefficients of similar magnitude; here we take for simplicity s = son = -soff > 0. We then obtain a crater landscape,In a simple case, there are just two relevant cellular states. The ρoff and ρon flanked by two sigmoid thresholds; see fig. r) where binding occurs and the gene is expressed in the on state but not in the off state. Sequences with large Hamming distance r >ρon can bind the factor neither in the on nor in the off state, while sequences with r <ρoff lead to binding in the on and the off state. Both cases lead to misregulation of the downstream gene, and hence to a lower fitness. We note that the key feature of these fitness landscapes, the sigmoid thresholds, is independent of the particular choices of son and soff.with a high-fitness rim between on state contributes significantly to selection, i.e., if s = son > 0 and soff = 0. The crater landscape then reduces to the mesa landscape discussed in [An even simpler fitness landscape is obtained if only the ussed in ,32,ρ and one sigmoid threshold; see fig. r <ρon) have a high fitness. In both cases, the parameters of the binding model have a simple geometric interpretation: ε gives the slope and the ρα give the positions of the sigmoid thresholds in the fitness landscape. Eqs. (3) and (4) are again to be understood as minimal models of fitness landscapes for binding sites, representing target sequence selection for a given level of binding (ρoff <r <ρon) and for sufficiently strong binding (r <ρon), respectively. Despite its simplicity, this type of selection model based on biophysical binding affinities is nontrivial from a population-genetic viewpoint since it leads to generic correlations between frequencies of nucleotides ai and aj within a site, see the Results section below. We will also study generalized models with correlations between two sites generated by cooperative binding. On the other hand, these models neglect the context dependence of the binding process through cofactors and chromatin structure. However, they are a good starting point for order-of magnitude estimates of the adaptive evolution of binding sites.which has a high-fitness plateau of radius μ ~ 10-4 per site and generation for RNA viruses to values several orders of magnitude lower in eukaryotes, e.g. μ ≈ 2 × 10-9 in Drosophila [quasispecies theory [n(r,t) of individuals at Hamming distance r from the target sequence at time t. Along with a generalisation for two binding sites, it has been analysed in detail in ref. [nstat(r) has been found exactly [s/μ and describes a stable polymorphic population, i.e., several sequence states coexist. The mean-field approach is valid as long as the stochastic reproductive fluctuations are leveled out by mutations. This requires absolute population numbers Nnstat(r) ≫ 1/μ for all relevant r, a stringent condition on the total population size N.The rates of nucleotide point mutation show a great variation, ranging from osophila .  and selection -36. AccoF ≪ 1 [N is the effective population size . Eq. (5) has three well-known regimes. For substantially deleterious mutations (NΔF ≲ - 1), substitutions are exponentially suppressed. Nearly neutral substitutions (N|ΔF| ≪ 1) occur at a rate u ≈ μ approximately equal to the rate of mutations in an individual. For substantially beneficial mutations (NΔF ≳ 1), the substitution rate is enhanced, with u ≃ 2μNΔF for NΔF ≫ 1.in a diffusion approximation valid for ΔF ≪ 1 . Here N T ~ N for nearly neutral and T ~ 1/ΔF for strongly beneficial substitutions. The picture is thus self-consistent for Tu ≪ 1, i.e., for μN ≪ 1. Asymptotically, it describes monomorphic populations moving through sequence space with hopping rates u.In this picture, a population has a monomorphic majority for most of the time and occasional coexistence of two sequence states while a substitution is going on. The time of coexistence is ensemble of independent populations, this stochastic evolution takes the form of a Master equation. For a single binding site, we obtainIntroducing an P(r,t) denotes the probability of finding a population at Hamming distance r from the target sequence, and ur,r' is given by (5) with ΔF = F(r') - F(r). The combinatorial coefficients arise since a sequence at Hamming distance r can mutate in  different ways that increase r, and in r ways that decrease r, where c = 4 is the number of different nucleotides. The stationary distribution isHere Pstat(r) ~ exp[S(r) + 2NF(r)].     (7) is the mutational entropy (the log fraction of sequence states with Hamming distance r) [ur+1,r/ur,r+1 = eN - 1)ΔF2(. To derive (7), we then simply approximated N - 1 by N. The form of Pstat(r) reflects the selection pressure, i.e., the scale s of fitness differences in the landscape F(r). For neutral evolution (2sN = 0), the stationary distributionHere tance r)  and we hsN ~ 1), Pstat(r) results from a nontrivial balance of stochasticity and selection. For strong selection (2sN ≫ 1), Pstat(r) takes appreciable values only at points of near-maximal fitness, where F(r) ≳ Fm - 1/2sN. In this regime, the dynamics of a population consists of beneficial mutations only, i.e., the system moves uphill on its fitness landscape.is obtained from a flat distribution over all sequence states. For moderate selection  and the mean-field quasispecies equation thus describe opposite asymptotic regimes, e genome .) We conr because it is a convenient parameterization of the binding energy in the two-state model. In order to compare this approach to standard population genetics, it is useful to recast eq. (7) for the elementary sequence states ,In the previous sections, we have expressed the fitness landscape and the resulting population distributions as a function of the Hamming distance r. At neutrality, the distribution over sequence states factorizes in the single nucleotide positions,where the sum runs over all sequence states at fixed ν0(ai) is simply a flat distribution over nucleotides but it is obvious how this form can be generalized to arbitrary nucleotide frequencies.In the specific case of the two-state model, According to eq. (7), the stationary distribution under selection takes the formF(r) is generically a strongly nonlinear function of r due to the sigmoid dependence of the binding probability on r. An analogous statement holds beyond the two-state approximation for the dependence of F on the binding energy E. Hence, even if  factorizes in the single nucleotide positions,  does not. The selection introduces specific correlations between the nucleotides: the fitness differences and, hence, the nucleotide frequencies at one position i depend on all other it l - 1 positions in the motif.The salient point is that sN = 100) in the crater fitness landscape (3) with parameters  = 10, ε = 2, ρon = 3, ρoff = 1 (implying that the factor concentrations differ by a factor of 50), and a case of moderate selection (sN = 7) in the mesa landscape with parameters  = 10, ε = 1, ρ = 3.6.  The fitness landscapes for both cases are shown in fig. s. Substantially beneficial mutations occur only on their sigmoid slopes, i.e., in narrow ranges of r. The upper boundary of this region is given by rs = ρon + log[sN(eε - 1)]/ε which takes typical values rs = 5 - 7. In fig. r = 5 to lower values of r, which are close to the point rm of maximal fitness. The statistics of this adaptation is governed by the ensemble P(r, t); the average  and the standard deviation δr(t) appear also in fig. We now apply the dynamics (6) to the problem of adaptively generating a binding site in response to a newly arising selection pressure. We study a case of strong selection  ~ exp[S(r)] shown as empty bars in fig. rmin in the entire region is given by the distribution , where  is the cumulative distribution for a single site.  is found to be strongly peaked, taking appreciable values only in the range  around its average. We assume selective evolution sets in as soon as at least one site has a Hamming distance r ≤ rs. This is likely to happen spontaneously if , leading to a joint condition on , L, and rs. For , there is a neutral waiting time before the onset of adaptation. Its expectation valueCan such a selective process actually happen? This depends on the initial state of the promoter region in question Ts, rendering the effective generation of a new site less feasible.is calculated in the appendix. It is generically much larger than the adaptation time Pstat(r) under selection is given by (7) and shown as filled bars in fig. rm of maximal fitness. For moderate selection, it takes appreciable values for r = 0 - 4: the binding site sequences are fuzzy. Assuming that the CRP sites at different positions in the genome of E. coli have to a certain extent evolved independently, we can fit Pstat(r) with their distance distribution (data taken from [ε and ρon chosen, the two distributions fit well, see fig. The stationary distribution ken from ). At thekBT [E1/kBT = εr1 and E2/kBT = εr2 for the binding of a single factor and  for the simultaneous binding of both factors. The cooperativity gain is assumed to result from mutations at  positions in the DNA sequences of the factors, which encode the amino acids at the protein-protein contact points. These mutations define a Hamming distance  from the target sequence for optimal protein-protein binding, and 2γε/ is the binding energy per nucleotide. Here we use the values ε = 2,  = 6 and γ = 1 but the qualitative patterns shown below are rather robust.The cooperative binding of transcription factors involves protein-protein interactions which may be specific to the DNA substrate. These interactions often do not require conformational changes of either protein involved and depend only on few specific contact points. They result in a modest energy gain of order 3 – 4kBT . Hence, The resulting equilibrium probabilities for the four thermodynamic states (--) (both factors unbound), (+-) and (-+) (one factor bound), and (++) (both factors bound) areq--,q+- = q-- exp[-ε(r1 - ρ1)],q+- = q-- exp[-ε(r2 - ρ2)],     (14)q++ = q-- exp[-ε(r1 + r2 - ρ1 - ρ2 - 2γ)],q-- + q+- + q-+ + q++ = 1. The scaled chemical potentials ρ1 and ρ2 are independent variables if the two sites bind to different kinds of factors and are equal if they bind to the same kind. As before, the binding probabilities determine expression levels and, therefore, the fitness. Here we study only pairs of sites contributing additively to the expression level in each cellular state, where we havewith the normalisation lac operon [q+-. The stochastic dynamics of substitutions is straightforward to generalise; it leads to a Master equation like (6) for the joint distribution P(r1, r2, , t). This higher-dimensional equation can again be solved exactly for its steady stateOther important cases include activator-repressor site pairs such as the famous c operon , where tPstat(r1, r2, ) ~ exp[S(r1) + S(r2) + S + 2NF(r1, r2, )].     (16)genetic switch with a sharp expression threshold is favoured in a system with a single transcription factor having similar concentrations in its on and off cellular state. As can be seen from eq. (14), cooperative binding can sharpen the response of the binding probability to variations in factor concentration, q++ ~ 1/[1 + exp(-2ερ + ...)] versus p ~ 1/[1 + exp(-ερ + ...)] as given by (1) for individual binding. Figs. F(r1, r2, γ) obtained from (14) and (15) for ρon = 2.5, ρoff = 1.5, and s = son = -soff. A simple signal integration module responds to two different factors in four different cellular states, (on, on), (on, off), (off, on), (off, off). Individually weak but cooperative binding leads to expression of the gene only if both factors are present simultaneously. This case is favoured by a fitness function of the form (15) with selection coefficients s = -soff,off = -son,off = -soff,on = son,on/2. The resulting