The large amount of data that are currently produced in the biological sciences can no longer be explored and visualized efficiently with traditional, specialized software. Instead, new capabilities are needed that offer flexibility, rapid application development and deployment as standalone applications or available through the Web.) – that enables fast development of applications for protein analysis and visualization. The toolkit is written in Java, thus offering platform-independence and Internet delivery capabilities. Several applications of the toolkit are introduced to illustrate the functionality that can be achieved.We describe a new software toolkit – the Molecular Biology Toolkit . Recent scientific and technical advances in the field of experimental biology, particularly in genomics, have produced large amounts of biological data, which has posed new conceptual challenges. The visualization and visualization-driven analysis of these experimentally derived data has become a key component of the scientific process.Until recently, these needs were typically approached by designing applications specialized in a set of well-defined specific tasks. So, for example, popular applications for molecular visualization include, but are not limited to, Molscript , PyMol , Rasmol This paper, aimed at bioinformatics software developers, provides a concise presentation of the design and capabilities of the toolkit, presents a number of models for its usage, and illustrates its performance with several applications. The paper is organized as follows. The following section provides a general overview of the application programming interface (API). The Results and Discussion section presents details about core components of the toolkit and illustrates its capabilities with two applications. The Conclusions section summarizes the main functionality of the toolkit, discusses availability and documentation, presents performance data and outlines future development plans.The application-programming interface (API) that serves as the foundation of the toolkit has emerged from the exploration of the typical needs encountered by a researcher in the analysis of a documented biological molecule. The list of requirements includes reading the data file and converting the information to data objects that are easily accessible by applications, extracting subsets of chemical components according to different chemical or biological properties of interest , deriving information that is not explicitly present in the original input data  and visualizing chemical or physical properties of different subsets of the molecule, or the entire molecule.StructureFactory class offers a uniform approach to loading structural data, independent of the format of the source. The class makes use of a set of loaders that can import data from a variety of sources, either on the same machine or located on a remote server. Moreover, this allows the developer to write applications that do not have to uniquely specify the source of data. Instead, a number of methods in the StructureFactory class enable loading of structures based on a series of source descriptors: file name, PDB id code, URL location, etc. The first loader capable of handling the source descriptor is used thus providing a data access and retrieval mechanism that is transparent to the user.Based on the requirements identified above, we have arrived at the multi-layered design illustrated in Fig. StructureFactory is a Structure object, which is effectively an interface to a summary of raw primary and secondary structure data. The Structure object is not restricted to holding structural information and can contain any other data relevant to the organization of the molecular entity. For instance, it can store the description of single or multiple protein sequences preserving the ordering and alignment of the amino acid residues and their properties.The output of the load methods in the StructureMap class builds the internal data model of the simple or complex molecule and provides hierarchical access to both raw data and derived information generated from the input. This includes access to chains, residues, atoms, bonds, nucleic acid components, ligand atoms, fragments associated with secondary structure components, or features defined by the user.The StructureStyles class provides information about rendering, coloring and selection attributes associated with any structure component produced by the StructureMap. A wide variety of methods in this class allow any module of an application, in particular any viewer, to set and retrieve the information describing the visually represented parameters of any structure component.The StructureDocument is designed as a container class that maintains a log of all loaded structures and viewers that are instantiated by an application in a given session. It also has the role of generating events associated with the addition or removal of structures and viewers.The The next level of the API contains graphical user interface (GUI) elements. This portion of the package contains high level constructs, such as windows and panels designed to display 3D graphics, protein or DNA sequences or tree representations of the chemical components of the molecule, as well as lower level components that can be used to build a molecular scene according to a developer's preferences.StructureMap class to retrieve all secondary structures within a given molecule, or how to obtain a list of atoms or residues. This is the level that most developers would modify in order to create custom applications tailored to their specific needs.Finally, at the application level, a number of applications are provided that can be used to illustrate the features of the toolkit, or as starting templates for new application development. For example, the applications illustrate how to use methods in the Note that care has been taken to further enable application developers to use different components of the toolkit to build purely analytical applications that have no visualization component. For example, a developer could write a command-line tool that simply loads a molecule and gathers statistics about the structure data without presenting any graphical interface. Such an application could be part of a back-end process that runs in a web-server environment.The MBT was developed as an object-oriented Java-based environment and hence is flexible, modular and lightweight, which facilitates maintenance, web deliverability and limits the required computer resources. Moreover, the toolkit can be easily extended and having been written completely in Java is effectively platform-independent.Structure object  is built, which is then attached as a BranchGroup node to a SceneGraphObject  representing the three-dimensional image of the molecule. The PsGeometry (primary structure) and SsGeometry (secondary structure) classes provide a number of methods that build complete 3D scenes from a given set of primary or secondary structure data object  provides a set of pluggable and extensible classes for use by application developers interested in the visualization and analysis of macromolecular data. The MBT provides a set of pre-written data loaders, viewers, a common data model and the means to add to and customize the toolkit for specific applications delivered as applets through the Web or as standalone applications. Base functionality and comparable tools (where applicable) are as follows:• Classes to load raw data from a number of common protein structure and sequence data sources  and a means to easily add new "loader" modules independent of the applications they might serve.StructureComponent objects) are provided  and new data types are easily registered. Further the data model provides an extensible means to describe viewable or visible attributes  of these objects.• A common data model to which raw information is imported, mapped, and indexed. A number of data record types  directly inside web pages. This enables the deployment of tightly coupled interactive web content much like the popular MDL Chime plug-in.• Applications are not restricted to the features provided by the MBT APIs. The Programmers Guide details how to extend the system.• With source code provided, core features of the toolkit may be directly modified or extended for independent use . However, adding code within the existing framework and contributing it back for others to use is encouraged. The source code has been extensively commented to produce a rich and complete set of hyper-linked javadoc API documents.• A set of pre-written viewers  that can be extended, replaced, omitted, or augmented with completely new viewers that implement entirely new visualization techniques.StructureViewer module provides visual representations similar to RasMol and Molscript such as balls-and-sticks, CPK spheres, split-bonds, extrusion/ribbon-style backbone traces, and secondary structure cartoons . It also provides 3D labeling.• The 3D • A scripting interface is being developed for MBT to enable toolkit functionality to be command-line or even script-driven (similar to MDL Chime or PyMol).5 atoms require physical memory in excess of 500 MBytes and on a notebook computer with a 1.2 GHz processor can take nearly one minute. Efforts at optimization are on-going.Version 1.0 of the toolkit and sample applications, including those described here, are available for download from the project web page . The sammbt@sdsc.edu. Bugs may be reported to a bug tracker available on the project web site [New applications are on-going including the generation of high quality images for all structures in the Protein Data Bank and new ways of visualizing protein-protein interactions. We invite contributions to the MBT by sending mail to web site .Project Name: Molecular Biology Toolkit (MBT)• Project Home Page: • Operating System: Platform independent• Programming Language: Java• Other requirements: Java 1.3.1 or higher, Java3D• License: Free for educational, research and non-profit purposes• Any restrictions to use by non-academics: Contact the University of California at San Diego's Technology Transfer Office • JLM is one of the designers of the API and co-developer of the toolkit. AG designed and implemented the geometry generation modules, implemented the algorithms for secondary structure generation and bond detection, and drafted the paper. OVB developed the PKR Explorer. QZ developed the Ligand Explorer. PEB coordinated the whole project, suggesting the general functionality and scientific objectives of the toolkit.


Gene expression analysis based on comparison of electrophoretic patterns is strongly dependent on the accuracy of DNA fragment sizing. The current normalization strategy based on molecular weight markers has limited accuracy because marker peaks are often masked by intense peaks nearby. Cumulative errors in fragment lengths cause problems in the alignment of same-length fragments across different electropherograms, especially for small fragments (< 100 bp). For accurate comparison of electrophoretic patterns, further inspection and normalization of electrophoretic data after fragment sizing by conventional strategies is needed.Here we describe a method for the normalization of a set of time-course electrophoretic data to be compared. The method uses Gaussian curves fitted to the complex peak mixtures in each electropherogram. It searches for target ranges for which patterns are dissimilar to the other patterns  and for references  in the set of resultant approximate patterns. It then constructs the optimal normalized pattern whose correlation coefficient against the reference in the range achieves the highest value among various combinations of candidates. We applied the procedure to time-course electrophoretic data produced by HiCEP, an AFLP-based expression profiling method which can detect a slight expression change in DNA fragments. We obtained dissimilar ranges whose electrophoretic patterns were obviously different from the reference and as expected, most of the fragments in the detected ranges were short (< 100 bp). The normalized electrophoretic patterns also agreed well with reference patterns.The normalization strategy presented here demonstrates the importance of pre-processing before electrophoretic signal comparison, and we anticipate its usefulness especially for temporal expression analysis by the electrophoretic method. Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique using electropherograms . AFLP anMspI and MseI. Consequently, the HiCEP method can detect a slight expression change of transcript-derived fragments (TDFs) with high coverage. The estimated 30,000 transcripts expressed in a cell are divided into 256 subgroups (16 MspI-NN primers * 16 NN-MseI primers) containing approximately 120 PCR-amplified TDFs. This number is small enough to be separated by fluorescent capillary electrophoresis using an automated DNA sequencer such as the ABI Prism 310 (Applied Biosystems). We can achieve higher throughput by using several fluorescent dyes at once [Recently, we developed an AFLP-based gene expression profiling method called HiCEP (High Coverage Expression Profiling) . The exp at once ,15.MseI) tends to produce many small TDFs (< 100 bp) and in our experience this range is prone to errors of fragment sizing. Cumulative errors of fragment sizing interfere with normalization across different electropherograms and lead to the mis-assignment of valid TDFs. Hence, more detailed analysis such as observation of gradual expression changes in the time series of a TDF still counts in subjective visual examination [Normally, digitized electropherograms are imported into image analysis software such as GeneScan (Applied Biosystems), which outputs each fragment (band) together with its length (in bp), area and height , carrying out accurate fragment sizing and background subtraction for most of the operations. GeneScan is capable of separating the signal from each fluorophore to provide higher throughput analysis. However, it should be noted that intense signals from abundant TDFs can breed into each other, potentially confusing the fragment sizing ,15. Furtmination . FurtherThe purpose of the present study is to develop a normalization method for the automated analysis of temporal electrophoretic data. We assume the samples to be compared are identical, that TDFs have similar fragment lengths across electropherograms and that expression changes can be detected as variations in peak height using the HiCEP technique. The performance of the method is demonstrated by analyzing a large set of time-course data obtained from mouse embryonic stem (ES) cells, using HiCEP.We analyzed a total of 2560 HiCEP electropherograms (256 sets of ten), containing time-course data of embryonic stem (ES) cells 0, 12, 24, 48, and 96 h after adding stimulation for differentiation. Reproducibility was confirmed by the duplication. We applied the current method to each of the 256 sets.0 h-1, 12 h-1, and 48 h-2) in the range (35–50 bp) appear to be compressed on the short side. This is probably because another intense peak just under 35 bp is mistaken for the 35 bp marker peak. This reduces the overall similarity between lanes and makes it difficult to recognize identical TDFs such as red filled peaks in Fig. When a set of electrophoretic data is arranged and surveyed, one can often find ranges  in which peak fragment lengths are incorrectly measured. For example, in Fig. Ptarget and the other lanes within a certain range using equation 3. By using the average correlation coefficients, we can make a quality score function Qk (t) for all lanes  at arbitrary length t (see Methods). An example of the calculation for lane 0 h-1 is shown in Table Q(t) take the place of detailed visual evaluation of dissimilar ranges  and at 75 bp in two lanes (0 h-2 and 96 h-1).To this end, we first developed a method for displaying dissimilar ranges. The method is based on a moving-fragment approach that continuously determines the average correlation coefficient between particular lane ges Fig. . UndoubtNext, we applied a simple method for the automated detection of dissimilar ranges to 256 sets of electrophoretic data (see Method). The method identified a total of 362 dissimilar ranges. Most 289, 79.8%) of the ranges were of 100 bp or less. This is reasonable because the main source of fragment sizing errors is the presence of intense peaks near the marker 9.8% of t,15 and t0 h-1, 0 h-2, 12 h-1, 12 h-2, and 48 h-2) in range (35–50 bp) and two lanes (0 h-2 and 96 h-1) in range (50–100 bp). Of these, we at first suspected that two lanes (0 h-2 and 12 h-2) in range (35–50 bp) were false-positives (mistakenly identified as dissimilar). However, we observed that the range in the two lanes is worthy of being normalized: the fragment lengths on the short side of the range deviate gradually from the mean lengths of lanes 24 h-1, 24 h-2, 96 h-1, and 96 h-2 [see Visual examination revealed many of those ranges to be genuine, but not all. The set of ten electropherograms shown in Fig.  h-2 see .Visual examination of all the electropherograms did not reveal any false-negative errors (overlooked dissimilar ranges). Recall that the samples to be compared are identical and that the measure of the quality of fragment sizing is based on a calculation of the average correlation between electropherograms. These results suggest that the normalization strategy we present here is useful, especially for temporal expression analysis.T in equation 3 in the Methods section, which is the number of consecutive fragments making up the quality profile examined by the program. Quality profiles using the shortest span (T = 1) are noisier than those using a moderate span, and runs using spans of less than four fragments were found unsatisfactory in our investigation. On the other hand, long spans (T = 10) tended to miss small dissimilar ranges. These trends are essentially the same as those in the delineation of hydropathy plots of proteins using a moving-window approach and in the detection of transmembrane regions [T = 5 throughout the analysis, further improvement in the choice of parameters as well as the method for the detection of dissimilar ranges remains to be studied.The effectiveness of the method depends on the choice of the parameter  regions . Althoug96 h-2 as the best reference in ranges (35–50 bp) and (50–100 bp). We cannot, of course, reject the possibility that accurate fragment sizing is performed in the minority group  in Fig. To normalize dissimilar ranges across a set of electropherograms, it is necessary to select one as a reference. In conventional algorithms the reference is selected manually ,18. For D × 100% expansion (or - D × 100% compression) to D × 100% compression of the short side of the original profile at intervals of d bp. The best approximating profile is one of the candidate profiles with {x × d - D × (Ce - Cs)} / (Ce - Cs) × 100% compression of the side in a given range (Cs - Ce bp), where x = {0, 1, ..., 2 × (Ce - Cs) × D / d}. There is of course a trade-off between the computation time and the normalization accuracy in the choices of parameters. In Model 1, we set D = 0.4 and d = 0.2. We expected that the normalization would be achieved by a linear expansion of the short side of the dissimilar range (35–50 bp) by anchoring the long-side in the target lane 12 h-1. Indeed, the best approximating profile that achieved the highest correlation coefficient against the reference 96 h-2 was the case of x = 9 (28% expansion).We prepared two models for accurate normalization of various types of fragment sizing errors. Model 1 is the case of an incorrect fragment sizing at the shortest (or longest) marker peak. Figure Mj in a dissimilar range (Mj-1-Mj+1 bp) (see Methods). Accordingly, the program can easily determine the length of 75 bp because there is only one marker length inside of the range (50–100 bp). We can directly apply the normalization procedure for Model 1 to Model 2 by considering two hypothetical dissimilar ranges, (50–75 bp) and (75–100 bp). The main difference from Model 1 is that the two ranges cannot be normalized independently in Model 2: {x × d - D × (100 - 50)}/(100 - 50) × 100% compression (resp. expansion) of the long-side of the original profile in range (50–75 bp) and {x × d - D × (100 - 50)}/(100 - 50) × 100% expansion (resp. compression) of the short side in range (75–100 bp) affect on each other. In Model 2, we set D = 0.1 and d = 0.2 as a maximal realistic displacement. The best approximating profile is the case of x = 13 and is consistent with the reference profile.Figure 0 h-2 has two normalized ranges) are normalized nearly perfectly  which is a possible false-positive error are normalized as 2.7% compression of a short side of the range. The correlation coefficients between the target 0 h-2 and the reference 96 h-2 in the range before and after normalization are 0.674 and 0.798, respectively.Figure tly Fig. . For exa48 h-2 indicates that an incorrect normalization is performed in range (35–50 bp) of the lane. The low correlation coefficient (0.4) between the normalized profile and the reference 96 h-2 in the range, compared to values (> 0.7) between four other normalized profiles  and the reference in the corresponding range, strengthens this suspicion [see 48 h-2 should be extended on the long side. We searched for the best range to be normalized and chose (35–53.6 bp). The correlation coefficient of the normalized profile, expanded by 26.3% on the short side in the range (35–53.6 bp), was 0.9. Undoubtedly an exhaustive search for edges in dissimilar range might yield better normalization for some cases. However, it also dramatically increases the possible combinations of normalization candidates. It is a balance between the computation time and the number of analyzable TDFs.A quality profile for lane cion see . After vQk (t) for lane k indicates greater consistency with the other lanes around arbitrary length t if the sample is identical . The quality scores after normalization overall were higher than before  which indicates a relative similarity at t (bp) in lane k to the other lanes. We demonstrate two other sets of electrophoretic data and discuss the feasibility of the method.A critical step in the analysis of 1-D electrophoretic data is the assignment of the correct size to each TDF. In time-course data, one expects that the same TDFs should have quite close fragment lengths across electropherograms and that temporal expression changes are reflected as differences in peak height. We developed the current method aimed at temporal expression analysis by the electrophoretic method and used a scoring system for an objective evaluation of experimental reproducibility using Figure 24 h-2, 48 h-2, and 96 h-1) detected by the method. Of these, 24 h-2 and 96 h-1 were normalized using Model 1 and 48 h-2 was normalized using Model 2. Visual evaluation of the electrophoretic patterns and the quality scores after normalization verified the choices of the models as appropriate  where both Models 1 and 2 are applicable. A set of ten electrophoretic patterns and their quality scores shown in Figure 5 bp wherQ(t) necessary for the accurate alignment of valid TDFs across lanes is about 0.7  ≥ 0.7 in the range (35–700 bp) before and after normalization are 202,204  and 205,829 (92.7%), respectively. Furthermore, 3,334 (92%) of the 3,625  new high-quality peaks were < 100 bp, which corresponds to the biased distribution of the detected dissimilar ranges (nearly 80% of which were 100 bp or less).We also estimated the feasibility of the method with regard to an increasing number of peaks with certain quality score or more. The minimum value of 0.7 Fig. . Accordii.e., corresponding TDFs across electropherograms should have nearly the same fragment lengths). The monitoring of temporal expression change by the HiCEP technique has great potential for screening of genes related to chemotherapeutic drug resistance, circadian rhythm, and so on [When we apply the method to HiCEP time-course data, we assume that the set of electrophoretic data to be compared is identical  cells at 0, 12, 24, 48, and 96 h after removal of Leukemia Inhibitory Factor (LIF) from the culture medium. The samples subjected to HiCEP reaction were duplicated. We designated each sample as MspI combined with MseI) and ligated with the corresponding adaptors. The resulting HiCEP templates, MspI-MseI-poly(A) mRNAs, were amplified by fluorescently labelled primers; for labelling, FAM, HEX, and NED were used. In total, 256 primer combinations  were used in the HiCEP analysis. For example, a primer combination of MspI-TA and GC-MseI is capable of amplifying particular transcript-derived fragments (TDFs) corresponding to that combination. The details of the protocol of the HiCEP reaction are described elsewhere [.mRNAs prepared from each sample were digested with two 4-bp-cutting endonucleases (lsewhere . An animL (in bp), peak height H, and area A (in arbitrary units). Accordingly, the subsequent normalization procedure accepts these three-tuples as input for detected TDFs between 35 bp and 700 bp. TDFs smaller than 35 bp or larger than 700 bp were omitted from the analysis because the range was outside the size calibration range.The PCR products were denatured and loaded on an ABI Prism 310 (Applied Biosystems) for capillary gel electrophoresis. The digitized images were analyzed by the GeneScan software (Applied Biotech). The size of the fragments was calculated by the software, according to internal molecular size markers of 35, 50, 75, 100, 139, 150, 160, 200, 300, 340, 350, 400, 490, 500, 600, and 700 bp, on each gel. The fragment sizing and baseline subtraction were performed by the software. The software quantifies each peak by the fragment length MspI-CT combined with tt-MseI (designated as CT-tt)' because the ten electropherograms have some ranges for which fragment sizing is obviously inappropriate (we therefore designated such ranges as "dissimilar ranges").The starting point of normalization is a set of lanes  in each of 256 primer combinations. We explain the procedure using data from the primer combination of 'Fi in lane P is originally characterized by the three-tuples . If lane P consists of n fragments , the approximation of the lane at length t is given by:The first step starts from the Gaussian approximation of each lane. The use of the approximating lane is the same as described in Aittokallio et al. -27. Brieσi is obtained from the following equation:where CT-tt are shown in Fig. The approximation is performed independently for each lane. The ten approximate profiles of time-course data in the primer combination of , we next assign quality scores to each of the fragments , where the fragments are originally numbered with respect to their lengths. By using the ten approximate profiles, relative similarity scores  for intervals from fragment i to fragment (i+T-1)  in lane Ptarget  are calculated from the following equation:For the automated identification of 'dissimilar ranges' from the expression profiles of ten lanes  is the Pearson correlation coefficient between the target lane Ptarget and one of the other lanes Pk in the interval (start-end bp) which always includes T fragments from fragment i to fragment (i+T-1) . The interval is defined as: start = Li - 2.5σi and end = Li+T-1 + 2.5σi+T-1. In this analysis, the number of fragments T is held constant at T = 5; other numbers are of course possible. By applying a moving window of T fragments, most of the fragments  have T relative similarity scores. Finally, the relative quality value Q(Li) for fragment Fi is defined as the average of the similarity scores which satisfy start ≤ Li ≤ end. An example of the calculation is given in Table t, Q(t), are interpolated by the use of cubic splines to . The procedure is applied to each of the ten lanes  and then the quality profiles  corresponding to the expression profiles are created  have a clear interpretation. The high (or low) score for Qk(t) in lane k indicates a high (or low) level of relative similarity between the lane and the others around the length t.The quality profiles delineated from Now we have information  for the automated detection of dissimilar ranges. Here we adopt a simple method for detecting the range. Briefly,Cseed_s - Cseed_e bp) which satisfy two conditions: a) Q(t) ≤ thresseed and b) they contain at least two peaks.1) Seek 'seed' ranges  Seek   to Ctmp_s (resp. Ctmp_e) for Cs (resp. Ce); accordingly, both Cs and Ce =  and Cs <Ce3) Substitute the nearest marker length thresseed is set to 0.3 empirically. Forexample, P9 has the following parameters in Fig. Cseed_s = 57.04, Cseed_e = 89.98, Ctmp_s = 52.60, Ctmp_e = 104.60, Cs = M2, and Ce = M4. Although fine tuning might be necessary, the procedure enables us to display dissimilar ranges.Aparameter Cs - Ce bp), we have to select the "reference" . One method is to choose lane Preference satisfying max {}, where  is the average of Qk in the range (Cs - Ce bp) in lane . For example, the algorithm selects P10  as a reference in a particular range (M1-M2 bp) and also in range (M2-M4 bp).When we want to correct a dissimilar range (L) and the areas (A) of peaks in a dissimilar range so that the similarity between the normalized electrophoretic pattern and the reference pattern in the corresponding range can be maximized. To normalize a particular lane Ptarget against the reference Preference, we now consider the following two models. Model 1 is the case of an incorrect fragment sizing at the shortest (or longest) marker peak, i.e, Cs = M1 = 35 (or Ce =  = 700). The peak lengths deviate more and more from the reference length moving from Ce to Cs (or from Cs to Ce). Model 2 is the case of an incorrect fragment sizing near marker length Mj ). Roughly, the deviation of peak lengths from the reference length gradually increases starting from Cs; the maximum deviation is reached at Mj (Cs <Mj <Ce); the deviation decreases gradually; and finally disappears at Ce bp.The meaning of the word "normalization" here is to correct the fragment lengths (P3 (12 h-1) in range (M1-M2 bp) against the reference P10 (96 h-2). Undoubtedly, the profile displays a systematic deviation from the reference. The degree of the deviation gradually increases starting from M2 bp to M1 bp probably because an intense peak generated near the shortest marker peak for the correction of M1 bp is used mistakenly. We expect the normalization will be achieved by a linear expansion of the short side (M1) of the range (M1-M2 bp) by anchoring the long side. The best approximating profile is found by considering various combinations of normalization candidates starting from D × 100% expansion to D × 100% compression of the short side at intervals of d bp. We set D = 0.4, as a maximal realistic displacement and d = 0.2. Accordingly, in practice, the number of combinations is 2 × (Ce - Cs) × D/d + 1 ) in Model 1.Normalization is performed by either expanding or compressing. Consider, for example in Model 1, normalization for the expression profile of x  × D/d)}, we make a candidate profile Px by changing three parameters  accompanied by fragments (Fi) in the dissimilar range (Cs - Ce bp), according to the level of correction (expansion or compression). Those parameters are calculated as follows:For each combination  in a given range (Cs - Ce bp) into eq. (1). The best approximate profile is the one that achieves the highest correlation coefficient between Preference and Px  × D / d}) in the range (Cs - Ce bp). In the normalization for the expression profile P3 in the range (M1-M2 bp) against the reference P10, the best normalized profile by our method matches well with the reference  in range (M2-M4 bp) with the reference P10 (96 h-2); there is no possibility of Model 1 (Cs ≠ M1) and the number of incorrect marker lengths is only one (M3). Model 2 is a mixture of Model 1. The normalization is also done by one of the {2 × (Ce - Cs) × D / d + 1} combinations starting from D × 100% compression of the long side in (M2-M3 bp) and D × 100% expansion of the short side in (M3-M4 bp) to D × 100% expansion of the long side in (M2-M3 bp) and D × 100% compression of the short side in (M3-M4 bp) at intervals of d (= 0.2) bp. Unlike Model 1, we set D = 0.1 as a maximal realistic displacement. In the normalization for the expression profile P2 in the range (M2-M4 bp) against the reference, the best normalized profile by our method is matches well with the reference  is very wide , there are two or more possibilities for incorrect marker lengths in Model 2. Of these cases, we only consider cases with j = 1 in Model 1 because such cases are the only realistic ones. For the remaining cases , the experiment should be redone rather than trying to normalize them by considering numerous possibilities. It should also be noted that there is a case of a dissimilar range (M1-M3 bp) to which both Models 1 and 2 are applicable. In this case, the best approximate profile is decided by comparing the two best possible profiles determined using Models 1 and 2.It should be noted that when a dissimilar range  in Fig. 0 h-2 and 12 h-2) confirmed the identification.Click here for fileMagnified electrophoretic patterns and the quality profiles in range (35–50 bp) in Fig. 0 h-2 and 12 h-2) which are suspected false-positive errors.Click here for file


We describe a simple approach we used to identify barriers and tailor an intervention to improve pharmacological management of hypertension and hypercholesterolaemia. We also report the results of a post hoc exercise and survey we carried out to evaluate our approach for identifying barriers and tailoring interventions.We used structured reflection, searched for other relevant trials, surveyed general practitioners and talked with physicians during pilot testing of the intervention. The post hoc exercise was carried out as focus groups of international researchers in the field of quality improvement in health care. The post hoc survey was done by telephone interviews with physicians allocated to the experimental group of a randomised trial of our multifaceted intervention.A wide range of barriers was identified and several interventions were suggested through structured reflection. The survey led to some adjustments. Studying other trials and pilot testing did not lead to changes in the design of the intervention. Neither the post hoc focus groups nor the post hoc survey revealed important barriers or interventions that we had not considered or included in our tailored intervention.A simple approach to identifying barriers to change appears to have been adequate and efficient. However, we do not know for certain what we would have gained by using more comprehensive methods and we do not know whether the resulting intervention would have been more effective if we had used other methods. The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial. Much research has been carried out with the aim of influencing the performance of clinicians. The results have varied ,2. As wiThe reasons why clinical practice sometimes is not consistent with current best evidence varies across clinical problems and from one clinician to another. A logical consequence of this is to tailor quality improvement strategies to address specific barriers . SeveralIn this article we describe a simple approach we have used to identify barriers to changing professional practice. This was done as the first step in a process of developing an intervention to improve the pharmacological management of hypertension and hypercholesterolaemia . The int• Contrary to recommendations, physicians seem to rarely estimate the risk of cardiovascular disease before initiating treatment • Sales of thiazides are low, despite these drugs being recommended as first-line medication • Relatively few patients reach recommended treatment goals ,12We also report the results of a post hoc exercise and a survey we carried out to evaluate our approach to identifying barriers and interventions.We developed the intervention through a process of identifying barriers to implementation of recommendations and measures specifically addressing these barriers ("tailoring"). The methods we used were structured reflection, searching for other relevant trials targeted at improving the management of hypertension or hypercholesterolaemia, conducting a survey among general practitioners and discussion with physicians during pilot testing of the intervention.The three authors reflected over possible barriers based in part on our own experience as physicians working in primary care in Norway. We used a worksheet to structure our reflection . The worksheet included factors that might act as barriers in the practice environment, the professional environment, and related to physicians' knowledge, skills and attitudes. One worksheet was completed for each targeted behaviour: increasing the use of cardiovascular risk assessment before initiating treatment for hypertension or hypercholesterolaemia, increasing the prescribing of thiazides for the treatment of uncomplicated hypertension, and increasing the proportion of patients on medication for hypertension and hypercholesterolaemia that reach recommended treatment goals. The worksheet was used to facilitate our group discussion of possible interventions to address the identified barriers.Our research group had recently completed a trial of a strategy for guidelines implementation when we were planning this study . In that trial register for trials of interventions targeted specifically at the management of hypertension or elevated cholesterol in general practice.We considered systematic reviews of interventions to improve professional practice when we designed our strategy . We searWe surveyed general practitioners about some of the interventions about which we were uncertain after our structured reflection. The details of the survey have been described elsewhere . Briefly1. Do physicians assess cardiovascular risk before prescribing antihypertensive or cholesterol-lowering drugs?2. If not, would physicians be more likely to do so it they received a fee for this?3. Do physicians comply with current regulations limiting the reimbursement of cholesterol-lowering drugs?The last question was asked for two reasons. Firstly, we were considering making risk assessment a condition for reimbursement of the drugs. Secondly, the existing regulations were a possible barrier to adhering to our recommendations because they conflicted with these.During pilot testing of the intervention at two practices, which were selected for convenience, comments from physicians relevant to possible barriers were noted. We also informally evaluated each component of the intervention., December 2003. Each participant was asked to complete a worksheet to identify barriers and possible interventions related to the low use of thiazides among general practitioners. They were randomly allocated to four different groups where they collaborated on completing the worksheet. They were also asked to grade the importance of each barrier or intervention as minor, moderate or major. We disregarded those rated as minor. We compared the results from the four groups with the barriers and interventions we had identified.After we had finished designing the intervention we had the opportunity of testing our method of structured reflection at a gathering of international researchers in the Research Based Education and Quality Improvement group (ReBEQI) While conducting the randomised trial to test the effectiveness of our multifaceted intervention we carried out telephone interviews with physicians allocated to the experimental group. They were asked if they adhered to our recommendations and, if not, why. The responses where noted down during the interviews.Figure Many of the barriers were related to a lack of knowledge and could be addressed through educational interventions. The use of educational outreach visits was logical since we had planned to use an active intervention, based on our previous experience, and since this type of intervention has consistently lead to improved professional behaviour in randomised trials . SimilarThe search (July 2001) of the EPOC trial register for randomised trials with the word "hypertension" in any field yielded 58 references. Most were excluded after reading the abstracts, leaving eight, for which the full text was reviewed -22. ThisThe survey results did not indicate that a fee for estimating cardiovascular risk before initiating drug therapy would affect practice . The surWe did not identify additional barriers during pilot testing of the intervention with five physicians in two practices, but several of those already identified were confirmed, particularly barriers to prescribing thiazides.Based on our findings and an assessment of the feasibility and evidence of effectiveness for various interventions, we designed a multifaceted intervention. The elements of the intervention are described in table We also considered a number of interventions that we excluded. For example:• We considered placing computers in waiting rooms so that patients could assess their cardiovascular risk before seeing the physician, but concluded this would be costly and difficult to implement.• We considered providing pre-printed prescriptions, but found this would not to be relevant because most physicians use computerised systems for prescribing.• We considered exposing conflicts of interest among clinical specialists who advocated using other first line drugs than thiazides, but elected not to do so.• We considered exposing techniques used in pharmaceutical advertisements, such as using relative risk reductions rather than absolute risk reductions , but conNineteen researchers were divided into four groups. All groups considered advocacy by drug companies to be a major barrier to change. Routines or habits were also included as an important barrier by all the groups, as well as lack of knowledge concerning the effectiveness of thiazides, their favourable adverse effects profile, and their low cost. All the groups also mentioned competing guidelines or diverging opinions as part of the problem. Three of the groups considered local or national opinion leaders as potential barriers to change. Patients' expectations or perceived expectations were also mentioned by three of the groups.The interventions recommended by the groups to address the identified barriers are presented in table Among the 195 physicians exposed to the intervention, 149 (76%) were contacted during the trial period and agreed to answer our questions. No major additional barriers were identified. However, some physicians questioned whether adhering to the recommendations would represent a good use of resources, specifically the recommended treatment goals.Addressing barriers to change with tailored interventions makes sense and there is some empirical support for this . It is uSeveral qualitative methods can be used to identify barriers, such as interviews, focus groups and observation. These methods may be valuable, but they are relatively labour-intensive. We used a simpler approach to identifying barriers to change. Would the use of other methods have provided us with important additional information? Pilot testing and discussions with five physicians in two practices and interviews with 140 participating physicians did not indicate additional barriers. The post-hoc focus groups with international experts did not add much with regards to barriers and interventions. Several of these groups included "routines and habits" as a potential barrier, which was not explicitly mentioned among the barriers identified by the investigators. However, all interventions that were mentioned by more than one of the groups in the post-hoc focus group exercise were included in our multifaceted intervention. Our use of computerised reminders was based on the assumption that this would help to establish new routines, although we did not record routines and habits as a barrier when we developed the intervention.There are inherent weaknesses in our approach. One is that the investigators undertaking the structured reflection were few and we were prejudiced by our own experiences. The lack of patient involvement is another limitation, which possibly lead to an under-emphasis of patient-mediated interventions. A weakness with the group of international researchers who participated in the post-hoc focus groups is their lack of familiarity with the Norwegian context.A number of trials of tailored interventions have been conducted. The methods used to identify barriers to change have varied. Some investigators have simply hosted a meeting ,26, othe.Our simple approach to identifying barriers to improving practice appears to have been effective in identifying all of the important barriers, and it was efficient. However, we do not know for certain what barriers other methods would have identified or whether the intervention could have been more effective, if we had used other methods. Further work to address these questions is planned, including direct comparison of alternative methods and evaluations of theory-based approaches The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial.None declared.All the authors participated in the process of structured reflection and in conducting the survey of physicians. AF was responsible for reviewing the results from previous research and pilot testing of the intervention. AF drafted the article while SF and ADO contributed to critical revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The key to a rational treatment of elementary Mendelian genetics, specifically to an understanding of the origin of dominant and recessive traits, lies in the facts that: (1) alleles of genes encode polypeptides; (2) most polypeptides are catalysts, i.e. enzymes or translocators; (3) the molecular components of all traits in all cells are the products of systems of enzymes, i.e. of fluxing metabolic pathways; (4) any flux to the molecular components of a trait responds non-linearly (non-additively) to graded mutations in the activity of any one of the enzymes at a catalytic locus in a metabolic system; (5) as the flux responds to graded changes in the activity of an enzyme, the concentrations of the molecular components of a trait also change.It is then possible to account rationally, and without misrepresenting Mendel, for: the origin of dominant and recessive traits; the occurrence of Mendel's 3(dominant):1(recessive) trait ratio; deviations from this ratio; the absence of dominant and recessive traits in some circumstances, the occurrence of a blending of traits in others; the frequent occurrence of pleiotropy and epistasis. AA + 2Aa + aa) and the dominant, hybrid and recessive traits written as (AA + 2Aa + aa). This assumption  to call alleles dominant or recessive, and to represent them by the same letters used by Mendel to represent traits . on iunction" ).Beadle  expresseThis lack of attention to the implications of Garrod's work is all the more surprising when it is recalled that Bateson  indirectly by a system of enzymes  most of which, but not all, are enzymes . All polypeptides, catalytic and non-catalytic, are synthesised in this way.Figure X1, X2 and X3 in Figure X3 stands for those substrates that lead, through a series of enzyme-catalysed reactions, to the synthesis of nucleoside triphosphates (NTPs) and their subsequent incorporation into mRNA. Note that mRNA is a terminal product of this pathway. It is a coding entity, a proxy for DNA. Each mRNA specifies the order of incorporation of individual amino acids into a polypeptide, but no individual mRNA molecule participates as a substrate in the subsequent steps of the catalysed formation of a polypeptide. The control of the overall expression of a gene as a polypeptide is therefore necessarily treated in Metabolic Control Analysis as a cascade of two fluxing metabolic pathways, one that starts at X3, the other that starts at X2 [ts at X2 .X2 stands for those extracellular substrates that lead, through a series of enzyme-catalysed reactions, to the synthesis de novo of amino acids (AAs) and their subsequent incorporation, along with any existing amino acids, into a polypeptide (P). In a haploid cell, one polypeptide is synthesised from each gene locus. In a diploid, one polypeptide is synthesised from each of two alleles at a gene locus. If these pairs of polypeptides are catalytically active, each enzyme in a diploid cell  consists of a pair of allozymes, one of each pair specified by the allele derived from the male parent, the other specified by allele derived from the female parent. Each pair of allozymes, whether normal or mutated, exhibits only one measurable activity (v) at a catalytic locus in a metabolic pathway. If the pairs of polypeptides (P) synthesised by a diploid cell are not catalytically active they will not, of course, play a direct role in catalysing a metabolic pathway. They may have other important functions (e.g. as hormones) and may be components of traits.X1 stands for all those initial extracellular substrates feeding the matrix of inter-dependent biochemical pathways that typify all functioning cells. It is these pathways that generate the non-protein, non-polyribonucleotide, molecular products of all cell traits.Each of these three major fluxing pathways Figure  is catalX1 for the simple reason that this pathway stands for all the matrix of interdependent biochemical fluxes that generate such a wide range of the non-protein (and non-polyribonucleotide) molecular components of cell traits .Most attention is concentrated on the pathway initiated by X1.If any one of the three major pathways shown in Figure If the implications of the work of Beadle and Tatum  were notX1, X2, X3in Figure R) in permitting extracellular effectors to modulate intracellular metabolism (and thus the effects of a mutation) will be found elsewhere [Note that changes in the concentrations of external metabolites , the question then arises: how do we account for Mendelian segregation of traits during sexual reproduction? The answer lies in the fact that a mutation at a biochemical locus, within the matrix of interdependent pathways, has its most obvious effect on the most closely associated pathways. Distant pathways  will be less obviously affected. Kacser and Burns  The explanation advocated in this article for the origins of dominant and recessive traits from normal and mutant alleles in a diploid is based on:(i) An obligatory distinction, by notation and nomenclature, between the variables (traits) and the parameters  of genetic/biochemical systems.(ii) The contention that the molecular components of all traits are the products of fluxing metabolic systems Figures , 4, 5, 6(iii) Experimental evidence for an inevitable non-linear response of a flux (through a metabolic system of enzymes) to graded changes in the activity of any one of those enzymes , evidenc(iv) A demonstration that dominant and recessive traits arise from changes in the concentration of the normal molecular components of a defined trait.(v) The argument that changes in concentration of a trait component may nevertheless be revealed as a qualitative change in that trait.(vi) A demonstration that both alleles  at a locus in a diploid are generally expressed. If the normal allele expresses a catalytically active polypeptide, many mutants of this allele will express an enzyme with lower activity; a mutated enzyme with zero activity is an extreme case.uu, 2uU, UU) one of which is a unique heterozygote (uU).(vii) The demonstration that an explanation of Mendel's observations cannot be based on an allele series containing only three terms (e.g. (viii) A demonstration that dominant and recessive traits cannot be generated by those polypeptides that are not enzymes embedded in a system of enzymes.H) expresses a dominant trait (A) because the  recessive allele (u) in a heterozygote (Uu) is always completely ineffective or because the allegedly dominant allele (U) suppresses the allegedly recessive allele (u) in the heterozygote [(ix) Rejection of the unjustified traditional claim that a hybrid (rozygote .(x) Rejection of the traditional, unsubstantiated and implausible claim that one so-called dominant allele in a heterozygote is as effective as two such alleles in the wild-type cell .It was also shown that pleiotropy and epistasis can be explained by taking a similar system approach to that used in explaining the origin of dominant and recessive traits.It is then apparent that, to account rationally for Mendel's observations of dominant and recessive traits, a minimum of four conditions must be met.(i) Alleles must be distinguished by notation, nomenclature and concept from traits; functions of components of the genotype must be distinguished from properties of components of the phenotype. Traits alone may be dominant or recessive.(ii) Alleles cannot be called "dominant" or "recessive". .(iii) It must be shown how dominant traits become distinguishable from recessive traits in the same cell or organism Figure , 3.(iv) It must be shown how a hybrid trait sometimes becomes indistinguishable from the dominant trait and sometimes does not Figures , 3. The If all four conditions are be met; the first two conditions must first be met. The treatment given in sections 2–5 meets each of these requirements.origin of dominant and recessive traits that arose from mutations of alleles at any one gene locus in a diploid or polyploid cell . Inherent in this explanation, as set out above , are further explanations for the occurrence of the 3(dominant):1(recessive) trait ratio in some situations in a diploid  may produce a trait that is indistinguishable from the trait expressed from the "homozygous" allele pairs (UU).Kacser and Burns  providedsections .3, 2.4. In other words, provided:(a) all current misrepresentations of Mendel's paper  are firs(b) alleles are distinguished by notation and nomenclature from the traits they specify,(c) alleles are regarded as normal or mutant (but not dominant or recessive), it is possible to provide a rational and internally consistent explanation for the origin of Mendel's dominant and recessive traits, for the occurrence of his 3:1 trait ratio, and for exceptions to these observations noted by later investigators. The same systemic approach is applicable to current problems in biotechnology and medical genetics (section 4). It also explains the origins of pleiotropy and epistasis (section 5); and challenges the assumption that a mutation in a non-catalytic protein provides an example of Mendel's dominant and recessive traits [A + 2Aa + a). In the present paper these proportions have been written as (A + 2H + a). If the symbol (H) for a hybrid in Figure Aa), it will be clear why Mendel postulated that his hybrids (Aa) displayed trait (A) and not trait (a). If the same exercise is repeated in Figure H) temporarily by (Bb), it will be clear why Mendel observed an anomalous blending of flower colours in the hybrids when he crossed parental bean plants bearing different flower colours.Mendel found, by experiment, that the proportions of plant forms in each of his F2 populations was represented by  Persistent misrepresentations of Mendel's paper, and incorporation of these distortions into currently favoured explanations of Mendel's observations [ Persistespecify the kind (and order of incorporation) of amino acids into polypeptides . Dominance and recessivity are a reflection of changes in the concentration(s) of the molecular component(s) of a trait when an enzyme is mutated within a fluxing metabolic pathway.(2) A failure to recognise the consequences of not distinguishing between the function of the alleles and the properties of traits in attempting to explain Mendel's results. Normal and mutant alleles variables of a biological system to perturbations of the magnitude any one system parameter.(3) Tardy recognition of the need to adopt the systemic approach of Metabolic Control Analysis in explaining the response of the (4) A reluctance to accept a change in concepts even when currently accepted representations of Mendel's results are demonstrably untenable.(5) Elucidation of the double helical structure of DNA Figure  and all (6) A determination in some quarters to regard genetics as an autonomous subject. It has been obvious at least since the work of Beadle and Tatum  that sucIt is significant that Kacser & Burns were also one of two sets of authors who initiated the systemic approach to the control of metabolite concentrations and fluxes ,40. ThisA) and (a). These statements are here formally withdrawn. They were consistent with textbook treatments of Mendelian genetics [In an earlier paper  it was sgenetics  but a sugenetics  will be


Until now, no specific therapies are available to inhibit pancreatic fibrosis, a constant pathological feature of chronic pancreatitis and pancreatic cancer. One major reason is the incomplete knowledge of the molecular principles underlying fibrogenesis in the pancreas. In the past few years, evidence has been accumulated that activated pancreatic stellate cells (PSCs) are the predominant source of extracellular matrix (ECM) proteins in the diseased organ. PSCs are vitamin A-storing, fibroblast-like cells with close morphological and biochemical similarities to hepatic stellate cells . In response to profibrogenic mediators such as various cytokines, PSCs undergo an activation process that involves proliferation, exhibition of a myofibroblastic phenotype and enhanced production of ECM proteins. The intracellular mediators of activation signals, and their antagonists, are only partially known so far. Recent data suggest an important role of enzymes of the mitogen-activated protein kinase family in PSC activation. On the other hand, ligands of the nuclear receptor PPARγ (peroxisome proliferator-activated receptor γ) stimulate maintenance of a quiescent PSC phenotype. In the future, targeting regulators of the PSC activation process might become a promising approach for the treatment of pancreatic fibrosis. Excessive production of connective tissue molecules forming the extracellular matrix (ECM) is a pathological process relevant to diseases of many organ systems, including liver, lung, kidney, bowel and pancreas. The resulting fibrosis frequently leads to a progressive loss of specific organ functions. In the past two decades, fibrogenesis has been intensively studied by a large number of laboratories, and a great deal of scientific information has been accumulated regarding the pathogenesis of fibrosis in various organs. Until a few years ago, pancreatic fibrosis, however, remained an exception: although known for a long time as a central pathological feature of both chronic pancreatitis and pancreatic cancer ,2, its cBoth chronic pancreatitis and pancreatic cancer are accompanied by an organ fibrosis ,2. The pin vitro data gave rise to the hypothesis that PSCs might play a pivotal role in pancreatic fibrogenesis.Fibroblast activation has been reported to be a common event in pancreatitis already more than a decade ago -9, but tin vivo studies using experimental models of pancreatic fibrosis: Infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct of rats causes a pancreatic necroinflammation followed by fibrosis . In rectivation : Culturetivation . Althougsystematic elucidation of signaling pathways in PSCs will also be one of the most important issues for future research. So far, research regarding intracellular signaling in PSCs has focused on two main aspects: the role of enzymes of the mitogen-activated protein kinase (MAPK) family and the transcriptional control of PSC activation.In the past two years, analysis of signal transduction pathways regulating PSC function has become a focus of attention. As detailed below, identification of signaling molecules that play a crucial role in PSC activation is a promising approach for the development of therapeutic strategies to inhibit pancreatic fibrosis. It is therefore envisaged that the MAPKs are a family of serine/threonine specific protein kinases with a wide range of biological functions in the regulation of fundamental cellular processes, including gene expression, proliferation and cell survival/apoptotic cell death -46. In mThe other two major MAP kinase pathways, involving JNK and p38, are well-established mediators of signals induced by pro-inflammatory cytokines and cellular stressors  . In PSCsin vitro by regulating the actin cytoskeleton [Two other intracellular signal transduction pathways that have recently been studied regarding their role in PSC activation are the phosphatidylinositol 3 (PI 3)-kinase and the Rho-Rho kinase (ROCK) pathway. The results suggest that PI 3-kinase activity is required for PDGF-stimulated PSC migration but not proliferation ,55. The skeleton .Cytokine and growth factor receptors exert their effects on the expression of target genes through signaling cascades that regulate the activity of a characteristic set of transcription factors. Recently, the group of the author has analyzed the activation profiles of activator protein (AP)-1 ,58, signIn the transduction of TGF-β receptor-derived signals into the nucleus, Smad transcription factors play a central role ,64. Stud2 and troglitazone (an antidiabetic drug of the thiazolidinedione group) act as antagonists of PSC activation in vitro that decrease cell proliferation and expression of α-SMA [Recent studies have implicated the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in the inhibition of stellate cell activation in liver -69 and pof α-SMA . In Wistof α-SMA . The antof α-SMA . Thus, tWhile the role of activated PSCs in pancreatic fibrosis is well established, the physiological functions of their quiescent precursors are less well understood. Importantly, PSCs are not only a source of ECM but also of matrix-degrading enzymes of the MMP  family and their inhibitors . Thus, PSCs have been shown to secrete MMP-2, MMP-9 and MMP-13 and to express TIMP-1 and TIMP-2 . It appeThe embryonic origin of PSCs still remains to be determined. Very recently, Seaberg et al.  reportedUntil now, the physiological consequences of vitamin A-storage in PSCs remain unclear. It has recently been shown by the group of the author that the vitamin A derivate all-trans retinoic acid has complex effects on PSC function and acts, at least in part, as an antagonist of the activation process . It is, In the past, research regarding PSC biology has almost exclusively focused on the molecular basics of the activation process. However, given that participation in regeneration after pancreatic injury is an important function of activated PSCs, it is apparent that a disturbance of the inactivation or elimination of activated PSCs, rather than PSC activation itself, is the pathological process that leads to fibrosis. So far, it has not been systematically studied whether activated PSCs are capable of returning into a quiescent stage after fulfilling a repair function. Alternatively, elimination by apoptosis might be important in terminating the wound-healing response after pancreatic injury .Finally, work on the complex relationships between PSCs and pancreatic tumor cells is still in its infancy. Very likely, activation of PSCs not simply accompanies tumor progression but plays an active role in this process. Thus, it has been recently been shown that pancreatic cancer growth and progression is accelerated through complex functional interactions between carcinoma cells and PSCs . FurtherStudies on PSC biology are still hampered by the limited availibility of primary cells. Possibly, recently established pancreatic stellate cell lines ,82 will hepatic stellate cell activation, such as interferons.Given that activated PSCs are the principle effector cells in pancreatic fibrosis, targeting PSCs might become a promising therapeutic approach. Principle strategies that can be envisaged include an interruption/reversion of the activation process as well as an elimination of activated PSCs, e.g. through an induction of apoptosis. So far, potential antifibrotic drugs have been mainly tested in models of liver fibrosis (reviewed in ). The exAs described above, studies on the regulation of PSC activation at the intracellular level have identified key mediators of stimulatory and inhibitory signals. Targeting molecules such as PPARγ, MAP kinases, PI 3-kinase, or Smad proteins might become an important approach for the treatment of pancreatic fibrosis in the future. Further progress in the development of antifibrotic therapies can be expected from the ongoing elucidation of the molecular principles of PSC activation.PSCs play a crucial role in pancreatic fibrogenesis Figure . Ethanol


The 12-item General Health Questionnaire (GHQ-12) is widely used as a unidimensional instrument, but factor analyses tended to suggest that it contains two or three factors. Not much is known about the usefulness of the GHQ-12 factors, if they exist, in revealing between-patient differences in clinical states and health-related quality of life.We addressed this issue in a cross-sectional survey of out-patients with psychological disorders in Singapore. The participants (n = 120) completed the GHQ-12, the Beck Anxiety Inventory, and the Short-Form 36 Health Survey. Confirmatory factor analysis was used to compare six previously proposed factor structures for the GHQ-12. Factor scores of the best-fitting model, as well as the overall GHQ-12 score, were assessed in relation to clinical and health-related quality of life variables.The 3-factor model proposed by Graetz fitted the data better than a unidimensional model, two 2-factor models, and two other 3-factor models. However, the three factors were strongly correlated. Their values varied in a similar fashion in relation to clinical and health-related quality of life variables.The 12-item General Health Questionnaire contains three factors, namely Anxiety and Depression, Social Dysfunction, and Loss of Confidence. Nevertheless, using them separately does not offer many practical advantages in differentiating clinical groups or identifying association with clinical or health-related quality of life variables. Recent studies of disease burden have demonstrated the importance of psychological disorders. For instance, depression was the fourth leading cause of disease burden, accounting for 4.4% of total disability adjusted life years in the world in 2000 . The 12-While the longer versions of the GHQ are normally considered multidimensional, the GHQ-12 is often regarded as measuring only a single dimension of psychological health. For example, Corti  analyzedUsing confirmatory factor analysis (CFA) to analyze the BHPS data, Cheung  comparedWhile the structure of the GHQ-12 has been studied using factor analysis methods, the construct validity and usefulness of those resulting factors are not often tested. The question is whether the additional information provided by the 2 or 3 factors, if they exist, is clinically useful. In other words, will multiple scores be more useful than a total single score in helping us to understand respondents' health status?The purpose of this study was therefore two-fold. First, we aimed to compare the previously proposed models of the GHQ-12 in an oriental population and identify the best-fitting one. It was not our objective to assess their absolute level of fit or to derive new model or version of the GHQ. Second, we aimed to assess whether the factors identified relate to clinical and health-related quality of life variables in different ways.A consecutive sample of outpatients with anxiety disorders and/or depressive disorders was recruited from a psychiatric clinic at a tertiary hospital in Singapore. Inclusion criteria were the presence of any anxiety disorder and/or major depressive disorder, literacy in English or Chinese, and completion of an informed consent form. Patients with organic brain syndrome or psychosis were excluded.During routine consultation visits, diagnoses of recruited patients were ascertained by a psychiatrist using DSM-IV criteria and the severity of their psychiatric disorders was assessed using a Clinical Global Impression (CGI) scale, which ranges from 1 (very mild) to 5 (very severe). Patients were then given a questionnaire containing the General Health Questionnaire (GHQ-12) , the BecThe General Health Questionnaire (GHQ-12) consists of 12 items, each assessing the severity of a mental problem over the past few weeks using a 4-point scale (from 0 to 3). The score was used to generate a total score ranging from 0 to 36, with higher scores indicating worse conditions . The ChiThe Beck Anxiety Inventory (BAI) is a valid and reliable self-report checklist for anxiety symptoms . This inThe Short Form 36 Health Survey (SF-36)  is a 36-Various factor structures of the GHQ-12 were tested by confirmatory factor analysis. Model I was unidimensional. Model IIA contained 2 factors: General Dysphoria and Social Dysfunction . Model IMethodologists have emphasized that it is desirable to use different indicators to examine a model's goodness-of-fit . The fitThe best-fitting model was examined in detail. The Kruskal-Wallis test was used to compare the GHQ-12 overall and factor scores of patients with different diagnosis. Pearson's correlation coefficient (r) was used to assess the association between GHQ-12 scores and various variables, namely Beck Anxiety Inventory, Clinical Global Impression and SF-36 scores. The Fisher's Z transformation was used to produce 95% confidence interval.A total of 120 participants (63 man and 57 women) were included in the analysis (Table Table Figure Having established that Graetz's 3-factor model fitted the data better than the other models, we calculated the factor scores as unweighted sums of the items concerned. From figure Table Several previous confirmatory factor analyses found that the 3-factor model of Graetz gave better fit to survey data from Australia , BritainThe one-dimensional model was the worst according to all three goodness-of-fit indices.The three factors in the model proposed by Graetz were found to be strongly correlated with each other, with correlation coefficients in the neighborhood of 0.8 to 0.9. Such strong correlations suggest that even if there were indeed three different factors, in practice it is quite difficult to differentiate them. The study of French and Tait  also shoTwo limitations of the study should be noted. Firstly, the sample size was somewhat small for confirmatory factor analysis. Secondly, the participants were clinical cases. This homogeneity might have made it more difficult to detect variations in GHQ-12 scores. We believe that the question about the relative plausibility of various factor models have been sufficiently answered by this and several previous studies -12. NeveSeveral studies, including the present one, have found that Graetz's 3-factor model of the GHQ-12 is more plausible than other models. However, the factors were strongly correlated and difficult to discern. Our analysis of the three GHQ scores in relation to clinical variables and aspects of health-related quality of life did not appear to be more informative than analysis of a single overall GHQ-12 score. As such, from a pragmatic point of view we consider it acceptable to use this instrument as a one-dimensional measure. Unless one has specific questions that are best answered by a subset of the three factors, there is no need to consider the multi-dimensionality.FG carried out the confirmatory factor analysis, interpreted the findings, and drafted part of the manuscript. NL designed the study, participated in the development of the statistical framework and interpreted the findings. JT participated in the study design, discussion of the statistical framework, and the interpretation of findings. CF participated in the study design and carried out the data collection and clinical assessments. SCL participated in the study design and discussion and interpretation of findings. YBC conceived of the study, developed the statistical framework, carried out part of the statistical analysis, and drafted part of the manuscript. All authors read and approved the final manuscript.


Diagnosing chronic heart failure is difficult, especially in mild cases or early in the course of the disease, and guidelines are not easily implemented in everyday practice. The aim of this study was to investigate general practitioners' diagnostic reasoning about patients with suspected chronic heart failure in comparison with recommendations in European guidelines.Think-aloud technique was used. Fifteen general practitioners reasoned about six case vignettes, representing authentic patients with suspected chronic heart failure. Information about each case was added successively in five steps. The general practitioners said their thoughts aloud while reasoning about the probability of the patient having chronic heart failure, and tried to decide about the diagnosis. Arguments for and against chronic heart failure were analysed and compared to recommendations in guidelines.Information about ejection fraction was the most frequent diagnostic argument, followed by information about cardiac enlargement or pulmonary congestion on chest X-ray. However, in a third of the judgement situations, no information about echocardiography was utilized in the general practitioners' diagnostic reasoning. Only three of the 15 doctors used information about a normal electrocardiography as an argument against chronic heart failure. Information about other cardio-vascular diseases was frequently used as a diagnostic argument.The clinical information was not utilized to the extent recommended in guidelines. Some implications of our study are that 1) general practitioners need more information about how to utilize echocardiography when diagnosing chronic heart failure, 2) guidelines ought to give more importance to information about other cardio-vascular diseases in the diagnostic reasoning, and 3) guidelines ought to treat the topic of diastolic heart failure in a clearer way. Chronic heart failure (CHF) is a major cause of morbidity and mortality and it has a considerable impact on the health care system . In a reThe European Society of Cardiology adopted guidelines for diagnosing CHF in 1995, and these were revised in 2001 -9. Swedia) one or more typical symptoms (at rest or during exercise), b) objective evidence of cardiac dysfunction (at rest), and c) response to treatment directed towards CHF (in cases where diagnosis is in doubt). Criteria a and b should be fulfilled in all cases. Echocardiography (ECHO) is mentioned as the single most effective tool in widespread clinical use for objective assessment of cardiac dysfunction. In the algorithm for the diagnosis of CHF, a sequence of investigations is recommended: suspect CHF because of symptoms and signs; assess presence of cardiac disease by electrocardiography, X-ray or Natriuretic peptides (where available); imaging by echocardiography; assess aetiology, degree, precipitating factors and type of cardiac dysfunction; additional diagnostic tests where appropriate; choose therapy. Table The full versions of the above-mentioned guidelines are comprehensive documents, covering epidemiology, aetiology, pathophysiology and diagnostic methods, but may be difficult to apply to specific diagnostic situations . HoweverRelatively few studies on how patients suspected of having CHF are diagnosed have been performed in primary health care settings, and most of them report over-diagnosis ,4,14-16.Process-tracing techniques are used to study the cognitive processes involved in decision-making such as, for example, how judgements change over time as new information is presented, and which decision rules are used . A methoThe value of conclusions reached in such studies depends on the validity of the think-aloud method, and on the reliability of the coding process. Thinking aloud while performing a task often lengthens the time for completing the task, but does not seem to change the accuracy of task fulfilment or the cognitive processes . In a ren = 61) were listed and contacted in a random order. The distance from central Stockholm was chosen for practical reasons. In each health care centre the GPs were contacted in a random order by one of the authors (YS). Only one GP at each centre was included in the study, and this person had to be a specialist in family medicine. We contacted the GPs during their regular telephone hour, during the period October 2001 to October 2002. Our goal was to include 15 GPs in the study.All health care centres in northern Stockholm within a distance of 20–30 km from the city centre  years, they were on average 52.7 (range 42–62) years of age, and six of them were men. The non-participating GPs were on average 52.7 (range 35–62) years of age, and seven of them were men.Six case vignettes (CV), based on authentic patients, were presented to the participants. The information presented in the case vignettes was obtained from the patient records and included information about relevant diseases , lifestyle factors , symptoms, signs, electrocardiography (ECG), chest X-ray findings, and ECHO. Chest X-ray and ECHO results were presented in the same format as in the patient records. ECHO results could contain information about ejection fraction (EF), valvular disorders and ventricular wall motility. The diagnoses made by the attending cardiologists  were used as a reference standard when assessing the participants' diagnostic accomplishments.The six cases were selected to represent patients with various types of potential diagnostic problems: A "prototypical" CHF patient (CV2), a patient with both CHF and chronic obstructive pulmonary disease (COPD) (CV6), a patient with CHF, tachycardia and mitral valve insufficiency (CV3), an obese non-CHF patient with normal ECG and EF (CV5), a non-CHF patient with COPD (CV4) and a non-CHF patient with alcohol abuse and a metabolic syndrome (CV1). For one of the cases (CV3) there was a disagreement between the diagnosis according to the cardiologists and the diagnosis that could be deduced from a simplistic interpretation of the guidelines. This patient had typical clinical findings including gallop rhythm, cardiomegaly, and pulmonary congestion, but normal left ventricular function according to ECHO. It could therefore be categorized as not CHF according to the definition given in the guidelines. However, this patient also had a mitral valve insufficiency, which can give a "false normal" ejection fraction value: the left ventricle is emptied both forward (cardiac output) and backward .Before the sessions the GPs had received written information about the aim of the study  and about the method , but not about the kind of medical problems that would be presented to them. The study was conducted at the GPs' offices. All visits and recordings were made by one of the authors (YS). The participants were instructed that six authentic patients, suspected by GPs to have CHF, would be presented, and that their task was to say aloud their thoughts about the case, and to try to decide whether the patient had CHF or not.The order of the cases was the same for all participants. The order in which the information was presented was arranged to be as realistic as possible in relation to clinical practice . Each vignette was presented on a computer screen in five successive steps using QA software . All preThe doctors first got a test case (not recorded) in order to get acquainted with the think-aloud method, and then continued with the six study cases. The only intervention from the researcher during the think-aloud session was that a participant who was silent for more than about 15 seconds was reminded to say his or her thoughts aloud about the information presented . All sesAll information in the case vignettes that was of relevance for the diagnosis and that could take on different values was considered to be variables. Fifty variables were defined: 19 of them were included in all six vignettes , six in five, one in four, six in three, one in two, and 17 in one vignette . For each case vignette, the presented variables were coded for content and value Table .2) as an argument against CHF. For each participant, a specific evaluation of each variable value was only counted once for each case vignette in order not to give more weight to thoughtful repetitions of an argument than to a single, firm statement. However, if a participant used the same variable value as an argument both for and against the diagnosis of CHF, both evaluations were coded. Ten percent of the 90 case vignette protocols were selected at random and coded independently by two of the authors  to estimate the interrater agreement of the coding process. The rest of the protocols were coded by one of the authors (YS).For each participant, every mention of a variable was coded for how the GP seemed to use it: as an argument for the diagnosis of CHF, as an argument against CHF, or as not being of any explicit use for the diagnosis (mentioned only). "He has basal rales. This guy has CHF!" is an example of a participant using the variable "rales"  as an argument for CHF. "So I'm not really sure that he has got CHF. Just a moderate cardiac enlargement, no, I wouldn't think so" is an example of a participant using the variable "relative heart volume"  in three categories: CHF or probably CHF; uncertainty about diagnosis; probably not CHF or not CHF.The participants were not forced to express their diagnostic judgements in a specific format, and their free verbal statements therefore had to be interpreted and coded. Two of the authors  independently classified all the diagnostic judgements (Stata 8.0 was used for the statistics. Cohen's kappa test (κ) was used to determine interrater agreement regarding the coding of the think-aloud protocols and the classification of the diagnostic judgements. Kappa values are classified as follows: <0, worse than chance; 0 to 0.2, poor; 0.21 to 0.4, fair; 0.41 to 0.6, moderate; 0.61 to 0.8, good; and >0.8, very good .The research ethics committee of Huddinge University Hospital approved the study.The randomly selected test protocols contained 322 segments of propositions, 36 of which were excluded since they contained variables that were not going to be investigated in this study (e.g. treatment suggestions). There was disagreement between the two coders about the content of variables in 14 of the remaining 286 segments (4.8%). The remaining 272 segments were then tested for interrater agreement on argument values , which was 95% (κ 0.85).The interrater agreement was 92% (κ 0.85). For the few diagnostic judgements where there was initial disagreement, it was possible to agree upon an interpretation.The information that was used most frequently in diagnostic arguments was the ejection fraction value on ECHO, pulmonary congestion, and cardiac volume Figure . The mosSymptoms and signs were not often used as arguments in the GPs' diagnostic reasoning Figure . SymptomAccording to the guidelines, a normal ECG opposes the diagnosis of CHF Table . Only onAccording to the guidelines, information about cardiac enlargement or pulmonary congestion on chest X-ray gives some support for CHF if there are any pathological findings, and is of some importance as an argument against CHF if the findings are normal Table . Chest XEach of the 15 GPs judged 6 case vignettes, which resulted in 90 judgement situations. In 48 of them, the EF value (or the information about left ventricular function in CV3) was used as an argument for or against CHF . In three judgement situations, the final diagnosis was in the same direction as the X-ray argument (three CHF). In seven judgement situations, the GP was uncertain about the diagnosis.In our study, the GPs used other diseases as an argument in a total of 70 judgement situations, mostly as arguments for CHF (91%). Atrial fibrillation, emphysema, history of myocardial infarction, and hypertension were the diagnoses most commonly used in this way.For certain variables, the same information value was used by some GPs as an argument for and by others as an argument against CHF. The presence of emphysema was sometimes seen as increasing the risk of CHF , or as an alternative explanation for symptoms .Diabetes could also be seen as increasing the risk of CHF , or as an alternative explanation for symptoms .Advanced age could be seen as increasing the probability of CHF , or as an alternative explanation for symptoms . Age was used as a diagnostic argument only for the two patients over 80 years of age.For relative cardiac volume, the reasoning could be compatible with GPs using different threshold values in their reasoning. The two lowest values were only used as arguments against CHF, the two highest values only as arguments for it, and the two intermediate values were used as arguments in both directions.There was total agreement among the GPs only for the prototypical CHF case; otherwise there was a large variation among GPs regarding diagnoses. Case vignettes representing CHF patients were more likely to be correctly diagnosed than those representing non-CHF patients Table .When comparing the GPs' diagnostic reasoning with guidelines, we found that the clinical information in the case vignettes was not used to the extent recommended in the guidelines. It is true that information about the ejection fraction value on ECHO was the single most frequent diagnostic argument, and it was the most common argument against CHF. This is in line with the guidelines, which emphasize the need for objective evidence of cardiac dysfunction. However, in more than one third of the judgement situations, the information about ECHO that was presented was not used as an argument. Over-diagnosis of CHF in primary health care has been demonstrated in a number of studies, with ECHO findings as the gold standard ,4,15. LiIn the diagnostic algorithm, symptoms and signs are the entry criteria. However, the GPs did not seem to use them consistently in this way, except when diagnosing the prototypical CHF and non-CHF cases. One reason for this might be that most symptoms and signs considered typical for CHF are fairly non-specific as regards the diagnosis CHF.Information about other relevant diseases, which was important in the GPs' diagnostic reasoning, is not included in the list of assessments to be performed routinely Table  9]. How. How9]. The case vignettes represented authentic patients referred by GPs to a cardiology department for problems related to heart failure. This may have led to a selection of more complicated patients than the "typical" heart failure patients in primary health care. The reason we chose this group of patients was that we wanted to include patients who were thoroughly investigated, with a well-founded clinical diagnosis, and for whom information about all variables of interest could be found in the patient records. Selecting GPs only from health care centres in, or relatively close to, the city centre may have biased the results, since differences in catchment areas, working conditions, and access to echocardiography may influence GPs' diagnostic habits. This could make it difficult to generalize the results to other GPs. Only 50% of the GPs who were contacted agreed to take part in the study, which could bias the results. However, since the age distribution was the same in the two groups, it seems unlikely that the drop-out group would differ from the study group regarding clinical experience.The full version of the guidelines is difficult to apply to individual diagnostic situations and it is also difficult to use it for assessment of diagnostic behaviour . In thisGPs' tendency to over-diagnose CHF has been explained by their relying on symptoms, signs and less specific investigations such as chest X-ray, and by limited access to ECHO in the primary health care. However, this study indicates that a substantial minority of GPs seem to be less familiar with the use of ECHO and EF. Thus, access to ECHO ought to be accompanied by education about how to integrate this information better in the diagnostic reasoning.Guidelines ought to include search of information about other cardio-vascular diseases in the list of assessments to be performed routinely Table  and in tThe information in the case vignettes was underused as arguments for and against the possibility of CHF as compared with the guidelines. Information about the EF value was the single most frequently used argument for or against CHF; nevertheless, in one third of the diagnostic judgements the GPs did not consider any information about the ECHO in their diagnostic reasoning. Information about symptoms and signs were not used to to the extent suggested in the guidelines. Information about other relevant diseases was frequently used in the GPs' diagnostic reasoning, indicating that they often relied on illness scripts. Some implications of our study are that 1) GPs should be taught how to use ECHO information better in their diagnostic reasoning, 2) guidelines ought to give more importance to information about other cardio-vascular diseases in the diagnostic reasoning, and 3) guidelines ought to treat the topic of diastolic heart failure in a clearer way.CHF Chronic heart failureCOPD Chronic obstructive pulmonary diseaseECHO EchocardiographyECG ElectrocardiographyEF Ejection fractionGP General practitionerThe author(s) declare that they have no competing interests.All authors conceived of the study and participated in the design. YS carried out the data collection, performed the statistical analyses and drafted the manuscript. All authors participated in the interpretation of the results and the discussions of the drafts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Case vignette characteristics The additional file shows some important characteristics of the case vignettes. Format: Word-table.Click here for file


No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality.Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries.There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain. We understand that conservative opinion is of the view that this interpretation is in conflict with Article 18(2) of the Convention itself. However, neither the Convention nor the Protocol on Cloning provide any sanctions for violation.The United Nations' struggle to agree on a cloning treaty exemplifies both the variation of approaches and the challenges associated with seeking consensus in a morally contested area . In DeceThere has, however, been little consensus on how to proceed. Though all countries agree that RC should be banned, there is deep division regarding NRC. Neither the Ad Hoc cloning committee nor the UN's Legal Committee could reach a consensus on which proposal to support and bring before the General Assembly. In November 2003 the Legal Committee voted (80-79) to recommend a two-year deferral on a General Assembly decision – a compromise that was put forward and supported by most of the members of the Organization of the Islamic Conference. This decision was largely seen as a victory for those countries supporting a more permissive approach to cloning policy . Indeed,The fact that the deep division at the UN is primarily about NRC reflects the lack of any consistent approach to cloning policy. For example, one would expect an emerging trend toward the banning of NRC, as suggested in the quote by Senator Morin, to be reflected in the building of consensus or, at least, a degree of flexibility at the UN General Assembly.Few areas of regulation are as evidentially driven by ethical views as the regulation of cloning and cloning research. This is not the place for in-depth analysis of the underlying debate. Elsewhere we have both argued that existing regulatory attempts to prohibit RC have rarely been underpinned by thoughtful exposition of underlying ethical principles ,10,34. PLawyers rarely look to regulatory outcome to understand ethical debates. Unfortunately, in the area of cloning many commentators do that very thing. As our starting quotation demonstrates, politicians and commentators are all too ready to find support for their ethical views in regulatory positions adopted elsewhere. There are, however, varying levels of ethical agreement. Agreement on the appropriate regulatory position does not imply agreement on the underlying ethical principles.full status position would grant the embryo the same level of moral duties as you or I. The no status position would grant the embryo no more status or dignity than your hair or nails. The limited status position would grant the embryo a fixed or gradualist level of intrinsic moral value between these two extremes. The full status position will require the prohibition of NRC (destructive use of embryos is considered murder) and the no status position will usually require NRC to be permitted . The limited status position is, however, potentially compatible with either regulatory position, depending on the particular status given to the early embryo and the weight given to potential benefits of NRC. It follows that the fact that the regulatory position permits or prohibits NRC does not do even tell us what status the embryo is considered to have. This is further complicated by the fact that supporters of the no or limited status position might be prepared to accept more restrictions than are strictly required by their position to protect a more important moral goal. Only supporters of the full status position cannot coherently make such pragmatic compromises [Consider the relationship between ethical positions on the moral status (or dignity) of the cloned embryo and NRC. The cloned embryo could be considered to have full, no, or limited moral status ,35. The promises .Similarly, the existing positions on RC could be supported by radically different ethical views. Prohibitions could be supported by those who hold that RC is absolutely wrong  and by those who hold that cloning at present would be wrong. Even time-limited prohibition does not enable us to discern whether it is underpinned by, for example, the view that RC is wrong because of current safety issues or the view that RC is not wrong but the most effective way to get there is by initially prohibiting it.In sum, the majority of regulatory outcomes could be coherently explained by reference to one or more underlying ethical positions. Thus, similar or even identical regulatory outcomes imply less by way of ethical agreement than some may be inclined to believe.Cloning laws and policies are far from uniform across the globe and the legal position in some countries remains uncertain. This will give little comfort to scientists and policy makers hoping to gain clear direction from the international position. For the time being at least, policymakers must accept the reality of international "dissensus" and scientists wishing to undertaken research on NRC are best advised to consider conducting their research in only a handful of countries. Even where there is agreement as to the regulatory outcome, policy-makers should not confuse this with agreement on underlying ethical principles. Like many topics concerning the developing genetic and reproductive technologies, cloning remains controversial.The author(s) declare that they have no competing interests.Both authors contributed to the original concept for the paper, the writing and revision of the manuscript and the analysis of the law.The pre-publication history for this paper can be accessed here: