There has been increasing interest in enhancing accountability in health care. As such, several methods have been developed to compare the quality of home care services. These comparisons can be problematic if client populations vary across providers and no adjustment is made to account for these differences. The current paper explores the effects of risk adjustment for a set of home care quality indicators (HCQIs) based on the Minimum Data Set for Home Care (MDS-HC).A total of 22 home care providers in Ontario and the Winnipeg Regional Health Authority (WRHA) in Manitoba, Canada, gathered data on their clients using the MDS-HC. These assessment data were used to generate HCQIs for each agency and for the two regions. Three types of risk adjustment methods were contrasted: a) client covariates only; b) client covariates plus an "Agency Intake Profile" (AIP) to adjust for ascertainment and selection bias by the agency; and c) client covariates plus the intake Case Mix Index (CMI).lower than the WRHA. In the WRHA, individual agencies were likely to experience a decline in their standing, whereby they were more likely to be ranked among the worst performers following risk adjustment. The opposite was true for sites in Ontario.The mean age and gender distribution in the two populations was very similar. Across the 19 risk-adjusted HCQIs, Ontario CCACs had a significantly higher AIP adjustment value for eight HCQIs, indicating a greater propensity to trigger on these quality issues on admission. On average, Ontario had unadjusted rates that were 0.3% higher than the WRHA. Following risk adjustment with the AIP covariate, Ontario rates were, on average, 1.5% Risk adjustment is essential when comparing quality of care across providers when home care agencies provide services to populations with different characteristics. While such adjustment had a relatively small effect for the two regions, it did substantially affect the ranking of many individual home care providers. In both Canada and the United States efforts are underway to develop systems to assess the quality of health care as a first step to improving services. In the US nursing home sector, the implementation of the Minimum Data Set has been linked to improvements in quality of care -4. RecenWhen comparing health care providers across a set of QIs there is a concern that they may differentially admit clients with a greater likelihood of triggering on quality issues. Since the indicators are defined as events that are preferable to avoid (e.g., skin ulcers, untreated pain, weight loss), in the absence of risk adjustment, these providers would appear to be delivering poorer quality of care. Risk adjustment attempts to adjust for different populations of clients who may be at greater risk for experiencing quality issues that is a function of their clinical status rather than the quality of care.In the US nursing home sector, the CMS recently funded a large-scale project to review all potential long-term care QIs and the risk adjustment process. The evaluation of risk adjustment included a review of both client-level and agency-level covariates. The research team revised the original set of client covariates to create a set of new models and assessed the effects of using an agency-level covariate, the Facility Admission Profile (FAP). The FAP represents the prevalence of the quality issue for residents newly admitted to the facility and was intended to adjust for potential selection bias  and ascertainment bias . However, after extensive analyses, the research team did not recommend the use of the FAP, given that the FAP did not appear to be a particularly useful measure of ascertainment bias .Nevertheless, in October 2002, the CMS included the FAP in three quality measures reported on the Nursing Home Compare web page. As partThe current research was part of a project to develop a set of 22 Home Care Quality Indicators (HCQIs) based on items in the Minimum Data Set for Home Care (MDS-HC). Implementation of the MDS-HC is complete or underway in 15 states and in 7 Canadian provinces and territories, where it is being used mainly as a clinical assessment instrument. In Michigan, the MDS-HC is being used as part of the MI-Choice waiver program to reduce nursing home admissions. In Ontario, Community Care Access Centre (CCAC) case managers use the instrument to determine needs, allocate services and make placement decisions.The HCQI derivation was accomplished using data from Ontario and Michigan since these were the regions with the largest scale implementation in Canada and the US, respectively. In their paper describing the HCQI derivation, Hirdes et al. recommenRAI, a non-profit multinational organization dedicated to the development and refinement of assessment instruments for older adults and persons with disabilities, and their related applications. The HCQIs represent outcome measures that document an agency's rate for triggering on a quality issue. For example, one indicator measures the prevalence of weight loss among individuals who are not considered to be palliative clients. The HCQIs include a mix of prevalence measures (i.e., measured on a cross-section of clients at one point in time) and incidence measures (i.e., failure to improve or incidence of an event measured across two points in time). All HCQIs are defined as events to be avoided such that a higher rate on the indicator is indicative of poorer performance.The set of HCQIs used in this study were developed by members of interRAI HCQIs. It explores the effects of risk adjustment at both the level of the region and at the level of the individual home care provider.This paper uses a dataset from two Canadian provinces to further explore these three types of risk adjustment for the interThe RAI Health Informatics Project was a two and a half year research study begun in 1999 in which fourteen of Ontario's 43 CCACs used the MDS-HC as part of usual practice for all adult (18 years and older) home care clients. CCACs provide several different services: information and referral, case management and placement in long-term care facilities. CCACs purchase in-home services from external contracted agencies through a "request for proposal" process. Services provided include in-home physiotherapy, occupational therapy, personal support and homemaking, medical supplies and equipment, and care by other professional such as social workers, dietitians and speech-language pathologists. Case managers oversee the assessment process, make referrals to service providers and then monitor the care provided in the home. Funding for CCACs is based on an annual budget provided by the Ontario Ministry of Health and Long-term Care. CCACs are governed by independent, non-profit boards of directors, accountable to the Ministry of Health and Long-term Care.In Ontario, it is now mandatory for all long stay home care clients to be assessed with the MDS-HC. However, at the time of the RAI Health Informatics project, the MDS-HC was not mandated for use. As such, some clients (e.g., those expected to be on service less than two weeks) did not receive an MDS-HC assessment and were not included in the current project.At this same time, Manitoba conducted a pilot implementation of the MDS-HC in the fourteen offices of the Winnipeg Regional Health Authority (WRHA). The WRHA is one of twelve health authorities in the province, and is responsible for providing health services to the approximately 646,000 residents of Winnipeg and the surrounding suburban area. The WRHA receives annual funding from the government of Manitoba. The home care program of the WRHA was established in 1974 with a mandate to provide effective and responsive health care services in the community to support independent living and facilitate admission into LTC facilities when independent community living is no longer an option. Home care services include personal care, nursing, counselling, occupational therapy and physiotherapy assessment, referral to other agencies and coordination of services.In the WRHA region, an MDS-HC assessment was completed by care coordinators only if the client was anticipated to be on service for at least 90 days. As a result, the study sample was focused on a long stay population of home care clients.All participating sites in the WRHA and in Ontario identified a set of case managers who received a two-day training session led by a member of the research team. They then used the MDS-HC instrument as part of their usual in-home assessment.The data used for this study represent a cross-sectional cohort of home care clients assessed between November, 1999 and December, 2002. Two CCACs in Ontario and four WRHA sites, were removed from the database because they submitted fewer than 20 assessments, resulting in a total of ten WHRA and twelve Ontario sites. Of these, three Ontario CCACs and eight offices of the WRHA also submitted client reassessments at approximately 90 days, which allowed for the calculation of failure to improve/incidence HCQIs.The protocol for data collection was reviewed and received ethics clearance through the Office of Research Ethics at the University of Waterloo, Canada.Risk adjustment attempts to adjust for differences in client populations that may bias the HCQI rates. Organizations that provide care to more impaired clients will tend to have higher unadjusted rates, regardless of the quality of care they provide. As such, risk adjustment methods are used to maximize the ability to make fair comparisons across providers. With any type of risk adjustment, caution must be exercised to prevent over adjustment (i.e., adjusting away poor practice). The choice of risk adjustment covariates is therefore highly important and should not include variables that would be considered to reflect suboptimal clinical care.There are two generic types of risk adjustment that have been recommended for the HCQIs. The first adjusts for differences in the population at the client-level. PotentiThe second type of risk adjustment was intended to control for two types of bias at the agency level: a) the agency's ability to identify differences in clients' clinical characteristics and b) differences in who an agency selects for admission. These risk adjustments are performed at the agency level after the individual-level risk adjustments are applied.The Agency Intake Profile (AIP) was used following the methodology outlined by Morris et al., for the MDS 2.0 quality indicators. The AIPMore recently, an alternative form of agency-level risk adjustment has been proposed, to control for potential selection and ascertainment bias. This adjustment employs the Case Mix Index (CMI) associated with the RUG-III/HC methodology. In thisThe process followed for adjusting HCQI rates was the same as that used by Morris et al. in adjusting the US nursing home QI rates. Three sThe final adjusted value can be thought of as an estimate of an agency's HCQI rate if the agency had clients with an average level of risk. This riFor each participating agency, the unadjusted HCQI rates were calculated for all 22 indicators. These rates represent the average rate across all eligible clients for a given agency. For prevalence indicators, the rates were calculated only for clients who had been on service for at least 30 days to avoid penalizing an organization for quality issues that were newly recognized.Several methods were utilized to assess the impact of risk adjustment, both at the regional and at the agency-level. The unadjusted and three adjusted rates were compared between Ontario and the WRHA, to assess the effects at the regional level. At the level of the individual agency, ranks were calculated for each HCQI and a count was created to examine how often a given agency was among those with the four highest rates. Since higher rates on each HCQI were indicative of higher prevalence or incidence of undesirable outcomes, agencies ranked within the four highest rates were considered to be among the "worst performers." The range between agencies with the highest and fourth highest rates was also examined for both unadjusted and adjusted rates to assess the degree of variation among the group of worst performers.The two regions were very similar on average