fitness landscape F(r1, r2, γ) is shown in figs. ρon = 3, ρoff = 1 (for each factor).Here we discuss two simple examples of fitness landscapes where binding cooperativity evolves by adaptation to specific functional demands. A r1, r2 = 3 - 4) and cooperativity (γ = 1) is seen to have a higher fitness than an optimal pair (r1 = r2 = 2) without cooperativity, as expected. Adaptive pathways  and  for strong selection (sN = 100) are shown in fig. Ts are again a few times 1/(sμN). A closer look reveals that this fast adaptation sometimes leads to a metastable local fitness maximum with some degree of cooperativity. Compensatory mutations (see below) are then required to reach the global maximum, a process that may be considerably slower. The fuzziness δr1,2(t) and δγ(t) observed in fig. In both cases, a pair of sites with weaker individual binding  generations, where Δr is the number of adaptive substitutions required. This number is given by the Hamming distance between the onset of selection and the point of optimal fitness, Δr = rs - rm, and takes values 2 – 3 for typical fitness landscapes; see fig. Drosophila melanogaster, with μ ≈ 2 × 10-9 [N ≈ 106, the resulting Ts is of the order of 106 generations or 105 years even for sites with a relatively small selection coefficient s = 10-3. Such selective processes are faster than neutral evolution by a factor of about 1000 and would allow for independent generation of sites even after the split from its closest relative Drosophila simulans about 2.5 × 106 years ago. Notice that new sites are more readily generated in large populations. As discussed above, generating a new site may also require a neutral waiting time to T0 until at least one candidate site in the promoter region of the gene in question reaches the threshold distance rs from the target sequence, where selection sets in. For site formation to be efficient, however, selection must be able to set in spontaneously, i.e., T0 must not greatly exceed the adaptive time Ts. This places a bound on the relevant length  of the binding motif that can readily form in a promoter region of length L. Given L ≈ 300, for example, a motif with  = 8 and rs = 3 could still allow for spontaneous adaptive site formation. (For longer motifs, corresponding to groups of sites with fixed relative distance, this pathway would require promoter regions of much larger L.) A more general case has recently been treated numerically in [G/C ratio of the initial sequence has been investigated. One may speculate that this adaptive dynamics is indeed one of the factors influencing the length of regulatory modules in higher eukaryotes. Clearly, the present model also allows for pathways of negative selection leading to the elimination of spurious binding sites in regulatory or non-regulatory DNA where the binding has an adverse fitness effect. This is important since under neutral evolution, candidate sites with a distance of at most rs from the target sequence occur frequently on a genome-wide scale. A recent study has indeed found evidence for such negative selection from the underrepresentation of binding site motifs over the entire genome [Despite this simplicity, the evolutionary dynamics of binding sites is far from trivial, since it is governed, in the generic case, by the interplay of three evolutionary forces: selection, mutation, and genetic drift. Here we have focused on the dynamical regime appropriate for eukaryotes, where the evolution can be approximated as a stochastic process of substitutions. We find the possibility of selective pathways generating a new site in response to a newly arising selection pressure, starting from a neutrally evolved initial state and progressing by point substitutions. Such a selective formation takes roughly 2 × 10-9  and N ≈ cally in , where te genome .i ≠ j and can be computed exactly from the joint distribution (11). We emphasize that these correlations refer to an ensemble of independently evolving (monomorphic) populations and are not to be confused with linkage disequilibria within one population. This finding limits the accuracy of bioinformatic weight matrices, which are often assumed to factorize in the nucleotide positions even in the presence of selection.We have shown that under stationary selection the frequencies of nucleotides at any two positions of the binding sequence are correlated. For the two-state model, the correlations are the same for any pair of positions μN ≪ 1), populations should be monomorphic at most positions of their binding site sequences and polymorphic at a few. On the other hand, the quasispecies model discussed in refs. [μN ≫ 1) may be most appropriate in viral systems. The intermediate regime μN ~ 1 with frequent polymorphisms and genetic drift could be realized in some bacterial systems and presents a challenge for theory. Thus it would be very interesting to compare the statistics of single-nucleotide polymorphisms at binding sites in eukaryotes, bacteria, and viruses. Polymorphism data are expected to contain evidence for adaptive evolution. However, statistical tests of selection must be modified for promoter sequences [The predictions of our model lend themselves to a number of experimental tests. In the dynamical regime appropriate for eukaryotes  However, the data are also compatible with strong selection if the selection coefficients sα, and hence the value of rm, vary between different genes. Clearly, comparing Pstat with the distribution of sites in a single genome requires the assumption that the evolutionary histories of sites at different positions are at least to some extent independent. Future data of orthologous sites in a sufficient number of species will be more informative. Thus, further experimental evidence is needed to clarify the role of mutational entropy in the observed fuzziness.For weaker selection, site fuzziness increases further since of refs. ,11.  and the coupled evolution of binding sites in different genes.The present work was aimed at obtaining some insight into the molecular mechanisms and constraints underlying the dynamics of complex regulatory networks, thereby quantifying the notion of their Ts ~ 1/(sμN) much smaller than T0 ~ 1/μ, the time scale of neutral evolution. The presence of these two time scales has a further interesting consequence. If the selection pressure on an existing site ceases, that site will disappear on the larger time scale T0. It is possible, therefore, that large existing networks have accumulated a considerable number of redundant regulatory interactions acquired by selection in their past. This may be one factor contributing to their robustness against perturbations.The rapid evolution of networks hinges upon the existence of adaptive pathways for these formative steps with a characteristic time scale T0, we study the mutation dynamics in the restricted range r = rs + 1,...,, allowing mutations from rs + 1 to rs but suppressing mutations from rs back to rs + 1. We evaluate the time-dependent solution P(r, t) of the Master equation (6) with the initial condition P(r, 0) = Pstat(r), and the resulting cumulative probability . The current across the lower boundary, J(t) = μ(rs + 1)P(rs + 1,t) = -dQ/dt, determines the waiting time for a single site,To estimate the average neutral waiting time r ≥ rs. Hence, P(r,t) ≈ Pstat(r) exp(-λt) with λ = J(0)/Q(0) = μ(rs + 1)Pstat(rs + 1)/Qstat(rs + 1). We conclude that the waiting time for a single site is positive with probability Qstat(rs + 1), following a distribution ~exp(-λt), and 0 otherwise. The resulting expectation value is T0 = Qstat(rs + 1)/λ. For L1 independent sites, the distribution of positive waiting times is still exponential, and to is given by an expression of the form (17) with a total boundary current . This yields  as given by (13). The average waiting time (in units of 1/μ) becomes large for values of rs in the tail of the distribution , where . This is the case for .This is formally solved by expanding in eigenfunctions of the mutation operator. In the case relevant here, the system remains close to equilibrium since the boundary current is much smaller than typical currents for JB carried out analytical and numerical work, SW performed numerical work and data processing. ML conceived of the study, carried out analytical work, and coordinated the project. All authors read and approved the final manuscript.


Ultrasounds and ionizing radiation are extensively used for diagnostic applications in the cardiology clinical practice. This paper reviewed the available information on occupational risk of the cardiologists who perform, every day, cardiac imaging procedures. At the moment, there are no consistent evidence that exposure to medical ultrasound is capable of inducing genetic effects, and representing a serious health hazard for clinical staff. In contrast, exposure to ionizing radiation may result in adverse health effect on clinical cardiologists. Although the current risk estimates are clouded by approximations and extrapolations, most data from cytogenetic studies have reported a detrimental effect on somatic DNA of professionally exposed personnel to chronic low doses of ionizing radiation. Since interventional cardiologists and electro-physiologists have the highest radiation exposure among health professionals, a major awareness is crucial for improving occupational protection. Furthermore, the use of a biological dosimeter could be a reliable tool for the risk quantification on an individual basis. Over the last 30 years, medical cardiology imaging has rapidly grown, becoming an essential part of the cardiology clinical practice. Imaging procedures include conventional imaging tests such as echocardiography, radionuclide imaging, and angiography as well as a newer imaging techniques such as emission computed tomography and magnetic resonance imaging which promise to expand diagnostic capabilities . These tMany cardiac procedures can deliver high radiation doses to the clinical staff . This exin vitro and in vivo. In the same study, no increase in chromosomal aberrations was observed during and after ultrasound therapy [Ultrasound imaging, also called sonography, is a method of obtaining human body images through the use of high frequency sound waves. Ultrasounds are mechanical vibrations with frequencies above the human limit of audibility. The use of ultrasounds in order to obtain images for medical diagnostic purposes, typically employs frequencies ranging from 2 MHz to about 12 MHz . Ultraso therapy . SubsequIndeed, ultrasound sources do not transmit acoustic energy into air, and only low level ultrasound reaches medical personnel through handling of the probe . Probablerythema or cataract, have a threshold dose below which the biological response is not observed [Ionizing radiation is known to cause harm. High radiation doses tend to kill cells, while low doses tend to damage or alter the genetic code (DNA) of irradiated cells. The biological effects of ionizing radiation are divided into two categories: deterministic and stochastic effects. Deterministic effects, such as observed -25. Someobserved ,27.A stochastic effect is a probabilistic event and there is no known threshold dose. The likelihood of inducing the effect, but not the severity, increases in relation to dose and may differ among individuals.In fact, the effect of low doses of radiation -less than 50 mSv- do not cause an immediate problem to any body organ, but spread out over long periods of time after exposure. The biological effects are at DNA level and they may not be detected -25. The Genetic effects are the result of a mutation produced in the reproductive cells of an exposed individual that are passed on to their offspring. These effects may show up as birth defects or other conditions in the future children of the exposed individual and succeeding generation. Indeed, studies with laboratory animals have provided a large body of data on radiation-induced genetic effects . RecentlThe use of radiation in medicine is the largest source of man-made radiation exposure. According to the latest estimation of the United Nations, an average of 2.4 mSv/year comes from natural sources . In westCardiac ionizing procedures expose both patients and medical staff to the highest radiation levels in diagnostic radiology, and recently, as the number of diagnostic and interventional cardiac catheterisation procedures has greatly increased, serious radiation induced skin injuries and an excess of cataract development have been reported in exposed staff -39. FurtToday, interventional cardiologists represent, indeed, the most important group of exposed among professionally exposed physicians ,42.As known, the limit on effective dose for exposed workers should be 100 mSv in a consecutive five year period, subject to a maximum effective dose of 50 mSv in any single year. Radiation dose limits to adult occupational workers provided by the International Commission on Radiological Protection (ICRP) are shown in table As a matter of fact, the head dose sustained by cardiologists may reach 60 mSv per year, and may in some cases exceed the occupational limit of 150 mSv per year recommended for the lens of the eye .However, the correlation between occupational doses and staff radiological risks is not simple, and it is very dependent on equipment, the specialist, and protocols followed throughout the procedure . Many faImportantly, a recent survey showed that that most of cardiologists do not correctly evaluate the dose exposure, the medico-legal regulation, the environmental impact and individual bio-risks of the radiological investigations . As showProbably, this unawareness has its root in the difficult perception of a long-term risk associated to radiation exposure. In particular, the perception of cancer risk, which can have a latency period of many years after exposure, is often elusive. Furthermore, the exact risk at very low doses to a specific individual is further complicated by many factors, such as carcinogenic agents in our environment, cigarette smoke, diet and genetic background.However, a recent study has estimated that from 0.6% to 3% of all cancers are due to medical X-rays . These fWith regard to occupational exposure for radiologists and radiotherapists, available epidemiological studies have been recently reviewed by Yoshinaga et al . An excebiomarkers as predictors of delayed health outcomes [To the fatal cancer risk, one must add the risk of non-fatal cancer and major genetic damage transmitted to the offspring. It is relevant to underline that the long-term damage may not include only cancer but also other major degenerative diseases, including atherosclerosis ,48. Howeoutcomes .Damage to deoxyribonucleic acid (DNA), which carries the genetic information in chromosomes in the cell nucleus, is considered to be the main initiating event by which radiation damage to cells results in the development of cancer and hereditary disease. Four biomarkers Figure  -analysiDuring the last years, the micronucleus assay has become popular since it is fast and inexpensive, and it is considered to be a "biological dosimeter" for exposure to ionizing radiation .The importance of cytogenetic study of peripheral lymphocytes in subjects exposed to ionizing radiation has been reported for more than 20 years, especially in radiologists -68. The As matter of fact, our results and a recent monitoring of personnel working in angiocardiography laboratories in Iranian Hospitals showed a high frequency of chromosome aberrations in cardiologists s and technicians compared to unexposed subjects ,69.Taken together, these evidences highlight that the use of a biological dosimeter could complement the data obtained by physical dosimetry and reduce the uncertainties of low-dose radiation risk assessment . The anaMoreover, the method is laborious, time consuming and requires expert skills. Scoring of micronuclei has been proposed as an alternative to conventional chromosome aberrations analysis, being more sensitive and faster . AlthougHowever, in many countries the application of cytogenetic dosimetry has yet medical-legal recognition, and it is complementary to physical dosimetry. On the other hand, the usefulness of biomarkers as early biological effects, with special concern for the prediction of cancer, has been recently emphasized . TherefoOccupational exposure can occur in cardiological procedures which employ ultrasound and ionizing radiation. Today, there are no consistent adverse biological effects on operators caused by exposures to ultrasound. However, it is clearly necessary to continually monitor both the potential risks and safety of ultrasound exposure. In contrast, exposure to ionizing radiation may result in adverse health effect on both cardiologists directly and on their progeny. Although the current risk estimates are clouded by approximations and extrapolations, most data from cytogenetic studies have reported an enhanced DNA damage in hospital workers exposed to chronic low doses of ionizing radiation. The occupational dose of interventional cardiologists, and electrophysiologists tend to be higher compared to other medical specialists as a result of the recent increasing use of interventional techniques. On the other hand, physicians are dramatically unaware of dose, long-term risks and populations health impact caused by the use of medical ionizing radiation. Thus, a major awareness appears to be crucial in order to improve both one's knowledge on the appropriateness of protective tools and also in trying to reduce the number of unnecessary procedures. The use of a biological dosimeter could be a reliable tool for risk quantification on an individual basis.


Though such partnerships create a powerful mechanism for addressing difficult problems by leveraging on the strengths of different partners, they also package complex ethical and process-related challenges. The complex transnational nature of some of these partnership arrangements necessitates that they be guided by a set of global principles and norms. Participation of international agencies warrants that they be set within a comprehensive policy and operational framework within the organizational mandate and involvement of countries requires legislative authorization, within the framework of which, procedural and process related guidelines need to be developed. This paper outlines key ethical and procedural issues inherent to different types of public-private arrangements and issues a Global Call to Action.The need for public-private partnerships arose against the backdrop of inadequacies on the part of the public sector to provide public good on their own, in an efficient and effective manner, owing to lack of resources and management issues. These considerations led to the evolution of a range of interface arrangements that brought together organizations with the mandate to offer public good on one hand, and those that could facilitate this goal though the provision of resources, technical expertise or outreach, on the other. The former category includes of governments and intergovernmental agencies and the latter, the  Public-private partnerships are being increasingly encouraged as part of the comprehensive development framework. The need to foster such arrangements is supported by a clear understanding of the public sectors inability to provide public goods entirely on their own, in an efficient, effective and equitable manner because of lack of resources and management issues. These considerations have necessitated the development of different interface arrangements, which involve the interfacing of organizations that have the mandate to offer public good on one hand, and those that could facilitate this goal.Within the health sector, public-private partnerships are also the subject of intensely fueled debate . SeveralThis paper focuses on public-private partnerships that are intended to address broad questions of providing sustainable health outcomes rather than on the day-to-day interaction that occurs when the government buys a health service from a private supplier or where it leaves the entire matter of health service supply to the private sector.public sector in this paper refers to national, provincial/state and district governments; municipal administrators, local government institutions, all other government and inter-governmental agencies with the mandate of delivering 'public goods'. The word private denotes two sets of structures; the for-profit private encompassing commercial enterprises of any size and the non-profit private referring to Non Governmental Organizations (NGOs), philanthropies and other not-for-profits. The word partnership in this paper refers to long term, task oriented, and formal relationships. There has been ample critique relating to the convention of using the word partnership to describe such arrangements; much of this debate is valid, given that there are certain requisites for coining such an association. For the same reasons it also needs to be differentiated from privatization, which involves permanent transfer of control through transfer of ownership right or an arrangement in which the pubic sector shareholder has waived its right to subscribe. A distinction also needs to be made between partnerships and contractual arrangements, particularly with regard to the relationship between the public sector and NGOs. Although such arrangements can be used for strategic purposes, they are inherently distinct from partnerships.The Initiative on Public-Private Partnerships for Health of the Global Forum for Health Research lists 91 international partnership arrangements in the health sector, which can qualify to be called public-private partnerships. Of these, 76 are dedicated to infectious disease prevention and control, notably AIDS, tuberculosis and malaria; four focus on reproductive health issues, three on nutritional deficiencies whereas a minority focus on other issues [the database of the ract {4}). This capublic and private sectors, it can be stated that public-private arrangements are fostered either when governments and inter-governmental agencies interface with the for-profit private sector to tap into resources, or the non-profit private sector for technical expertise or outreach. Several varieties of arrangements of various sizes, forms and scope at a global, regional or country level qualify to fall within this categorization. Transnational partnerships involving a visible role of the for-profit sector are at one end of the spectrum. These usually involve larger partnerships and a complex grouping; depending upon their structure, they may bring together several governments, local and international NGOs, research institutions and UN agencies in transnational programs, often also involving the non-profit sector. Such partnerships can be housed and coordinated by different sources [Several classifications have been proposed to conceptualize and categorize public-private partnerships. These may be based on the terms of the constituent membership or the nature of activity ,5. Howev sources . They ca sources , Roll Ba sources , Stop TB sources , Safe In sources , Global  sources , the Spe sources , and the sources . Partner sources , and can sources , Medicin sources , Global  sources , and the sources . Large p sources , the Mec sources , and the sources .At the other end of the spectrum, there are examples of individual governments forming partnerships with the for-profit private sector . There aPartnerships in the health sector can be for various purposes; categories as stated by the Initiative on Public-Private Partnerships for Health have been summarized in Table In many countries, there are long established links of the public sector with NGOs. Theoretically, since NGOs are not driven by a profit generating motive, many of the ethical challenges that potentially exist in partnering with the for-profit are not of relevance in this case. However, it could also plausibly be argued that NGOs, who though objective and altruistic, may, in fact, have quite complex motives. In promoting public-private partnerships therefore, several issues need to be clearly flagged in an attempt to address them in tandem with efforts that aim to foster such relationships. Within that context, a set of ethical and process related challenges are summarized hereunder:1. Global norms and principals: many of the large partnerships involving a variety of players are of a transnational nature. However, against this backdrop, there are no global norms and principals, to set a framework within which global public health goals can be pursued in a partnership arrangement.2. Impartiality in health: if public-private partnerships are not carefully designed, there is a danger that they may reorient the mission of the public sector, interfere with organizational priorities, and weaken their capacity to uphold norms and regulations. Such a shift is likely to displace the focus from the marginalized and may therefore be in conflict with the fundamental concept of equity in health.3. Social safety nets: it has been increasingly argued that engaging in a partnership mode provides the public sector an opportunity to renounce their responsibilities; this in a sense may lead to withdrawal of social safety nets. Failure to commit to maintain the role of the state in such partnerships may result in a laissez-faire attitude, prejudicial to the interest of the most vulnerable groups.4. Conflict of interest: many partnerships are initiated on the premise that they fulfill a social obligation, and can involve good intentions on part of individuals and organizations. However the basic motive that drives the 'for-profit' sector demands that these involve a financial pay off in the long term. In such cases, the difference between corporate sponsorships and scientific philanthropic donations with long term visible public health goals needs to be clearly separated. This issue has been further complicated in recent years as many global health initiatives funded by endowments generated by foundations have partnerships with the private sector as a key feature [ feature . Such do5. Redirecting national health polices: there are also concerns that such partnerships redirect national and international health polices and priorities and have the potential to defeat crucial local and national efforts.6. Fragmentation of the health system: partnerships generally tend to aim for short term high profile goals and tend to pick the lowest lying fruits. Partnerships do have the mandate and cannot be held accountable to synchronize their activities with emerging processes within countries aimed at developing their health systems. Therefore if they are instituted in countries with weak health systems they have the potential to fragment the healthcare system by instituting independent vertical programs. The changing global agenda around 'vaccines' helps to highlight many of these issues. Previously polices around vaccination were grounded in the general principal that promoted equitable access to few vaccines around the world. However new initiatives and their vertical systems have less of a focus on sustainability, may not contribute to strengthening of the health system and have the potential of redirecting national health policies, which focus on equitable care [ble care .7. Contribution to common goals and objectives: it is common for partners to have different objectives while pursuing a relationship though it may be implicit that partnerships are contributing to common goals.8. Lack of outcome orientation: many a times, partnerships exist in form and do not contribute to improvements in quality and efficiency.1. Legislative frameworks, polices and operational strategies: many developed countries have legislation to interface with the private sector [ad hoc and opportunistic basis and may have questionable credibility; as a results of this failure, polices and specific operational strategies fail to develop.e sector . However2. Participatory approach to decision making: the expression 'partnership' gives the impression of equality. However many a times, a participatory approach to the decision making process is usually not optimally accomplished. This has implications of varying degrees. Almost all the large 91 transnational partnerships referred to earlier are focused on the developing world. However, among these, 85 have their secretariats in Europe and North America; the United States and Switzerland being the commonest host countries. Cleary this lack of proximity to the intended beneficiaries has a bearing on the manner in which the beneficiaries have a role in the decision making process [ process . The dec3. Governance structures: workable partnerships require a well-defined governance structure to be established to allow for distribution of responsibilities to all the players. Public-private partnerships may run into problems because of ill-defined governance mechanisms. Recent evaluation of the RBM project while acknowledging the successes of the partnership in drawing global attention to the scale of the problem posed by Malaria has outlined serious governance-related issues [d issues . More red issues .4. Power Relationships: skewed power relationships are a major impediment to the development of successful relationships. Governments in developing countries usually tend to assume core responsibility of the joint initiative and take charge of the weaker partner. In case of NGOs with outreach-related strengths, this usually takes the form of a 'contractual relationship' without much regard to the participatory processes, which should be key to a public-private partnership arrangement. In case of relationships with NGOs with technical strength, there are issues relating to power relationships of a more serious nature with regard to who assumes the leadership role.9. Criteria for selection: the criteria for selection are an important issue both from an ethical and process-related perspective as it raises the questions of competence and appropriateness. In many instances the public sector is vague about important issues related to screening potential corporate partners and those in the non-profit sector.10. Sustainability: the question of long-term sustainability is often ignored in public-private partnerships. An analysis of the operation of GAVI has concluded that it overemphasizes high technology vaccines, lacks sustainability, relies too heavily on the private sector and consequently, runs the risk of compounding health inequities in the poorest countries [ountries .11. Accountability: many partnerships do not ensure that all players are held accountable for the delivery of efficient, effective and equitable services in a partnership arrangement. Often in public-private relationships it is unclear as to whom are these partnerships accountable to, according to what criteria, and who sets priorities? To hold partners accountable for their actions, it is imperative to have clear governance mechanisms and clarify partner's rights and obligations. Clarity in such relationships is needed in order to avoid ambiguities that lead to break up of partnerships. A case in point is the recent breakup of GAEL with the exit of the International Association of Anti-leprosy [-leprosy .In the world we live today, global agendas are being increasingly shaped by the private sector. The 'for-profit' private sectors' immense resources make it an irresistible partner for public health initiatives. These arrangements can also be mutually synergistic. Governments and international agencies can tap into additional resources to full fill their mandate whereas the commercial sector can fulfill its social responsibility, for which it is being increasingly challenged. Additionally, the recent SARS epidemic and bio-terrorist threats should help to make the private sector understand the value of investment in health for reasons beyond fulfilling their social obligations. Active involvement of the 'non-profit' sector and donor coordination in country goals is also being increasingly encouraged within comprehensive development frameworks; this approach is synchronous and in harmony with the Poverty Reduction Strategy Paper Framework .The development and health actors have highlighted the need to harness the potential that exists in collaborating with the private sector to advance public health goals. This is also becoming increasingly essential as both the public and the private sector recognize their individual inabilities to address emerging public health issues that continue to be tabled on the international and within country policy agendas. Public-private partnerships therefore seem both, unavoidable and imperative. However in building such collaborations, certain measures must be taken at a global level to assist global partnerships and set a framework within which efforts at a country level can emanate.As a first step, there is a need to develop a set of global norms and ethical principles; a broad-based agreement over these must be achieved. The transnational nature and global outlook of emerging partnerships necessitate that these stem from a broad-based international dialogue.It is critical that the driving principles for such initiatives be rooted in 'benefit to the society' rather than 'mutual benefit to the partners' and should center on the concept of equity in health. Norms must stipulate that partnerships contribute to strengthening of social safety nets in disadvantaged settings and should be set within the context of 'social responsibility' as the idea is not meant for private funds to be put to public use nor to privatize public responsibilities.outcome, but a process and an output; it is important for partnerships not to just exist in form but to contribute to improvements in health outcomes.Global principles must specify that partnerships should be in harmony with national health priorities; they should complement and not duplicate state initiatives and should be optimally integrated with national health systems without any conflict of interest. Norms must make it mandatory for all partners in a 'partnership' arrangement to contribute to common goals as a true partnership is one in which the partners, though having different motivation and values have a shared objective. Global norms must outline that partners be committed to making contributions, sharing risks and the decision making process. Principles should emphasize an outcome orientation. Development of a public-private partnership in itself should not be seen as an It must be made binding for international agencies to develop transparent policy and procedural frameworks. Many international agencies have established guidelines on interacting with the private sector -45. Howewithin countries [Global efforts should demand, encourage and assist the development of policy and legislative frameworks shaping public-private partnerships ountries . HoweverWithin stipulated legislative and policy frameworks, support must be provided to developing countries to develop specific guidelines to steer such relationships. Guidelines can assist with the development of selection criteria and help specify roles of the public and the private sectors. They can also assist with the development of models that outline combined governance structures, clearly aimed at improved systems of governance. Guidelines must also articulate a clear policy on a participatory approach to the decision making process. In addition, they should assist with assigning responsibilities to various levels of Government and then hold people and institutions both within Governments and those in the private sector that partner with them accountable for their performance.Though an evidence-based approach and ethical considerations must never be compromised in such endeavors and every effort should be made to ensure that goals are mutually compatible, guidelines also need to be flexible in order to accommodate each partner's organizational requirements and integrity. Moreover they need to be pragmatic. The public sector needs to recognize the basic legitimacy of the private sector and the profit motive that drives it. It is also essential for the public sector to respect the organizational autonomy and priorities of the non-profit sector. In this context, partnerships and contractual relationships need to be carefully differentiated.Partnerships must also be the subject of noteworthy empirical research, which would enable a detailed assessment of the specific issues inherent to the various types of public-private partnership arrangements from an ethical and methodological perspective.The impetus for driving global and national efforts in creating a transparent and conducive environment for public-private partnerships needs to come from the public sector. This raises the issue of capacity within countries; the gap needs to be bridged by assistance from UN agencies, which have the mandate of harnessing and coordinating support among a variety of players for global actions. However, the results of such actions will only be as good as Governments make them; weak and poorly informed Governments cannot remedy their own deficiencies by seeking to yolk the private sector to their own uncertain cart.In conceptualizing a framework that assists with setting global norms and guidelines and within-country legislative actions, it needs to be recognized that the dynamics of public-private partnership arrangements are generic across social sector. It may therefore be useful to allow this commonality to prevail in initiating global and country-specific actions.


Xenopus frog embryo that could induce the differentiation of a whole new axis, including a new central nervous system, when grafted into an abnormal location. (Gastrulation is the process that establishes the basic body plan of the organism as cells arrange themselves into three embryonic germ layers: the endoderm, mesoderm, and ectoderm.) From these and later experiments, the notion emerged that neural induction in Xenopus takes place at gastrulation and requires signals from the mesoderm. (The Spemann–Mangold organizer is itself derived from the endomesoderm.)Formation of the central nervous system has long been thought to result from an induction process, whereby signals emanating from a portion of the dorsal endomesoderm (the inner middle layer of the developing embryo), known as the Spemann–Mangold organizer, instruct cells of the overlying dorsal ectoderm (outer layer) to become neural instead of epidermal. The Spemann–Mangold organizer was itself defined in Spemann and Mangold's seminal 1924 publication as a portion of the dorsal “vegetal” half  of a gastrulating Now Hiroki Kuroda, Oliver Wessely, and Edward De Robertis challenge this model by demonstrating that a group of cells in the dorsal region of the prospective ectoderm is fated to become neuronal as early as the blastula stage (which precedes gastrulation) and that these cells can express their neural character in the absence of any mesodermal influence. The authors call this group of cells the BCNE (blastula Chordin- and Noggin-expressing) center, based on their previous observation that this center expresses the proteins Chordin and Noggin at the blastula stage. Chordin and Noggin are also expressed later in the Spemann–Mangold organizer and are among the key signals that mediate neural induction by the organizer. The presence of the neural inducers in blastula ectodermal precursor cells prompted the authors to test these cells' neural potential. They first demonstrated that BCNE cells normally give rise to the anterior portion of the brain, which confirms these cells' neural fate. Moreover, when cultured in vitro, BCNE cells taken from tissue begin to express neural protein markers, even when extra care is taken to prevent any contact with mesodermal precursors. It therefore appears that BCNE cells are already specified to become neural by the blastula stage, before the Spemann–Mangold organizer forms.To further demonstrate BCNE cells' independence from mesodermal signals, the authors generate embryos without a mesoderm. Having previously observed that such embryos do develop a central nervous system, Kuroda et al. now demonstrate that this intrinsic neuronal potential depends on Chordin and Noggin expression in BCNE cells. The model that emerges from these experiments suggests that neural induction begins at the blastula stage, with Chordin and Noggin signaling within the BCNE center and may later be consolidated or modulated by signals emanating from the organizer.What of the endodermal portion of the Spemann–Mangold organizer? It expresses a secreted protein called Cerberus that is involved in development of the head. The authors show that abolishing Cerberus function in the prospective endoderm results in headless embryos. Complete brain removal can also be achieved by partially inhibiting Cerberus function, so long as Chordin is simultaneously inhibited in the dorsal ectoderm. It is therefore likely that while BCNE cells harbor an intrinsic neural potential, neural induction in a living organism occurs via cooperation between the germ layers.