Protein evolution and protein classification are usually inferred by comparing protein cores in their conserved aligned parts. Structurally aligned protein regions are separated by less conserved loop regions, where sequence and structure locally deviate from each other and do not superimpose well.Our results indicate that even longer protein loops can not be viewed as "random coils" and for the majority of protein families in our test set there exists a linear correlation between the measures of sequence similarity and loop structural similarity. Results suggest that distance matrices derived from the loop (dis)similarity measure may produce in some cases more reliable cluster trees compared to the distance matrices based on the conventional measures of sequence and structural (dis)similarity.We show that by considering "dissimilar" loop regions rather than only conserved core regions it is possible to improve our understanding of protein evolution. Globular proteins are considered to be structurally similar if their regular secondary structure elements can be superimposed well and are connected in the same order. The loop regions connecting secondary structures demonstrate less regularity in their conformations even though short loops linking specific secondary structures can be classified into distinct classes -6. The sRecently it has been observed that structural variation in the core of homologous proteins is linearly correlated with sequence changes ,9. As waTo check this hypothesis we performed an analysis of structural variation in the loop regions within different homologous protein families using a recently introduced new measure of loop similarity . This neTo select sets of homologous proteins the Conserved Domain Database (CDD) version 1.62 was taken, which can be accessed at . The CDDEach CDD family was decomposed into a set of pairwise structure-structure alignments. Structural alignments were computed by the VAST algorithm  and only- at least one structure in a pair had X-ray resolution of greater than 3.0 Å- the Blast E-value calculated for the VAST alignment exceeded 0.01- at least one structure in a pair contained a chain discontinuous domain inconsistently aligned between VAST and CDD- at least one structure in a pair contained more than 25% of its nonaligned loops with missing residues.To ensure that protein families span a wide range of sequence similarity, all families were examined and those having less than 30% sequence identity span were not considered in further analysis. The redundancy between protein families was checked by using the procedure implemented in the CDART algorithm  and not To measure the sequence similarity between homologous proteins from the same family we used a Blast bitscore normalized by the alignment length. Among structure similarity measures used in this paper, two of them, RMSD and alignment-based Hausdorff measure (AHM) were computed by comparing the proteins in structurally aligned regions, while the loop-based Hausdorff measure (LHM) quantified the difference in the loop regions.A = {a1,..., am} and B = {b1,..., bn} be finite point sets in a Euclidean space. The Hausdorff distance between the sets A and B is then defined by:The root mean squared deviation (RMSD) was calculated using the superposition algorithm due to McLachlan . The AHMdH (A, B) = max {min j d(a1, bj),..., min j d(am, bj), min i d(ai, b1),..., min i d(ai, bn)}     (1)d(ai, bj) denote the usual Euclidean distance between the points. In other words, the Hausdorff distance between the sets A and B is the smallest distance such that every point ai ∈ A is within this distance of some point bj ∈ B and vice versa. Hausdorff distance can be calculated under the assumption that the Cα atoms for both structures are in a common coordinate frame which is defined by the structural alignment between two domains. The Hausdorff measure for loops (LHM) was calculated as an average of Hausdorff distances over all loops in the protein pair, where ns is the number of aligned secondary structure elements:Here the terms The "loop" was defined as a region between two consecutive aligned secondary structure elements and:hi = 0, if the i-th loop regions do not have any unaligned residues;hi = dH (Ai, Bi), where Ai contains the set of Cα coordinates of non-aligned residues in the i-th loop of the first structure in a pair, the last aligned residue from the preceding aligned region and the first aligned residue from the following aligned region. Similarly, Bi is defined for the second structure in a pair. The sets (Ai, Bi) are defined to include two aligned residues so that the measure can be defined even if one of the sets of non-aligned residues is empty. The Hausdorff measure for the structurally aligned regions (AHM) was defined similarly. In this case, instead of the sets that contain the coordinates for the Cα atoms in the loops, we use the coordinates for the Cα atoms in the aligned segments and average over the number of aligned segments.The correlation analysis between the measures of sequence and structural similarity, linear/nonlinear regression analyses and cluster analysis were performed using Splus version 6. Pearson (ρ) and Spearman correlation coefficients were calculated to quantify the accuracy of linear correlation. The P-value under the null hypothesis that the correlation coefficient between two variables is equal to zero has been estimated and those families with the P-values less than 0.01 were considered as having statistically significant correlation. The cluster analysis was done using the complete linkage clustering  where thTables ) and nonlinear  models . In the overall test set only 12 families have r2 – ratio smaller than 0.9  indicating that for these cases adding the non-linear term improves the performance of modeling by about 10%.The dependence of structural similarity on sequence similarity in some cases can be more accurately described by the nonlinear regression model taking into account higher order quadratic terms. To quantify how much the nonlinear terms improve the data fitting, we use the ratio of squared correlation coefficient for linear . The sequence-structure correlation for loop regions of this family is not statistically significant (the Pearson correlation coefficient is close to zero) whereas the sequence-structure correlation for the protein core is very high (ρ = -0.85 with AHM). Indeed, different proteins of this family show distinctly different loop conformations and evolutionary analysis of ADF family argued that the insertions present in the vertebrate ADF/cofilins (and not present in non-vertebrate cofilins) might be important for nuclear function of mammalian cofilins . TherefoAHM = -0.90) and loop regions (ρLHM = -0.94). On the other hand, the protein core structure of the RnaseA family changes very little with sequence whereas the loop structure gradually diverges as sequence becomes more and more dissimilar (ρAHM = -0.48, ρLHM = -0.87).Among families with particularly high LHM correlation are the families of Xylose isomerase (Xylose_isom), Class I Histocompatibility antigen , Protein tyrosine phosphatase (PTPc) and others. Figure AHM = -0.48, ρLHM = -0.78), using different measures of sequence and structural similarity. Figure To understand whether significant sequence-structure correlation for loop regions has an underlying biological meaning, we performed a cluster analysis of proteins from two diverse families, Ribonuclease A (RnaseA), and SH2 domain (SH2, ρRana ribonuclease (RR) although angiogenin has a very weak enzymatic activity and is a tumor-growth promoter while Rana ribonuclease P-30 has ribonuclease activity and antitumor effects. In contrast to the RMSD cluster tree, distance matrices based on the loop (dis)similarity measure correctly cluster the representatives of the five major groups of the Ribonuclease family as per Rosenberg et al [The RnaseA family represents a very interesting example to study as it is characterized by considerably different catalytic efficiency and substrate preferences among family members and the different aspects of its activity is not well understood. Although cysteines that form disulfide bonds, catalytic histidines and lysine residues are mostly structurally and sequence conserved, there is a great variability in sequence between other regions of RnaseA proteins ,24. We crg et al . Althougrg et al .SH2 domains represent phosphor-tyrosyl peptide binding modules which are found in many signaling proteins. The specificity of phosphate interaction with a protein has been attributed to the hydrophobic pocket which is mostly formed by two loop regions . Our anaHere we have presented an analysis of how the structure of protein loops changes in evolution as homologous proteins diverge from each other. We showed that for the majority of protein families there exists a statistically significant linear correlation between measures of sequence similarity and average loop structural similarity. This in turn suggests that loops change in evolution via a stepwise insertion or deletion process and clearly one can not portray even longer loop regions as "irregular conformations" or "random coils". Indeed, our results imply that, in general, loops are under constant evolutionary constraints which, apparently, are weaker than those for a protein core but still strong enough to preserve the loop overall structure. Since loops do not contribute much to the protein core stability, these constraints predominantly arise from the importance of loops in interacting with ligands, other proteins and cells, as well as a possible role of loops in protein folding.Modeling of insertion and deletion events in evolution poses a lot of difficulties and protein evolution is usually reconstructed based only on the aligned regions of proteins. We demonstrated that loop regions which usually correspond to the non-aligned protein regions can be very important in inferring the phylogenetic history of a protein family. Moreover, it was shown, that sometimes sequence and structure similarity measures comparing proteins in their core are not sensitive enough to detect subtle (dis)similarities between the subfamilies. Loop-based measures which emphasize the dissimilarities between different protein members can shed light on the evolutionary relationships between homologous proteins.AP and TM contributed equally to this paper.


The evidence on early childhood strongly suggests the need to shift child health policy from the current focus on social welfare to a socio-ecologically based approach. This paper reviews three governing instruments, exhortation, expenditure and regulation, that have been used by governments in Australia and discusses the relative effectiveness of these approaches in shifting the child health policy paradigm. There can be no keener revelation of a society's soul than the way it treats its children .Research evidence has demonstrated that the experiences of early childhood can have a profound lifelong impact on a child's health, wellbeing and competence . The impEducation, literacy and other social determinants of health can influence the coping skills of children, which provide the basis of learning, behaviour and health throughout life . PovertyConsistent with the increasing evidence, many governments have identified support in early childhood as a lifelong determinant of health, wellbeing and competence, as a matter for policy development, initiating actions to ensure comprehensive child development strategies for their societies. This approach requires a whole of government response, integrating health, welfare, education and other relevant parts of government. The evidence suggests that healthy public policy for infants, children and their parents is dependent on understanding of the socio-ecological factors supported by integrated, multidisciplinary and intersectoral policy and programs.'Sure Start', has been positively reviewed by the UK Audit Office and is considered by many to be the standard for the whole of government approach [In Canada, Britain and the United States, targeted interventions in the antenatal period, infancy and childhood, including parenting skills programs, are recognized for their potential to support healthier families. A socio-ecological model of health is increasingly perceived to be the most appropriate approach for the early years of life agenda. Consistent with this approach, the United Kingdom program approach . In addiapproach .The Canadian experience is widely quoted as best practice , with boThe adoption of healthy public policy for children based on this socio-ecological framework has been inconsistent throughout Australia. In an attempt to explore these inconsistencies, this paper reviews the use of three governing instruments, that is exhortation, expenditure and regulation, by national and state governments in Australia. Governing instruments are the major mechanisms governments use to seek compliance, support and implementation of public policy. Governing instruments range from minimum coercion by exhortation, through expenditure, taxation, regulation, to maximum coercion through public ownership . The folDuring the 1990s the Australian Government identified the health of children and young people as a key policy area, with a series of policy documents:The National Health Goals and Targets for Australian Children and Youth (1992)• The National Health Policy for Children and Young People (1995) and associated Implementation Plan (1996)• The National Health Policy for Young Australians (1997).• These documents provided broad national goals for children and young people:• Reducing preventable premature mortality• Reducing the impact of disability• Reducing the incidence of vaccine preventable disease• Reducing the impact of conditions occurring in adulthood with their origins or early manifestation in childhood or adolescence• Enhancing family and social functioningAlthough the evidence supporting a broader definition of child health was strong, the focus of these National Health Goals and Targets remained heavily focused towards surveillance and the reduction of injury and illness, perhaps reflecting a comfort with current and past approaches.To date there has been little evidence of an integrated, multidisciplinary approach to child health at the national level. The 2003/04 federal budget did not provide the broad whole of government approach recommended for child health, with only a few targeted interventions, such as the National Meningococcal C Campaign, and a much greater focus on the health needs of the aging population. In 2003, the Australian of the year, Fiona Stanley suggested that the social and economic policies of the Government were not effective in tackling the issues associated with ensuring healthy children and young people .Towards the Development of a National Agenda for Early Childhood signaled a changing paradigm, with a whole of child and life course approach addressing promotion, prevention and early intervention for all children.The platform for a paradigm shift was established in 2001 with the appointment of the Minister for Children and Youth Affairs and the subsequent statement in 2002 of the intent to develop a National Agenda for Early Childhood. The consultation paper Better Child Public Health: A Strategic Approach to Building Capacity – A National Action Plan 2004–2007 has been developed and is being used in consultation and capacity building initiatives. In October 2002 the Minister for Children and Youth Affairs referred an inquiry into improving children's health and well being to the Standing Committee on Family and Community Affairs. The Australian Council for Children and Parenting (ACCAP), an advisory body to the Minister for Children and Youth Affairs, was granted a two year term from July 2003, with a focus on strategic advice in the areas of early childhood intervention and prevention, parenting and child protection, foster care and emerging early childhood initiatives, including advising about the continuing development of the National Agenda for Early Childhood.The last years have seen the creation of ever more advisory groups, partnerships and inquiries with a mandate to influence child health policy. The Child and Youth Health Intergovernmental Partnership  was convened in December 2001 to develop a national child public health strategy and advise on the National Agenda for Early Childhood. Their draft strategy framework As described above, the policy approach at the national level has focused almost entirely on exhortation, the least coercive instrument, where support and compliance are sought voluntarily through persuasion and discussion. In comparison with other countries, such as Britain and Canada, the lack of a common and shared understanding of the socio-ecologic approach and its implications has made it difficult to show any significant advances in this area. In fact, the recent demise of the Child Health Unit within the Australian Government Department of Health suggests less focus on child health.'Communities for Children' initiative as part of the Stronger Families and Communities Strategy. Communities for Children will directly fund 35 Australian communities between $1 and 4 million over four years to support parents, neighbourhoods and the wider community to give children the healthy start they need [Communities for Children has not been set up to respond to the greatest need.Nationally, child health has not been heavily addressed through other policy instruments, such as expenditure, taxation, or regulation, although more recently the Commonwealth Government Department of Family and Community Services (DFaCS) established the hey need . ImportaIt has been suggested that system change can be accomplished by motivating institutions, systems and actors to move in common directions and develop structures that sustain these efforts over time . This reThe use of exhortation may be successful at motivating common approaches but will be much less effective at ensuring the sustaining structures are developed. This is apparent in the existing committee structures, which still operate from within the government structures and are thus unable to cross the 'silos' to promote the needed whole-of-government approach. To be effective in changing the paradigm in this area, the exhortation process will require back up by more coercive governing instruments.In comparison with other Australian states, Victoria has been slow to provide visible translation of the socio-ecological model of health for children and young people in a coordinated and systematic way to state policy. A recent review of Victorian paediatric services suggested that Victoria needed to establish a child and young people focus ensuring appropriate mechanisms to plan, coordinate and monitor across government departments and service providers . It was 'Best Start' program and will pilot it as demonstration projects in 10 communities across the State with an investment of $7.6 million. Best Start is auspiced by the Departments of Human Services and Education and Training and is focused on reducing the impact of disadvantage (from any cause) and enhancing the life chances of all children by strengthening the universal preventative system [Best Start are multi-level including the social, emotional and physical well-being of children, capacity building of parents and carers and communities to assist them to become more child friendly, while focusing on specific interventions for socially disadvantaged families [The lack of coordinated focus on child health in Victoria is perhaps the result of a lingering policy focus on health surveillance. Despite the increasing evidence that surveillance and screening programs have limited effectiveness in child health ,25, it ie system . The aimfamilies . The demBest Start, existing government funding and reporting in the area of maternal and child health is still largely focused on surveillance [Best Start signals intent to change the child health policy paradigm, the incentives established through the broader expenditure pool suggest, for the moment, maintenance of the status quo in Victoria.Despite the intentions of eillance . The Vic'Families First' initiative targets families with children 0 to 8 years, with the aim of helping parents give their children a good start in life. Demonstrating the commitment to a whole of government approach, the Office of Children and Young People (OCYP), located within The Cabinet Office, reporting directly to the Premier, has played a lead role in the development and implementation of the Families First strategy. This evidence-based approach is delivered jointly by five NSW government agencies – Area Health Services, Community Services, Education and Training, Housing and Disability, Ageing and Home Care in partnership with parents, community organisations and local government. NSW Health supports 'the ongoing development of partnerships at policy, planning and service delivery levels to enable improved co-ordination and intersectoral collaboration in the delivery of child health services' [[In New South Wales the rvices' [ pg. 44].NSW has also successfully translated much of the evidence into coordinated service planning and delivery at the regional level. Paediatric networks, associated with the Area Health Services, were established in 1997 and today provide designated primary, secondary and tertiary level services for families with children aged 0 to 5 years . In addiFamilies First has been guided by a series of policy and practice guidelines. Recently, an independent review of Families First implementation within three regions, (Orana Far West, Illawarra and South West Sydney) found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill, with considerable effort to develop structures and processes that sustain interagency collaboration [The implementation of boration . This reRegulation involves the imposition of requirements to meet specific obligations. Often regulation is seen to exist within legislation outlining strict rules of behaviour. However, in health policy guidelines are considered effective means of imposing regulation, recognising the inherent uncertainty in safe practice in health care . The impHowever, it is yet to be seen whether the policy will be supported with the necessary resources for compliance. While regulation involves shifting costs of compliance from government to other participants, enforcement and monitoring can be expensive and difficult . WithoutA variety of instruments have been used by government to change the child health policy paradigm from that focused on social welfare to 'healthy' public policy predicated on a socio-ecological foundation. NSW has chosen regulation to implement a child health policy framework that is built upon the international evidence of the effectiveness of integrated, multidisciplinary and intersectoral policy and programs. A little slower to change paradigms, Victoria has established demonstration programs through targeted expenditure, without an overarching whole-of-government child health policy framework. Nationally, there is a move to change the paradigm to a broader definition of child health almost exclusively through exhortation.Families First implementation found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill to develop the necessary structures and processes – a suggestion that without other approaches to structural reform, exhortation is unlikely to be successful.Instrument choice is influenced by a variety of factors. The use of exhortation by the Commonwealth Government is a relatively risk-free easy approach, which can counteract the divided accountabilities among federal and state governments in the area of health and social services. Exhortation is easy to implement; it is the least coercive and relies on voluntary goodwill. While it may be successful in building a common understanding, and even this is debatable, an independent review of the The expenditure policy of Victoria illustrates an approach that is compromised by the lack of an underlying agreed evidence-based policy framework. This lack of coordinated government approach is reflected in conflicting expenditure policy in this area, with the potential to confound outcomes.While the regulatory approach of NSW suggests bold steps to change the paradigm, in fact, because regulation is not subjected to the same level of scrutiny of other instruments, such as expenditure, and even exhortation , it is aThe evidence for a new policy paradigm is strong. But the use of these different policy instruments underscores the lack of shared understanding and policy agenda. Oberklaid suggests that while there are similarities in the rhetoric throughout Australia, there has been relatively little investment in child health . The chaThe authors declare that they have no competing interests.


Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment.Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry.Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes.We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. With the improved long-term outcome of allograft recipients in the cyclosporine or tacrolimus era, malignant tumors have become increasingly important. Malignant tumours develop in 15–20% of graft recipients after 10 years, and thus contribute substantially to the morbidity and mortality of these patients [During the last years, the novel immunosuppressive drug mycophenolate mofetil (MMF) has been introduced into the clinical protocol to overcome severe side effects associated with cyclosporine or tacrolimus. Meanwhile, it has become part of the immunosuppressive regimen after liver, kidney or heart transplantation . Still, MMF effects are based on the inhibition of inosine monophosphate dehydrogenase (IMPDH) and prevention of guanosine monophosphate synthesis from inosine monophosphate, a rate-limiting step in the purine biosynthesis in lymphocytes. Consequently, MMF blocks the proliferation and clonal expansion of T and B lymphocytes, and prevents the generation of cytotoxic T cells, as well as other effector T cells . AdditioThe integrins constitute a family of transmembrane receptor proteins composed of heterodimeric complexes of noncovalently linked alpha and beta chains. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. For various types of cancers, different changes in integrin expression are closely associated with tumor growth and metastasis. Based on the knowledge that MMF modulates integrin expression, we postulated that MMF might not only suppress leukocyte recruitment to the donor graft, but also prevent integrin-dependent tumor dissemination. To explore how far MMF might serve as a metastasis-blocking agent, we investigated the beta1 integrin subunit expression pattern of colon, kidney, pancreas and prostate tumor cells before and after MMF treatment, as well as MMF effects on tumor cell adhesion to human endothelium in vitro.The present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium.2 incubator.Kidney carcinoma Caki I cells, pancreatic carcinoma DanG cells and colonic adenocarcinoma HT-29 cells G were obtained from the tumor cell bank of Johannes Gutenberg University, Mainz, Germany. Prostate carcinoma DU-145 cells were purchased from DSMZ (Braunschweig, Germany). Tumor cells were grown and subcultured in RPMI1640 medium (Seromed, Berlin, Germany) supplemented with 10% FCS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified, 5% CO4 cells/scan). Cell cultures with a purity > 95% were serially passaged. Subcultures from passages 2–4 were selected for experimental use.Endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. HUVEC were grown in Medium 199 (Biozol, Munich, Germany), 10% fetal calf serum , 10% pooled human serum (Blood Bank of The German Red Cross, Frankfurt am Main, Germany), 20 μg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin (Roche, Basel, Switzerland), 100 ng/ml gentamycin (Gibco) and 2% 1 M HEPES-buffer (Seromed, Berlin, Germany). To control the purity of HUVEC cultures, cells were stained with fluorescein isothiocyanate (FITC)- labelled monoclonal antibody against Factor VIII-associated antigen  and analyzed microscopically or by FACscan  channel histogram analysis; 1 × 10Tumor cells were pretreated with MMF (Roche Bioscience, Grenzach-Wyhlen, Germany) (0.1 μM, 1 μM). Before adding the MMF-treated tumor cells to HUVEC (monolayer adhesion assay), cell cultures were washed to remove MMF from the medium. Results were compared to untreated controls.Viability of tumor cells in presence of MMF was assessed by propidium iodide dsDNA-intercalation or quantitative fluorescence analysis of enzyme-catalyzed fluorescein-diacetate metabolism.6 tumor cells of each entity/well were carefully added to the HUVEC monolayer for 60 min. Subsequently, non-adherent tumor cells were washed off using warmed (37°C) Medium 199. The adherent cells were fixed with 1% glutaraldehyde and counted in five different fields (5 × 0.25 mm2) using a phase contrast microscope (20 × objective) to calculate the mean cellular adhesion rate.HUVEC were transferred to six-well multiplates  in complete HUVEC-medium. When confluency was reached, 0.5 × 104 cells/scan) and expressed as mean fluorescence units (MFU).Tumor cells were washed in blocking solution (PBS, 0.5% BSA) and then incubated for 60 min at 4°C with the FITC-conjugated monoclonal antibody anti-alpha2beta1 , anti-alpha4beta1 , anti-alpha5beta1 , anti-alpha6beta1 , or with the PE-conjugated monoclonal antibody anti-alpha1beta1 , or anti-alpha3beta1 . Integrin expression of tumor cells was then measured using a FACscan  channel histogram analysis; 1 × 10A mouse IgG1-FITC was used as an isotype control for FITC conjugated antibodies. To evaluate background staining of PE conjugated antibodies, goat anti mouse IgG-PE was used .To analyze integrin beta1 distribution on the cell membrane, tumor cells were transferred to round cover slips (pretreated with 2% 3-aminopropyl-triethoxysilan) placed in a 24 well multiplate. Upon reaching confluency, cell cultures were washed and fixed in cold (-20°C) methanol/acetone (60/40 v/v). Subsequently, cells were incubated for 60 min with unconjugated anti-integrin monoclonal antibodies. Indocarbocyanine  conjugated goat-anti-mouse IgG was then added as the secondary antibody. To prevent photobleaching of the fluorescent dye, cover glasses with stained cells were taken out of the wells and the residual liquid was removed. These were then embedded in an antifade reagent / mounting medium mixture (ProLong™ Antifade Kit, MoBiTec, Göttingen, Germany) and mounted on slides. The slides were viewed using a confocal laser scanning microscope  with a plan-neofluar ×100 / 1.3 oil immersion objective.2 growth area; Falcon Primaria, Becton Dickinson) and cultured with or without MMF. Total RNA was extracted by using RNeasy kit (Qiagen, Hilden, Germany) and RNA samples were then treated with 80 U/ml of Rnase-free Dnase I (Boehringer Mannheim, Mannheim, Germany) for 60 min at 37°C, to eliminate amplifiable contaminating genomic DNA. Subsequently, samples were incubated for 10 min at 65°C to inactivate Dnase. Complementary DNA was synthesized from 1 μg of total RNA per sample with a 60 min incubation at 42°C, using the Moloney murine leukaemia virus reverse transcriptase (Invitrogen, Karlsruhe, Germany) and oligo-(dT) priming (Boehringer Mannheim). Amplification was carried out using gene specific primers and Platinum-Taq polymerase (Invitrogen) in a Mastercycler Gradient thermocycler (Eppendorf, Hamburg, Germany). Reactions were performed in the presence of 0.5 μl cDNA, with an initial incubation step at 95°C for 2 min. Cycling conditions consisted of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec over a total of 30 cycles. The reaction was completed by another 10 min incubation step at 72°C. The specific sequences for sense and anti-sense primers are shown in table mRNA expression of beta1 integrins was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Tumor cells were seeded in 50 ml culture flasks  > alpha2beta1 (175.7 ± 24.3) > alpha6beta1 (54.8 ± 9.2) > alpha1beta1 (27.3 ± 3.2).Caki I: alpha3beta1 (601.3 ± 82.0) > alpha1beta1 (139.2 ± 24.0) > alpha5beta1 (22.9 ± 4.2).HT-29: alpha3beta1 (202.0 ± 33.8) > alpha6beta1 (119.6 ± 14.5) > alpha2beta1 (69.2 ± 9.0) > alpha1beta1 (25.4 ± 3.4).DU-145: alpha3beta1 (942.5 ± 112.9) > alpha2beta1 (95.4 ± 12.4) > alpha5beta1 (69.1 ± 8.9) > alpha6beta1 (50.9 ± 7.2) > alpha1beta1 (31.4 ± 4.8).Mean IgG1-FITC isotype control was 8.6 ± 1.8 MFU, mean IgG1-PE isotype control was 9.9 ± 1.7 MFU.Analysis of the mRNA expression level confirmed the flow cytometry data (see below).The distribution pattern of those integrins which were predominantly expressed on the respective tumor cell line was further explored by confocal microscopy figure . alpha2bMMF evoked distinct alterations of the beta1 integrin expression pattern figure . MMF onlTo allow a clear interpretation of the strong effects of MMF on adhesion and beta1 integrin surface expression of HT-29 and DU-145 cells, MMF evoked alterations of gene activity was also evaluated in these cell lines figure . ControlAlthough MMF has become part of the standard regimen after organ transplantation its impact on tumor development and dissemination is still not clear.Our adhesion experiments demonstrate that MMF down-regulates binding of tumor cells to endothelium, which might argue for anti-tumoral properties of this compound. Notably, HT-29 and DU-145 cells responded well to MMF (-70% adhesion reduction), while Caki I and DanG tumor cells were influenced only modestly. From a clinical viewpoint, distinct adhesion-blocking properties of MMF might be limited to colon and prostate carcinoma cells. Interestingly, HT-29 cells were more susceptible to MMF than DU-145 cells: 0.1 μM MMF was sufficient to significantly diminish adhesion of HT-29 cells, whereas 1 μM MMF was necessary to evoke maximum effects on DU-145 cells. The different sensitivity of the tumor cell lines to MMF might be caused by an unequal metabolic activity, coupled with variable IMPDH levels. Recent data have shown that the level of expression of IMPDH mRNA and protein differ among several cell lines , and thaIt should also be considered that MMF might switch on/off different intracellular signaling cascades in colon versus prostate tumor cells. Indeed, adhesion blockade of HT-29 cells was accompanied by reduced alpha3beta1 and alpha6beta1 surface expression, while adhesion blockade of DU-145 cells was accompanied by a dose-dependent up-regulation of integrins alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1 on the cell membrane.Studies on integrin receptors presented evidence that beta1 integrin expression by colon carcinoma cells qualifies these cells to successfully adhere to secondary sites. Recent experiments have demonstrated that colon cancer cells adhere to endothelial cells via beta1 integrins and that addition of beta1 integrin blocking antibodies reduces tumor cell adhesion ,16. Basehigh and alpha3beta1low expressing prostate carcinoma cells were injected into immunocompromised SCID mice, only those cells with a drastically reduced integrin level were found to form tumors at the primary sites and to be highly invasive and metastatic [A converse scenario might be created during prostate carcinogenesis, as levels of beta1 integrins have been found reduced in neoplastic versus normal prostate tissue ,19, and tastatic . This isWhen discussing the relevance of integrins in tumor recurrence and malignancy, we should keep in mind that integrin receptors serve as mechanistic binding as well as differentiation triggering elements. Therefore, up-regulation of the same integrin type might either lead to enhanced cell adhesion by coupling the receptor to its ligand, or to a reduced cell adhesion by activating integrin driven differentiation signals. Based upon our in vitro assay, we conclude that MMF blocks adhesion of colon and prostate carcinoma cells by two different mechanisms: a) Loss of alpha3beta1 and alpha6beta1 surface expression directly contributes to the reduced adhesive behaviour of HT-29 cells, b) Enhancement of integrin beta1 subtypes might cause re-differentiation of DU-145 cells towards a low-adhesive phenotype. However, it still remains to be determined if MMF indeed acts as a differentiation inducing drug in prostate tumor cells. Beside the hypothesis that beta1 upregulation might activate differentiation inducing signals, selective inhibition of tumor-promoting pathways should also taken into consideration.Presumably, down-regulation of alpha3beta1 and alpha6beta1 surface expression on HT-29 tumor cells might be caused by inhibition of receptor glycosylation and/or receptor de novo synthesis. The latter hypothesis seems to be more likely because MMF's effects at the cell surface were also observed at the mRNA level. This was not the case with DU-145 cells where MMF evoked up-regulation of membranous beta1 integrins was not paralleled by similar modifications of the beta1 integrin coding mRNA. There is still no clear concept why MMF causes integrin up-regulation in one tumor entity but down-regulation in another entity, both coupled with reduced tumor cell adhesiveness. Presumably, HT-29 and DU-145 tumor cells might be equipped with different enzyme systems, the intracellular signaling cascade might be activated differentially in colon versus prostate tumor cells, or sensitivity of specific pathways to MMF might differ between both tumor types.Speculatively, alterations of post-translational events might change the receptor surface presentation in prostate carcinoma cells. Elegant experiments by Liang and coworkers demonstrated that over-expression of alpha5beta1 or beta1 integrin induced the decrease of protein kinase B (PKB) phosphorylation and subsequent accumulation of cyclin-dependent kinase inhibitor p21 . A yeastThe present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium. MMF might also act on further adhesion proteins which are relevant for tumor recurrence and dissemination. An in vitro study published recently refers to the sLeX-selectin pathway targeted by MMF . CD44 glThe author(s) declare that they have no competing interests.TE performed parts of the in vitro studies, contributed toward the design of the study and drafted the manuscript. JM carried out confocal microscopy, BR and IN performed FACS-analyses. IM designed PCR primers and carried out the PCR studies. WDB contributed to the manuscript design and finalisation. DJ participated in the conception and design of the study. RAB carried out the adhesion assays, participated in the conception and design of the study and its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The International Network for the Availability of Scientific Publications (INASP) was established in 1992 to bridge the information divide between the developed and developing world Since 1992 INASP has worked, in response to requests, to develop its activities for capacity building in information production, access, and use, with an overarching vision that all people are able to access and contribute information, ideas, and knowledge necessary for sustainable and equitable development.To ensure that INASP activities are appropriate for the communities and cultures of the countries in which INASP works, local partners are used to build networks and to provide advice and support. Enhancing capacities is central to all activities, and local ownership and sensitivity to local conditions and opinions are of paramount importance. Here I highlight two aspects of our work: increasing access to information and supporting the visibility of regional journals within the global research community.The growth and acceptability of the internet during the 1990s opened up tremendous possibilities for the dissemination of research information, providing many nations with access to information that had previously been out of their reach. Although internet connectivity remains problematic in many countries, it still offers great potential for bridging the information gap.Arising from discussions with librarians and researchers in 1999 and 2000, the Programme for the Enhancement of Research Information was formally launched in 2002 after a two-year pilot programme. The Programme for the Enhancement of Research Information works in a two-phase manner: (1) providing and supporting access to international research information and (2) supporting and promoting access to nationally published research.To support access to international information, INASP negotiates for heavily discounted or free access to online information from publishers and information providers in developed countries. Enabling access, however, does not guarantee that resources are used; both training and promotion are needed so that researchers and downstream information providers know how to make the best use of what is available. With this in mind, INASP has set up four series of training workshops, which have trained over 1,000 people from over 200 institutions in over 17 countries during the last two years. Although some of the training is undertaken by INASP staff, most is facilitated by in-country trainers.Planning for long-term sustainability, INASP aims to hand over the tasks of negotiation, purchasing, and training to local consortia, associations, or networks.Although much needed, access to international resources can discriminate against nationally published scholarly information. This may be due to one of the following: a perception that local publications are lower quality, distribution problems and irregular publishing, or lack of online visibility. However, national publications provide vital access to potential collaborators and information about research on topics of local relevance—since 1998, INASP has provided support to indigenous research publications to enable them to survive and coexist with international information.One specific project that exemplifies these aims is known as African Journals OnLine (AJOL). AJOL launched in 1998 in response to requests from African journals. Starting with only ten titles, it now includes 189 from 21 African countries. Access to the site, which includes tables of contents, article abstracts, and a homepage for each journal with information about editorial boards, guidelines for authors, and more, is entirely free. Participating journals report increased international submissions and increasing contact with international researchers.Researchers make wide use of this service, and over 8,000 people have registered to use AJOL since it launched. Registration is optional and until March this year did not provide the user any benefit—it simply provided an indication of the number of people visiting and from which countries. However, since March 2004 anyone who registers can sign up to receive a free E-mail table of contents alert. Over 500 people have chosen to receive E-mail alerts from an average of four journals each, and the effect of these alerts is felt through the increase in document delivery requests.AJOL does not currently host full text, but can provide full-text articles through a document delivery service (there are plans to load full-text articles on AJOL in the future). This service is provided to researchers from developing countries for free and to researchers elsewhere at a minimal charge . In the first six months of 2004, over 700 articles were ordered and sent out.Most journals publishing out of Africa are not run by commercial publishers, and most of them operate at a loss, subsidised by universities, associations, funding agencies, or a mixture of all three. Paid subscriptions to print journals are frequently a vital part of their economics, and the financial viability of many journals is constantly under threat. Originally, it was hoped that AJOL would increase subscriptions, thereby providing greater financial security to the journals. This has not been achieved and is unlikely to occur in the future. However, with increasing visibility, the value of the journals increases along with, hopefully, their importance to the supporting agencies and long-term sustainability.Since AJOL is set up to provide support for the participating journals, the journals' opinions are constantly sought before any developments are undertaken, meaning AJOL is effectively “owned” by them. Although the service is not actively run by the journals themselves, all participating journals are considered to be part of a community, receiving regular E-mail contact and skills support. Many journal editors  feel isolated and unskilled, and even though AJOL does not operate as an association for the journals, it encourages communication between the editors, and a sharing of experience.The AJOL website was relaunched in March 2004 using open-source software called Open Journal System, originally developed in Canada by the Public Knowledge Project at the University of British Columbia and further developed for AJOL at Bristol University, United Kingdom. This software was written to enable a single journal to manage the entire publishing process online from submission to publishing. Being open source, the software can also easily be adapted and modified. The Public Knowledge Project set up the system with the developing world in mind: it operates efficiently at low bandwidths and is easy to use, with many guides and help functions built in.For the users, this software offers sophisticated searching, E-mail alerts, and a space for each journal within AJOL for journal-specific information. It also now provides a range of management tools, which makes the service more efficient and enables it to grow.Another important consideration for choosing this system was that individual journals can take over the responsibility for maintaining their own journal areas within AJOL via the Internet. Over 50 journals have expressed an interest in taking this on, and one publisher is already successfully maintaining its material. To support this, training workshops are currently in preparation. This development will assist the long-term sustainability of AJOL and also give the participating journals experience in managing and publishing content online.In the near future, INASP will be including full text on AJOL and hopes to move the management of AJOL to an African organisation so that it will truly become an African gateway for published research. In addition, INASP continues to work with journals to strengthen their quality and sustainability, providing advice, training workshops, study tours, and a coordinating point for discussion and collaboration.Outside Africa, INASP is working on similar developments in Nepal and the Caribbean and has received expressions of interest from Bangladesh, Sri Lanka, and Vietnam.It is vital that researchers everywhere in the world have access to reliable, relevant information. At the same time, providing access to international literature needs to be balanced with supporting local publications to ensure that indigenous knowledge is not lost, but can take its place in the research community and contribute to the continuing development of science. Worldwide access to information is central to all INASP activities, and INASP supports policies and activities that work towards this.http://www.inasp.infoINASP: http://www.ajol.infoAJOL: http://pkp.ubc.caPublic Knowledge Project:


In vitro, TRAIL-mediated apoptosis of prostate cancer cell lines can be enhanced by doxorubicin and correlates with the downregulation of the anti-apoptotic protein c-FLIP. This study evaluated the effects of doxorubicin on c-FLIP expression and tumor growth in combination with Apo2L/TRAIL in a xenograft model.Prostate cancer is a significant health problem among American men. Treatment strategies for androgen-independent cancer are currently not available. Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a death receptor ligand that can induce apoptosis in a variety of cancer cell lines, including androgen-independent PC3 prostate carcinoma cells. In vitro cytotoxic effects of TRAIL were measured using a MTS-based viability assay. For in vivo studies, PC3 prostate carcinoma cells were grown subcutaneously in athymic nude mice and tumor growth was measured following treatment with doxorubicin and/or Apo2L/TRAIL. c-FLIP expression was determined by western blot analysis. Apoptosis in xenografts was detected using TUNEL. Statistical analysis was performed using the student t-test.In vitro experiments show that PC3 cells are partially susceptible to Apo2L/TRAIL and that susceptibility is enhanced by doxorubicin. In mice, doxorubicin did not significantly affect the growth of PC3 xenografts but reduced c-FLIP expression in tumors. Expression of c-FLIP in mouse heart was decreased only at the high doxorubicin concentration (8 mg/kg). Combination of doxorubicin with Apo2L/TRAIL resulted in more apoptotic cell death and tumor growth inhibition than Apo2L/TRAIL alone.in vivo than either agent alone and could present a novel treatment strategy against hormone-refractory prostate cancer. The intracellular mechanism by which doxorubicin enhances the effect of Apo2L/TRAIL on PC3 xenografts may be by reducing expression of c-FLIP.Combination of doxorubicin and Apo2L/TRAIL is more effective in growth inhibition of PC3 xenografts  Prostate cancer is a significant health problem among American men. This year about 230,110 men will be diagnosed with the disease and about 30,000 will likely die in this country alone [in vitro and in vivo. Apoptosis is initiated by binding to receptors DR4/DR5 (TRAIL-R1, 2), which is followed by assembly of the death inducing signaling complex and activation of caspase-8. Subsequent activation of caspases-3/7 (in a mitochondria-dependent or independent fashion) leads to execution of apoptosis [TRAIL is a type II membrane protein that can induce apoptosis by binding to death domain containing receptors DR4 and DR5 . TRAIL iActive caspase-8 tetramers are generated by cis- and transcatalytic cleavage from pro-caspase-8 homodimers -11. In ain vivo model using xenografts of PC3 prostate carcinoma cells.We have previously shown that prostate cancer cells are relatively resistant to recombinant TRAIL but can be sensitized by pretreatment with the chemotherapeutic agent doxorubicin ,14. The Our results show that doxorubicin reduces c-FLIP expression in xenografts and that combination of doxorubicin with TRAIL is more effective in tumor growth inhibition than either agent alone.2. Apo2L/TRAIL was generously provided by Genentech Inc., San Francisco CA. KillerTRAIL was purchased from Alexis, San Diego, CA. Doxorubicin was obtained from the MUSC pharmacy. The CellTiter Aqueous One Solution Cell Proliferation Assay and DeadEndTUNEL kits were purchased from Promega, Madison WI. The c-FLIP antibody NF-6 was kindly provided by Dr. Marcus Peter, University of Chicago. The Dave-2 (c-FLIP) antibody and anti-actin were purchased from Alexis, San Diego, CA and from Sigma, St. Louis, MO, respectively. Supersignal DuraWest was obtained from Pierce Biotechnology Inc., Rockford, IL.PC3 cells were purchased from the ATCC and were maintained in RPMI1640 supplemented with 10% FBS at 37°C with 5% CO4 cells/well, incubated overnight and subsequently treated with doxorubicin and/or Killer-TRAIL/Apo2L/TRAIL. The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent was added 24 hours after initiation of treatment and plates read at an absorbance of 490 nm one to two hours later using a Vmax kinetic microplate reader . All treatments were performed in triplicate. Background absorbance was determined by incubating media with substrate alone and subtracting the values from wells containing cells. Percent cytotoxicity was calculated as follows: % cytotoxicity = 1 - [ × 100]. Experiments were repeated three times with similar results. There were no discrepancies between results obtained in the MTS assay and visual assessment of the cells prior to adding the MTS reagent.Cells were seeded into 96-well plates at 1 × 106) were injected subcutaneously into the flanks of mice. Animals bearing tumors were randomly assigned to treatment groups (five or six mice per group) and treatment initated when xenografts reached volumes of about 100 mm3. Tumors were measured using digital calipers and volume calculated using the formula: Volume = Width2 × Length × 0.52, where width represents the shorter dimension of the tumor. Treatments were administered as indicated using vehicle (PBS containing 0.1% BSA), doxorubicin (2–8 mg/kg), Apo2L/TRAIL , or a combination of 4 mg/kg doxorubicin followed by 500 μg Apo2L/TRAIL. Doxorubicin was administered systemically whereas Apo2L/TRAIL was given either intra-tumorally  were purchased from Harlan, Indianapolis IN and were housed under pathogen-free conditions according to Medical University of South Carolina animal care guidelines. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at MUSC. PC3 cells  prior to performing protein assays on the supernatant. Protein (50 μg) was separated on 4–12% Bis/Tris NuPage gels in MES buffer and transferred to nitrocellulose for 60–90 minutes at 30 V. After transfer and blocking (5% milk), blots were probed with Dave-2 (1:500 in 0.5 % milk) or NF-6 (1:5 in TBS-Tween) overnight at room temperature. Following three washes with TBS-Tween, membranes were incubated with the anti-mouse (1:5,000) or anti-rat (1:15,000) HRP-conjugated secondary antibodies for 1 hour at room temperature in 5% milk in TBS-Tween. Membranes were washed three times in TBS-Tween followed by chemiluminescent detection of the secondary conjugates with DuraWest Supersignal. Membranes were reprobed with anti-actin (1:2000) to ensure equal loading.Tumors were excised and fixed in 10% formalin and processed by standard procedures. Sections were analysed for apoptotic cells using the DeadEND Tunel kit according to the manufacturers instructions. Stained slides were examined for TUNEL positive cells using a Zeiss Axiovert 200 microscope (20×). TUNEL positive cells from 4 fields/slide were counted by two investigators to calculate the mean ± SEM. P-values were calculated using the student's t-test.We have previously shown that the TRAIL apoptotic response in prostate cancer cells can be enhanced by doxorubicin ,15. Thesin vitro. As shown in Figure Initially we compared the susceptibility of PC3 prostate carcinoma cells to KillerTRAIL and Apo2L/TRAIL in parallel assays in vivo experiments, mice bearing PC3 xenografts were injected with 2, 4 or 8 mg/kg doxorubicin and tumor volume was measured over time  was not diminished following doxorubicin treatment, whereas the signal of the upper band (c-FLIPL) was slightly reduced following administration of 8 mg/kg doxorubicin. Since 4 mg/kg doxorubicin reduced c-FLIP in PC3 xenografts as effectively as 8 mg/kg without affecting endogenous mouse c-FLIP, this dose was chosen for combination therapy.Previously, we have examined possible targets of doxorubicin that may be responsible for increasing the susceptibility of prostate cancer cells to TRAIL d c-FLIP . In PC3 in vivo following treatments, mice bearing bilateral PC3 xenografts were injected systemically with vehicle or 4 mg/kg doxorubicin, followed by intratumoral injection of vehicle (left tumor) or Apo2L/TRAIL (right tumor). Twenty-four hours after Apo2L/TRAIL injection, tumors were harvested and analysed for apoptosis by TUNEL staining , which was not significantly different from the control group (1025 ± 118 mm3, p = 0.41). Tumors in animals treated with Apo2L/TRAIL were reduced compared to either untreated or doxorubicin treated groups (833.5 ± 150 mm3, p = 0.03). However, tumors that had been exposed to both doxorubicin and Apo2L/TRAIL were significantly smaller (224 ± 145 mm3, p < 0.001) and remained smaller at day 33 (490 ± 197 mm3) when the experiment was terminated. Analysis of tumor volume over time suggests that a single round of combination treatment delays growth until day 26  declare that they have no competing interestsAZ carried out the animal experiments using doxorubicin and Apo2L/TRAIL and TUNEL staining. JM carried out the animal experiments using doxorubicin. CVJ carried out viability assays and western blot analysis, conceived of the study, participated in its design and coordination, and prepared the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


PK2) is a clock-controlled gene that may function as a critical SCN output molecule responsible for circadian locomotor rhythms. As light is the principal zeitgeber that entrains the circadian oscillator, and PK2 expression is responsive to nocturnal light pulses, we further investigated the effects of light on the molecular rhythm of PK2 in the SCN. In particular, we examined how PK2 responds to shifts of light/dark cycles and changes in photoperiod. We also investigated which photoreceptors are responsible for the light-induced PK2 expression in the SCN. To determine whether light requires an intact functional circadian pacemaker to regulate PK2, we examined PK2 expression in cryptochrome1,2-deficient (Cry1-/-Cry2-/-) mice that lack functional circadian clock under normal light/dark cycles and constant darkness.The suprachiasmatic nucleus (SCN) contains the master circadian clock that regulates daily rhythms of many physiological and behavioural processes in mammals. Previously we have shown that prokineticin 2 (PK2 expression exhibits transients in response to phase advances but rapidly entrains to phase delays. Photoperiod studies indicate that PK2 responds differentially to changes in light period. Although the phase of PK2 expression expands as the light period increases, decreasing light period does not further condense the phase of PK2 expression. Genetic knockout studies revealed that functional melanopsin and rod-cone photoreceptive systems are required for the light-inducibility of PK2. In Cry1-/-Cry2-/- mice that lack a functional circadian clock, a low amplitude PK2 rhythm is detected under light/dark conditions, but not in constant darkness. This suggests that light can directly regulate PK2 expression in the SCN.Upon abrupt shifts of the light/dark cycle, PK2 in the SCN is regulated by both the circadian clock and light. PK2 is predominantly controlled by the endogenous circadian clock, while light plays a modulatory role. The Cry1-/-Cry2-/- mice studies reveal a light-driven PK2 rhythm, indicating that light can induce PK2 expression independent of the circadian oscillator. The light inducibility of PK2 suggests that in addition to its role in clock-driven rhythms of locomotor behaviour, PK2 may also participate in the photic entrainment of circadian locomotor rhythms.These data demonstrate that the molecular rhythm of  Per1 and Per2, as well as immediate-early gene c-fos. Exposure to light pulses at night induces expression of these genes in the SCN, and this light induction mechanism has been suggested as a critical pathway for the resetting of circadian clock in response to changes in light/dark conditions [Light is the principal zeitgeber that entrains circadian rhythms of physiology and behaviour ,2. The mnditions -19. Intenditions .Cry1-/-Cry2-/-) mice, which are behaviourally arrhythmic under constant darkness. The contribution of masking to normal locomotor activity rhythms is unclear, as is the participation of the SCN in masking effects of light. Vitaterna et al (1999) first observed a light-driven Per2 rhythm in the SCN in Cry1-/-Cry2-/- mice, and have suggested that the light-driven molecular rhythm in the SCN may be related to the preservation of their locomotor rhythm [In addition to the effect of light on circadian entrainment, light also has a direct effect on physiology and behaviour, generally termed as "masking" ,22. For r rhythm .PK2) is a first order clock-controlled gene, whose expression in the SCN is regulated by CLOCK and BMAL1 acting on the E-boxes in the gene's promoter [PK2 expression is rapidly induced by light pulses administered at night [PK2 expression in the SCN. In particular, we investigated the photoreceptive mechanisms responsible for the light-induced PK2 expression in the SCN. Utilizing Cry1-/-Cry2-/- mice, we also determined whether light can drive PK2 expression in the SCN independent of a functional circadian clock.We previously found that prokineticin 2 , then subjected to either a 6 hour delay (6hrD) shift or 6 hour advance (6hrA) shift of light/dark cycles. We measured PK2 mRNA in the SCN of these animals to examine how quickly the PK2 mRNA rhythm re-entrains to the shifted light/dark cycles. Under LD, PK2 mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), the PK2 mRNA rhythm responds quickly: the rising phase of PK2 expression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles . During the first cycle of the advance shift, the PK2 oscillation pattern remains similar to that of the unshifted LD , 16 hour light: 8 hour dark (16L:8D), or 20 hour light: 4 hour dark (20L:4D). During 12L:12D, PK2 mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 , as PK2 mRNA rises before lights on and persists after lights off  is very similar to that observed under 12L:12D  than in shorter days (8L:16D)   Figure , further) Figure . We alsoe Figure . Under td Figure . As it hoperiods -35, we hdies see . The PerPK2 molecular rhythm is normally entrained in melanopsin-deficient (Opn4-/-) mice. Figure PK2 in the SCN of Opn4-/- mice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile of PK2 mRNA corresponds with the normal locomotor rhythm of Opn4-/- mice under light/dark conditions [Opn4-/- mice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light [PK2 is blunted in Opn4-/- mice. Figure PK2 in the SCN of Opn4-/- mice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively.As melanopsin has been implicated in circadian photoreception -11, we enditions ,8. As Opht light , we alsoOpn4-/- light pulse studies show that a residual PK2 expression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induce PK2 expression. Thus, we decided to examine the light inducibility of PK2 in triple knockout mice lacking melanopsin, rod and cone phototransduction systems (Opn4-/- Gnat1-/- Cnga3-/- mice), as these animals free run under light dark conditions (LD) and lack masking responses to light [PK2 in the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light [PK2 mRNA followed the free-running locomotor rhythms in these triple knockout mice  and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of PK2, and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of PK2.The to light . Figure to light . In addie Figure , with hiPer2 rhythm is maintained in the SCN of cryptochrome-deficient (Cry1-/-Cry2-/-) mice that lack functional circadian clock [PK2, Per1, Per2 and Bmal1 expression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes in Cry1-/-Cry2-/- mice under both light/dark (LD) and constant dark (DD) conditions. Figure Per2 remained largely intact in Cry1-/-Cry2-/- mice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of the Per2 oscillation profile was similar to that observed in wild type mice [Per1 rhythm in Cry1-/-Cry2-/ mice was also apparent under LD, but not DD . As PK2 remains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplitude PK2 rhythm in Cry1-/-Cry2-/- mice under LD is directly driven by light.We also examined the molecular rhythm of hase see . Similar Figure. . No PK2 D Figure . Further4 Figure . NeverthPK2 rhythm may be related to the maintenance of behavioural rhythms observed in Cry1-/-Cry2-/- mice under LD, we studied the responses of Cry1-/-Cry2-/- mice to a 6 hour advance of lighting schedule. In contrast to the transients of entrainment of locomotor rhythms in wild type mice (which takes about 4-5 days to re-entrain to phase advance), the locomotor activity of Cry1-/-Cry2-/- mice adjusted rapidly to 6 hr advance   Figure . As the L:4D see , it is lCry1-/-Cry2-/- mice revealed the presence of a light-driven PK2 molecular rhythm in the SCN under LD, indicating that light can drive PK2 rhythm independent of functional circadian clock. Interestingly, the molecular rhythms of some clockwork genes such as Per2, Per1, and Bmal1 were also partially maintained in the SCN of Cry1-/-Cry2-/- mice under LD  in the promoter of the PK2 gene [PK2 in mutant mice that lack functional clock may indicate that CRE-dependent pathway and CLK/BMAL1 transcriptional factors may interact in the light-induced PK2 expression in the SCN. Accumulative data have implicated the photic regulation of the transcription of clock genes such as Per1 and Per2 in the entrainment of behavioural rhythms [Per1 promoter, whose activation in the SCN shifts rapidly when the LD cycle is advanced [PK2 exhibits transients during phase advance, more similar to those of Cry1 and Cry2 [Per1 or Per2. The presence of E box motifs in the PK2 promoter suggests that light-regulated Per1 (and perhaps Per2) expression can influence PK2 expression. However, the light inducibility of PK2 indicates that PK2 may have a more direct and central role in entrainment in addition to its putative role as an SCN output signal. In other words, whether PK2 functions completely outside the central circadian loops or partly within them has yet to be determined. It is well established that the activation of glutamate receptor and its downstream actions are critical for the retinohypothalamic inputs of light to the SCN [PK2 may be relevant to both the phase resetting of the core circadian loops and critical SCN output signals.The light inducibility of 2 Figure . The samPK2 gene . CRE-depPK2 gene -48. The  rhythms ,34. The advanced , PK2 exhand Cry2 ,31. This the SCN . As rece the SCN  and acti the SCN , it is p the SCN , furtherPK2 is predominantly driven by the circadian clock, as PK2 expression exhibits circadian transients in response to phase advances. Furthermore, shortening of the light period does not result in corresponding reduction of the phase of PK2 rhythm, also consistent with the dominant control from the circadian clock on PK2 expression. However, light also modulates PK2 rhythm. Nocturnal light pulses can directly induce PK2 expression in the SCN. Studies with Cry1-/-Cry2-/- mice revealed that light can drive a low amplitude PK2 molecular rhythm in the SCN in the absence of functional circadian oscillators. These studies demonstrate that PK2 molecular rhythm in the SCN is controlled by dual mechanisms: dominantly by the circadian transcriptional loops but also directly by light. The light inducibility of PK2 in the SCN suggest that in addition to PK2's role as a SCN output signal, PK2 may also participate in the photic entrainment of circadian clock and perhaps in masking.Our studies demonstrate that PK2 expression two days after the shift, animals were placed in two additional light/dark cycles and brain samples were collected. All animal procedures were approved by the Institutional Animal Care and Use Committee and consistent with Federal guidelines. In situ hybridization was used in all studies to examine PK2 mRNA expression in the SCN [PK2 (accession number AF487280 1-528 nt), mouse Per1 (accession number AF022992 340-761nt), mouse Per2 (accession number AF035830 9-489 nt) and mouse Bmal1 (accession number AB015203 864-1362 nt) were generated.Male adult C57BL/6 mice (Taconic Farms, New York) were entrained under 12 hour light: 12 hour dark (12L:12D, lights on at 0700 h) cycle for two weeks with food and water available ad libitum. Light phase was either delayed by 6 hours (lights on at 1300 h) or advanced by 6 hours (lights on at 0100 h) and samples were taken every three hours for the 24 hour period (Zeitgeber time, ZT, ZT1-22). To examine  the SCN . AntisenAnimals were initially entrained under 12L:12D for one week, followed by placement in different photoperiods (light intensity ~400 lux) for three to four weeks: 8 hour light:16 hour dark (8L:16D, lights on at 0900 h, lights off at 1700 h), 16 hour light: 8 hour dark (16L:8D, lights on at 0500 h, lights off at 2100 h). For the 20 hour light: 4 hour dark (20L:4D, lights on at 0300 h, lights off at 2300 h), animals were first placed in 14L:10D for one week, transferred to 16L:8D for another week, followed by two weeks in 20L:4D. All brain samples were taken every two hours throughout the 24 hour cycle, except the first and the last two time points which were sampled every three hours.Opn4-/-) mice (on C57BL/6:129 hybrid background) [Opn4-/- mice and triple knockouts (Opn4-/- Gnat1-/- Cnga3-/- mice) that lack melanopsin, rod and cone phototransduction systems were used [Wild type and melanopsin-deficient  mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type and Cry1-/-Cry2-/- mice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group of Cry1-/-Cry2-/- mice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of PK2, Per2, Per1 and Bmal1 were measured in the SCN. For light pulse experiments, Cry1-/-Cry2-/- mice received a 15 min light pulse (~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark control Cry1-/-Cry2-/- mice did not receive a light pulse. For the shifting experiments, wildtype and Cry1-/-Cry2-/- mice were initially entrained under 12L:12D, then subjected to an acute 6 hour advance of lighting schedule. Running-wheel activities of these mice were monitored 10 days before and 10 days after the 6 hour advance shift. The 6 hour phase advance was then repeated and brains were collected at ZT4 and ZT16 on the day of the shift.Cryptochrome-deficient  and Per2 (b) mRNA under 8L:16D, 16L:8D, 20L:4D. Open and filled bars indicate light and dark periods, respectively. The zeitgeber time (ZT) on the x-axis reflects the timescale for each photoperiod. Each value represents the mean ± SEM of 3–4 animals.Click here for filePK2 mRNA expression in Cry1-/-Cry2-/- and wildtype mice. Representative autoradiograms of PK2 mRNA in the SCN of Cry1-/-Cry2-/- mice (Cry) and wild type mice (WT) under LD (ZT1-22) are shown (top and bottom row, respectively). Scale bar = 1 mm.Click here for file


The Medical Library Association converts access to information into access to knowledge in a networked environment of digital resources Access to PMC is free and unrestricted. Recently, NLM, working with MLA headquarters, made the full-text archives of BMLA from 1911 onward available online through PMC. This is an excellent resource for the study of health information sciences and the management of knowledge-based information, putting into practice MLA's belief in open access. The association has supported open access to information in several other ways, including memberships in the Scholarly Publishing and Academic Resources Coalition (SPARC) and the Information Access Alliance (IAA). MLA's statement on open access, found at http://www.mlanet.org/government/info_access/openaccess_statement.html, defines the association's position on this important topic.For over 100 years, the Medical Library Association (MLA) has upheld the belief that quality information is essential for improved health and has worked to ensure that health sciences librarians have the skills, knowledge, and leadership necessary for the delivery of information in a biomedical setting. The association has also promoted the concept of unrestricted, affordable, and permanent access of health information worldwide. For example, MLA's peer-reviewed User's Guide to Finding and Evaluating Health Information on the Web for the Pew Internet and American Life Project. The guide provides access and evaluation guidelines and MLA's top ten most useful Web sites, as well as lists of top Web sites for cancer, diabetes, and heart disease.However, open access increases the need for more sophisticated information management tools and systems, such as quality filtering and customization of clinical and research information at the point of need and decision-making. MLA is pursuing a number of initiatives that address the specific information needs of clinicians, healthcare students, biomedical researchers, and institutional leaders. Our members are excited to be in a unique position to develop tools, resources, and advice on how to find relevant information on the Internet. For example, MLA members have developed a http://www.mlanet.org/resources/expert_search/) and the provision of continuing education opportunities in this area (see http://www.mlanet.org/education/telecon/ebhc/index.html). These techniques identify best practices and cutting-edge clinical and research knowledge and cull through a sometimes overwhelming amount of medical literature that continues to grow exponentially. MLA's work in the area of expert searching was prompted by the increased emphasis on evidence-based practice by the Institute of Medicine. This, along with the publicity following the unfortunate death of a healthy research volunteer at Johns Hopkins about the need for more vigilance in maintaining the quality of literature searching, has created a renewed interest in the knowledge base and skill set required for expert literature searching and expert consultation. The use of evidence- or knowledge-based information retrieved through the expert searching process can help insure the clinical, administrative, educational, and research success and positive performance of the individual healthcare provider as well as the hospital or academic health center.MLA is furthering the concept of evidence-based medicine through its exploration and definition of expert searching techniques (see http://www.mlanet.org/research/informationist/). Librarians are being recruited to join clinical and research teams as clinical medical librarians and information specialists in context and to provide expert consultation on issues ranging from informatics literacy to evidence-based medicine classes.In addition to retrieving the best evidence, it is also important to deliver knowledge and services within the specialized context to patient care, research, and learning. MLA's exploration, along with NLM, of the informationist concept, i.e., specialist librarians who blend the knowledge and skills of both the clinical and information sciences, is defining new roles for librarians for providing filtered and customized clinical/research information at the point of need and decision-making  to stress the importance of “information” in health literacy. The association defines health information literacy as the set of abilities needed to recognize a health information need; to identify likely information sources and use them to retrieve relevant information; to assess the quality of the information and its applicability to a specific situation; and to analyze, understand, and use the information to make good health decisions. MLA has also developed a resources Web site for health consumers at http://www.mlanet.org/resources/consumr_index.html and http://caphis.mlanet.org/consumer/index.html that helps them find quality health information on the Web. These tools are publicly available to anyone in the world at any time.Besides health-care providers, millions of consumers search for health information on the Web every year. Recognizing the documented difficulties and frustrations health professionals and consumers face in coping with the barrage of available information in a way that results in informed healthcare decisions, MLA has established its health information literacy program (see MLA recognizes that this is a time of rapid change in our society in which the availability of digital resources in a networked environment provides unprecedented opportunities for more open access to the scientific and medical literature. As health sciences librarians, we are excited about the potential to serve a much wider group of international consumers, ranging from medical researchers to patients and their relatives. We will continue to work to convert access to information into access to knowledge.


Exhaustive methods of sequence alignment are accurate but slow, whereas heuristic approaches run quickly, but their complexity makes them more difficult to implement. We introduce bounded sparse dynamic programming (BSDP) to allow rapid approximation to exhaustive alignment. This is used within a framework whereby the alignment algorithms are described in terms of their underlying model, to allow automated development of efficient heuristic implementations which may be applied to a general set of sequence comparison problems.The speed and accuracy of this approach compares favourably with existing methods. Examples of its use in the context of genome annotation are given.This system allows rapid implementation of heuristics approximating to many complex alignment models, and has been incorporated into the freely available sequence alignment program, exonerate. George Box  once rem"All models are wrong, but some are useful."useful in the context of large scale analyses.This statement bears much relevance to biological sequence alignment, where there is no guarantee that the alignment model will accurately represent the evolutionary (or other) processes which separated the two sequences. All that is certain is that exhaustive dynamic programming (DP) algorithms (such as Smith-Waterman ) will yiTransformation between a finite state automata describing an alignment model and the recurrence relations used in DP is a powerful technique ,5 as it The availability of vast amounts of sequence data has generated a need for faster alignment algorithms . Many ofThe alignment program FASTA , generatautomated generation of heuristics in terms of the underlying model. This allows the development of heuristics for complex models such as those used in GeneWise.The aim here is to combine the strengths of Gapped BLAST and Dynamite, in a system which allows Firstly, we describe bounded sparse dynamic programming (BSDP), a novel heuristic for sequence alignment, then we describe the system for automated implementation of the complex alignment algorithms required. We present proof of principle results, but this paper is primarily focussed on developing a framework for the general case.The basic strategy of seeding alignments used here is the same as for BLAST, in that alignments seeds are generated, and then extended to form High-scoring Segment Pairs (HSPs), which are then joined together to form the alignments. The alignments are seeded using an Aho and Corasick  type finHowever the methods for seeding HSPs are independent from those used for building the alignments, and this paper only deals with algorithms involved in the generation of gapped alignments from sets of HSPs, and not in the calculation of the HSPs themselves.The following strategies are employed to enable alignments to be built efficiently from sets of HSPs:portal describes a set of states in the model which correspond to a set of HSPs, a span refers to a looping state for large alignment features such as introns, and a SAR (Sub-Alignment Region) describes a rectangular region on an HSP to which DP is applied.• To connect the underlying alignment model to the heuristics, a upper bounds are generated for the best alignment score for each SAR, and BSDP (Bounded Sparse Dynamic Programming) exploits these bounds to yield alignments in an efficient manner.• To avoid DP in every SAR, • To perform various types of DP in these SARs, the required models are generated automati cally, including C code to produce an efficient viterbi implementation for each model.sparse dynamic programming [sub-alignments improve the quality of the overall alignments, and they allow complex alignment models, and large gaps such as introns to be integrated into a sparse DP framework. Secondly, as it would take too long to apply DP to every sub-alignment region (SAR), upper bounds are calculated for the DP scores for each of these SARs. This allows the sub-alignment DP to be avoided in cases where it joins HSPs which cannot feature in the final alignment, so that alignments can still be generated very rapidly even when large numbers of HSPs and SARs are involved.Dynamic programming  requires quadratic time, and hence is the most computationally expensive part of building an alignment. For pairs of sequences more than a few kilobases long, DP becomes prohibitively slow. The approach used here is similar to gramming , and thegramming , in thatBefore the BSDP can be performed, a single point on each HSP is selected which will feature in any alignment generated using that HSP. This point corresponds to a pair of equivalenced symbols which must feature in any alignment to include that HSP. A point is chosen where half the HSP score is generated by equivalenced symbols on either side of it, as this is likely to be in the highest quality portion of the HSP. As shown in the example in Figure The five types of region used for sub-alignments are classified in Figure Regions for the sub-alignments are selected within the area between the centre points of the two HSPs to be joined, or in the case of terminal HSPs, between the HSP and the ends of the sequences. In addition, the positions of the SARs must be constrained to limit their size, and so that the HSPs correctly intersect with the corners of the SAR. In the case of overlapping HSPs, where there is a choice of positions for placement of the SAR, the position is chosen such that the highest scoring parts of the HSPs are outside the SARs.Once the SARs have been selected, an upper bound is placed on the score for each sub-alignment. The calculation of these bounds is described in a later section describing automation of this method. The BSDP approach can then utilise these upper bounds to avoid application of DP in some SARs, as demonstrated by the example in Figure The BSDP is mediated through a set of priority queues, one of which is associated with each HSP, and will contain an entry for each partial alignment that ends at the HSP. The key for these priority queues is the highest score for any partial alignment ending in that HSP. Additionally, there is one global priority queue containing the highest scores from each of the other priority queues. The upper bound scores are confirmed by DP in the SARs in the current highest scoring putative alignment path. The highest scoring path will change as the scores are updated during this process. DP is applied to SARs in this way until the highest scoring path does not contain any bound scores, and then the alignment may be extracted. Upper bounds dictate that there can be no better alignment using these HSPs.admissibility property of A* search, such that the result of the BSDP computation is guaranteed to be the same as if DP had been performed on every candidate SAR prior to calculation of the alignment. This is because no alignment can be extracted until all the alternative sub-alignments (which have upper bounds that indicate they could contribute to a higher-score) have been eliminated.This algorithm is similar to A* search, (but differs in that many different points may be the start or end of the search), and retains the The BSDP alignment process can be iterated to generate sub-optimal alignments similar to those generated by the Waterman-Eggert algorithm , with onAfter the first alignment has been reported, the scores for any SARs which have already been confirmed by DP, but which are not yet included in a reported alignment, are then considered as an upper bound. SARs are disallowed before recalculation when the region between the centre of the HSP and its SARs overlaps with a previously reported HSP, in which case, the SARs are disallowed. Otherwise the DP is recomputed for SARs which contain part of an alignment which has been reported since the DP for the SAR was last calculated.As illustrated in the previous section, BSDP becomes quite complex and requires a large number of DP algorithms for computation of the alignment through the SARs. We have build a system to facilitate implementation of these models and the integration of the sub-alignments which they produce. To allow generalisation of the BSDP, everything must be defined in terms of the underlying alignment models. The alignment models are described as finite state machines, consisting of states and transitions, similar to those used in Dynamite .Briefly, to convert these models into DP implementations, each state must correspond to a score in each cell of the DP matrix, and the scores for each cell are calculated by taking the maximum of the score from transitions arriving at each cell. In addition, a topological sort is required to satisfy dependency ordering for silent states.However, in addition to automated generation of code from alignment models, the generation of the models required for DP in the SARs is also automated, as described below.Table To enable DP in the SARs for the BSDP, a heuristic model is generated from the original alignment model. This model is not used directly for calculation of alignments, but a derived model is generated corresponding to each transition in the heuristic model. Each of these derived models correspond to a type of SAR used in the BSDP.portal states or span states. A portal defines a group of states which can share a set of HSPs (High-scoring Segment Pairs); these are the match states. A span is a state which has sequence independent looping transitions .The model is first annotated, labelling certain states as either portal which corresponds to the match forward and match reverse states, and the intron forward and intron reverse states are span states.The heuristic model is build using states corresponding to each portal and span state, with transitions between these states in cases where there is a valid path between the corresponding states in the original model. An example model is shown in Figure Derived models are produced for the DP in SARs automatically from each transition in the heuristic model. The source and destination states from each transition in the heuristic model become the start and end states in each derived model. All reachable states and transitions from these states in the original model are recursively copied to the derived model. An example of this process is shown in Figure match forward and match reverse states, shown in Figure For some cases, such as between the portal states and two span states in this model, and therefore, a derived model is generated for each of the five cases in Figure Ten different derived models are generated from the model shown for cDNA to genomic alignment in Figure For each derived model, an additional model is created which is used for the pre-calculation of upper bounds for all possible sizes of SARs. For each transition in the model which has a position dependent score associated with it, the upper bound is also supplied. For example, in a match transition, the upper bound is the maximum value from the substitution matrix. A special model is created using these transition upper bounds, instead of the normal sequence-dependent transition weights. As this removes the sequence-dependent components of the algorithm, it allows pre-calculation of the upper bound for alignment score of any sequences up to the maximum permitted size of SARs. These bounds are then stored in a table for retrieval during the BSDP.The model used in Figure This system is used for alignment of ESTs within the Ensembl gene-building pipeline , and a pFigure Another example is the alignment of the Collagen alpha 1(IX) precursor to a region of chromosome 6 containing the corresponding gene. This is a large gene, containing 38 exons over about a 90 Kb region of the genome. Using GeneWise, the alignnment required 4260 seconds (about 1 hour, 11 minutes) using exonerate to perform full DP alignment required 2700 seconds (about 45 minutes), and using exonerate with the heuristic BSDP approach required only 7 seconds, with all three methods generating identical alignments.This approach represents an advance from previous methods for automatic implementation of sequence alignment algorithms -6, in thThis has allowed development of heuristics with sub-quadratic running times. This makes their application practical to a much larger set of problems, while retaining the much of the simplicity of their implementation.We recognise that this approach is limited to the subset of sequence comparison problems which can be represented by a regular grammar. For example, it is not possible to use this system to model stochastic context free grammars as used in some types of RNA secondary structure such as pseudo-knots . This apThis framework has already been used for many different alignment models ranging from simple models such as Smith-Waterman-Gotoh, to more complex models such as those for protein to genome alignment as used by GeneWise . It is aAlthough this method is useful in many cases, for some types of distantly related sequences, the method can breakdown. For example, when long regions of the correct alignment contain many gaps with intervening sequences too short to be an alignment seed, the alignment cannot be extended beyond the boundary of the SAR, and the correct alignment may be missed. These cases can be avoided by increasing the size of the SARs, but this results in higher bound scores, so DP become necessary in more of the SARs. Such gap rich alignment are the type where the gapped HSP extension used by Gapped BLAST excels, however such gapped extension necessitates DP surrounding all HSPs, which becomes time consuming when allowing both short exons and long introns during alignments of cDNAs or proteins to genomic sequences.As this system separates the development of the underlying alignment model from the heuristics which are built on top of them, we expect that this framework will prove useful for evaluation of the quality of the heuristics, as comparison between the alignments from the two techniques can be automated, and training may be performed to select optimal parameter sets., and in the exonerate module of the Ensembl CVS repository from . It is built using the glib portabilility library available from  Both exonerate and glib are available under the GNU lesser general public license. The code has been tested extensively on Linux and OSF1/alpha but has been written to be portable to many UNIX systems.The implementation of the system described here is called C4 (to reflect the aim of producing something more powerful than Dynamite ), and isCode generation for the DP is performed to exploit compile time optimisations such as loop un- rolling and and efficient handling of edge conditions in the viterbi matrix, which is particularly beneficial for the small DP calculations required by SARs. This generated code is typically about five times faster than using a generic viterbi implementation, but produces about a million lines of code for the current set of models used by exonerate, and hence compilation takes longer.