age and sex. The WRHA clients were significantly less likely to have some level of cognitive impairment, as measured by the Cognitive Performance Scale (CPS), compareWhen comparing the two regions, there were statistically significant differences for five of the 22 unadjusted HCQIs .Ontario also had a significantly (p < 0.0001) higher Case Mix Index (CMI) on intake (i.e., the CMI covariate), at 0.84 (95% CI: 0.81, 0.86), compared with the WRHA at 0.70 (CI: 0.68, 0.71).higher in Ontario than in the WRHA. Following the +AIP adjustment, Ontario had a rate that was 1.4% lower than in the WRHA.In general, the risk adjustment process minimized the differences between the two regions compared with the unadjusted rates. For example, the unadjusted difference between regions for the prevalence of disruptive/intense daily pain was 9.1%, however, the difference was reduced to 8.0% with the CC adjustment, 5.7% with the +CMI adjustment and 2.8% with the +AIP adjustment Table . The +AIA summary measure was created to count the frequency with which an agency was ranked among the worst performers. Overall, only three of the ten offices within the WRHA did not change their ranking when rates were adjusted Table . For exaIn Ontario, only CCAC 900 experienced no change in their ranking following risk adjustment Table . In anotExamining only the ranking of agencies may be misleading if very small changes in the actual rate resulted in an increase or decrease in the rank for a given agency. For example, the change in the rate for a given agency could be very small, say 5%, but could result in the organization moving from the fifth highest rate (i.e., fifth worst performer) to the third highest rate. Examining only the ranking tells one little about the actual magnitude of change among the worst performers. Therefore, it is also important to explore the range in the rates across the four highest agencies.The unadjusted and CC adjusted rates had the largest degree of variation between the highest and fourth highest agency, with a mean of 11% across the 19 HCQIs. The +CMI adjustment had the next largest amount of variation at 10% and the +AIP adjustment at 8% Table . These rOntario CCACs exhibited several key differences when compared to the WRHA. Ontario sites had significantly higher unadjusted rates across four HCQIs. However, the degree of variation between the sites was small, with a mean difference across indicators of less than 1%. Ontario as a region had significantly higher AIP values for eight HCQIs, indicating a greater propensity in Ontario to admit clients exhibiting quality issues, and they also had a higher mean Case Mix Index.Overall, the change in the actual rates at the regional level was small following risk adjustment. When risk adjustment was applied, for the client covariates alone or the client covariates with the CMI covariate, there was little effect on the mean difference between the two regions. In each case, the mean difference was positive (i.e., Ontario had a higher rate on average) and less than 1%. The +AIP adjustment, however, resulted in a negative mean difference (i.e., WRHA higher than Ontario on average) across the set of risk-adjusted HCQIs.At the agency level, there was a greater influence of risk adjustment, as assessed by agency rankings across the set of indicators. In general, sites in Ontario benefited from risk adjustment and were less likely to be ranked among the worst performers. The opposite was true within the WRHA. Again, the +AIP adjustment had the largest influence when compared to the other two types of risk adjustment.It is possible that the level of variation between agencies on the AIP covariate was greater than the level of variation between clients on the individual-level covariates. Although a detailed analysis is beyond the scope of this paper, use of a single HCQI example may prove beneficial. When examining the prevalence of falls (an HCQI that showed the largest changes in the agency rankings following risk adjustment), the coefficient of variation (CV) for the AIP value was 23.0. The CV is a relative measure of dispersion about the mean and is calculated by dividing the standard deviation by the mean and multiplying by 100. This CV value was much lower than any of the corresponding CV values for the various MDS outcome scales that reflect differences at the level of the individual client. For example, the CV for the Cognitive Performance Scale was 158.5 and for the ADL Self-performance Hierarchy Scale, the CV was 195.6 (data not shown).Clearly, the +AIP adjustment had the effect of minimizing differences between the individual providers and it also resulted in rates that had less variability, as demonstrated by the drop in the CV value when comparing the unadjusted and +AIP adjusted rates for the prevalence of falls indicator . However, this effect cannot be explained by differences in the variances of the adjusters since the AIP had a lower level of variance than the individual covariates.It is also possible that the AIP covariate serves as a proxy for regional differences in practice patterns. If the Ontario agencies are grouped into four main geographic regions, the AIP value for the falls HCQI ranged from 24.1% to 34.0%. The degree of variation appears modest, but cannot be discounted as one factor in the ability of the AIP adjustment to minimize differences between providers and between regions.Although there continues to be support for the conceptual notion of risk adjustment, the potential to over adjust remains a concern. In a recent publication, Mor et al recognizIn the current project, the AIP covariate represents a continuous, numeric value that can range from zero to one. Thus, it does not simply serve as an agency identifier, but represents the rate among an intake cohort. Furthermore, in the US, Morris et al. determined that the Facility Admission Profile, the conceptual cousin to the AIP, did not substantially improve the risk adjustment models and they did not recommend its use. This decision was not based on a fear of over adjustment, but rather a concern that the FAP added increased complexity without significantly improving the risk adjustment process.inappropriate risk adjustment where variance is due to poor practices  and "over adjustment" due to the excessive use of spurious risk adjusters resulting in suppression of variance. Another possible example of over adjustment is the use of individual level covariates that are too closely related to the quality indicator. For example, one might argue that using dressing of the upper body as a risk adjuster for a QI on dressing the lower body is a form of over adjustment because the dependent variable is represented on both sides of the regression equation.It is also important that to develop a clearer understanding of what is meant by "over adjustment". For example, it would seem reasonable to differentiate between instances of truly This area of research continues to present many challenges. Given the current results and those from the long-term care sector, it appears advantageous to undertake some form of risk adjustment, even though the definitive method may not yet be best understood. Given that these HCQIs are new and so little is known about the risk adjustment process, it seems appropriate that all three type of risk adjustment be considered by researchers and policy makers. The +AIP adjustment represents the most conservative approach and may therefore be most appropriate for public reporting of HCQI results.The assessment of quality of care, and ongoing refinement of risk adjustment methods, is ultimately intended to provide information to different audiences to assist in continuously improving the quality of care. To date, many initiatives in the US have taken place to provide additional information on the quality of LTC and home health services. SimilarSeveral important issues should be addressed prior to moving towards more public reporting of these types of quality data. For example, there needs to be a discussion of the relative importance of the HCQIs. The current project made no attempt to prioritize the indicators, although clearly some would have higher clinical priority than others. To some degree, individual agencies must determine their own priorities. However, a simple system based on prevalence, severity and modifiability may be useful in determining relative importance. For example, pain is a prevalent condition in this population, can be severely debilitating for clients and is clearly modifiable. One might argue that it is of higher importance to address than declining cognitive performance, which is less prevalent and in many cases not modifiable. On the other hand, cognitive decline can have extremely serious consequences for the individualIn addition, a decision is needed regarding the calculation of the HCQIs, in particular, which pairs of assessment are to be used in the calculation of failure to improve/incidence HCQIs and what time period in between assessments is acceptable (e.g., a minimum of 90 or 120 days). There will need to be further analysis to explore the relationship between the length of stay and triggering on the HCQIs. It is anticipated that clients who have been on service longer will show important clinical differences from short-stay clients and would therefore be expected to have differential HCQI rates.Tracking of the HCQI rates over time will be essential to determine the level of stability in the indicators. It would be useful to measure the amount of variation and to assess methods to summarize the rates over time in order to maximize their stability. For example, it may be important to report annual HCQI rates for a given organization or province if in fact the rates are found to be highly variable over shorter periods of observation. A shorter time frame may also lead to unstable rates if the number of observations is small. In this paper, a minimum of 20 observations was required and a decision would also be needed for the minimum sample size for public reporting of HCQIs.Finally, it will be essential to assess possible methods to create summary measures across the set of HCQIs. This is not a simple task and one for which little research has been conducted to date. Previous research has pointed to the lack of correlation between measures of quality, but has also suggested that summary measures would be useful for consumers who would likely prefer less complicated information.Knowledge about the home care sector in Canada remains rudimentary, and our knowledge and understanding of quality assessment in this sector is still in its infancy. The results from the current project serve to enhance the overall understanding of the issues involved in quality assessment and our ability, through the risk adjustment process, to create fair comparisons across providers.RAI, provides an important first step in assessing quality and the variable effects of different types of risk adjustment.Risk adjustment of quality indicators is important to enable fair comparisons across geographic regions or across home care providers. To date, little research has examined the quality of home care services. This project, using a set of HCQIs developed by interThe author(s) declare that they have no competing interests.DD participated in the coordination of the study, assisted in the conceptual design of the study, carried out the data analysis and took the lead in developing the manuscript. JH designed the original study, oversaw the development of the indicators, gave input into the data analysis and development of the draft manuscript. BF was the Co-Principal Investigator in the development of the indicators and provided feedback in terms of data analysis and choice of statistical methods. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Traditionally, developing small molecules for research or drug treatments has been a painstaking enterprise. Drugs work largely by binding to a target protein and modifying or inhibiting its activity, but discovering the rare compound that hits a particular protein is like, well, finding a needle in a haystack. With a specific protein target identified, scientists typically either gather compounds from nature or synthesize artificial compounds, then test them to see whether they act on the target.The birth of combinatorial chemistry in the early nineties promised to revolutionize this laborious process by offering a way to synthesize trillions of compounds at a time. Though molecules still had to be evaluated one by one, high-throughput screening technology could manage up to a million molecules a day. Despite these technological advances, few drugs have emerged from combinatorial chemistry approaches, leaving the promise largely unfulfilled.Another strategy looks to nature as a model. The immune system fights disease and infection by generating billions of antibodies, each primed to recognize a specific pathogen. Antibodies recognize antigens (protein fragments of pathogens) with an exacting specificity that develops through an iterative process. The body first produces a diverse, random collection of antibodies. Every antibody is encoded with a unique DNA blueprint in a B-cell. Antibodies that happen to bind to a pathogen are “selected” to pass on their blueprints: successful binding stimulates cell division, during which blueprints are copied, varied by mutation, and used to create a new generation of antibodies. Specificity is refined over multiple generations.Over the past fifteen years, biologists have developed techniques to recreate this process in a test tube. Today, it's common practice to “evolve” collections of as many as a quadrillion different proteins or nucleic acids to bind a molecular target. These techniques are called molecular breeding, because like traditional livestock and crop breeding techniques, they combine sets of genotypes over generations to produce a desired phenotype. Molecular breeding has so far been restricted only to applications that involve materials encoded by DNA. Drugs produced by conventional synthetic organic chemistry, for example, cannot be bred.PLoS Biology, David Halpin et al. describe a strategy that addresses this limitation. By inventing a genetic code that acts as a blueprint for synthetic molecules, the authors show how chemical collections of nonbiological origin can be evolved. In the first article, Halpin et al. present a method for overcoming the technical challenge of using DNA to direct the chemical assembly of molecules. In the second, they demonstrate how the method works and test its efficacy by creating a synthetic library of peptides (protein fragments) and then showing that they can find the “peptide in a haystack” by identifying a molecule known to bind a particular antibody. The third paper shows how the method can support a variety of chemistry applications that could potentially synthesize all sorts of nonbiological “species.” Such compounds, the authors point out, can be used for drug discovery or as molecular tools that offer researchers novel ways to disrupt cellular processes and open new windows into cell biology. While medicine has long had to cope with the evolution of drug-resistant pathogens, it may now be possible to fight fire with fire. To learn more about the DNA display method described here, see the primer “Translating DNA into Synthetic Molecules,” also in this issue.In a series of three articles in this issue of


The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.When hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Cα-H···O=C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface.The occurrence of weak Cα-H···O=C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition. The Arg-Gly-Asp (RGD) sequence is one of the most easily recognised motifs in molecular biology . DiscovecRGD) [As a consequence of their role in so many fundamental processes, integrin defects have been implicated in many common diseases, from cancer to pathogen invasion. An ability to block a particular integrin-ligand interaction may be a possible route to the control of certain pathological states, hence it is not surprising that some integrins have become attractive targets for drug design. Understanding the molecular bases of the interaction of integrins with their ligands is therefore essential for effective protein-based design of inhibitors or activators of their function. A milestone was reached in 2002 with the determination of the crystal structure of the extracellular segment of αVβ3 integrin in complex with a cyclic peptide containing the prototypical RGD sequence . In thatcRGD) .cRGD-αVβ3 structure and in the structures of αIIbβ3 in complex with ligand-mimetics . Both Hα atoms from the collagen Gly residues are in hydrogen bonding position (Cα–Hα···O > 90°), and their Cα-Hα···O=C hydrogen bonds adopt a three-centred and bifurcated configuration . Positional errors inevitable at that resolution may affect the precision of the fitting of the cRGD and EFB peptides and the accuracy of the hydrogen bonding geometries for both weak and strong hydrogen bonds. For example, a close look at the salt-bridge interactions between the Arg guanidinium group from the cRGD peptide and two Asp side chains on the αV integrin surface (Asp150 and Asp218), shows less than "ideal" hydrogen bonding orientation, especially for Asp150 (not shown). Yet, the accumulated knowledge of hydrogen bonding geometries in high-resolution crystal structures and their significant variability leaves no doubt about the existence of these strong hydrogen bonds and their contribution to the specificity of binding.An obvious 2 hydrogen bond from ab initio quantum calculations [cRGD-αVβ3 and EFB-αIIbβ3 structures respectively, even though the accuracy of the values shown in Table cRGD and EFB peptides and the main chain carbonyl group from Arg216 in the integrin surface bear all the characteristics of Cα-Hα···O=C hydrogen bonding. This observation is consistent with the exceptionally high frequency of intermolecular Gly-Cα-H···O=C hydrogen bonds recently reported in high resolution crystal structures of protein-ligand complexes [A similar level of confidence can be achieved for the Gly-Cα-Hα···O=C hydrogen bond by analysing the metrics of equivalent interactions in high-resolution crystal structures of small organic and organometallic molecules. Figure ulations . Figure omplexes .cRGD-αVβ3 and EFB-αIIbβ3 crystal structures), forming one or two Cα-H···O hydrogen bonds that complement the main metal-coordination and salt-bridge interactions from the Asp and Arg side chains.A simple molecular docking analysis further supports the occurrence of Gly-Cα-H···O=C hydrogen bonds between RGD-containing ligands and integrin binding sites. In a first set of calculations, an RGD tripeptide was docked into the binding sites of both αVβ3 and αIIbβ3 integrins using constrained molecular dynamics (MD). Two constraints were imposed in the docking calculations: the carboxyl group from the Asp residue had to complete the metal coordination on the β3 subunit, and the Arg side chain had to form a salt bridge with appropriate Asp residues in the αV and αIIb subunits (see Methods), as observed in the crystal structures of αVβ3 and αIIbβ3 with different ligand-mimetics. Ten slightly different RGD models were obtained from the NMR structures of the adhesion domain of fibronectin , and wercRGD-αVβ3 and EFB-αIIbβ3 complexes? A quantitative analysis of C-H···O hydrogen bonding at protein-protein interfaces has shown that they have an important contribution to the association and stability of protein complexes, accounting for about one third of the total hydrogen bonding interaction energy [How important are these weak C-H···O hydrogen bonds in stabilising the n energy . In factn energy .N-terminal to the conventional N-H···O=C hydrogen bond in β-sheets [N-terminal to the one carrying the N-H donor group, and both share the same C = O group as acceptor, an arrangement sometimes referred as "bifurcated" hydrogen bond [N-terminal to the one carrying the N-H donor group is Gly, with one or two Hα from Gly being in hydrogen bonding position. The bifurcated hydrogen bond scenario also occurs in the cRGD-αVβ3 and EFB-αIIbβ3 structures, where the N-H group from the Asp residue in the RGD peptide donates a hydrogen bond to the main chain carbonyl group from Arg216 of β3. This hydrogen bond has very bad geometry in the cRGD-αVβ3 structure (distance H···O 2.69 Å, angle N-H···O 133°), but looks better in the EFB-αIIbβ3 structure (distance H···O 2.39 Å, angle N-H···O 144°). These deviations from ideal hydrogen bonding geometry might be consequence of the resolution of the crystal structures, but all the MD docking simulations described above result in N-H···O=C hydrogen bonds that are slightly longer  and slightly less linear  than the average hydrogen bonds between peptide groups in protein secondary structures. Automatic computational docking calculations of known integrin ligands on structural models of αVβ3 and αVβ5 consistently predict this N-H···O=C hydrogen bond to occur whenever an N-H group is present in the proximity of the carboxylate moiety [The recurrent appearance of some weak hydrogen bonding topologies in many structures of proteins and at protein-protein interfaces also reinforces the notion that they have a significant contribution to macromolecular stability. The most common occurrence of C-H···O hydrogen bonds in protein structures is a widespread Cα-H···O=C hydrogen bond β-sheets ,17 and iβ-sheets . In thisgen bond ,27. Thise moiety ,36. Had cRGD and EFB peptides and the αVβ3 and αIIbβ3 surfaces suggests an additional candidate for classification as C-H···O=C hydrogen bond, between the Cβ-Hβ group from the Asp residue of the cRGD and EFB peptides and the main chain C = O group from Asn215 in the β3 subunit. This weak hydrogen bond is adjacent to the stronger, conventional hydrogen bond between the main chain N-H group from Asn215 and Oδ2 from the Asp residue in the RGD motif. Thus, a total of four hydrogen bonds, weak and conventional, aligns the bottom of the cRGD and EFB peptides against the integrin surfaces , PDB accession code 1L5G [cRGD or EFB were also maintained. Hydrogen atoms were built with standard stereochemistry for the cRGD and EFB peptides and for the integrin RGD-binding sites as defined above, using the program REDUCE [The following crystal structure coordinates were downloaded from the Protein Data Bank : αVβ3 inode 1L5G ; αIIbβ3 ode 1L5G ; αIIbβ3 ode 1L5G ; αIIbβ3 m REDUCE . For thecRGD-αVβ3 and EFB-αIIbβ3 structures for guidance. Then each docked model was pulled away to about 10 Å from the integrin surfaces, and was docked back into the integrin binding site via molecular dynamics (MD) simulations using the program CNS [cRGD-αVβ3 and EFB-αIIbβ3 structures. The side chain of the Asp residue group was restrained to coordinate the bound metal ion in the RGD-binding sites and to receive a hydrogen bond from the amide group of Asn215, in the β3 subunit. The side chain of the Arg residue was restrained to form hydrogen bonds with residues Asp150 and Asp218 on the αV subunit or residue Asp224 on the αIIb subunit. Additional restraints were imposed in the MD simulations with the VTGRGDSPAS peptide: the ring of Pro172 was restrained to hydrophobic contact with the side chain of Lys125, in the β3 subunit, and the Cα atoms of the N- and C-terminal residues in the peptide model were restrained not to separate more than 5 Å from each other. The coordinates of the integrin binding sites were kept fixed in all the simulations, and only the peptides were allowed to refine by restrained MD and energy minimisation. All molecular models were analysed with the program CHAIN [For the molecular docking calculations, conformational models for RGD and VTGRGDSPAS peptides were obtained from the NMR structures of the adhesion domain in fibronectin . Ten congram CNS . Five siam CHAIN  in a SilA survey in the Cambridge Structural Database  July 20.cRGD, cyclic pentapeptide with sequence Arg-Gly-Asp-D-Phe-N(Me)-Val; EFB, eptifibatide; PDB, Protein Data Bank; CSD, Cambridge Structural Database; MD, molecular dynamics.ECM, extracellular matrix; RGD, Arg-Gly-Asp sequence; J.B. conceived the study and carried out the analysis of the structural data and molecular docking calculations. Both authors participated in the design, coordination and writing of the manuscript. Both authors read and approved the final manuscript.


Many medical errors occur during the laboratory testing process, including lost test results. Patient inquiry concerning results often represents the final safety net for locating lost results. This qualitative study sought to identify, from a patient perspective, specific preferences and factors that influence the process of communicating normal (negative) laboratory test results to patients.We conducted 30-minute guided interviews with 20 adult patients. Patients were recruited from two practice-based research networks in Colorado that were participating in a medical errors study. A semi-structured interview elicited the participant's experience with and preference for laboratory test result notification. Quantitative descriptive statistics were generated for demographic and preference data. Qualitative results were analyzed by a team of experienced qualitative researchers using multiple styles of qualitative analyses, including a template approach and an editing approach.Ninety percent of participants wanted to be notified of all tests results. Important issues related to notification included privacy, responsive and interactive feedback, convenience, timeliness, and provision of details. Telephone notification was preferred, followed by regular mail. Electronic notification was perceived as uncomfortable because it was not secure. While 65% preferred being notified by a provider, participants acknowledge that this may be impractical; thus, they wanted to be notified by someone knowledgeable enough to answer questions. Participants do not normally discuss their preferences for test result notification with their providers.Privacy, responsive and interactive feedback, convenience, and timeliness with detailed information may be critical for patient satisfaction and for improving patient safety, and are features that may be incorporated into emerging communication channels. Understanding how patients would like to be notified of laboratory test results is important for improving provider-patient communication, patient satisfaction, and patient safety. Data from Applied Strategies for Improving Patient Safety (ASIPS), a primary care practice-based study of medical errors, indicate a high frequency of errors in laboratory testing and patient notification. Other sPatients want to be notified of all test results, regardless of whether the results are abnormal -8 A few This study was conducted within the Colorado Research Network (CaReNet) and High Plains Research Network (HPRN), two primary care practice-based research networks. CaReNet practices are located mostly in urban / suburban cities in Colorado; HPRN practices are in rural northeast Colorado. We intentionally used these settings as the most optimal example of the setting (primary care clinics) in which we would see the need for normal lab result notification. We purposively sampled study participants based on emerging themes from our analysis. Thus, we intentionally recruited participants who were 18 years of age or older, able to speak and understand English or Spanish, and who had at least one laboratory test at a participating practice within the last year. Laboratory tests were defined as blood or urine tests, pap smears, and biopsies. The aim of our strategy was not to maximize generalizability, but rather to understand the context and conditions under which normal lab result notification does or does not occur. We recruited to the point of saturation or replication of data. All finPatients were recruited via posters and business cards placed in 12 primary care practices participating in the ASIPS study. ASIPS was a multi-institutional, primary care practice-based project to collect, codify, and analyze data on medical errors[This study was approved by the University of Colorado Health Sciences Center's institutional review board.We collected the patient's age, gender, race, ethnicity, highest level of education, if they had a permanent home address, and access to the Internet and e-mail. Patients were asked if they had personally received results of a test by message left on answering machine, through phone conversation, mail, e-mail, automated telephone call-up system, or via web-based system. Patients ranked these notification methods by their preference.The interview began with questions about the patient's most recent experiences with test result notification (who, what, when, etc.). The interview then shifted to preferences of notification and patient-doctor discussion about notification preferences. Inquiry also elicited patient factors that may affect test result notification.An in-depth interview provides a narrative understanding of how particular individuals arrive at their experience. Our purpose was to construct a meta-narrative of the interviewees' many stories. This on-going interpretive process informed each subsequent interview in an iterative fashion. For example, during early interviews it appeared that different preferences might be expressed by people with different educational levels. We then stratified our subsequent sample by educational level (more than a high school education vs. high school education or less).We used mixed methodology to analyze the data. Quantitative descriptive statistics (frequencies, proportions, etc.) were generated for our demographic and preference variables. All coding and analysis was done by a three-member team that included a physician, a doctorate researcher experienced in qualitative methodology, and a professional research assistant. The use of a multidisciplinary team approach helped limit any personal biases, subjectivity, and preconceptions as well as enhanced our reflexivity process. A commiIn the end, we found no differences qualitatively or quantitatively across participants' educational levels. Thus, all results are reported as a single group. Most (75%) participants were female  we found no differences in preferences by age groups, educational levels, or access to e-mail and the Internet table . Many paPatients' preferences seemed to reflect their most recently experienced method of notification. Table Important Characteristics of Notification: most notable factors influencing patient preferences in notification; (2) Patient/Provider Discussion: the lack of communication between patients and providers around notification preferences; and (3) Communication Frustration: challenges encountered during attempted communication between the patient and the practice regarding notification of their results. Below we briefly describe these themes with illustrative quotations from the participants.We identified three emerging themes: (1) Important factors that defined patients' concerns around notification were always being notified of results, timeliness, details of the results, responsive and interactive feedback, who should provide the notification, convenience, and assured security/confidentiality.Not surprising, almost all patients responded that they wanted to be notified of all results. The "no news is good news" approach is unacceptable to patients."Obviously there was a reason to have that diagnostic test, so I'd at least like to know whether it was normal or what.""To me, no news is worrisome."Patients wanted to receive their results in a timely manner – shortly after the physician or provider receives the results."Let me know right away. Don't keep me hanging. Do the test on Friday and if I don't know till Tuesday, I want to know Monday morning. As soon as you find out the results, you let me know the results because this is my body."The amount and detail of information is important in providing the context in which the patient can interpret the results."I would like to know what normal means in relation to the general population, so I would certainly like some reference ranges...If there weren't any reference ranges, then I would certainly like to know that a test...came back negative and what negative meant."The test result was only half of the information participants want. They also wished to discuss what the results mean for them. In some cases, patients are left wondering about the "next steps." A patient may feel confused if someone is not available during the notification process who can discuss what the test means."Well, the information on what I can and what I can't do, you know. I mean I don't know what I can do at this point.""The thing I do like is that we can actually talk about the numbers and I can see where it's at because he just gives me, we just look at the sheet together...I like that."Most patients recognize that providers are too busy to attend to all normal test results. They were comfortable having someone else notify them, but preferred that this person be knowledgeable enough to answer questions. Many patients indicated that notification by receptionists, who they felt were not knowledgeable enough to answer questions, is unacceptable. When asked specifically the preferred role of a notification person the responses were: 65% provider and 15% nurse; 20% were not concerned with who notifies."Ok, this test result is normal but I still have this pain, what alternative do I have now. Where do I go from here and a physician would be the best person to be able to explain it to me, rather than a physician assistant or a nurse or an administrator or something like that.""Well, it doesn't have to be the actual doctor. It could be the RN...Somebody who knows about what is going on and if I have a question I could ask that person."Patients identified convenience as important to their satisfaction. Calling the office for results can mean long waits to reach a person who notifies them of their result and then longer waits when the results prompt a patient question that cannot be immediately answered."That way (using web-based system) I could do it at anytime and it seems more personal and confidential to me because...you can draw up that information at any time when you're ready.""That's why the mail is so good because I always get the mail everyday."The most persistent issue we uncovered was a patient's privacy and assured confidentiality of test results and diagnoses. Participants seemed hesitant to experiment with alternative notification methods (i.e., web-based methods) if they perceived a possibility of a breach in this trust."Who wants the public to read what their values are?...Even if it's normal, I'd rather have it personalized in a sealed envelope."Most patients assumed that their office used a specific system for notification; however, they were not aware of the details of this system. Often patients were told to call for results if not received within specified time, leaving the patients to close the feedback loop. Other patients were told to rely on "no news is good news," although they were uncomfortable with this policy.The majority of patients indicated that they had not discussed their notification preferences with their provider."No, I haven't. I just assumed that was normal and customary procedure...That they only notify you if something is wrong.""I didn't know they were just going to send me a letter. In the past, I've had the doctors call me. Well, they automatically call [ed]."Two areas of frustration related to communication were identified: lack of follow-through and confusion within the office.Communicating how the provider will notify the patient is important, but equally important is follow-through. Unmet expectations result in patient frustration."I told him that he could call me, or the nurse. Either one would be ok, but...it doesn't happen. Even though I asked them face to face, it still hasn't happened."Patients are also frustrated and worried when they try to complete the feedback loop, but find that the practice is unable to provide them the information they need."I have to sometimes call in to find out about my results...I wasn't actually notified of the results...so obviously it was my responsibility. I felt it was my responsibility anyway. But...they [the results] seemed to have [been] misplaced. The parties concerned didn't seem to be aware that I had had a particular test and so they couldn't provide me with information...It wasn't until I went back recently and asked about that result, that I was told it was normal."Our results reinforce other literature suggesting patients want timely and detapersonalized to their situations.A number of our findings, however, suggest that more than preferring a particular channel of communication, patients prefer a specific manner of communication, features of which could be incorporated into newer communication channels. For example, our participants overwhelmingly preferred a personal telephone call – but only from someone who was knowledgeable enough to answer their questions. They wanted responsive and interactive feedback, Many of the patient identified characteristics of notification could be more easily incorporated into a system that allowed for asynchronous communication (communication that doesn't rely on immediate person to person transfer of information.) Nonetheless, few of our participants were willing to try the computerized asynchronous communication methods we asked about: an automated telephone call, e-mail, or web-based system. This finding is similar to others, althougOur patients emphasized the importance of receiving results in a timely fashion. Timeliness is a critical feature of notification systems that can significantly affect patient safety; timely recognition of mishandled or misplaced results will increase the practice's ability to correct or mitigate an error. However, timeliness is not an inherent feature of patients' preferred channel of communication: a telephone call. In our experience, providers' and patients' hectic schedules often mean it can take days for successful telephone contact between providers and patients. Again, our patients' focus on timeliness suggests that they are not wedded to the concept of telephone communication; rather, they prefer the perceived timeliness of communication by telephone. Similarly, our participants indicated that convenience was important to them. Again, waiting for a telephone call is not intuitively convenient, though the dramatic increase in cell phones may help alleviate this problem. Perhaps our study participants could not envision a convenient communication method other than phone calls that provide secure, personalized, interactive communication.A traditional mail-based system (a low-tech asynchronous communication system) was ranked second highest among preferences for notification. This finding detracts from our assumptions that patients focus on notification methods that are timely and interactive; however, it supports our idea that patients want convenient, private, personalized information. Perhaps the three patients who preferred mail notification were more concerned with privacy and convenience than with timeliness and interactivity. Further study is needed to elucidate these findings.Finally, we found that patients do not discuss with their provider their preferences for notification. Our patients indicated that it never occurred to them that their health care provider lacked a standard procedure for communicating test results to patients. Our findings may be a manifestation of poor communication between patients and providers, which has been shown to be a related to poor patient outcomes and safety issues. More stA potential limitation of this study relates to the recruitment, which was limited to patients who had access to call our research line after seeing an advertisement. However, qualitative inquiry rarely uses random sampling. Rather, samples are selected more purposefully and not by the need to generalize or predict but by a need to create deeper understanding or meaning. Thus, sThe results of this study provide us with a better understanding of how patients experience notification of laboratory tests within the primary care setting. Notifying patients of test results is important for laboratory information management, and ultimately, patient safety. We believe patients can play an important role in ensuring that laboratory tests results are obtained and reviewed by providers. Asking for all laboratory tests results is a recommended strategy for imprThe author(s) declare that they have no competing interests.All authors contributed to the study design and conceptualization, discussed the drafts of the paper, and read and approved the final manuscript. Additionally: DB developed the study idea and conducted the data analysis. JQ conducted the interviews. CD provided technical assistance in protocol development and data analyses, and contributed to the development of the manuscript. ES contributed to the development of the manuscript. WP obtained funding for the study, helped develop the study methods, and contributed to the development of the manuscript.The pre-publication history for this paper can be accessed here:


Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules.Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals.This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants. The discovery of cyclic 3'5'-adenosine monophosphate (cAMP) by Earl Sutherland in the late 1950s was one of the most significant paradigm shifts in biochemistry .The ubiquitous presence of cyclic nucleotides may be due to several characteristics which make it an ideal second messenger. Cyclic nucleotides are derived in a energetically favourable reaction from common metabolites (ATP and GTP), and can be broken down into non-toxic products (inorganic phosphate and AMP/GMP). The synthesis and degradation of cyclic nucleotides are controlled by enzymes termed adenylate (or guanylate) cyclases and cyclic nucleotide phosphodiesterases, respectively ,4.In plants, cyclic nucleotides have endured a checkered research history fraught with complications and setbacks. Despite this, recent work has shown unequivocally that cyclic nucleotides are present in plant cells ,6, and tCyclic nucleotides are able to bind to two distinct protein domains, CNB domains and GAF domains. CNB domains were first identified in the regulatory subunit of mammalian cAMP-dependent protein kinase (RI and RII). Since several CNB domain containing plant proteins have been shown to be directly modulated by cyclic nucleotides, this indicates that the CNB domain in plants is functionally similar to CNB domains in other organisms -15.Anabaena cyclic nucleotide stimulated adenylate cyclase and several other proteins [GAF domains were initially identified as conserved domains in light sensing molecules but are known as small molecule binding domains in cyclic nucleotide regulated phosphodiesterases, the proteins . GAF domproteins  and cGMPproteins ,19. Receproteins ,20. TherArabidopsis thaliana and Orzya sativa genomes [In order to further explore the roles of cyclic nucleotides in plants, we performed a bioinformatics based analysis of the completed  genomes -23 in orAnabaena adenylate cyclase shows that these residues may only be strictly conserved in mammals. Further complicating our analysis is the fact that GAF domains are known to bind other small molecules such as 2-oxoglutarate, formate and bilins [Based on the PDE2 crystal structure 11 residues were proposed to be involved in cyclic nucleotide binding , but comd bilins -26. GAF Phytochromes are light sensing signal transduction molecules which function to control several aspects of plant biology. Interestingly phytochromes were found to function upstream of cyclic nucleotides in their signal transduction pathways since their functions can be mimicked by cGMP and calcium in phytochrome knockout cells -29. The Ethylene responses have been documented for nearly a century in plants. This gaseous hormone is involved in many aspects of plant physiology, including fruit ripening, organ development, germination, seedling growth, flowering and response to challenges such as pathogens and stress . There ain silico model. We chose the plant protein which showed highest similarity to known crystal structures  and based our model on the solved crystal structures of RIα, RIIβ, HCN2, CAP and Epac2 . Connecting these sheets are exposed loops, the most important of which is the phosphate binding cassette . It is iThe hydroxyl and amide groups of Ser 92, as well as the hydroxyl group of Tyr 91 are all within proximity of the cNMP phosphate and could play a role in stabilizing the cyclic nucleotide , long distance transport (AKT2), transport into growing pollen tube (AKT6), secretion into xylem sap (SKOR) and transport during guard cell opening either into the cell (KAT1, KAT2) or out of the cell (GORK) [Plant potassium channels fall into two classes, the KCO channels and the shaker-type channels . In addil (GORK) .Shaker-type channels are voltage dependent outward (GORK, SKOR) or inward (KAT and AKT) rectifying channels. Analysis of heterologously expressed channels have shown that cyclic nucleotides function to adjust the activation potential of these channels ,12. SincE. coli [Initially we detected a short CNB containing protein which was only slightly larger than the domain itself. Sequencing of the EST provided by the Arabidopsis Biological Resource Center  showed tE. coli .E. coli, however after extensive trials we were unable to express and purify soluble protein.Fatty acid synthesis requires the use of acyl-CoA's as building blocks for incorporation into lipids. It is therefore possible, that these thioesterases function as scavengers which remove "irregular" fatty acids from the pool of available building blocks . FurtherWe found no PKG or PKA regulatory subunit homologs in the Arabidopsis genome. There has been a long standing controversy in the plant field as to the existence of a plant cyclic nucleotide dependent kinase -52. As PArabidopsis thaliana using the PKA substrate Kemptide. Kemptide is a peptide which has a motif which was confirmed as the optimal substrate for PKA [A. thaliana extracts with polyclonal antibodies raised against mammalian PKA subunits  reveals that no structurally similar proteins are present in this extract  all cyclic nucleotide binding proteins had been previously identified indicates that the previously attained biochemical data agrees with our bioinformatic evidence.The identification of no transcription factors or protein signal transduction molecules with CNB domains implies that cyclic nucleotides may be unable to directly modify the proteome of plant cells. This is in stark contrast to bacterial, yeast and mammalian systems. The only common domain context of CNB domains in animals and plants is the CNGC channels, however, even these channels appear to have evolved independently ,59. TherE. coli CAP [pir: E86000]). This yielded no new inclusions to our list of proteins, but did confirm each of our previous entries. For examination of the Oryza sativa spp. Japonica genome we performed BLAST searches using the aforementioned baits, as well as each of the Arabidopsis proteins. This search was performed using the Blast utility of the TIGR rice database [Orzya sativa, we named them so that they agreed best with the nomenclature of Maser et al. [In order to identify the proteins which contain CNB or GAF domains, we initially used the Simple Modular Architecture Research Tool  to sdatabase . The crir et al.  [see addr et al. , 2, 3. Sr et al.  or T-COFr et al. and then r et al. , then wer et al. . Trees gOne protein, which appeared to contain only a cyclic nucleotide binding domain and no other motifs was found in the Arabidopsis database. We obtained the clone corresponding to this putative gene from the Arabidopsis Biological Resource Center and sequenced it. Sequencing was performed at the University of Calgary Core Sequencing Facility. We determined that the gene prediction algorithm which scanned the genome improperly predicted the intron/exon structure of this gene. The new gene, which we named cyclic nucleotide regulated thioesterase 1 (atCNTE1) was deposited in the NCBI database [GenBank: AY874170]. A subsequent BLAST search using this gene found another isoform of this gene in Arabidopsis (atCNTE2) and one isoform in Rice (osCNTE1) which we also included in our analysis.We generated a model of the atCNTE1 cyclic nucleotide binding domain (residues 28–117) based on an alignment of atCNTE1 with the CNB domains of RIIβ [pdb: 1CX4] , RIα pd, CAP pd, HCN2 p and Epac32P-γ-ATP (Amersham-Pharmacia), 30 μM Kemptide substrate, 50 mM HEPES pH 7.4, 1 mM dithiothreitol and 10 μM cyclic nucleotide as specified. Reactions were allowed to occur for 10 minutes at 30°C and assays were terminated by spotting onto squares of P81 paper followed by extensive washing with 75 mM phosphoric acid [Unless otherwise indicated all chemicals were purchased from Sigma-Aldrich. Assays were performed on extracts of Arabidopsis cells grown in suspension culture  or isolaric acid . Assays ric acid .Bovine heart PKA catalytic subunit and RII were purified to homogeneity ,79. PuriCAP catabolite activator proteincAMP 3'5'-cyclic adenosine monophosphateCGK2 cGMP dependent protein kinase 2cGMP 3'5'-cyclic guanosine monophosphatecNMP 3'5'-cyclic nucleotide (cAMP or cGMP)CNB cyclic nucleotide bindingCNGC cyclic nucleotide gated channelCNTE cyclic nucleotide dependent thioesteraseEpac exchange protein directly activated by cAMPGMP-phosphodiesterases, adenylyl cyclases and FhlAGAF domain found in cGORK guard cell outward rectifying potassium channelPKA protein kinase ASKOR stellar potassium outward rectifying channelSMART simple modular architecture research toolDB performed the bioinformatic analysis, modeling and biochemical studies and drafted the manuscript. MEF participated in the modeling. GBG conceived of the study and participated in its design and co-ordination. All authors have read and approved the final manuscript.Co-ordinate file of model generated for Figure Click here for fileArabidopsis thaliana and Oryza sativa. Phylogenetic analysis of CNGC and shaker-type channels from Un-rooted neighbor-joining trees were constructed for (A) shaker-type and (B) CNGC channels using full-length sequences. For the shaker-type channels, numbers at nodes indicate number of trees out of 100 in which the node occurred. These are omitted in (B) for clarity. Scale indicates number of differences per residue. Trees were generated using ClustalX [ClustalX  and visuClustalX .Click here for fileRelevant Sequence Alignments Protein sequence alignments upon which phylogenetic analyses in Figure Click here for fileArabidopsis thaliana and Oryza sativa. Cyclic nucleotide binding proteins in Proteins containing CNB domains including species, name, aliases, accession number, other domains present , sequence length, residues encompassing the CNB domain and E-value for the CNB domain as determined by SMART [.by SMART . All accClick here for fileProtein sequences of all sequences analysed in this manuscript. Sequences are named according to details in the text and are in FASTA format.Click here for file


Direct admission to Coronary Care Unit (CCU) on hospital arrival can be considered as a good proxy for adequate management in patients with acute myocardial infarction (AMI), as it has been associated with better prognosis. We analyzed a cohort of patients with AMI hospitalized in Rome  in 1997–2000 to assess the proportion directly admitted to CCU and to investigate the effect of patient characteristics such as gender, age, illness severity on admission, and socio-economic status (SES) on CCU admission practices.Using discharge data, we analyzed a cohort of 9127 AMI patients. Illness severity on admission was determined using the Deyo's adaptation of the Charlson's comorbidity index, and each patient was assigned to one to four SES groups (level I referring to the highest SES) defined by a socioeconomic index, derived by the characteristics of the census tract of residence. The effect of gender, age, illness severity and SES, on risk of non-admission to CCU was investigated using a logistic regression model (OR, CI 95%).Only 53.9% of patients were directly admitted to CCU, and access to optimal care was more frequently offered to younger patients , those with less severe illness  and the socially advantaged .In Rome, Italy, standard optimal coronary care is underprovided. It seems to be granted preferentially to the better off, even after controversial clinical criteria, such as age and severity of illness, are taken into account. The Italian National Health Service is supposed to provide universal coverage of standard care to all citizens, no social or economic selection bias should limit access to high technology resources as far as they are available.Direct admission to Coronary Care Unit (CCU) on hospital arrival can be considered as a good proxy for adequate management in patients with acute myocardial infarction (AMI) because it has been associated with better prognosis and shorter hospital stay ,2.Timely access to advanced diagnostic and therapeutic options, thorough cardiovascular monitoring, provision of primary angioplasty or thrombolytic therapy when indicated, and prompt defibrillation when necessary all contribute to favorable outcomes.With regard to reperfusion therapies, they have been shown to be effective in reducing short and mid-term mortality in patients with ST-segment elevation AMI -8. The tIntensive coronary care should be administered without delay, ideally within 60 minutes of the onset of symptoms -14.Previous studies have investigated the role of demographic characteristics (such as age, gender and ethnicity), clinical characteristics (such as time from symptoms, presence of ST elevation and Killip class at presentation), and hospital characteristics (such as location, teaching status and level of invasive capability) on the probability of being admitted to CCU and the probability of receiving initial thrombolysis ,15-18, bWe used discharge abstract data, routinely collected by the regional Hospital Information System (HIS), to identify a cohort of patients with AMI , aged 18 years of age or more, residing in Rome, admitted to one of the 11 city hospitals equipped with an emergency department and a CCU, and surviving the Emergency Room, from 1 July 1997 to 31 December 2000.From the above defined cohort, we then excluded:• patients who had been hospitalized for AMI in the previous six months, identified through record linkage within the HIS file,• patients transferred from other acute care facilities,• ruled-out AMI ,• episodes of care with a diagnosis of trauma, with an important surgical operation, or with DRG non compatible with an AMI diagnosis.We categorized patients into five age-intervals: (less than 50, 50–64, 65–74, 75–84 and over 85 years). The Deyo's adaptation of the Charlson's comorbidity index  was calcFor each patient, vital status 30 days after hospital admission was obtained through an automatic record linkage with the Municipal Registry of Rome.The main outcome measure was the indication of CCU as admission ward in the discharge abstract. For the purposes of this study we considered admission to Intensive Care Unit (ICU) in hospitals equipped with CCU equivalent to direct admission to CCU, because both represent an intensive care for AMI patients.t test.As a first step, a logistic regression analysis was performed in order to confirm the association between direct admission to CCU and 30 days mortality. after adjusting for patient characteristics (gender, age, SES, Charlson's index) and admitting hospital (ORs and 95% CI). Moreover, we compared the average length of stay among patients admitted to CCU and among those admitted to other wards, after excluding deceased and transferred patients, using the Student's We then assessed the extent of differences among Rome hospitals with respect to the number of AMI patients admitted, the size of CCU, and the proportion of AMI patients directly admitted to CCU. For each hospital the ratio between AMI patients admitted and number of CCU beds was calculated as a proxy of the pressure on CCU resources.The effect of personal characteristics, i.e. gender, age, illness severity and socio-economic status, on risk of non-admission to CCU was then investigated using a logistic regression model. Since important differences among hospital rates of CCU admission had been found, and we wanted to take into account the possible effect of patients clustering by hospital, we used a random effect model with admitting hospital as clustering variable.The area under the receiver operator characteristics (ROC) curve was estimate as a measure of the overall predictive ability of each model, while the Hosmer and Lemenshow (H-L) statistics was used to assess models' calibration.The possible effect modification of gender on the other variables was tested by forcing interaction terms in the multivariate models.All statistical analyses were performed using STATA version 7.0 .Of 9127 patients hospitalized with an incident AMI diagnosis , 53.9% were directly admitted to CCU. The crude 30-day mortality was 13.2% in patients admitted to CCU and 25.0% in those admitted to other wards. The protective effect of CCU admission on 30-day mortality remained strong after adjusting for potential confounders . Moreover, among 3765 AMI patients admitted to CCU or ICU, and discharged to home, the length of stay was significantly shorter than among those admitted to other wards (12.2 versus 14.4 days, p < 0.0001).a  to 87.5% in hospitals l . The ratio between AMI patients admitted and number of CCU beds varied from 37 to 166. This two indicators were not slightly correlated (r = 0.52). Patients characteristics with respect to CCU admission status are presented in table The CCU admission proportion varied widely among hospitals Table , from 27In the bivariate analysis it emerged that lower odds of being directly admitted to CCU were associated to female gender, older age, and higher Charlson's index, while we found no difference with respect to SES level  influence the physician referral decision, we think that, if the percentages of non-ST segment MI in the groups under study are the same, our results should not strongly be biased.We used a small area-based SES index, because direct individual data on social class were not available. This index has been shown to be a strong predictor of differences in mortality,  and assoAge and admitting hospitals were the variables responsible for a negative confounding effect on the association of socio-economic status with direct CCU admission. Patients with low SES levels are younger than patients with high SES levels and tend to be admitted to hospital with higher provision of CCU care.In Rome, where high-technology resources are available, they do not seem to be efficiently and fairly used. Our results suggest that access to optimal care is offered selectively to the socially advantaged, as well as to younger patients (even well under the 75 years threshold) and to those with less severe illness. The National Health Service, its policy of unrestricted access notwithstanding, falls short of providing equal opportunities to all citizens. Delivery of effective services to the underprivileged should be actively promoted.The author(s) declare that they have no competing interests.CA conceived of the study, participated in its design, planned the statistical analysis, drafted the manuscriptMA participated in the study design and coordination, drafted the manuscriptCS participated in the study design, drafted the manuscriptNA participated in the study designDF performed the statistical analysisVT assisted in data managementCAP conceived of the studyThe pre-publication history for this paper can be accessed here:


The design of a randomized multicenter trial is presented on the effectiveness of a prolonged conservative treatment strategy compared with surgery in patients with persisting intense sciatica .Patients presenting themselves to their general practitioner with disabling sciatica lasting less than twelve weeks are referred to the neurology outpatient department of one of the participating hospitals. After confirmation of the diagnosis and surgical indication MRI scanning is performed. If a distinct disc herniation is discerned which in addition covers the clinically expected site the patient is eligible for randomization. Depending on the outcome of the randomization scheme the patient will either be submitted to prolonged conservative care or surgery. Surgery will be carried out according to the guidelines and between six and twelve weeks after onset of complaints. The experimental therapy consists of a prolonged conservative treatment under supervision of the general practitioner, which may be followed by surgical intervention in case of persisting or progressive disability. The main primary outcome measure is the disease specific disability of daily functioning. Other primary outcome measures are perceived recovery and intensity of legpain. Secondary outcome measures encompass severity of complaints, quality of life, medical consumption, absenteeism, costs and preference. The main research question will be answered at 12 months after randomization. The total follow-up period covers two years.Evidence is lacking concerning the optimal treatment of lumbar disc induced sciatica. This pragmatic randomized trial, focusses on the 'timing' of intervention, and will contribute to the decision of the general practictioner and neurologist, regarding referral of patients for surgery. One of the greatest advantages of publishing the design of a randomized controlled trial (RCT) before results are available is the accessibility to criticism of the methodological quality irrespective of the results. Firstly the scientific reader must be enabled to search for epidemiological shortcomings when the results differ from the expected outcome as compared to results in line with one's expectations. Secondly, it is possible to more extensively elaborate the background and rationale of the research question, the study population, the chosen treatments and outcome measures, as compared to publications describing the trial results. Thirdly, but not less important, publishing the design of a RCT is instrumented in preventing publication bias in subsequent meta-analyses. Studies with non-significant results are less likely to be published than those with significant results ,2. It isThe lumbosacral radicular syndrome  is typically characterized by radiating pain in the dermatome of a lumbar or sacral spinal nerve root. Occasionally more than one root is involved. Contained in the syndrome pain may be accompanied with lumbar fixation, reflex abnormalities motor and sensory disturbances. In diagnosis includes stenosis of the spinal and/or root canal, infection, multiple sclerosis, autoimmune or metabolic neuropathy, and tumour. This study will be restricted to herniations at the lowest three lumbar disc levels, since these represent the most common sites. In the vast majority of cases LSRS is the result of a herniated disc. In the Netherlands annually between 60,000 and 75,000 new cases of LSRS are diagnosed by the General Practitioner (GP) . The preThe natural history of LSRS is in general favourable. In 60–80 percent of patients, the leg pain decreased or disappeared within 6–12 weeks after onset -9,51. ThSince the first publication on lumbar disc surgery by Mixter and Barr  many stuThe only study, which compared surgery with conservative care directly in a RCT, was performed by Weber more than 20 years ago ,8. He foSince 1983 a few cohort studies have been published on non-surgical treatment of patients with at least six weeks of leg pain with good short-term results at one-year follow-up ,22. ThesWhether particular demographic findings, symptoms, physical signs and/or MRI findings either separately or combined do have prognostic value has not been investigated scientifically yet. It would be of great value if one were able to identify early in the course of the disease those patients who will have an unfavourable outcome without surgery.In spite of the known favourable natural course the surgical rate in the Netherlands is quite high . We perfBecause of the observation that most people recover from their complaints in the first 6–8 weeks ,51 this It is not known whether the relative high rate of disc surgery in the Netherlands is cost-effective or not, compared to other countries ,16.In summary, consensus is missing on the preferred timing of disc surgery, due to insufficient evidence that a prolonged conservative care strategy is effective. More insight is needed into the potential short-term effects of a relative early surgery strategy, as compared to an extended wait-and-see period. In particular the effects on the return to work or resumption of previous daily activities as well as the complications of both strategies have not yet been investigated.The main goal of this comparative study is to investigate whether the completion of a 6–12 weeks period of lasting radicular pain constitutes a solid indication for surgery and is superior to prolonged conservative care. A secondary goal is to identify possible subgroups of patients who will substantially benefit from one of the proposed treatment strategies. The cost-effectiveness results will be a trade-off between a quicker relief of leg pain in the surgery group versus the advantage of lower costs and avoiding the negative effects of surgery in the conservatively treated group. The difference in disease related quality of life depends on the duration of persisting pain and disability after randomization in the prolonged conservative care group.This study to investigate this scientific gap in our understanding of the effectiveness of surgery for LSRS is in line with a recommendation by the Dutch Health Council in 1999 to the Minister of Health  and the The results of this trial will lead to a more rational use of the existing guidelines if the hypothesis is rejected. If the latter is accepted and prolongation of the conservative treatment policy is more cost-effective than surgery after 6–12 weeks, the current guidelines for the timing of surgery need correction.To answer the main research question the investigators propose to conduct a multi-centre comparative randomized clinical trial with parallel group design. The main research question will be answered after a follow-up of six months Figure . The comAll patients between 18 and 65 years with sciatica of less than 12 weeks duration are eligible for this study. Because of the multi-centre  design the patients in a large region in the western part of the Netherlands can be included in this trial if they meet the in- and exclusion criteria  will register the MRI findings. The MRI will be performed according to a standardized protocol and including Gadolinium series for the intended subgroup analysis.Patients will randomly be allocated to either surgery within 1–2 weeks or prolonged conservative treatment by their GP. Patients, their doctors and research nurses can obviously not be blinded for the allocated treatment. Blinding of the outcome measurements is not possible, due to the fact that mainly self-reported outcomes are used. A randomization list is prepared for every participating hospital. Permuted blocks of random number patients are formed to ensure near-equal distribution of patients over the two randomization arms in the hospitals. Using random number tables generates the random sequence of the permuted blocks. The data manager, who is not involved in the selection and allocation of patients will prepare coded, sealed envelopes containing the treatment allocation. During the second patient visit the research nurse will open the envelope together with the patient and appointments will be made for the allocated treatment, either surgery or referral back to the GP, to ensure that treatment is started as soon as possible after randomization. This will be done after checking all the criteria and especially the persistence of pain with disability in daily functioning. A letter about the allocated treatment arm informs all caregivers. Although the principal investigator will not include and operate upon trial patients he may be biased with a preference for surgery, which could theoretically influence analysis. Therefore the principal investigator is blinded for the allocated treatment. As he is not involved in treatment of the study population blinding during later analysis is only possible after blinding during the randomization and follow-up period.After randomization two groups of patients will exist.Group A; the surgically treated patients and group B; the conservatively managed patients.(A) will be performed in the conventional manner with microscope or loupe magnification. The investigators prefer the standard surgical approach because the other  surgical approaches have limited indications, are not more cost-effective, and have a long learning curve. During the transflaval approach care is undertaken to minimize bony removal and on the other hand to prevent overstretching of the compromised nerve root. In addition to removal of herniated disc material as much as possible nuclear material will be removed with pituitary forceps, curettes and rongeurs in order to prevent recurrence. The participating treating doctors are 2 orthopaedic- and 12 neurosurgeons with large experience in the standard approach with loupe magnification or microscope. A standardized CRF will register the findings of the surgeon and the herniated disc material will be investigated histologically for granular infiltration.Surgery will take place as soon as possible and within a maximum of two weeks after randomization. Hospital admission will be 2–7 days, including the day of surgery. During the immediate post-operative period the patients will be mobilised with the help of a physiotherapist. At home guidance is confirmed by their own physiotherapist. The frequency will be 2 times a week for 8 weeks.(B) will be conducted by the general practitioner (GP) or neurologist when necessary. The GP will provide ample information about the favourable prognosis of LSRS. The treatment of LSRS is aimed primarily at pain relief and maintenance/restoration of normal day-to-day activities. Unfortunately, the effect of giving information and counselling has not been studied specifically among LSRS patients. However, various studies have evaluated the effect of such support for people suffering from other pain syndromes . InferenAfter the first consultation the GP will make a follow-up schedule. During the next visit the patient and doctor will look at the changes since the first visit to determine whether there is any improvement in the ability to perform normal activities. The doctor will check the efficacy of the prescribed pain medication and may adjust the dose or sort of analgesics according to the NHG guidelines. In these guidelines paracetamol is the first choice. If not effective, NSAID's (ibuprofen, diclofenac or naproxen) are to be prescribed. Only in the event of severe disabling pain morphine may be given for a restricted period of time. By preference all analgesics should be taken at fixed times of the day rather than on a 'if necessary' basis. If the GP and the patient conclude that there is considerable kinesiophobia because of the fear that the radicular or low back pain will increase, the help of a physiotherapist can be recommended. Guided by the GP (and physiotherapist) the patient will upgrade his or her activities according to the agreed time schedule ,26. The In the LSRS the most common complaints are pain and disability to perform normal daily activities. We will use below described validated outcome parameters, which will be assessed by means of questionnaires. Patients are not informed about their earlier scores. Follow-up examinations by the research nurse will take place 8, 26 and 52 weeks after randomization and the patients will keep a diary (table 1) Roland Disability Questionnaire for Sciatica.' This illness-specific 23-item functional assessment questionnaire is frequently used for low back pain and sciatica ,39. Scor2) Perceived recovery.' This is a seven-point Likert scale measuring the perceived recovery, varying from 'completely recovered' to 'worse than ever'. This outcome scale has been used in previous studies and appears to be valid and responsive to change . Next to3) VAS pain in the leg. This parameter will measure the experienced intensity of pain in the leg during the week before visiting the research nurse. Pain will be assessed on a horizontal 100 mm scale varying from 0 mm, 'no pain in the leg', to 100 mm, 'the worst pain ever'. Patients do not see the results of earlier assessments and will score the pain experienced at the visit. -32.1) EuroQol classification system and VAS rating personal health. A cost-utility analysis will be performed using QALY's based on the EuroQol questionnaire, which has been validated in many studies and is easy to fill out ,42,51. T2) Short-Form 36 (SF-36). Quality of life was also assessed using the RAND-36 questionnaire. This is a generic health status questionnaire, which can easily be filled out at home. The questionnaire consists of 36 items on physical and social functioning has 8 domains; 1) physical functioning, 2) physical restrictions, 3) emotional restrictions, 4) social functioning, 5) somatic pain, 6) general mental health, 7) vitality, 8) general health perception. This questionnaire has been used frequently and was validated in studies on low back pathology and surgery -37. From3) Sciatica Frequency and Bothersome Index (SFBI). This is a scale from 0 to 6, which can assess the frequency  and bothersomeness (0 = not bothersome to 6 = extreme bothersome) of back and leg symptoms. The sum of the results of four symptom questions yields both indexes, ranging from 0 to 24: leg pain; numbness and/or tingling in the leg; weakness in the leg or foot; pain in the back or leg while sitting. .5) PROLO-scale. This scale measures the evaluation of the research nurse of the functional-economic status of the patients. This parameter has been used in studies on the difference in functional outcome between different techniques of lumbar spine fusion .6) VAS pain in the back. This parameter measures the intensity of the pain in the back experienced during the week before visiting the research nurse. Assessment will be based on a horizontal 100 mm scale varying from 0 mm, 'no pain in the back', to 100 mm, 'the worst pain ever'. Patients do not see the results of earlier assessments and will score their pain during the visit. This parameter is included because a lot of patients with LSRS also have back pain in varying intensities, which can change after surgery or conservative treatment.1) Costs. The societal costs during the first year will be estimated in accordance with the recent pharmacoeconomic guideline ,48. The Patients will register other health care needs in a diary . Each diary covers a period of 3 months and will be discussed with the patient during the follow-up visits to the research nurse. The volume of health care will be assessed using standard prices .In the diary the patient will also register direct non-medical costs (including time costs, travel expenses and domestic help). To estimate productivity costs the patients will also report absenteeism in the diary. At the follow-up visits, the research nurse will register the work situation, work efficiency and gross wages. Absenteeism will be valued according to the friction-cost method.2) Incidence of (re-) surgery. One of the goals of the policy for group B is to avoid surgery while achieving at least the same effects. The surgical rate is therefore an indication of the success or failure of this policy. The incidence of re-operation at the same disc level in group A will be an indication of the failure rate for surgery.3) Side-effects or complications that are ascribed to the treatment are recorded by the patients, their treating physicians and the research nurses.4) MRI findings. The results of the differences between the baseline MRI and the MRI made 52 weeks after randomization are important secondary outcome measures. The difference in size of the disc herniation (in mm), nerve root compression, and amount of scar tissue will be registered. Failures of surgery can be recognized by inadequate disc removal or decompression of the nerve. The data will be gathered, using a standardized CRF, which will be filled out by the local radiologist, orthopaedic- or neurosurgeon and (neuro-) radiologistThe result of this study is based on the short-term success of surgical intervention and will be a trade-off between a quicker relief of leg pain versus an advantage in cost-effectiveness for conservatively managed patients. The sample size is calculated on the basis of the Roland Disability Questionnaire for Sciatica averaged during the 12 months follow-up period. The numbers used for this sample-size are drawn from the Maine Lumbar Spine Study 1 year and recently published 5-year results ,55. The Baseline comparability will be investigated by descriptive statistics to examine if randomisation was successful. Differences in success rates between both groups are calculated, together with 95 per cent confidence intervals. In addition to an analysis of the difference in recovery between the two groups (as explained under the paragraph sample size) analyses of the difference in time to recovery will be carried out. Due to lack of data in the literature we could not base our sample size calculations on these differences. Survival-analysis is used to calculate differences in median time to recovery. Continuous outcomes are evaluated as change scores (differences between baseline measurement and each follow-up measurement). Multivariable analyses are performed to adjust for the eventual differences between the groups at baseline in prognostic indicators. All the analyses are performed according to the intent-to-treat principle. An additional per protocol analysis is performed comparing patients in the wait-and-see group who received surgery with patients in the same group who had not and with patients in the surgery group. To compare the actual treatment sec instead of strategies an explorative analysis will be performed in subgroups off all patients who actually received surgery and who did not receive surgery in both groups. All patients who withdraw from the study are included in the analysis until the time of withdrawal.The result of this study will be a trade off between the disadvantages of surgery  versus the possible advantages (earlier relief of pain and return to work). For that reason recovery, measured as an 11 point difference in score when compared to baseline (Roland Disability Questionnaire for Sciatica), is the clinically most relevant patient outcome. Quality of Life (SF-36) and perceived recovery are important to compare the reduced quality of life from surgery to the possibly prolonged pain from conservative therapy and also to be able to compare cost-effectiveness with that of other spine interventions. The EuroQol is important to obtain cost-utility ratio's that can be compared with those of a wide range of other interventions. Utilities are obtained from the descriptive classification system of the EuroQol, using the model described by Dolan ,53. ConsFinally, to answer the second research question explorative analyses are conducted to investigate whether the treatment effect after two, six and twelve months varies in specific subgroups of patients Table .Using logistic regression for success rate and linear regression for severity of the disability, each prognostic indicator is checked for interaction with treatment. If the interaction term is significant, a stratified analysis will be performed.In this article the rationale and design of a pragmatic RCT on the cost-effectiveness of timing of disc surgery for LSRS is described. The only randomized trial  so far oGP = General PractitionerLSRS = Lumbosacral Radicular SyndromeRCT = Randomized Controlled TrialVAS = Visual Analogue ScaleThe author(s) declare that they have no competing interests.WP designed the study is responsible for the protocol. HH is responsible for the calculation of the sample size and contributed to the design of analysis. WH is responsible for the design of the cost-effectiveness analysis. RB has contributed in the case record forms and is responsible for the database ProMIse. JE contributed to the involvement of the GP's. JT structured the ideas about the diagnostics of LSRS and intake by neurologists. RT is the neurosurgical supervisor of WP. BK is the epidemiological supervisor of WP. All authors participated in the trial design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: