Health policy and systems research (HPSR) is an international public good with potential to orient investments and performance at national level. Identifying research trends and priorities at international level is therefore important. This paper offers a conceptual framework and defines the HPSR portfolio as a set of research projects under implementation. The research portfolio is influenced by factors external to the research system as well as internal to it. These last include the capacity of research institutions, the momentum of research programs, funding opportunities and the influence of stakeholder priorities and public opinion. These dimensions can vary in their degree of coordination, leading to a complementary or a fragmented research portfolio.The main objective is to identify the themes currently being pursued in the research portfolio and agendas within developing countries and to quantify their frequency in an effort to identify current research topics and their underlying influences.HPSR topics being pursued by developing country producer institutions and their perceived priorities were identified through a survey between 2000 and 2002. The response to a call for letters of intent issued by the Alliance in 2000 for a broad range of topics was also analyzed. The institutions that were the universe of this study consisted of the 176 institutional partners of the Alliance for Health Policy and Systems Research producing research in low and middle income countries outside Europe. HPSR topics as well as the beneficiaries or issues and the health problems addressed were content analyzed. Topics were classified into 19 categories and their frequency analyzed across groups of countries with similar per capita income. Agendas were identified by analyzing the source of funding and of project initiation for projects under implementation.The highest ranking topic at the aggregate level is "Sector analysis", followed by "Disease burden" and "Management and organization". Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". "Disease burden" is more often funded than other topics for which there is more demand or perceived priority. Analysis suggests few although important differences across priorities, demand for funding and actual project funding. The donors' agenda coincides most with the ranking of research topics overall.Ranking across country income groups shows important differences. Topics that gain prominence in low income countries are "Disease burden" and "Accessibility". In lower middle income countries "Insurance" gains prominence. In upper middle income countries "Decentralization/local health systems", "Equity" and "Policy process" are more prominent. "Program evaluation" is the most consistently ranked topic across income regions, showing a neutral influence by donors, governments or researchers.The framework proposed offers a basis to identify and contrast research needs, projects and products at the international level and to identify the actor agendas and their influence. Research gaps are suggested when comparing topic ranking against the challenges to health system strengthening and scaling up of disease control programs. Differences across per capita income groups suggests the need for differentiated priority setting mechanisms guiding international support. Data suggests that stakeholders have different agendas, and that donors predominate in determining the research portfolio. High-level consensus building at the national and international levels is necessary to ensure that the diverse agendas play a complementary role in support of health system objectives.The Ministerial Summit for Health Research to be held in Mexico in November 2004 should be an opportunity to analyze further data and to commit funding for priorities identified through sharing and discussion of agendas. Countries and international agencies have made a qualitative leap in the funding of the global disease challenges. The Global Fund for AIDS, TB and Malaria has received pledges totalling over US$ 2 billion. Bilateral donors are also making important funding contributions. In this context, strengthening of health systems has become a critical issue. Research can play a major role to identify the best policies to channel massive efforts, to ensure that vertical approaches do not fragment fragile health systems and to monitor and evaluate progress. How relevant is the research effort being undertaken in developing countries, and how is the agenda being driven?WHO is organizing the Ministerial Summit on Health Research, to be held in Mexico City, 23 to 26 November, 2004. The main theme will be the role of health research in meeting the Millennium Development Goals. Health policy and systems research (HPSR) will have a prominent role in the context of the scaling up of efforts against major diseases and child and maternal mortality. Looking towards the Summit, WHO established a task force to identify HPSR priorities as an effort to advocate for major funding in this area. The Alliance for Health Policy and Systems Research, an initiative of the Global Forum for Health Research in collaboration with WHO, has been promoting since its inception in 2000 the identification of research priorities among producer institutions in developing countries.This paper proposes a conceptual framework and a methodology to think about the HPSR research portfolio, the agendas that influence it and the priority setting process. The paper makes the case for the formulation of HPSR priorities at national, international and global levels. A previous article  looked aPriority setting efforts are often bogged down because of inadequate methods of categorization of the research that is undertaken and the influences shaping it. These influences, in turn, are often not adequately understood, including the nature and role of priorities. The conceptual framework proposed here strives to offer some simple definitions of the research portfolio and its various influences, as well as indicators to measure and relate these concepts.The nature of overall health research has been defined in terms of basic, applied and strategic research. These categories are useful to guide investment decisions which might maximize benefit ,3. HowevTo identify the object of research the concept of the research portfolio can be useful. The HPSR portfolio can be defined as the current set of research projects on health system structures, functions, processes and results at national, international and global levels. Projects, as distinct from plans or priorities, would include the commitment of resources towards a specific, time-bound aim and a set of objectives.It has been proposed that the research portfolio should be analyzed along three dimensions of strategic importance: where to make investments, that is, the identification of the object of research or thematic areas in which investments are made; the type of investment research and development (R & D) instrument used and the resources spent through each area and type of instrument . While oThe Ad Hoc Committee on Health Research proposed three types of R & D instruments: discovery oriented research to develop new health products and interventions; innovation research to adapt efficacious but unaffordable interventions to make them cost-effective, and implementation R & D to achieve greater efficiency in the use of existing interventions . HarrisoWith regard to funding, there is a need not only to consider investments, but also funding sources and mechanisms. Four broad types of sources can be recognized: bilateral and multilateral donors, government commissioning, private commissioning, and funding through resources available to research institutions as part of their budgets. Each will have different implications for the kind of knowledge produced and for its possible influence on the health system . This suPrevious analyses have revealed a complex heterogeneity along which researchers classify HPSR in developing countries, which is not surprising in an interdisciplinary field . However• concepts reflecting the health system, such as policy and financial structures, regulatory functions, processes such as technology evaluation and quality monitoring, and results such as satisfaction and health gain• the levels of the health system, such as the households and the community, first level facilities and hospitals• the issues or problems pertaining to the health system such as priorities, equity and the public private mix• the populations addressed by the system, such as children, mothers and the elderly, or rural and urban populations• the health needs addressed, whether in terms of risks or disease.While these dimensions can be useful to characterize the research portfolio, it is clear that there will be overlaps; for example, equity is both an issue and an attribute of the health system, particularly if it has been integrated in monitoring and regulation. In order to make use of these dimensions it is proposed to consider as the project topic the first dimension of concepts pertaining to the health system structures, functions, processes and results. The topic could then be classified following normative or theoretical frameworks or by using the categories researchers apply in their own research. The other four dimensions can be used to qualify the research topic as to provide a more detailed description. These four dimensions could be selectively used or aggregated to facilitate description according to the needs at hand.Each of the five project dimensions can be analyzed in terms of the range of items considered. The ranking and emphasis of each item can also be revealed by analyzing its frequency. A research portfolio at any level can be very focused and comprise a narrow set of topics. Or it can be wide-ranging across many health system functions, structures, processes and results. It has been argued that research portfolios should be more focused on improving immediate health problems through operations research in low income countries, where funding and human resources are very limited . HoweverThe level at which the portfolio is analyzed is important, as it will have different characteristics and uses. At national level, the HPSR portfolio would be the set of projects addressing health and health system problems within the confines of national borders and governmental authority as well as sector-wide and inter-sectoral issues. Examples would be the impact on equity of decentralization policies, or the roles played by conflicting policy actors in scaling up of services. The international research portfolio would be topic areas which are common across a number of countries or regions. Identification of the international portfolio could serve, among other purposes, to fund research at regional or international levels, to strengthen the critical mass of research available to inform country policy making and lesson learning across countries, and to extend the range of methodological approaches through comparative research . The HPSThe HPSR portfolio at any level is influenced by factors within and outside the research system . Within It is clear that if actor agendas have few areas in common there will be no consensus and therefore no overall priorities. Significant overlap of interest, on the other hand, can lead to the identification and formulation of shared research priorities. Research priorities are therefore defined as the explicit areas of agreement on, and ranking of, the object of research across diverse actor agendas. Priorities can then become policy instruments to coordinate diverse agendas towards a common end without forcing a single research agenda.The characteristics of agendas and of priorities can be identified through the same kinds of dimensions and indicators as the research portfolio. That is, preferences can be classified in terms of topics, issues or beneficiaries, and health problems. The characteristics of the HPSR agendas and priorities can also be studied in terms of the range of topics, issues, levels, populations and health problems. They can also be ranked and their emphasis revealed by frequency analysis. In this way, the agendas can be compared across themselves, priorities can be identified as common topics and issues and with similar ranking and emphasis, and the influence of agendas and priorities on the actual research portfolio can be assessed.It has been argued that co-ordination of the various influences shaping the HPSR portfolio can increase the impact of research on equity and can contribute to its strategic role for development . Co-ordiHPSR portfolio change through co-ordination would involve a gradual process of adjustment to new priorities, project completions, maturation of research capacity and funding opportunities. Coordination requires interfaces and mechanisms such as Research Forums and Essential National Health Research Mechanisms  to develIn sum, the HPSR portfolio can encompass a differing range of topics with diverse rankings and emphases; can be more or less coherent with respect to researcher, funding and policy maker agendas; and can be more or less co-ordinated along a set of shared priorities. The interplay of these dimensions can give rise to a number of scenarios, of which three are here illustrated.a) Co-ordination could lead to focusing the HPSR portfolio on a few, highly cost-effective topics in a situation of few resources and well identified, high priority needs. There would be, eventually, a high degree of coherence across the portfolio and agendas held by various actors. The risks here would be lack of diversity to foster innovation and healthy criticism.b) In a situation of plentiful resources the research portfolio and its driving agendas could be wide-ranging and have little overlap. Specific portfolio segments would correspond with particular agendas, thus satisfying multiple interests. However, co-ordination through overarching mechanisms could ensure integration of knowledge around high level priorities. In this manner a unified, although highly diverse, HPSR field of enquiry would be obtained.c) In a situation of lack of coordinating mechanisms, with low resources, the portfolio could focus on a reduced set of topics, each satisfying a particular agenda and thus fragmenting resources and hindering support to health system development. If resources are more plentiful, lack of coordination could lead to a rich but highly dispersed and inefficient research portfolio with little impact on development.Irrespective of the availability of resources, co-ordinating mechanisms are likely to be important to ensure an efficient use of research resources.Two sources of information were used to illustrate the conceptual framework proposed. The first was a survey of Alliance-HPSR partner institutions in developing countries detailing research priorities and project information. Researchers reported here on the priorities they had received from policy makers in the course of diverse consultations in the past year. The second source was a database of letters of intent (LOI) submitted to the Alliance for funding, where projects were justified on the basis of priorities negotiated by researchers and policy makers or service managers.This proceeded in several steps. A preliminary list with 24 research topics was identified through an inductive analysis using the research statements expressed in the LOI, which were the most detailed. This list was then used to classify research topics in the projects and priorities expressed in the Alliance partners' profiles. Beneficiaries/issues and diseases/health problems addressed by the LOI, projects and priorities were also categorized and classified at this stage. While projects may have contained more than one topic or beneficiary/issue, the most prominent one was selected. In a few cases where several topics were considered this was indicative of a sector-wide analysis and classified accordingly.The beneficiaries/issues of the research were classified to include any of four alternative dimensions: a) the demographic group: elderly or children/adolescents, b) level of care: community, primary or hospital, c) the geographical focus: urban or rural, and d): gender, equity/poverty, indigenous populations, and public-private mix. Whenever more than one dimension or aspect was applicable , a decision was made to include the most prominent.The concept "equity" was classified both as a topic and as an issue. It was assigned as a topic whenever equity was the main objective of the research and it was addressed through a number of health system attributes such as financing, access, and service delivery. Equity/poverty was considered as an issue when the poor were identified as the main subjects of research or when the equity implications of research directed mainly to another topic were highlighted as a major concern.The distribution of statements was analyzed according to country income group: low income, lower middle income and upper middle income. These income groups correlate highly with geographical regions, with LI being mostly in Africa and Asia, LMI mostly in Asia and with a particular weight by China, and UMI in Latin America and the Caribbean table .Two researchers classified all statements independently and disagreements were discussed and resolved. The 24 topic categories were reduced to 19 to avoid groups with less than 2% of the total number of statements while maintaining topic coherence. Table The frequency of responses by country for all types of statements is generally proportional to country population, with China, India, Brazil and Bangladesh at the top of the frequency. However, countries with a strong health systems research presence are over-represented, such as Colombia, Argentina, Philippines, Thailand, South Africa, Uganda, Ghana, Cuba, Costa Rica, Benin, Jamaica and Tanzania.The range and emphases of the HPSR portfolio and agendas were mapped through topic content and frequency analysis Figure . ProjectTwo methods were used to assess the coherence between the HPSR portfolio and the agendas held by researchers, policy makers and international donors and partners. The first method used the survey data to infer the agendas by observing the topic frequency of: projects proposed and funded by researchers without external assistance; projects initiated and funded exclusively by government, and projects initiated and funded exclusively by international stakeholders or research partners.The second method compared the research portfolio against the agenda expressed by policy makers. The policy maker agenda was observed through the survey as reported by researchers and through the LOI as negotiated with researchers. A negligible influence by the donor, in this case the Alliance-HPSR, would be expected in the LOI given that the call requested priorities within the generic definition of HPSR presented above.Each of the three modes of identification of agendas could have method-specific biases. In the first method, preferences are derived from the portfolio itself, that is, from research projects in implementation. Furthermore, the method isolates the preferences expressed by each actor. As such, this method could be deemed to reveal most objectively preferences behind each actor funding or initiating a project. However, projects under implementation may hide topic preferences that are not translated into projects or topics that were generated and funded through joint actor participation.The observation of negotiated priorities expressed in LOI captures the mix or balance of researcher-side influences and policy maker needs. It will therefore reflect a consensus position across each actor. However, it will exclude the influence of funding opportunities, will not reveal actor-specific preferences and will be limited by the constraints placed on the LOI (see below).Priorities based on consultations between researchers and policy makers and expressed by researchers through a survey will reveal the understanding and conceptual framework of researchers and may underplay policy-maker needs. Furthermore, these priorities will be influenced by the research projects under execution and reported in the same instrument.The analysis of relationships or influences across the portfolio and each of the actor agendas, as well as of similarities or differences between agendas, was undertaken by correlating topic frequencies across lists and by undertaking a qualitative analysis of changes in rank order and emphasis.Analysis of the range and rank of topics across groups of countries by income was undertaken by aggregating project and priority topic data into a reference list representing the combined set of influences on the agenda-setting process, including the portfolio itself. The aggregation of data into a reference list was mainly a strategy to increase the observations and make the analysis more reliable, although it may have validity if it describes the overview of the agenda-setting factors at play. That is, the actual portfolio can be conceived as a force shaping the agendas, together with other factors.The survey of HPSR producer institutions in developing countries was described in detail elsewhere  and inclBiases in the partner database could have occurred as a result of preferences by certain type of institutions in joining the Alliance HPSR and in answering the questionnaire required from partners. Over-representation at both levels could have occurred of more competitive and productive institutions with larger project portfolios and funding, and more interest in international funding. On the other hand, larger institutions may have been discouraged from responding given the larger number of projects to be reported, although they would also have more capacity to respond. Furthermore, the response rate could have been lower among institutions where producing HPSR is not a main function.The LOI database has 403 submissions for research funding in response to a call by the Alliance-HPSR in 2000. Applicants requested funding for one year projects in high priority areas identified jointly by them and national policy makers and stakeholders. A limitation of this database is the exclusion of funding requests for projects over one year as well as topics that would be formulated solely by researchers. Analysis of the partners' database indicates that 24% of projects are of longer duration and that up to 34% of projects undertaken are initiated by the research institution without stakeholder collaboration.The frequencies of statements for each income region were expanded proportional to population to make comparisons across regions possible. The frequency of statements across the three types of statements (projects, LOI and priorities) was standardized to give each equal weight when aggregating them to analyze the combined representation of the research portfolio and the agenda-setting process as a whole.Responses show a distribution across income regions proportional to population in some cases and with significant biases in others table . The lowThe frequency of statements on priorities is as would be expected for the population in each region. However, project statements and demand for funding are biased in favour of UMI, with 25% and 27% of the statements, respectively, against 12% of population share.This was done for each topic or category within the topic by rounding percentage differences to integers and grouping in the same rank all categories falling within the same percentage.This article does not attempt to provide an exhaustive analysis given the fact that data is limited to Alliance partner producer institutions in the South. The purpose here is to illustrate the potential of the proposed methodology and to present the most robust findings. HPSR topics are first presented and analyzed aggregating in a single list the topics in the research portfolio as well as in the policy maker and researcher agendas. This aggregate representation is then analyzed by groups of countries according to their per capita income. The influence exerted on the HPSR portfolio by various actors is then analyzed.HPSR producer institutions are generally small with an average of 3 projects, 8 researchers and a project portfolio worth $155,226 [A total of 19 research topics were identified when aggregating portfolio (project) and priority (voiced preferences) data into the reference list. Topics ranged in frequency from 2% to 11% and were ranked in 8 classes Table . The higThe fact that "Equity" appears so low in the aggregated ranking could be partly attributable to the fact that this topic was defined to include only projects and priorities having equity as the central topic and measuring it through multi-dimensional approaches such as health conditions, access to services and financing. A subsidiary analysis was thus undertaken to include under "Equity" those projects or priorities addressing equity or poverty as a secondary, qualifying, role of research on other topics. This broadened topic "Equity" climbs to fourth rank, at the same level as "Accessibility", "Program evaluation" and "Research to policy".Public and private institutions show no significant changes in topic ranking (corr = 0.70). "Community participation" and "Accessibility" are the only topics with major differences, ranking higher among private institutions.Out of the total topics classified in the reference list, only 38% 404) were sufficiently focused or detailed to be able to attribute a beneficiary or specific issue (Table 04 were sThe topics with least identification of beneficiary or issue were "Costing and cost effectiveness", "Policy process" and "Research to Evidence", with 79% to 96% in this situation. By contrast, "Community participation" and "Management & Organization" were the topics least frequently unidentified with beneficiary/issue, at between 30% and 41%.The most frequent beneficiary/issues were Community, Equity/poverty Hospital, Gender Primary care and Rural areas. The three beneficiary/issues least identified were Urban areas, elderly and Indigenous peoples/traditional medicine.The analysis of the correlation between beneficiary/issue and topic is tentative at this stage given the low frequency in many of the cells of the 19 by 11 matrix. Two beneficiaries/issues account for a large part of the focus of research topics: Community and Hospital as a focus on levels of care. At the community level the topics of community participation, financing, health insurance, decentralization, policy process, information systems and human resources are all prominent. At the hospital level the topics of costing and cost effectiveness, pharmaceutical policy, quality of care and management and organization are most prominent. By contrast, the topic of program evaluation is fairly widely spread across several issues or beneficiaries.The following topics show also a fairly discreet relationship to beneficiaries/issues: Research on accessibility is mostly focused on rural areas. Research on disease burden is prominent among the elderly and children. Economic policy and health focuses on children. Gender is also an important component of these three topics. Equity is focused on indigenous populations. The topic of information, education and communication is prominent among children. Sector analysis focuses mainly on the public private mix.The differences in ranking of the topics in the reference list across income regions are shown in the first three columns of Table The overall ranking of topics in the reference list was compared against the ranking of topics in projects initiated by each actor. The relationship between actor preferences and the reference list was assessed through an analysis of their rank congruence.As described in a previous paper in more detail , the resThe agendas across actors differ substantially, and none can be said to be close to the other. As a result, the reference list shows marked difference with respect to each actor's agenda. Government initiation shows preference for financing and cost-effectiveness as compared to the overall ranking. Government initiation tends to give lower regard to disease burden studies and for research on accessibility. International donor initiation matches best the reference list but gives somewhat less preference to sector-wide analyses. Research institution initiation is more marked for equity and community participation and less so for management and organization, accessibility and program evaluation. International donor initiation preferences are associated to the top ranking topics in the overall listing, suggesting a predominance of their agenda on the reference list.The analysis of the research portfolio and priorities at the international level shows a widely diversified set of topics, ranging from sector wide issues to more focused program evaluation. The emphasis on sector wide issues reflects the challenges to health systems today and suggests that countries consider as important the macro-level analysis as the micro. Micro approaches with a focused attention to beneficiaries or specific issues are well identified, particularly under the topic of program evaluation.However, the evidence also suggests a gap between the research that is actually being undertaken and the challenges for strengthening and scaling up of disease control programs. Such a gap is evident in the low emphasis given to research on human resources, policy process, equity, economic policy and health and information systems.By contrast, the analysis suggests a high degree of attention at the community level, although much attention is also given at the hospital level. Primary care thus seems to be under-emphasized. Considering the disease focus, whenever this was made evident, the data do not suggest a bias towards problems that would not be evidently important at country level.The fact that the public or private character of research institutions is insignificant for the agenda suggests the capacity of diverse institutions to work within a common agenda.There are significant differences in the research portfolio across groups of countries based on per capita income, suggesting the need for priority setting mechanisms at both national and international levels that reflect such diversity. The greater congruence between donor preferences and the international research agenda highlight the importance of consensus building between national and international actors. While it is appropriate for governments and international donors to fund different aspects of the research portfolio, this requires high-level priority setting and consensus mechanisms to ensure they complement each other rather than lead to fragmentation.More research is required to establish the relationships between actors' agendas and the research portfolio at the international level. There is also a need to discuss the most desirable balance of influences and to increase the voice of developing country actors. Evidence-based HPSR priorities emerging through such a process would then be able to support scaling up of research efforts on a par with scaling up of health system strengthening and disease control. Regional and global meetings, such as the Ministerial Summit for Health Research to be held in Mexico in November 2004, are good opportunities to present and discuss the evidence and to commit funding accordingly. Attention must be given to encouraging consensus building on research priorities within regions comprising countries with similar needs. The interests of donors, governments, health workers, the community and researchers must all be taken into consideration so that research funding leads not only to fund relevant research but to build the necessary interfaces for utilization.None declared.


S100B belongs to a family of calcium-binding proteins implicated in intracellular and extracellular regulatory activities. This study of serum S100B in primary progressive multiple sclerosis (PPMS) is based on data obtained from a randomized, controlled trial of Interferon β-1a in subjects with PPMS. The key questions were whether S100B levels were associated with either disability or MRI findings in primary progressive MS and whether Interferon β-1a has an effect on their S100B levels. Serial serum S100B levels were measured using an ELISA method. The results demonstrated that serum S100B is not related to either disease progression or MRI findings in subjects with primary progressive MS given Interferon β-1a. Furthermore there is no correlation between S100B levels and the primary and secondary outcome measures. S100B belongs to a family of calcium-binding proteins implicated in intracellular and extracellular regulatory activities . IntraceSeveral studies suggest that S100B has a role in the pathogenesis of multiple sclerosis (MS). Phenotypically and functionally similar T cells specific against S100B can be detected in the peripheral blood of MS patients making S100B a putative candidate auto-antigen in MS . FurtherInterferon-β (IFN-β) is effective in reducing relapse rate in relapsing-remitting ,14,17 anIn this clinically negative phase II study , we asse®, Biogen) and were assessed three monthly over a study period of 2 years. Fifteen of these patients were treated with IFNβ-1a 30μg intramuscularly (im) weekly (IFN30), 15 received IFNβ-1a 60μg im weekly (IFN60) and 20 with placebo. IFNβ-1a was reduced to half dose in 5 subjects receiving 60μg im weekly, and in 2 subjects receiving IFNβ-1a 30μg im weekly. Seven subjects withdrew from treatment [Fifty patients with PPMS were recruited in a phase II trial of IFNβ-1a . Progression was defined as a sustained (3 months apart) increase of at least 1.0 on the EDSS scale between 0 to 5 and 0.5 for subjects with EDSS score of 5.5 and above.Fourteen healthy subjects served as controls.All subjects provided informed consent prior to their inclusion in the study. This study was approved by the ethics committee and has therefore been performed with the ethical standards laid down in the 1964 Declaration of Helsinki.MRI was performed at baseline and 6 monthly for 2 years. Only baseline and year 2 data were included in this study. Brain and spinal cord atrophy, ventricular volume, T1 and T2 lesion load were measured as described elsewhere .Serum samples were centrifuged and stored at -20°C. Serum S100B levels were quantified using a modified ELISA method as previously described by Green et al. . Ninety-Median, interquartile range and significance of group differences (Mann-Whitney U tests) were evaluated. Changes of serum level over time were examined using variance components regression models of serum response variable on time as predictor, with random subject-specific intercepts and fixed common slopes. Curvature was assessed using a quadratic term in time; modification of curve over time by treatment was assessed using additional terms for treatment and treatment by time interaction in the model. Two sets of treatment terms were used: i) indicators of assigned weekly dose ii) average weekly dose over follow-up (including changes to dose regime) as continuous variable. Modification of the curve over time by MRI variable values were similarly examined using terms for MRI variable and MRI variable by time interaction.Direct associations between serum level and MRI/clinical variables were examined by regression models of 24 month serum on 24 month MRI variable, adjusting for baseline serum and MRI values (this type of model takes into account change from baseline), with additional terms for treatment and treatment by MRI variable interaction, the latter to assess possible modifications of the relationship by treatment.Software used were the SPSS software package (version 11.0 for Windows) and Stata 7.0 .The median and interquartile ranges for all subjects are described in Table There was no change over time in the serum S100B levels. The shape of the serum trajectory did not vary between the treatment regimes, i.e. placebo vs. IFN30 vs. IFN60.There was no evidence that the 24-month serum S100B values were associated with either changes in the T1 or T2 loads, or ventricular or cord volumes at 24 months, after adjusting for the baseline values of each subject. There was no correlation with disease progression on the EDSS. There was also no evidence that these relationships were modified by treatment assignment  (Table These results suggest that serum S100B levels in patients with PPMS were not affected by intramuscular IFNβ-1a and that there was no observable change in S100B over time. Furthermore, we did not observe any correlation between S100B levels and clinical disability or between S100B and quantitative MRI measures.This study therefore suggests Although there is evidence that S100B elevation in MS is related to inflammatory activity ,13, thisIt would be valuable to identify surrogate markers of clinical progression in PPMS to aid the development of effective therapeutic intervention, since clinical trials with a disability endpoint are very large and resource consuming. It is possible that such markers would need to be less related to acute inflammation and more dependant on other neuropathology such as axonal loss and regeneneration.


The colour of animals' skin, fur, feathers or cuticula has been estimated in a large number of studies. The methods used to do so are diverse, with some being costly and not available to all researchers. In a study to measure plumage colour in a bird species, a new method of creating a colour chart was developed. While colour-charts have their own limitations, these can be minimised when they have the following properties: 1) being readily available to the majority of biologists, 2) containing a large array of colours to allow accurate recording and differentiation of subtle colour differences, 3) low cost, 4) adhering to a world-wide standard, and 5) being available in both hard-copy and digital formats to allow for various analytical methods. The method described below satisfies all of these requirements.Colour charts estimated to fit the range of the species' plumage colours were created on the computer screen using web software that allowed for HTML-coding (in this case Dreamweaver™). The charts were adjusted using feathers from dead specimens until a satisfying range of darker and lighter colours were found. The resulting chart was printed out and was successfully used in the field to determine the plumage colour of hand-held birds.Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit alphanumeric code. We believe this method is very useful as a low-tech method for future estimation of individual colour. Beagle expedition [® – Adobe Systems Inc., San Jose, CA, USA) has been used in estimating colour brightness, saturation and hue from digitised photos [Animal studies from diverse fields – such as morphology, physiology, behaviour, population dynamics, genetics, ageing and sexing – often require the estimation or classification of colour in skin, fur, feathers or cuticula. To make meaningful comparisons within and between studies, and effectively communicate these findings, a standardised method of assigning a colouration is required. Colour can be categorised, scored and ranked without recourse of any aid other than the observer's eye, as has been successfully demonstrated in many studies -4. Aids pedition . Newer epedition -14, havepedition . Lately,d photos ,16.The benefit of using highly technical methods is the ability to gain very detailed and precise information on different aspects of colour that may be impossible to detect using the human eye. However, in many studies such a high level of precision is not required, and the equipment may be impractical in the field or beyond the budget of the research group. Thus despite the option of high-tech measurement methods, visual comparisons to standardised colour charts are still practical and valuable for many field biologists. While colour-charts have their own limitations, these are minimised when they have the following properties: 1) availability to the majority of biologists, 2) large array of colours to allow accurate recording and differentiation of subtle colour differences, 3) low cost, 4) adherence to a world-wide standard, and 5) availability in both hard-copy and digital formats to allow for various analytical methods. Below we describe a method that satisfies all of these requirements.Petroica longipes) – where there was a need for a reliable, species-specific and cost-efficient method to estimate colour  and has since become the industry standard [®, by using the composer feature, or through web-design programmes (e.g. Dreamweaver™ – Macromedia Inc. or FrontPage© – Microsoft Corp.). The colours can be displayed on the computer screen and fine-tuned by adjusting the HTML code. For example, between the colours "0000FF" for blue, "00008B" for dark blue and "00C78C" for turquoise blue, a large number of other blues can be created and viewed. When a suitable set of colours have been decided upon, they can be printed out as they present themselves on the screen and saved for future use.The light emitted from a computer monitor is composed of a particular combination of red, green, and blue light. The proportion of each of these components in the visual colour spectrum can be expressed as a number unique for each specific colour or hue. These colours are coded for using the HTML (Hypertext Markup Language) computer language. This system of colour coding was developed for the Netscapestandard . Today, standard . Each HTstandard . The actThe focus species of the plumage study, the North Island robin, has a brown-grey-black plumage Fig.  19]. Us. Us19]. The method of colour-chart creation utilising HTML code satisfies the criteria listed above. Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours (and patterns) that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit hexadecimal code. Comparisons are not limited to a printout of a colour chart, digital images can also be compared and their colours scored using this method. Drawbacks may include identifying the right colours for the chart to accurately match the animals as encountered in the field, a problem equivalent to other printed colour charts. It is also possible that when printing, the printed colours differ from the colour range as displayed on the computer screen. With some people still using older computers, which are not able to display colours coded in newer machines, there is a risk that there is a discrepancy between computers in the colour presented on the screen. This may be a problem when the aim is to compare specific colours between different studies, but not an issue within studies. Fortunately, this problem will decrease with more computer system being able to display the full range of 16.7 million colours. When using the colour-charts in the field, the usual care of indexing individuals under the same lighting conditions should be taken . As the JM came up with the initial idea of using web designer tools for creating colour charts. ÅB developed the colour charts using HTML coding and used them in the field as a research tool. ÅB and JM wrote the manuscript, with ÅB doing the major part. All authors read and approved the manuscript.


Obesity is the most common health problem in developed countries. Recently, several physicians' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. The aim of this study was to examine if effective weight-reducing treatment can be given by a family physician. It compares regular treatment with intensive treatment that include close follow-up and orlistat treatment.The study was conducted in three primary care clinics. 225 patients were divided into three groups according to their choice. Group A received a personal diet with fortnightly meetings with the family physician and dietitian and orlistat treatment. Group B received a general diet, monthly meetings with the family physician only and orlistat treatment. Group C received a personal diet, monthly meetings with the dietitian only and no drug treatment. The primary endpoint was reduction of at least 5% of the initial weight during the study period.A greater percentage of patients in group A achieved their weight reduction goals than in other groups (51%, 13% and 9% in groups A, B and C, respectively, p < 0.001). There was a significant reduction in triglycerides in all groups, a significant reduction of low density lipids (LDL) in groups A and B and no significant difference in high density lipids (HDL) in any group.Significant weight reduction was obtained in a family physician setting. Further research is needed to evaluate if, by providing the family physician with the proper tools, similar success can be achieved in more clinics. It is aabove 25 . The ratabove 25  and has above 25 . In Israabove 25 .Obesity is associated with increased prevalence of many serious chronic diseases such as diabetes mellitus, hypertension, dyslipidemia, and coronary heart disease ,9. It maDiet and exercise have limited effectiveness on long-term maintenance of weight loss . Within The intervention of primary physicians during a ten minute physician/patient encounter and telephone consultation with a community dietitian resulted in a significant decrease in the weight of patients . RecentlThe purpose of this study was to examine if more efficient and effective weight-reducing treatment can be given in the family doctor setting. The study compare a non-pharmacological intervention with drug intervention (orlistat) and compare regular management with more intensive family physician based management..The study was conducted in three primary care clinics in an urban area in central Israel. The family physicians who took part in this study participated in 80 hours CME course dealing with obesity treatment in Israel.®) is a lipase inhibitor for obesity management that acts by inhibiting the absorption of dietary fats.The patients were divided into three groups according to their choice. Patients in groups A and B were treated with orlistat at 120 mg TID. Orlistat  patients of either sex or patients with a BMI above 27 plus two or more cardiovascular risk factors, aged 20–75 years, were eligible for the study. Patients were excluded if they were pregnant or lactating or if they had any contraindication against using orlistat .Before the intervention, patients underwent an initial screening that included recording of a complete medical history, measurement of vital signs, body weight and height, and calculation of the BMI. Laboratory analysis included a lipid profile. The readiness of the patient to receive treatment was assessed. At the beginning of the intervention, all participants were in the third stage of readiness ("the preparation stage") according to the Transtheoretical Model of Behavior Change.An adverse event of any dose of orlistat was considered serious if it resulted in death, was life-threatening, required hospitalization, or resulted in significant disability.The main measure was weight loss. Each patient was weighed during each meeting. The primary endpoint was reduction of at least 5% of the initial weight during the study period (six months). Achievement of this goal was defined as successful treatment. Another measure was improvement in the lipid profile.The second lipid profile was done to half of the patients, only those who had dyslipidemia in the first profile had been offered a second profile.Data on patients' background and weight-reduction results between groups were compared using the Chi square test. Continuous data of the groups, i.e., measurements at the beginning of the study and continuous background data, were compared using one-way tests (ANOVA). When significant statistical differences were found among the three groups, the Tukey Post Hoc test was used to examine the statistical difference between each group of two. ANCOVA was also used and, if there was a difference amongst the groups, the significance (for the measured parameter) was checked using the Bonferonni technique.Two hundred and twenty-five patients participated in the study. Their demographic characteristics, history of cardiovascular disease, and initial lipid profile are shown in Table p = 0.03).Patient-reported adverse events in the orlistat-treated groups were all related to the gastrointestinal tract. The most commonly reported events were flatulence with discharge (9.2%), fatty or oily stool with increased defecation (9.2%), feeling of fullness in the stomach (4.6%), and constipation (1.7%). There were no statistically significant differences in side effects between groups A and B. In group A, 11 patients (17.7%) stopped the treatment for the following reasons: cost of the medication (47%), lack of time (33%), or dissatisfaction (20%). In comparison, ten patients (8.9%) in group B stopped the treatment, mainly because of the cost of the treatment (65%) or low motivation (23.5%). The reasons for stopping treatment significantly differed between groups A and B  of their initial body weight, patients in Group B reduced 7.8 kg (range 10–12 Kg.) from their initial body weight and patients in Group C reduced 3.12 kg (range 5–6 Kg.) of their initial body weight.Obesity is a worldwide problem. Treatment of a patient for obesity involves two processes: evaluation of the severity of the obesity and general health condition of the patient, and management which includes guidance in how to gradually reduce weight and maintain the new weight together with imparting healthy lifestyle habits and keeping track of improvement.In this study, we examined weight-reduction techniques that can be carried out in the setting of a community family practice. Intense treatment, combining frequent counseling by the family physician and a dietitian with medications (group A), resulted in the best weight reduction and lipid profile improvement in the short period of this study, as was reported earlier in special weight reduction clinics -27The effectiveness of interventions in primary care setting are controversial -30. BeerHauptman et al  in a stuThere are many advantages to a program involving cooperation between the family physician and the dietitian. The family physician is acquainted with the patient for a longer time than a dietitian. The physician is familiar with the patient's health condition, medications taken by the patient, the patient's environment and lifestyle and can recommend a treatment suitable to the patient's personality and lifestyle. The family physician is knowledgeable about weight-reducing drugs and possible side effects. The patient trusts and has confidence in the family doctor. Together with the dietitian, a personal diet appropriate to the drug treatment can be created.Obesity is a chronic disease. The physician and the patient must recognize that obesity treatment is a prolonged process that extends a lifetime. Since family physicians are usually familiar with their patients as well as with the patient's family and environment for many years, family physicians know what changes the patient can achieve. They can recruit family members to support the necessary lifestyle changes. Also, because of their training, family physicians are the most suitable professional to holistically treat obesity and its complications.There were several limitations in our study. One limitation was the non-random division of patients into groups A, B and C. It is possible that the patients who chose drug treatment differed from those who chose a diet only. Perhaps they were more ready for the weight-reduction process and to expend money for the drug (in Israel, there is a patient co-payment for medications). The rate of women amongst the patients seeking treatment was higher than the rate of men, as in other reports ,27. HencThe study periods for groups A and B (which received orlistat) were shorter than for group C, partly because there is significant co-payment for orlistat and partly because the targeted weight had been achieved earlier. The drug was well tolerated with minimal gastrointestinal side effects.The patients were treated by family physicians that were orientated toward and trained in weight reduction. Results might have been less successful with other physicians. Hence, family physicians should be trained in the subject by increasing both their knowledge and their treatment skills. Healthcare funds must recognize the importance of weight reduction so that they will allocate the necessary additional time and resources of their physicians, clinics and multi-field staff.This study evaluated only the weight reduction period. Long-term results and whether or not the patient maintained the lifestyle change for a long period were not examined. Further studies should examine the best program for maintaining the new weight.In conclusion, this study showed that within the setting of the family practice it is possible to carry out an effective program of weight reduction and achieve significant weight loss. The addition of orlistat can further improve results. We believe that by providing family physicians with the proper tools, similar success can be achieved in many more clinics.The author(s) declare that they have no competing interests.AF and SP conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. MS and EF participated in the design of the study, data collection and interpretetion. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.The bacterium  Deinococcus radiodurans is able to repair radiation induced genome damage by virtue of a homologue of the eukaryotic Rad52 protein, which preserves genome integrity by protecting DNA ends Deinococcus radiodurans is a non-spore-forming bacterium notable for its capacity to tolerate exposure to ionizing radiation , gap (DR1343), and DR0423 genes were determined as previously described  relative to R1, indicating that DR0423p is not essential for the processes of DNA replication or homologous recombination. To establish whether DR0423p was necessary for DNA damage tolerance, TNK104 was evaluated for its ability to survive ionizing radiation and mitomycin C. Aliquots of exponential-phase cultures were exposed to these DNA-damaging agents. TNK104 exhibits increased sensitivity to both agents relative to the wild-type R1 strain (DNA damage response A (ddrA).The DR0423 gene was inactivated by deletion in lsewhere , and the1 strain , but culrecA gene was deleted from R1 and TNK104 (see (ΔrecA) and TNK110 (ΔrecA, ΔddrA), respectively. Deinococcal strains lacking recA function are considered the most ionizing-radiation-sensitive strains described for this species (ΔrecA strain, indicating that DNA damage response A protein (DdrA), at least in part, contributes to D. radiodurans' survival by a mechanism that is independent of RecA function.The K104 see B and 1C, species . However4, and irradiated. No carbon source was added. Restoration of the genome was monitored by pulsed-field gel electrophoresis, and aliquots retrieved from the recovering cultures were used to determine viability. Cultures were left in this medium and sampled at 24-h intervals over a 120-h time course.To determine if loss of DdrA affected genome restitution and stability postirradiation, we followed the recovery of cultures of R1 and TNK104 following a 5,000-Gy dose of γ radiation. Initially, exponential-phase cultures were harvested, suspended in 10 mM MgSOD. radiodurans genome, and when restricted, most of the resulting fragments can be separated by pulsed-field gel electrophoresis as seen in the lane (C) corresponding to the unirradiated control. Immediately after irradiation, the introduction of DNA double-stranded breaks results in the disappearance of the higher molecular weight NotI fragments, but the pattern of fragments is restored in 24–48 h, indicating that R1 is repairing double-stranded breaks under these conditions, in spite of the absence of nutrients. This pattern persists throughout the rest of the time course, indicating that once reformed the genome is stable. Despite genome restitution, R1 cultures held in MgSO4 are not as proficient at recovering from ionizing-radiation-induced damage as cultures that are allowed to recover in rich medium (4 exhibit a modest 2-fold reduction in viability when exposed to 5,000-Gy γ radiation relative to R1 cultures irradiated in rich medium (see 4 (4 for 5 d (data not shown).The gel depicted in dium see . The lonm . Consistent with the survival curve depicted in ddrA gene was cloned and expressed in E. coli, and the protein was purified to homogeneity (The ogeneity . The ideogeneity DdrA exhibited no ATPase, helicase, recombinase, or nuclease activity (data not shown). However, it bound to single-stranded DNA as determined by an electrophoretic mobility-shift assay (EMSA) . BindingE. coli, which digests single-stranded DNA from the 3′ end , DdrA would cap the strand breaks and help stabilize the genome until such time as conditions were more amenable to systematic DNA repair.The extraordinary resistance of iccation . In an addrA gene is deleted from R1, an otherwise wild-type cell becomes more sensitive to DNA-damaging agents (see ddrA deletion are amplified if nutrients are not provided after exposure to ionizing radiation, and cells held this way for 5 d display a 100-fold reduction in viability relative to the wild-type cells (see The results we have obtained both in vivo and in vitro are consistent with this hypothesis. When the ents see . We showents see , and purents see  and 8. Nells see . In thes4, there was no evidence of genome reassembly in similarly treated cultures of TNK104, the ΔddrA derivative of R1 (see 4, and we suggest that TNK104's inability to reconstitute its genome under these conditions is likely to be related to the DNA degradation that is observed in this strain following irradiation (see Even though the R1 strain was able to restore its genome following irradiation and incubation in 10 mM MgSOf R1 see . This retion see B.4. Since DdrA binds the 3′ ends of single-stranded DNA, we presume that this protein either has the same activity or is rendered unnecessary by a compensating activity possible only in a nutrient-rich environment (such as DNA synthesis to counter exonucleolytic degradation). If, instead, DdrA is part of a passive DNA protection system, this system may be critical under conditions in which active (energy-requiring) DNA repair is not possible, such as when cells are desiccated or held in a nutrient-free medium. DdrA may not be as important in a nutrient-rich environment, where active DNA synthesis and other DNA repair processes may compensate for the loss of DNA end protection.DdrA is not needed if cells are allowed to recover in a nutrient-rich medium see . This su(ΔrecA ΔddrA) relative to TNK106 (ΔrecA) indicates that DdrA participates in a process that complements RecA-mediated survival mechanisms .Since inactivation of DdrA reduces, but does not eliminate, the DNA damage resistance of D. radiodurans, and other bacteria with similar capacities to survive high DNA damage loads, employs multiple systems to repair its DNA. The DNA end-protection system we have begun to explore may be supplemented by special genome architectures (It seems likely that tectures , traditihttp://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gdr). All strains derived from D. radiodurans were grown at 30 °C in TGY broth (0.5% tryptone, 0.3% yeast extract, and 0.1% glucose) or on TGY agar (1.5% agar). E. coli strains were grown in Luria-Bertani (LB) broth or on LB plates at 37 °C. Plasmids were routinely propagated in E. coli strain DH5αMCR. D. radiodurans cultures were evaluated for their ability to survive exposure to DNA-damaging agents in exponential growth (OD600 = 0.08 − 0.15, 5 × 106 − 1 × 107 cfu/ml). All cultures were treated at 25 °C. Gamma irradiation was conducted using a model 484R 60Co irradiator  at a rate of 30 Gy/min. Resistance to mitomycin C was determined by adding 1 mg of mitomycin C (Sigma, St. Louis, Missouri, United States) to 1-ml broth cultures of the D. radiodurans strain. Aliquots of the treated culture were removed at one-half-hour intervals over the next 2 h, washed in 10 mM MgSO4, and plated on TGY agar to determine viability.Strains and plasmids used in this study are described in ddrA and recA were disrupted by targeted mutagenesis using techniques described previously  from pHP45omega-hyg (D. radiodurans katA gene (DR1998) (katA–hyg fusion product was joined to PCR fragments (ddrA. This hybrid fragment was cloned into pGEM-T (Promega, Madison, Wisconsin, United States), creating pTNK205. pTNK205 was propagated in E. coli DH5α-MCR. The deletion of ddrA was accomplished by transforming (The construction of TNK104 began with the creation of a drug cassette capable of conferring hygromycin resistance on mega-hyg  was spli(DR1998) , using pragments  derived sforming  an exponddrA, were used to generate PCR fragments from genomic DNA from hygromycin-resistant colonies and R1. The purified PCR products were restricted with EcoRI and EcoRV. The hyg gene contains an EcoRI site, but ddrA does not. ddrA contains an EcoRV site, but hyg does not. In the recombinant, designated TNK104, a single 1.3-kbp fragment, corresponding to the katA–hyg cassette was amplified, whereas there was no trace of the 0.85-kbp fragment, indicative of ddrA amplification  from pBC . This drug cassette was then spliced to PCR products corresponding to genomic DNA sequences 1.6 kbp upstream and 1.2 kbp downstream of recA by overlap extension, before being cloned into pGEM-T. The resulting plasmid was designated pTNK210. An exponential-phase R1 culture was transformed with the replacement cassette from pTNK210, and chloramphenicol-resistant recombinants were selected on TGY plates containing 3 μg/ml chloramphenicol. Genomic DNA of each recombinant was amplified to determine if the recA coding region was deleted. Purified PCR products amplified using primers that anneal to sequences flanking recA were treated with PvuII and BglII. The cat gene carries a PvuII site, but recA does not. recA contains a BglII site, but cat does not. A 1.3-kbp fragment, corresponding to the katA–cat cassette, was obtained from a recombinant designated TNK106, but DNA from this recombinant did not generate the 1.5-kbp fragment corresponding to recA  and resuspended in either TGY broth or 10 mM MgSO4 solution, before being placed in a shaking incubator at 30 °C for 24 h. Aliquots of these cultures were removed at various time points, and cells were washed in 0.9% NaCl and suspended in 0.125 M EDTA (pH 8.0) at a density of 5 × 108 cells/ml. The suspensions were mixed with low-melting-point agarose (Sigma) to obtain a final concentration of 0.8% agarose. Agarose blocks containing the cell suspension were incubated overnight at 37 °C in 0.05 M EDTA (pH 7.5) containing 1 mg/ml of lysozyme. After lysozyme treatment, agarose plugs were placed in ESP buffer (EDTA 0.5 M [pH 9–9.5], 1% lauroyl sarcosine, 1 mg/ml proteinase K) at 50 °C for 6 h, followed by a 2-d incubation at 37 °C. Prior to digestion with restriction enzymes, agarose plugs were washed once with TE buffer (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride and then four times with TE buffer (pH 7.5). DNA contained within the agarose plugs was digested with 10 U of NotI restriction enzyme (New England Biolabs, Beverly, Massachusetts, United States) overnight at 37 °C. Restriction digests were analyzed on 1% agarose gels in 0.5X TBE, using a CHEF-MAPPER electrophoresis system  at 6 V/cm for 22 h at 12 °C, with a linear pulse ramp of 10–60 s and a switching angle of 120°. Gels were stained with water containing 0.5 μg/ml ethidium bromide for 20 min and destained for 10 min in water.After irradiation at 5.0 kGy, cells were collected by centrifugation (6,000D. radiodurans cultures using TRI Reagent (Molecular Research Center, Cincinnati, Ohio, United States) following manufacturer's instructions. Cell disruption was accomplished by adding 100 μ1 of 0.1-mm zirconia/silica beads (Biospec Products, Bartlesville, Oklahoma, United States) and TRI Reagent to the cell paste from 1 l of cells and vigorously agitating this mixture for 6 min with a vortex mixer. Two micrograms of each DNase I–treated, purified RNA sample was converted to cDNA using SUPERSCRIPT II RNase H− Reverse Transcriptase  combined with 25 pmol of random hexamers to initiate synthesis. Conditions for this reaction followed the manufacturer's instructions.The protocol followed was the same as that described previously . Total R−4) of each experimental sample was generated and run in duplicate. Negative controls without a cDNA template were run on every plate analyzed. All assays were performed using the iCycler iQ Real-Time Detection System (Bio-Rad). All data were PCR-baseline subtracted before threshold cycle values were designated and before standard curves were constructed. Mean concentrations of the transcripts in each sample were calculated from the standard curves generated using the recA primer set. Induction levels were determined by dividing the calculated concentration of transcript from the irradiated sample by the concentration of transcript from the unirradiated sample for each strain. The mean concentration of the gap transcript, a housekeeping gene whose expression is unaffected by ionizing radiation, was also determined before and after irradiation for each strain.Approximately 100 bp of unique sequence from the genes encoding DdrA (DR0423), RecA (DR2340), and glyceraldehyde 3-phosphate dehydrogenase (DR1343) were amplified using the following primer sets: DR0423up (5′-GGTGCAGGACCGACTCGACGCCGTTTGCC-3′), DR0423down (5′-CCTCGCGGGTCACGCCGAGCACGGTCAGG-3′), DR2340up (5′-GTCAGCACCGGCAGCCTCAGCCTTGACCTC-3′), DR2340down (5′-GATGGCGAGGGCCAGGGTGGTCTTGC-3′), and DR1343up (5′-CTTCACCAGCCGCGAAGGGGCCTCCAAGC-3′), DR1343down (5′-GCCCAGCACGATGGAGAAGTCCTCGCC-3′). The PCR reaction (50 μ1) for amplifying these genes contained the appropriate primers at a final concentration of 0.2 μM, 1 μ1 of the cDNA template, and SYBR Green PCR Core Reagents . Amplifications were carried out by incubating reactions at 95 °C for 3 min prior to 40 cycles of 30 s at 95 °C, followed by 30 s at 65 °C and 30 s at 72 °C. Data were collected and analyzed at each 72-°C interval. Each 96-well plate consisted of standard curves for each primer set run in duplicate. Standard curves were constructed using cDNA obtained from the unirradiated wild-type organism. A dilution series . The remaining cultures were centrifuged for 20 min at 4 °C at 7,000 rpm. Bacterial pellets were washed twice and resuspended in 10 mM MgSO4 for γ irradiation. Cell suspensions were irradiated at 5,000 Gy and incubated at 30 °C for 120 h. Aliquots were removed immediately following irradiation, at 48 h, and at 120 h postirradiation. Cells were toluene-fixed as described above; 100 μ1 of DAPI (stock solution 3 μg/ml) was added to each dilution tube and mixed. The fluorescence intensity was determined after excitation at 350 nm by measuring emission at 450 nm.Overnight cultures growing in TGY medium were harvested at room temperature. Control culture aliquots were fixed with 1% toluene , shaken vigorously, and stored at 4 °C. The fixed bacteria were diluted (1/10, 1/100, and 1/1,000) in 3 ml  of dilution buffer: 10 mM NaCl, 6.6 mM NaddrA gene was amplified using the genomic DNA from D. radiodurans strain R1. PCR primers were designed according to the ddrA gene sequence annotated in the genomic bank (http://www.ncbi.nlm.nih.gov). The gene was cloned in E. coli overexpressing plasmid pEAW298. DdrA-overproducing cells were lysed with lysozyme, and the protein was precipitated from the supernatant by adding ammonium sulfate to 30% saturation. The protein was purified with DEAE and hydroxyapatite chromatography to greater than 99% purity. The identity of the purified protein was confirmed by N-terminal sequencing (Protein and Nucleic Acid Chemistry Laboratory, Washington University School of Medicine, St. Louis, Missouri, United States) and accurate mass determination (Biotech Center, University of Wisconsin, Madison, Wisconsin, United States). The protein was transferred into the storage buffer (20 mM Tris-acetate, 80% cation [pH 7.5]/50% glycerol [w/v], 0.5 M NaCl, 0.1 mM EDTA, and 1 mM DTT) and stored at −80 °C.The L-tyrosylglycine and N-acetyl-L-tryptophanamide in 6 M GND–HCl are 1,280 M−1cm−1 and 5,690 M−1cm−1, respectively (denat, 280 nm = 5 × 5,690 + 5 × 1,280 = 3.485 × 104 M−1cm−1. Absorbance spectra of native and denatured (6 M GND–HCl) DdrA protein were scanned at 25 °C, from 320 to 240 nm, for five different dilutions and with two different protein preparations. DdrA protein was diluted in storage buffer or storage buffer plus 6 M GND–HCl  in a total volume of 80 μ1 and was preincubated at 25 °C for 5 min before scanning. Each dilution was carried out in triplicate, and the absorbance values at 280 nm were averaged. The concentrations of native and denatured protein were equal to each other in each scan at each dilution. The extinction coefficient of native DdrA protein at 280 nm was determined according to the expression . The oligo was labeled at the 5′ end using [γ-32P] ATP and polynucleotide kinase. After labeling, hairpin formation generated a DNA with 18 bp in the hairpin duplex and a 15-nt 5′ extension. A blunt-ended duplex DNA fragment was prepared by annealing oligonucleotide C (5′-GGT CTT TCA AAT TGT TTA AGG AAG AAA CTA ATG CTA GCC ACG GTC CGA GCC-3′) 32P-labeled at its 5′ end, with unlabeled oligonucleotide D (5′-GGC TCG GAC CGT GGC TAG CAT TAG TTT CTT CCT TAA ACA ATT TGA AAG ACC-3′). The single-stranded oligonucleotide was the end-labeled oligo C. EMSAs for DNA binding were carried out in 15-μl reaction mixtures containing the reaction buffer (40 mM Tris-acetate [pH 7.5],10% glycerol [w/v], 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT) and 0.7 nM (60 nM nt) 32P-labeled duplex DNA. The reaction was initiated by adding the DdrA protein to the required concentration. The reaction mixture was incubated at 30 °C for 30 min and loaded onto a 10% native polyacrylamide gel. The electrophoresis was performed in 1X TBE (89 mM Tris-borate [pH8.3], 2 mM EDTA) at room temperature. After the electrophoresis was completed, the gel was dried and exposed with a Phosphoimager .The duplex oligonucleotide with a 3′ single-stranded extension was hairpin-forming oligonucleotide A (5′-TTA ACG ACC GTC GAC CTG CAG GTC GAC GGT CGT TAA CGT CTC TCA GAT TGT-3′), which was labeled at the 3′ terminus with [α-The general strategy of this experiment was to incubate a DNA duplex with a 3′ extension with DdrA protein, resolve the protein complex in native PAGE, excise the complex from the gel, extract the protein from the slice, and analyze the protein in SDS-PAGE. If the protein is DdrA, it will comigrate with DdrA protein in SDS-PAGE.32P-labeled oligonucleotide  was annealed with a longer unlabeled oligonucleotide . Annealing was carried out in a 40-μ1 solution containing 0.5 μM of each oligonucleotide in 25 mM Tris HCl (pH 8), 50 mM NaCl, and 12.5 mM MgC12. The solution was heated briefly at 100 °C, by transferring the closed tube to a beaker of boiling water, and allowed to cool slowly overnight. The tube was refrigerated for several hours and then stored at −20 °C until use.A The resulting labeled duplex DNA with a 3′ extension (0.7 nM) was incubated with 4 μM DdrA protein under the DNA binding conditions described above. The mixture was loaded onto a 10% native polyacrylamide gel. Electrophoresis was performed as described above. The gel was exposed with X-ray film to map the position of the protein–duplex complex. The complex was cut out of the gel. The gel slice was frozen in liquid nitrogen and crushed into a slurry with a plastic stick. The slurry was mixed with an equal volume of SDS-PAGE loading buffer and boiled for 3 min. The mixture was loaded onto a 12% SDS-PAGE gel and the protein present compared to molecular weight standards and purified DdrA protein.32P]ATP, and cold oligonucleotide B (5′-GGT CTT TCA AAT TGT TTA AGG AAG AAA CTA ATG CTA GCC ACG GTC CGA GCC-3′). The annealing generated a 14-nt 3′ extension at one end of the short duplex. Before adding the exonuclease, the 32P-labeled duplex (60 nM nt) was preincubated with the DdrA protein at the indicated concentration in 15 μl of the exonuclease reaction buffer (40 mM Tris-acetate [pH 7.5], 0.1 M NaCl, 10 mM MgC12, 0.1 mM EDTA, 1 mM DTT, 10% glycerol) at room temperature for 10 min. In the control experiment, the DdrA protein was replaced with bovine serum albumin. Exonuclease I was added to 200 U/ml and the reaction mixture was incubated at 37 °C for 30 min. After the incubation was complete, the reactions 5 and 6 were deproteinized with 0.2% SDS and 0.2 mg/ml proteinase K at 37 °C for 15 min. The DNA–protein complexes were resolved in the native polyacrylamide gel as above.The duplex with a 3′ extension was prepared by annealing oligonucleotide A (5′-CTA GCA TTA GTT TCT TCC TTA AAC AAT TTG AAA GAC C-3′), which was labeled at the 5′ terminus with [γ-http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers for the genes and gene products discussed in this paper are ddrA/DR0423 (NP_294146), Erf (P04892), gap/DR1343 (NP_295066), Rad52 (P06778), recA/DR2340 (NP_296061), RecT (P33228), and Redβ (P03698).The GenBank (