Various scales have been used to assess palliative outcomes. But measurement can still be problematic and core components of measures have not been identified. This study aimed to determine the relationships between, and factorial structure of, three widely used scales among advanced cancer patients.Patients were recruited who received home or hospital palliative care services in the south of England. Hope, quality of life and palliative outcomes were assessed by patients in face to face interviews, using three previously established scales – a generic measure (EQoL), a palliative care specific measure (POS) and a measure of hope (Herth Hope Index). Analysis comprised: exploratory factor analysis of each individual scale, and all scales combined, and confirmatory factor analysis for model building and validation.Of 171 patients identified, 140 (81%) consented and completed first interviews; mean age was 71 years, 54% were women, 132 had cancer. In exploratory analysis of individual means, three out of the five factors in the EQoL explained 75% of its variability, four out of the 10 factors in POS explained 63% of its variability, and in the Hope Index, nine out of the 12 items explained 69% of its variability. When exploring the relative factorial structure of all three scales, five factors explained 56% of total combined variability. Confirmatory analysis reduced this to a model with four factors – self-sufficiency, positivity, symptoms and spiritual. Removal of the spiritual factor left a model with an improved goodness of fit and a measure with 11 items.We identified three factors which are important outcomes and would be simple to measure in clinical practice and research. Measurement of the effect of illness and its treatment on patients is now an accepted part of clinical trial design . Such meIn response to these difficulties, different measures have been developed and tested among patients receiving palliative and hospice care in different countries and contexts ,11. ThesSecondary analysis of a prospective observational study.Patients living in Chichester in the South of England receiving home or hospital palliative care support, from community, hospice or hospital palliative care team staff, were approached to take part in the study. Local research ethics committee approval was obtained. The local hospice was planning to develop a day care unit and patients were recruited during this period. A historical group was recruited before the day care unit was established. Consecutive consenting patients were recruited for both series. Patients were eligible if they were in the care of the hospice home care team, or neighbouring home care teams, that had access to the day care unit. Patients were excluded if they were judged by staff to be too ill for interview, if staff felt it would distress them, or they lived outside the catchment area of the hospice day unit. Two concurrent groups were recruited after day care was established – patients who did (Group AD) and did not (Group AN) choose to receive day care.Data was collected using trained interviewers. Interviews took place in the patients' preferred location, usually their own home. Interviewers explained the background to the study and used a structured schedule to collect clinical, demographic and use of service data. They then administered three scales. All were short, taking less than 10 minutes on average to complete, and were acceptable to advanced cancer patients. Scales were administered in the order they are listed below.1. EQoL EQ-5D. This generic questionnaire defines health in five dimensions: mobility, self-care, usual activities, pain or discomfort, and anxiety or depression. Each dimension is divided into three categories – whether the respondent has no problem, a moderate problem, or an extreme problem. A sixth item scores the person's overall health on a visual analogue (0 – 100) scale. The questionnaire has been validated and applied in a wide range of patient groups -16.2. Palliative care Outcome Scale (POS). This 10 item scale (plus an open question) was specifically developed and validated for palliative care and covers physical symptoms, patient and family or caregiver anxiety/fears and well being. The effect of the items on the daily life of the patient over the past three days is scored on a five-point Likert scale ranging from 'none' (0) to 'overwhelmingly' (4). For example: "over the past 3 days, have you been feeling anxious or worried about your illness or treatment? (0) not at all – (4) overwhelmingly" ,18. In t3. Herth Hope Index (Hope). This 12 item instrument assesses hope in adults in clinical settings, and is designed to assess change. For example: "I have a positive outlook toward life? strongly disagree (1) to strongly agree (4) -". Patients are asked how much they agree with the statement right now .Full details of the scales are shown in the Appendix 1 see . PatientData were analysed separately for the historical and concurrent (post day care) groups. The relative factorial structure of the three scales was explored in two steps. First, we performed a preliminary exploratory factor analysis (EFA) on each individual scale and on all the items of the three scales combined, using Principal Component Analysis on the historical sample. Second, we performed further exploration and final validation using confirmatory factor analysis (CFA) on the combined historical and concurrent samples. The EQS software  was used171 patients were identified and asked to take part in the study, 82 in the historical group, and 89 in the concurrent group (40 were AD). Of these, 140 (81%) were successfully approached, agreed to take part in the study, and completed the first interview. As shown in Table Summaries of the distribution of scores on the three instruments assessing hope, quality of life and palliative outcomes for the combined sample as well as results of the exploratory factor analysis, are shown in Table 4 see . On prinfirst principal factor, explaining 25% of the total variability of the combined scales, was determined by the three items of positivity contained in POS , together with all the Hope items and the anxiety items in both, POS and EQoL. The EQoL items "General Health" and items of "self-sufficiency", which constituted the most important factor of the EQoL scale, loaded together into the second factor, explaining 10% of the total variability of the combined scale. The third most important principal component, explaining 8% of the variability, comprised a general measure of patient anxiety (measured by both EQoL and POS), and family anxiety (measured by POS). The fourth principal component explained 6% of the variability and was defined by pain (measured by both EQoL and POS). The POS items "information" and "time-wasted" loaded together into the fifth factor, explaining only 6% of the total variability. In addition, the POS item "symptom control" did not load into any of these five factors and appeared to be acting independently.The exploratoty factor analysis of EQoL, POS and Hope on the historical sample alone gave rather consistent results for different extraction methods. Table 4 see  shows thOne of the extractions explored was principal axis factoring with a varimax rotation. This provided a better definition of the structure, with items loading more exclusively onto one of the factors. The first factor that we had obtained with the unrotated matrix essentially separated into two. The first axis, explaining 29% of the variability, was defined by the POS item "life worthwhile" loading with those items of Hope that reflected positivity and direction: positive outlook, goals, inner strength, loving, sense of direction, days have potential and life has value. The second axis, explained 11% of the variability and contained the anxiety items of EQoL and POS, the "feel good" and "share feeling" items of POS and the items of Hope that reflected pessimism or anxiety: "alone", "scared of future" and "past memories". The third factor was the EQoL general health and self-sufficiency and explained 7% of the variability. The fourth factor was solely defined by the pain items in EQoL and POS and explained 6% of the variability. The rest of the items played only a minor role. The POS items: practical matters, information and time wasted loading in a minor factor while the POS item "family-anxious" and the Hope items "tunnel" and "faith" disappeared altogether from the rotated matrix. Therefore, the model derived from this data is one in which the following items are omitted: POS2 and POS4 from the POS scale and Hope4 Hope5 from the Hope scale, leaving the rest of the items to define four major latent factors in the following manner: Spiritual, positivity, symptoms and self-sufficiency.family-anxious did not particularly disrupt the identifiability of the model but its presence reduced the goodness of the fit. Similarly, four Hope items were discarded – alone, light at the end of the tunnel, faith and scared of future – to give a total of seven items discarded. We arrived to two models, exhibited in Table Several models were explored and the most relevant are presented in Table In all the models presented, the matrix was positively definite, the test of independence was significant and the frequency distribution of the standardised residuals was symmetrical around 0. Models 3 and 4, not only omit the superfluous items but also separate the factors on clinical considerations. Both provide a huge improvement over the first two models. Model 3 allowed for a high correlation between the positivity and spiritual factors. More remarkably, the results show that Model 4, which disposes completely of the spiritual factor defined by the remaining Hope items, is an enormous improvement on the other models. The chi-square statistic was greatly reduced and almost reached the threshold indicating that no lack of fit was detected.An important next step in quality of life measurement is the translation of measurement into clinical practice to improve patient care . One impA factor which we entitled 'positivity' appeared to be highly important among people with advanced illness. Spirituality/positivity has also been related to communication outcomes . In the When attempting to develop a reduced scale we identified two models, one with four factors (19 items) and another one with three factors which provided a good fit (11 items). All the Herth Hope items are excluded from the latter model, which captured self-sufficiency, symptoms and positivity. Positivity appeared very close to spirituality, as measured by the Hope index. Further work is needed to determine the relationship of these questions with specific spirituality scales -29.Symptom control was absent in the structures obtained by EFA. We suspected that this was because this item was not a structured question designed for any specific symptom, but elicited in an open way what symptoms had troubled the patient. In the CFA this item loaded with the General Health status factor. Measures which specifically address symptoms are available and have been used in palliative care populations ,30. WorkSpecial attention was given to the three items forming the practical factor in POS. The information item in POS (POS-5) was constant in the concurrent sample and only a few patients in the historical sample recorded non zero for this item. The time wasted item of POS was also essentially constant. It may be that the grading for these items needs to be reviewed to ensure that they give greater sensitivity to change. In our analysis this could have contributed to the poor fit shown when attempting to fit a general POS factor containing these items. It may also be because that none of the three items is an indicator of QOL; they are rather items of the provision of health care. In this study all of the patients were in receipt of a wide range of services, including specialist care teams and their practical needs were likely to have been met. Research among patients in different circumstances has shown deficiencies in these aspects of care ,33.The POS item family-anxious was intriguing. It did not disrupt the validity of the model but if kept and loaded in the positivity factor, it reduced the goodness of fit. This item also showed erratic behaviour in the exploratory factor analysis. Family anxiety may be related to a large number of factors, some of which are determined by the circumstances of the patient and some of which are determined by other events. Family needs often increase as patients deteriorate and are difficult to resolve. Further work is required directly targeting the needs of families .Our study was limited by the comparison of these scales among patients in one setting: we do not know if similar results would be found if patients were not in receipt of specialist palliative care available in the United Kingdom. However, our findings are consistent with other work assessing quality of life measurement in palliative care and in advanced cancer . CorrelaOur reduced model suggests that clinicians may sensibly target quality of life measurement in advanced cancer towards three main components, positivity, self-sufficiency, and symptoms. This might be achieved by the 12 items in model 4 of our factor analysis. Such a scale would be short and simple to use in both clinical practice and research, improving the measurement of outcomes in this population.Appendix 1: Full details of the three palliative care outcome scalesClick here for fileTable 4: Summary Statistics and Principal Component Analysis (unrotated) of the three scales (POS-EQoL-Hope) in the historical sample.Click here for file


Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders.ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage).We have used a new integrative approach that allows to incorporate AS detection into ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.This automatic combination of experimental data analysis and  Alternative splicing (AS) is a biological process that occurs during the maturation step of a pre-mRNA, allowing the production of different mature mRNA variants from a unique transcription unit. AS is known to play a key role in the regulation of gene expression and transcriptome/proteome diversity . First cintrinsic or ab initio, the only type of information used for gene prediction lies in the statistical properties of the various gene elements . On the contrary, so-called extrinsic approaches essentially rely on the existence of similarities between the sequence to annotate and other known sequences . Several existing gene finding tools are essentially intrinsic (or ab initio): this is the case for Genscan [k best predictions. This approach has been applied eg. in HMMgene or in FGENES-M (unpub.). Another way to obtain suboptimal solutions from a HMM is to do HMM sampling [a posteriori likelihood is larger than a given threshold.In the context of structural gene prediction, two classes of approaches are usually considered. In the first approach, usually denoted as  Genscan , HMMgene Genscan  or SLAM  Genscan . For sucsampling . This meintrinsic approaches cannot take into account transcript evidences, they suffer from two major problems of sensibility and specificity:In addition to the fact that all these exclusively eg. shifts from a strong to a weak or a non-consensus splice site or shows a complete coding exon skipping event, it is quite unlikely that the probability will remain in the neighborhood of the optimum since it will not be able to incorporate the corresponding splicing or coding score.First of all, these methods assume that predictions representing AS variants should have a probability which is very close to the optimal probability according to the underlying gene model. This is however quite arguable, especially when the alternative structure significantly differs from the optimal one. Actually, when an AS variant in silico false positives. To perform this, and as long as AS sites dedicated prediction tools are unavailable, the probability of a prediction alone cannot be sufficient and additional evidence is required.Moreover, a strong specificity problem has been observed for this approach. Since a very large number of alternative predictions can always be produced for any sequence, it is essential to be able to distinguish those reflecting real AS variants from intrinsic approach, the analysis of experimental data can provide useful information. More specifically, sequences of mature transcripts resulting from AS provide reliable evidence of the existence of the AS event. Large scale studies have already been undertaken to detect AS evidences from transcript alignments and to collect them in databases such as eg. HASDB [extrinsic annotation tools include GeneSeqer [In opposition to the purely g. HASDB , ASDB [9g. HASDB , ASD [11g. HASDB  or ProSpg. HASDB . Some soeneSeqer , ASPic [eneSeqer , TAP [16eneSeqer , and PASeneSeqer . Except eneSeqer , has beeeneSeqer  to identintrinsic methods, extrinsic approaches take advantage of transcript information. However, they also suffer from some limitations : first they entirely depend on the availability of transcribed sequences which bounds their sensitivity. With little exceptions , they cannot predict a splice site if it is not represented in a transcript-to-genome alignment and therefore require a total coverage of each gene with all exon-intron boundaries. This can be problematic considering the ESTs fragmented nature. Moreover, when such methods can take advantage of a total gene coverage, the CDS localization remains to be done and the pure transcript predictions may not respect elementary coding gene properties (such as the presence of an ORF w.r.t. a given frame). Furthermore, overlapping transcripts are sometimes assumed to come from the same mature mRNA and are therefore merged. This may lead to the fusion of two overlapping transcripts coming from exclusive inconsistent mRNA variants, thus forcing the prediction to respect a chimeric virtual assembly.Unlike intrinsic and extrinsic information are needed inside an annotation pipeline [Finally, and because experimental transcripts cannot exist for every existing gene, both pipeline . The prepipeline . A fullypipeline  seems topipeline ,18. The intrinsic/extrinsic integrative annotation method with the following aims:To extend the domain of application of gene prediction to alternatively spliced gene structure prediction, we have designed an • For a given genomic sequence, an optimal gene structure prediction is produced, as usual.• In addition to this optimal prediction, for every transcript sequence providing evidence of AS, an optimal prediction consistent with this splicing form is also provided.• Each additional or alternative gene structure prediction has to be supported by some biological evidence.• Full-length transcript coverage is not required for a complete gene structure identification.• Each prediction satisfies the usual constraints on gene structure. A correct proteic coding gene is defined by a succession of one or more exons separated by introns flanked by splice sites. It contains a CDS between a start and a stop codon, and no in-frame stop in coding exons.intrinsic and extrinsic approaches in an integrative system allowing for AS detection based on the analysis of genomic aligned transcript sequences. The method has been implemented inside EuGÈNE-M, a new version of the Arabidopsis thaliana EuGÈNE gene finder [Our aim is to combine the advantages of the e finder ,23, and spl7 Arabidopsis thaliana gene. This gene codes for the squamosa promoter-binding protein-like 7, has 10 exons and two known alternative mature mRNA variants, both supported by a distinct full-length cDNA  show incompatible alignments (see Methods), EuGÈNE-M computes two additional predictions, each being consistent with one of them. The first alternative prediction is the same as the optimal one and corresponds to one variant; the second corresponds to the other variant.Given only the genomically ESTs alignments, we applied EuGÈNE-M on the genomic sequence containing the Arabidopsis thaliana curated genes recently used in the assessment of GeneSeqer [alternative predictions, that were carefully analyzed. This is summarized in Table in silico expertise, require experimental verifications to be confirmed. If we assume the 7 remaining genes are effectively subject to AS, this yield to an AS rate of ~4.2%, a ratio in the same order as previously estimated, from 1.5% [For a more extensive test, we applied EuGÈNE-M on AraSet , a data eneSeqer . Since EeneSeqer , the aimrom 1.5%  to 6.5% rom 1.5% ).Arabidopsis thaliana Genome Database AtGDB [In the recent assessment of GeneSeqer on this AraSet data set, only three AS cases were reported . Howeverse AtGDB ,28. We nse AtGDB ,29 and ose AtGDB ,30. OnlyA. thaliana than in Homo sapiens. Nevertheless, this AS ratio estimation is expected to increase in the future with the growth of transcript data availability. Another interesting point is the nature of the variants: on this gene set, the majority of AS cases involves a simple acceptor or donor alternative splice site. Notice however that since EuGÈNE-M's underlying model allows arbitrary alternative gene structure to be predicted, it is not limited to the prediction of such simple AS events and can perfectly cope with complex AS events, as found in mammals. This methodology can also be integrated in other existing gene finders where the score of a gene structure is defined as the sum of elementary scores of the signals and nucleotides involved in the gene structure (this includes HMM-based gene finders).The analysis of these AS cases confirms that AS seems to be much less frequent in intrinsic and an extrinsic process to incorporate AS detection into ab initio gene finding. We showed that this method allows the discovery of new alternative spliced genes, with the reliability of extrinsic annotation and the potential exhaustiveness of ab initio gene prediction.In this paper we have presented a new method to deal with alternative splicing in annotation and gene prediction. This integrative approach combines the advantages of an The process that goes from the original genomic sequence and associated aligned transcripts to the AS prediction is composed of three steps which we rapidly describe here :• first, the set of genomically aligned transcripts is analysed to detect AS evidences on the basis of splicing inconsistency between transcripts variants.• Then, the graph-model used in EuGÈNE to model potential gene structures is modified to take into account these aligned transcripts. For each transcript variant, the graph used in EuGÈNE for gene structure prediction is connected to an additional parallel graph subunit where local constraints are injected according to the exon-intron information provided by the corresponding transcript alignment.• Finally, an extended version of the dynamic programming algorithm used for obtaining an optimal prediction allows to identify, for each graph subunit, the best prediction consistent with the corresponding transcript alignment.Since EuGÈNE already exploits transcripts information to improve the gene prediction process , the AS a priori imposed by the method. Transcript sequences in our analysis were extracted from the A. thaliana section of dbEST [Transcript sequences are first aligned against the genomic sequence using a spliced alignment tool. The choice of the source transcript database and the alignment tool is not of dbEST  .A first filtering step is performed on the basis of the transcript sequence and alignment quality. To be considered, an alignment has to satisfy some constraints defined by filtering parameters. For A is labeled as included in B if and only if for each genomic position of A the same genomic position in B shares the same alignment information (either gap or match). Every transcript included in another transcript is ignored by default. Transcript alignments A and B are labeled as incompatible if and only if there is a genomic position for which both ESTs are informative and give an inconsistent information, that is a gap (representative of the presence of an intron) is faced with a match . Examples of incompatible ESTs are displayed in Figure incompatible after all pairwise comparisons. Considering orientation of ESTs, the information on the clone-sequencing orientation that can be found with ESTs is totally ignored in this filtering process because of its unreliability. In practice, spliced EST can be reliably oriented by looking for splice sites on the hit-match frontier of the EST alignments and by choosing the strand for which such splice sites exist. The parameters of these two automatic filtering steps can be modified by the user through a simple text file. We will denote the resulting transcript alignments kept as alternative transcripts.The second filtering step depends on the relation between transcript alignments. To detect AS evidences, every pair of overlapping transcript alignments is analyzed. We consider two special types of pairwise relation : a transcript alignment EuGÈNE is a gene finding software based on a directed acyclic graph gene model . For eack tracks that represent the possible annotations that can be attributed to each nucleotide . Let ℓ be the genomic sequence length. For a given nucleotide's position i with (1 <i < ℓ) and for each track j with (1 <j <k) the two flanking vertices  and  are defined. Two edges are also built : a contents edge  linking  to  and a transition edge  linking  to   is present, as illustrated in Figure  and  are added at the extremities of the graph. They are respectively connected to all  and all . Initially, all edges are oriented from left (5') to right (3'). It is easy to see that all possible gene structure can be represented by a path from  to .Each path through the graph represents a potential gene structure prediction for the genomic sequence Figure . The gra  Figure . Additioc and t are penalized respectively by weights Wc and Wt according to a weighting function characterized by parameters specifically set for the corresponding source of evidence. The set of parameters is optimized on a learning dataset by maximizing the overall accuracy of the software. For more information about the weighting methods, please refer to [The weight of a path is the sum of all the weights of the edges in the path. The edges are weighted according to the evidences used. EuGÈNE can combine several sources of evidence such as probabilistic coding models, output of splice site or start codon prediction software and sequence similarities with transcripts, proteins, or other genomic sequences . Contentrefer to .A transcript-to-genome alignment can easily be taken into account by weighting the appropriate edges of the graph. To favor a gene prediction in the alignment region, the intergenic track edges included in this region can be penalized by increasing their weight. More finely, the exon and the intron tracks edges can also be penalized at all positions involved respectively in a gap and in a match in the alignment. Thus, all gene structure prediction inconsistent with the transcript alignment information tends to be penalized. More drastically, it is possible to force the prediction to be consistent with the alignment by applying infinite penalty weights. Note that there are several such predictions since the start codon used is unknown and the transcript may be incomplete. a variable  which contains the weight of the optimal path from  to  and a variable  which contains the vertex that precedes  in this optimal path. The weight of this path can be computed recursively from 5' to 3' as:To identify the optimal path defined by the lowest weight, EuGÈNE uses a dynamic programming algorithm inspired from Bellman's shortest-path algorithm , also us that minimizes this value provides the previous . At vertex , the best path is retrieved by a simple backtracing procedure through all π. This algorithm is linear in time and space in the length of the sequence (O(ℓ) complexity). It is important to note that the same algorithm can be used in a backward version (from  to ), by computing at each vertex  the weight  of the best path from  to  as .A short example is displayed in Figure alternative transcript genomic alignment, any prediction which is optimal among all the predictions that are consistent with the alignment evidence will be called an alternative prediction. Given the set of the previously detected alternative transcripts, we want EuGÈNE-M to produce a set of alternative predictions such that every alternative transcript has a corresponding prediction in this set. A simple way to produce such an alternative prediction would be to inject the exon-intron structure information given by the transcript alignment into the graph as described above , and then to execute EuGÈNE on the resulting graph. However, obtaining all alternative predictions would require one execution for each alternative transcript. n being the number of transcripts and l the genomic sequence length, this would result in a O(ln) time complexity, which is not appropriate for long genomic sequences and numerous transcripts.Given an Hopefully, this complexity can be drastically reduced. The general idea to achieve a realistic complexity is to duplicate the subsection of the graph region involved in an alignment to create a so called local "Parallel Graph Subunit" (PGS), connected to the main graph at its extremities. Each alignment information is taken into account as constraints in the corresponding PGS, in such a way that finding the optimal path going through the PGS provides a corresponding optimal alternative prediction.g to h on the genomic sequence, the entire subsection of the graph between g and h is duplicated to create a Parallel Graph Subunit (PGS)  Figure . This PG to  and applies the recursive formula described above to compute all , branching into each PGS  are reversed, and the backward version of the same shortest-path algorithm is used from A, if we now consider the vertices  at the rightmost extremity of A, then the weight of an optimal path that goes from  to  through A can be computed as . From the given vertex, backtracing in both directions provides an optimal path that represents an optimal prediction in accordance with the transcript alignment evidence.For a given PGS alternative prediction the name of the corresponding transcript is mentioned and the region that differs from the optimal prediction is displayed. Besides, if several predictions are identical , a single representative is displayed, along with the list of its associated transcripts.Predictions are produced in the standard GFF format. The entire optimal annotation is first displayed, followed by the alternative ones. To enhance the readability and to avoid redundancy, for each incompatible transcripts identification requires O(n2) pairwise comparisons. Each comparison is itself linear in the maximum number of introns in the transcript compared, which is typically bounded by a small constant and the whole process is therefore in O(n2).The initial filtering and L is the total nucleic sequences length , the weights of all  predictions can be computed in O(L).The step that corresponds to the two dynamic programming scans (application of the recursive formula) requires a time and space complexity which is linear in the size of the input data. Indeed, if For the backtracing and output step, since each alternative prediction has to be displayed in the region where it differs from the optimal one, and because this can extend beyond the alignment region, it is not possible to obtain an algorithm which is linear in the size of the input. However, it is possible to reach a linear complexity in the size of the output. This can be done by a simple modification of the standard backtracing procedure to avoid a full backtrace for each prediction. This is yet not implemented in the current version of the software.A typical run of EuGÈNE-M on an AMD Athlon 1.7 GHz takes 47 sec. for a 500 kb BAC .SF designed and implemented the filtering algorithm as well as the double dynamic programming algorithm for AS prediction. He also ran the experiments on the Araset dataset. TS directed the research. All authors read and approved the final manuscript.EuGène's predictions on AraSet. The additional file (SupplementaryFiles.tar.gz) contains gene structure predictions in the standard GFF text format for every AraSet sequence. ESTs detected as Incompatible by the method are displayed at the top of each prediction (if any).Click here for file


Drosophila robusta have been characterized by well-defined latitudinal, longitudinal, and elevational clines, but – until at least ten years ago – stable, local population frequencies. Recent biogeographical analyses indicate that D. robusta invaded North America from southeast Asia and has persisted in eastern temperate forests for at least 20–25 my without speciating. The abundant chromosome polymorphisms found across the range of D. robusta are thus likely to be relatively ancient, having accumulated over many well known climatic cycles in North America. Sufficient long-term data are now available such that we can now gauge the rate of these evolutionary changes in natural populations due to environmental change.Studied since the early 1940's, chromosomal polymorphisms in the deciduous woods species Recent local collections have revealed significant changes in the frequencies of several chromosomal forms. New data presented here extend the range of these changes to six states, three in the northeastern United States and three west of the Mississippi River. These data reinforce recent directional changes in which the frequencies of three gene arrangements have reached percentage levels typical of distant southern populations consistent with regional climatic changes. Another gene arrangement has been steadily decreasing in frequency at a number of the sites studied. Meteorological records from 1945 to 2003 indicate temperature increases at all study sites, particularly average minimum air temperatures.D. robusta are evolutionary responses to environmental change. Since these chromosomes are known to be sensitive to ambient temperature, regional climatic shifts associated with global warming are likely to be responsible.Observation of parallel genetic flux suggests that these long-term temporal frequency shifts in widely disparate populations of  AVTEMP and MAXTMP varied significantly from site to site in different years, but MINTMP significantly increased over the 58-year period (P < 0.0001) with no heterogeneity among sites. Thus, the most consistent change in climate across sites in this study was due to significant increases in minimum monthly temperatures.Regional patterns of climate change were revealed from ANCOVA analysis of temperature and precipitation data from 1945 – 2003. No long term tends were detected for precipitation except for a significant increase in Central Park, NY (data available from the authors). Significant long-term temperature changes were apparent at all six 2003 study sites . Causes for these decreases are obscure, but in New Jersey may be due to the exaggerated temperature fluctuations in the early 1990's . X chromosome combinations 1S and S1 show substantial frequency changes with time, with S1 doing so in a site-specific manner , tends to be higher in southern populations than in northern ones [Like 2L-1 and 3R-1, the frequency of gene arrangement XL , and 3R-1 from South Carolina, Alabama, and Mississippi were released in midsummer in the Englewood, NJ woods. In other years the released flies carried XL-1, 2L-3, and 3R from Minnesota and Michigan. Despite evidence of hybridization of the introduced flies with the local population, in each case the following spring saw return to frequencies of the previous year.Evidence of strong natural selection maintaining earlier observed genetic stability has also come from perturbation experiments in the wild . At fourTL and PP of D. pseudoobscura mentioned above, since high frequencies of inversion homozygotes would undermine the structural stability of the new adaptive complex. The sudden rises of D. robusta X-chromosome combination S1 in New York and New Jersey and of S2 in Missouri could be cases in point, but it would not explain the near doubling of S1 in Arkansas from a 35% base between 2001 and 2003 nor the three most consistent directional changes, those of 3R-1, 2L-1, and XL , Little Falls and Ridgefield for Englewood ; see Fig. Temperature and precipitation data were obtained online from the National Climatic Data Center ML collected the Allentown, New York, and New Jersey flies and carried out the chromosomal analyses. WJE contributed the Fayetteville flies. Both authors contributed to planning the study, analyzing the data, and writing and revising the manuscript.


Ever since the early Greeks recast humans as the center of the universe and remade God in their own image, Western philosophers and poets have grappled with the limits of human mortality. Philosophers found relief from Keats's “unwilling sleep” by dividing human existence into body and soul and asserting that the true essence of humanity lies in the immortal soul, not in the body. Ironically, as this decidedly nonscientific subject has lost favor with modern-day philosophers, it has captured the imagination of scientists. But, for now at least, the interest is in prolonging life rather than escaping mortality.SIR2. Now, Brian Kennedy and colleagues show that calorie restriction and SIR2 promote longevity through distinct genetic pathways—and that aging in yeast and higher organisms may be more similar than previously thought.Over the past twenty years, mounting evidence from a wide range of organisms indicates that a longer life awaits those who eat less. In yeast, calories can be restricted directly, by limiting yeast's glucose supply, or indirectly, by inhibiting yeast's ability to metabolize glucose. Either way, many studies have suggested that the increased longevity associated with calorie restriction is linked to increased activity of a gene called FOB1 gene, for example, dramatically reduce ERC levels and increase the reproductive life span of cells by 30%–40%. In contrast, mutations that disrupt SIR2 increase ERC levels and cut life span in half, while increasing SIR2 activity increases life span by 30%–40%.One of the causes of aging in yeast is the accumulation of coiled bits of DNA, called extrachromosomal ribosomal DNA circles (ERCs), in the nucleus of a mother cell . An overabundance of these rDNA circles wreaks havoc on a cell and eventually kills it. Genetic mutations that reduce their levels are linked to increased life span. Mutations that disrupt the SIR2, and the accumulation of ERCs. The idea is that calorie restriction somehow activates the protein encoded by SIR2, which in turn decreases ERC accumulation. Now, Kennedy's team has found that the combination of calorie restriction and FOB1 mutation increases life span more than either approach does alone. This finding was unexpected because previous studies showed that combining increased SIR2 activity with FOB1 deletion mutations did not extend life span. If calorie restriction extends life through SIR2, then combining either caloric restriction or SIR2 overexpression with FOB1 mutations should produce the same result.In previous experiments, several groups have identified a link between calorie restriction, SIR2. In support of this view, caloric restriction enhances life span to a greater extent in FOB1 mutants lacking SIR2 than in FOB1 mutants with an intact SIR2 gene. This and other genetic experiments indicate that calorie restriction does not always work through SIR2.This contradiction raised the possibility that calorie restriction operates through another mechanism, independent of FOB1 mutants is not related to ERCs, because these yeast strains already have low ERC levels. Since calorie restriction is the only demonstrated approach to increasing life span in a diverse range of organisms, including mammals, and since there's no evidence that ERCs affect the aging of any organism besides yeast, these results bode well for understanding how calorie restriction works in higher organisms. And the finding that calorie restriction and SIR2 operate through genetically distinct pathways in yeast, the authors conclude, suggests that certain aspects of both pathways might have been conserved through evolution. Working out the details of these pathways in yeast is the first step toward understanding which, if any, of these components might enhance longevity in humans. Of course, as any student of Greek mythology knows, longevity without eternal youth comes with a price.That suggests, the authors explain, that calorie restriction functions either by regulating ERC levels or by some still unknown molecular pathway. They conclude that the enhanced longevity seen in calorie-restricted


Migraine and tension-type headaches impose a tremendous economic drain upon the healthcare system. Intravenous and oral niacin has been employed in the treatment of acute and chronic migraine and tension-type headaches, but its use has not become part of contemporary medicine, nor have there been randomized controlled trials further assessing this novel treatment. We aimed to systematically review the evidence of using intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types.We searched English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004).Nine articles were found to meet the inclusion criteria and were included in this systematic review. Hypothetical reasons for niacin's effectiveness include its vasodilatory properties, and its ability to improve mitochondrial energy metabolism. Important side effects of niacin include flushing, nausea and fainting.Although niacin's mechanisms of action have not been substantiated from controlled clinical trials, this agent may have beneficial effects upon migraine and tension-type headaches. Adequately designed randomized trials are required to determine its clinical implications. Migraines and tension-type headaches impose an important burden upon society and the working public. According to the National Headache Foundation, some 45 million Americans suffer from chronic, recurring headaches and 28 million of these suffer from migraine headaches annually . FurtherEven though advances have been made with regard to the treatment of acute migraine headaches , many patients often discontinue their migraine interventions due to treatment dissatisfaction . Among iOne novel, but not really new treatment option, is the administration of niacin (nicotinic acid) through intravenous and/or oral routes. Niacin is a well-known over-the-counter (OTC) supplement primarily used for its ability to favorably influence cholesterol levels. Recently, there have been anecdotal reports demonstrating the effectiveness of niacin for aborting acute migraine attacks , and forWe conducted a systematic search of English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004). Articles were searched with the key search terms "Migraine" combined with the Boolean Operator "AND "Niacin" OR "Nicotinic Acid." Additional searches were conducted with the search terms "Headache" and "Tension." To supplement the search, we searched through the references of the articles we found from the databases.To be included in our final review, articles had to report on the use and administration of niacin for migraine or any other types of headache. We included articles assessing original reports in both peer-reviewed and non-peer-reviewed journals.We determined the evidence grade of each report found, based on the hierarchy of evidence developed by the Oxford Centre for Evidence Based Medicine . Table 1A total of 14 articles were screened ,7,9-20. This case series of 21 patients was limited as it was uncontrolled and involved a small number of patients. The methods that were used to evaluate efficacy of the treatment were primarily based upon a subjective questionnaire or medical record. The results would have been more meaningful if all the patients used the questionnaire to evaluate their treatment responses. Finally, although the symptomatic changes were witnessed immediately following niacin injection, the lifestyle recommendations may have had therapeutic benefits as well.This study involved 100 patients having headaches of multiple etiologies. This study was not properly controlled, but did at least provide some comparison against a group of patients that were not administered the flush forms of intravenous niacin. The fact that the control group did not substantially benefit from the intravenous niacinamide lends more therapeutic efficacy to the ability of intravenous sodium nicotinate or niacin to have a marked therapeutic effect. The sample size was sufficient in number (n = 100), but the determination of therapeutic effectiveness was purely subjective and did not include any questionnaire or standardized method of evaluation.Side effects were minimal; 2 patients experienced mild abdominal cramps, 1 patient vomited but was suspected as having a pathologic condition of the stomach, and 1 other patient with migraine vomited forty-five minutes after the injection. A few patients found the treatment to be worse than their headaches. The authors concluded that the relief of headaches seemed to be correlated with the degree of flushing from the sodium nicotinate or niacin, and that this therapy was most useful among the migraine, spinal tap, and idiopathic groups.A number of shortcomings were evident in this article. The first of which was that the study was not properly controlled and did not contain a placebo group or a group of patients acting as self-controls. However, some of the patients were given intravenous treatments on more than one occasion. This procedure helped in determining treatment reliability and reproducibility since niacin appeared to achieve therapeutic benefits on several occasions. There were no standardized methods of evaluating efficacy since the treatment responses were based upon the medical record and subjective reports. The results would have been more meaningful if all the patients used a standardized questionnaire prior to and after each treatment. In addition, there was only one male subject and 14 females in the study.In this study of 35 patients were given treatments of intravenous dihydroergotamine methanesulfonate, intravenous niacin, or oral combination tablets of ergotamine tartrate and caffeine. Although no control group was used in this study, the patients were given multiple treatments on several occasions. This offered an interesting comparison to be made between niacin and other treatments. Since no control or placebo group was included, it cannot be determined if the therapeutic results were due, in part, to chance or placebo effects. The results might have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to further substantiate patient responses.These cases reported were well described and clearly demonstrated therapeutic responses during the intravenous and oral niacin therapy. The results would have been more reliable if a comparison had been made to a control group or to a similar patient cohort that were not given the same treatments. Even if the patients served as self-controls, and were told to stop the niacin treatment for a specified period of time, more information could have been gained from their therapeutic responses to niacin. Overall, this report of five cases provides an interesting approach to patients having chronic tension headaches and depression. Its value is limited by the difficulty in extrapolating these findings to a greater number of patients.In this study involving 50 patients there was no data that listed pertinent identifying information, treatment response, past treatments, and duration of treatment for each patient. This would have strengthened the report by being more descriptive, and thus more amenable to critical analysis. If a comparison were made to a control group or a similar patient cohort not given the same treatments, more validity could be have been ascribed to this method of treatment. The patients could have also served as their own controls, thus providing more information about the therapeutic responses to niacin. It cannot be determined if the therapeutic results were due, in part, to chance or placebo. No method of evaluating efficacy was mentioned, except that the responses to niacin were "very satisfactory" in 44 of 50 cases. The results would have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to substantiate their responses to niacin.In this report, Hall describes the use of niacin for his migraine headaches remarking that the migraines resolved when intense flushing occurred. According to Hall, niacin's benefits and side effects might be due to its ability to release serotonin and histamine from the stomach. There is no reason to doubt Hall when describing his therapeutic response to niacin. However, his report was brief, had no control and was entirely subjective as he was the participant as well as examiner.In this report of 2 cases, it was found that in both cases migraines were relieved with oral niacin. The report would have been more rigorous if the patients had acted as their own self-controls. The value of the report is further diminished by the difficulty in extrapolating these findings to a greater number of patients.This case report was of a 62-year-old woman with a 40-year history of migraine headaches. The patient never acted as her own self-control, which would have made the findings of the case report more meaningful. The fact that the patient's migraine headaches increased in severity after a reduction in dosage does lend more support to niacin as being a migraine preventive agent. The hypothesized increase in serotonin from niacin administration cannot be proven given the limited amount of data contained in the case report. Like all case reports, its value is diminished by the difficulty in extrapolating these findings to a greater number of patients.The results of this systematic review indicate that niacin may have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other etiologies. The quality of the evidence at this point, however, is only hypothesis generating, and randomized trials are required to determine the clinical implications of this novel treatment.There are several important limitations to consider in the interpretation of this review. We did not find any randomized or controlled trials of niacin on these headaches. We cannot determine to what extent publication bias has on the results of this review. We are unable to draw clinical inferences on the results of the included studies as they were of low quality and have a low level of external generalizability. Despite these limitations, we attempted to conduct an exhaustive search and included all reports of relevance.2 (PGD2) in the skin, leading to a marked increase of its metabolite, 9α, 11β-PGF2, in the plasma [-1 M aqueous methylnicotinate on the forearm, PGD2 is markedly released in the skin and its metabolite appears in high amounts in the plasma [2 causes vasodilation of the intracranial arteries, but niacin's ability to abort acute migraine headaches suggests that this might be what is occurring. Old reports cited by Bicknell and Prescott [Reasons for niacin's effectiveness can only be considered hypothetical, and require clarification from future randomized controlled trials. In acute migraine headaches some of the symptoms arise from activation of the trigeminovascular complex. Activation of this complex leads to intracranial vasoconstriction causing the migraine aura, followed by headache due to vasodilation of the extracranial vessels and activation of the perivascular nociceptive nerves . When tae plasma . When nie plasma ,23. It iPrescott , demonstIn terms of tension-type headaches, it appears that intravenous niacin is of benefit acutely due to its presumed central vasodilatory properties. Like migraines, part of the underlying pathophysiology of chronic tension-type headaches involves central mechanisms, such as the trigeminal system . ChronicSome of the reports did demonstrate prophylactic benefits when niacin was administered orally every day. It is now recognized that a deficit of mitochondrial energy metabolism  plays a role in the pathogenesis of chronic migraine headaches . Niacin Niacin might also prevent tension-type headaches by improving mitochondrial energy metabolism within the skeletal muscles, and by increasing blood flow and oxygenation to the skeletal muscles. The overall net-effect could be a reduction in lactic acid concentrations, leading to reduced episodes of muscular tension and soreness. Niacin may reduce lactic acid concentrations since supplemental niacinamide (the amide of niacin) has been shown to reduce blood lactate and pyruvate concentrations by more than 50% in a patient with MELAS  syndrome by the third day of treatment . This poThe side effects of intravenous niacin were found to be minimal from the summarized articles. The most common side effects were abdominal cramping, vomiting, and uncomfortable sensations of the skin and burning. A few of the patients described found the treatments to be worse than their headaches. In the articles where blood pressures were evaluated, little-to-no change occurred among the individuals treated with intravenous niacin. In a more recent study, parenteral niacin given to hypertensive and normotensive patients demonstrated a significant decrease in systolic, diastolic, and pulse pressures among the hypertensive subjects .With respect to the oral administration of niacin, very few patients reported side effects. Even though niacin was well tolerated orally, we previously reported in a randomized placebo-controlled trial examining the safety of immediate-release or crystalline niacin, that it can be associated with intolerable side effects . The mosThe side effects of greater concern from oral niacin have to do with sustained- or slow-release preparations. These preparations are better in terms of compliance since patients experience less cutaneous flushing with them. However, the use of these preparations have been associated with reversible hepatic toxicity in doses equal to or greater than 1500 mg per day with an acute onset of clinical symptoms of hepatitis in a relatively short period of time (2 days to 7 weeks) . Other rEven though niacin's mechanisms of action have not been substantiated from controlled clinical trials. It is possible that this agent has a beneficial effect upon migraine and tension-type headaches and the prophylaxis of these headaches. It is imperative that properly designed controlled trials are developed in order to determine niacin's true therapeutic effects and adverse effect profile. In terms of its ability to abort acute migraine headaches, a placebo-controlled trial of parenteral niacin and sumatriptan seems to be worthy of consideration. In terms of prophylaxis, a placebo-controlled trial of oral niacin and riboflavin or coenzyme Q10 also seems worthy of investigation.Dr. Jonathan Prousky is associated with Swiss Herbal Remedies, Ltd., a company that sells nutritional supplements including niacin. They had knowledge of this manuscript and have not seen this manuscript.JP wrote the manuscript. DS contributed to the text, revised the results section, and assisted with the tables.


This study was carried out to assess the reliability of core biopsy in predicting the grade and type of cancer accurately as obtaining this information can influence initial therapeutic decisions.Core biopsy is a method of choice for the triple assessment of breast disease as it can reliably distinguish between benign and malignant tumours, between kappa statastics.A total of 105 patients who had invasive breast carcinoma diagnosed by core biopsy in year 2001 and who subsequently underwent surgical management were included. The core biopsy results were compared with final histology with the help of A moderate level of agreement between the predicted grades and final grades was noted (kappa = 0.585). The agreement was good between predicted and final type of tumour (kappa = 0.639).Core biopsy as a predictor of grade and type has limited use at present. We suggest that initial clinical decisions should not be based on the results of core biopsy. Collins et al have shown that majority (83%) of core biopsies and excisional procedures demonstrate exact histological agreement [Core biopsy is rapidly replacing fine needle aspiration cytology (FNAC) as a procedure of choice for the triple assessment of the breast problems. Where there is access to an experienced cytopathologist, the FNAC can provide a rapid and cost effective means of triage of patients who would benefit from more expensive core biopsy . Core bigreement . Core bigreement .As the range of options for the treatment of the breast cancer widens, it has become increasingly important that clinicians are provided with accurate prognostic information to base the initial therapeutic decisions on. Prognostic factors for breast cancer have been extensively studied. Histological grade and type can be used to predict biological behaviour as has been assessed by overall survival and local recurrence for women with primary breast carcinoma -9. HistoThe aim of this study, therefore, was to see how reliable core biopsy is in predicting the grade and the type of cancers, as that could influence the further management of breast cancer.in-situ diagnosed by core biopsy and patients who underwent neo-adjuvant therapy were excluded from the study. Level of agreement between core and excision biopsy was assessed using kappa statistics.All patients with invasive breast cancer diagnosed by the core biopsy and treated subsequently by surgical excision in the year 2001, at a district general hospital were included in the study. Of the 105 patients whose records were studied retrospectively, 47 lesions were palpable and 58 lesions were screen detected. The core biopsies were performed under ultrasound guidance as a part of triple assessment and at least four cores were obtained from palpable lesions and six or more from screen detected lesions with a 22 mm automated core biopsy gun. Two dedicated breast pathologists had authorised all the reports. Age of patients ranged from 35 to 84 with a median age of 62 years. The histology reports for the core biopsy and final histology were extracted and compared. Carcinoma Of 105 patients there was no prediction of grade in 2 patients and in 19, a prediction of grade 1 or 2 or grade 2 or 3 was made. This left 84 where a clear prediction was made. On final histology 35 (33.3%) were categorised as grade I, 45 (42.8%) were grade II and 25 (23.8%) were grade III. The predicted grades versus final grade results are detailed in table Of the 84 cores in which clear prediction of grade was made 63 (75%) were correct. All 21 of grade 1's were predicted correctly, 35 (71%) of grade 2's were predicted correctly but only 7 (50%) or grade 3's were predicted correctly on core biopsy. Of the predicted grade 2's which were reclassified, 5 (10%) were downgraded and 9 (18%) were upgraded. Of the reclassified grade 3's, 6 (43%) were downgraded to grade 2 and 1 (7%) was downgraded to grade 1.Of 105 patients, 101 patients had a prediction of type made. Of 84 cases predicted to be ductal, 81 (96%) were correct and one case was reclassified as mixed histology. Of the 14 predicted to be lobular 9 (64%) were correct and one reclassified as mixed Table . Of the In general the level of agreement between the predicted grades and final grades was moderate (kappa = 0.585) and between predicted and final types was slightly better (kappa = 0.639).et al reported percutaneous, image guided biopsy to be an accurate diagnostic alternative to surgical biopsy in women with mammographically detected suspicious breast lesions [Fajardo  lesions . The fal lesions . However lesions  or amoun lesions  has not  lesions .Histological grade and type, tumour size and presence or absence of axillary node metastases is well-recognised prognostic factors of breast cancer. Tumour grade, size and nodal involvement are three factors considered in Nottingham Prognostic Index . HistoloGreen Hough 1925) was the first to categorise the breast tumours into three grades according to its differentiation. He also assessed the association of grades with "cure" though the term cure was not clearly defined . Since t was the Of the two major histological types, lobular is known for its multifocality and multicentricity and its diffusely infiltrating nature . It is iOur results suggest that the prediction of grade and type of breast cancer from core biopsy has only limited use at present. The group of patients we would like to be predicted most accurately would have been those with a high-grade and lobular type, for the reasons stated above. Our results suggest that these patients are most difficult to predict in practice. However, present study being retrospective has its own drawbacks. A prospective study specifically aimed at Kappa statistics between core biopsy and final histopathology may be able to answer this question better. Further refinements are needed in technique of core biopsy and these technical innovations will ultimately improve the results of core biopsy.The author(s) declare that they have no competing interests.AD: Original idea, planning of study, background search, data compilation and drafting the manuscript.TG: Data collection, data compilation, help with the manuscript drafting.SH: Overall supervision and guidance with the study, helped in the analysis and helped with manuscript drafting and revisions.All authors read and approved the final versionNone


Rare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddle's syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system.Initially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy  Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests.Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean  plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048).Two commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males  and blood donors . βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined . When expressed in At least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels. Epidemiological studies have shown a significant correlation of blood pressure levels in close relatives and higher concordance values for occurrence of hypertension in monozygotic vs. dizygotic twins, and thus support the idea that genetic factors influence susceptibility to essential hypertension . While rDisappointments in the previous strategies justify alternative approaches in which a better phenotyping of the study individuals is connected to their targeted molecular genetic characterization. There are several features that collectively make the genes encoding the beta (βENaC) and gamma (γENaC) subunits of the kidney tubular epithelial sodium channel as serious candidates for susceptibility genes of low-renin human essential hypertension. First, gain of function mutations in β and γ ENaC subunits cause Liddle's syndrome, a well-known monogenic form of human hypertension associated with low renin activity and low plasma aldosterone level -15. SecoThe clinical records of all consecutive patients with hypertension n = 615) referred to the Hypertension Outpatient Ward, Helsinki University Central Hospital, between 1992–96 were reviewed. Moderate-to-severe hypertension, suspicion of secondary forms of hypertension, or hypertension resistant to drug treatment were causes to the admittance. A letter with request to donate a blood sample for genetic studies on hypertension was sent to those 598 individuals whose address became available in 1998. A total of 399 individuals (67%) of these responded and were subsequently examined at the Hypertension Outpatient Ward in 1998 to 1999. Clinical and family histories were recorded, and venous blood samples taken for DNA analysis. Based on the previous documents and current examinations, altogether 52 individuals were excluded from the present study: clinical records were missing or insufficient in four cases, 22 subjects were considered as normotensive, while 26 were judged to have a secondary form of hypertension. The latter group consisted of the following cases: renal artery stenosis (n = 12), adrenal cortical adenoma (n = 3), hydronephrosis (n = 2), pheochromocytoma (n = 1), IgA glomerulonephritis (n = 1), non-specific chronic glomerulonephritis (n = 1), LED nephritis (n = 1), diabetic nephropathia (n = 1), chronic pyelonephritis (n = 1), hypernephroma (n = 1), fibromuscular dysplasia (n = 1), unspecified renal failure (n = 1). The remaining 347 patients  comprised our final cohort of patients with moderate-to-severe essential hypertension. Antihypertensive drug treatment was in use in 283 (82%) of the patients . At least two concomitant drugs were used by 24% of the patients. A flow-chart of the study design is illustrated in Fig. 5 referreDNA was extracted from 301 randomly selected healthy blood donors aged 40–50 years  visiting the Finnish Red Cross Blood Transfusion Service. Their residences represent the same capital area from which the hypertensive patients originated.These individuals were selected from the participants in the Alpha-Tocopherol, Beta Carotene (ATBC) study  using thThe Ethics Review Committee of the Helsinki University Central Hospital approved this study, and all patients and controls gave their informed consent.The patients were advised to stop using estrogens and spironolactone at least 4 weeks before the tests, diuretics and prostaglandin inhibitors at least two weeks before the tests, and β-adrenergic antagonists and ACE inhibitors at least one week before the test. The only antihypertensive agents permitted at the time of the test were calcium channel blockers. Some of the patients were on oral potassium supplementation because of hypokalemia. The mean baseline blood pressure level at the time of captopril test was 139 ± 16/94 ± 10 mmHg in those without any drugs (n = 79), and 142 ± 16/95 ± 11 mmHg in those with calcium channel blockers (n = 234).Fasting blood samples were taken for determination of serum creatinine, uric acid, cholesterol, potassium, sodium and blood glucose concentrations. Urine samples for determination of the daily (24 h) excretion of potassium and sodium were collected. Most hypertensive patients (n = 298) underwent a test for the responsiveness of serum aldosterone level and plasma renin activity to postural change. To this end, the first blood sample was taken after at least 60 minutes of rest in supine position. After 2 hours of standing and moderate walking, a second blood sample was taken. This test was carried out at the inpatient ward in 220 cases and at the outpatient ward in 78 cases. Urinary electrolyte excretion rates were analyzed in 262 patients (26 ENaC variant carriers and 236 non-carriers) who did not use potassium supplementation.One to three days later, a captopril challenge test (CCT) was carried out as described earlier . This teGenomic DNA was extracted from peripheral venous blood using standard techniques. For targeted search for ENaC variants postulated to be associated with increased channel activity, we chose to sequence the exons 13 coding for the carboxyterminal domains of βENaC (amino acids 515–640) and γENaC (amino acids 524–649), as well as the 5'-flanking intronic regions, using oligonucleotide primers, PCR (polymerase chain reaction) conditions and sequencing instruments described previously by us . DNA samAluI , followed by analysis of the cleavage products on a 12% polyacrylamide gel. The wild-type (wt) allele results in longest fragments of 266 and 137 bp, while the variant allele produces fragments of 266 and 147 bp for βENaC -i12 -17CT, and 240 and 137 bp for βENaC G589S. For the γENaC V546I variant, 2.0 U of SfaNI (New England Biolabs) was used and the cleavage products were analyzed on a 2% agarose gel. The resulting fragment sizes were 357 and 77 bp for the wild-type allele and 279, 78 and 77 bp for the variant allele.Specific PCR-based methods were set up for assaying the three ENaC variants detected during the present study. After PCR of the βENaC fragment, the βENaC -i12 -17CT and βENaC G589S variants could be assayed simultaneously. An aliquot (8 μl) of the PCR product was digested with 3.0 U of in silico using GrailEXP v3.3  exon prediction program 27]), butrtension ,28. The rtension .Xenopus oocytes, these investigators were able to show a borderline 1.3 to 1.5-fold increase in activity for the G589S variant compared with the wild-type subunit. A similar trend was noticed in our experiments  and 175 normotensive males (p = 0.043). Patients with this variant allele displayed the lowest plasma renin levels and responses of all the subgroups examined .The authors declare that they have no competing interests.TH-H collected the clinical material, and participated in the DNA analyses and drafting of the manuscript. KK and TPH designed the study and drafted the manuscript. IT, TT, FF, KH designed the clinical chemical and hormonal assays, and participated in collection of the patient material. HF, HEM and KP participated in DNA analyses and bioinformatics. JV and TK collected the control populations and designed their studies. SS consulted in the statistical analyses and assisted in data handling. IG and LS carried out the electrophysiological studies and participated in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.XenoMouse8 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 102 fragments, or as a bispecific antibody.Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab') On thAlthough targeting of solid tumors with radiolabeled antibodies was first reported years ago ,5, RIT sColorectal cancers represent a high percentage of solid tumors and are dramatically in need of therapeutic progress. Surgery is the only potentially curative treatment. Despite recent developments in chemotherapy protocols, the overall median survival in metastatic colorectal cancer remains inferior to two years, and the recurrence rate after resection of a stage III tumor is up to 50% -18. For ® technology, we describe the generation and the characterization of two fully human anti-CEA antibodies, one IgG2κ and one IgM, designed for RIT of colorectal cancers.Different chimeric or humanized anti-CEA MAbs have been described and evaluated in experimental and clinical studies ,15,19,20®-G2 strain, engineered to produce fully Human IgG2κ antibodies, was described by Mendez et al. [®-G2 animals were immunized i.p. with 20 μg of human recombinant CEA (rhCEA) [Generation and characterization of the XenoMousez et al. . XenoMou (rhCEA)  emulsifi4κ subclass [1 [The mouse-human chimeric MAb X4 was used as positive control in all the experiments. MAb X4 was constructed using the variable domains from the murine MAb CE25 and the constant domains from a human IgGsubclass ,24. It isubclass . MAb F6 class [1 .The CEA-positive human colon carcinoma LS174T cell line  was obta®-1077 and HISTOPAQUE®-1119. Following centrifugation at 700 g for 30 minutes, cells of the granulocytic series were found at the 1077/1119 interphase.Granulocytes were obtained from heparin-treated human peripheral blood by using gradient density centrifugation methods. A double gradient was formed by layering an equal volume of HISTOPAQUEκ  and HRP-conjugated sheep anti-human IgG (γ chain)  were used as detection antibodies.The specificity of the antibodies in hybridoma supernatants was determined by ELISA using rhCEA to capture the antibodies (coating overnight at 2 μg/ml rhCEA at room temperature). Horse radish peroxidase (HRP)-conjugated goat anti-human IgG5 cells were incubated for 1.5 h at 4°C with hybridoma supernatants or controls (RPMI medium for background measurement and RPMI containing 20 μg/ml MAb X4 for positive control). After washing, the cells were incubated with an FITC conjugated goat anti-human IgG kappa light chain (Sigma) or with the murine anti-human μ chain DA4-4 (ATCC HB-57), FITC labelled in our laboratory, for 1 h at 4°C; then they were washed twice before analysis on a FACScanII . Each figure represents data obtained from analysis of 10000 cells.Determination of CEA or NCA specific antibodies in hybridoma supernatants was carried out by flow cytometry using CEA positive cells (CO115-5F12) and NCA positive cells (human granulocytes). About 5 × 10® NHS-anti-human κ chain MAb HP6053 (ATCC CRLC-1758).The percentage of fetal calf serum was gradually reduced in the culture medium before MAb purification . MAbs were purified from large volumes of hybridoma supernatants or ascites produced in nude mice using HitrapProteins separated on SDS-PAGE 6% polyacrylmamide gels were transferred to a nitrocellulose membrane . Non-specific binding sites were blocked overnight at 4°C by incubation with 5% (w/v) non-fat dry milk in TBS. The membrane was probed for 2 h at room temperature with serum diluted 1:1000 or the following antibodies: anti-human κ light chain-HRP conjugate , anti-human λ light chain-HRP conjugate , anti-human μ chain-HRP conjugate , rabbit anti-human J chain-specific antiserum . Bound sin silico methods.Antibody VH and Vκ cDNAs were recovered from hybridomas by RT-PCR and sequenced using the ABI-PRISM Big Dye Terminator Cycle Sequencing Kit . Determination of V, D, and J gene usage was performed using The affinities of the antibodies for CEA were determined by using surface plasmon resonance (SPR) technology . rhCEA  was immo125I or 131I, kindly provided by Schering-CIS Biointernational by the iodogen method . Free radioiodine was separated from the protein on a Sephadex G-25 column (Pharmacia) equilibrated in PBS, pH 7.4.Batches of 50 μg or 100 μg of MAb or rhCEA were labelled with 4.6 MBq or 11.5 MBq, respectively, of 125I-rhCEA  was incubated with increasing concentrations of antibody (68.3 × 10-12 to 33.3 × 10-15 M) for 2 h in 0.15 M phosphate buffer, pH 7.4. CEA-antibody complexes were precipitated at 4°C with 53.5% (v/v) saturated ammonium sulfate. Background binding to an irrelevant human IgG, MonoD, was subtracted. The radioactivity precipitated with a rabbit polyclonal anti-CEA serum was taken as 100%.About 1 ng 125I-human MAb (15 ng) was added to the wells and incubated for 1 h 30 at 37°C. The percentage of binding was determined by measuring the radioactivity bound to the rhCEA after two washings.A competitive binding assay of radiolabeled human MAb and unlabeled anti-CEA MAb was used to determine the CEA epitope recognized by the human MAbs. RhCEA (100 ng/well) in TBS was coated on microtiter wells overnight at room temperature. An excess (500 ng/well) of each of the different reference anti-CEA MAbs, specific for the Gold epitopes 1 to 5, was then added to the wells and incubated for 1.5 h at 37°C. Then, without washing, each 3 (100 to 300 mm3), mice were grouped according to tumor volume. Lugol iodine solution (10% solution) was added to the drinking water one day before the injection of radiolabeled MAbs. Groups of four mice were injected with a 125I-labeled human MAb (VG-IgG2κ or VG-IgM) together with 131I-labeled chimeric MAb X4 as positive control. The total amount of each injected antibody was adjusted to 8 μg protein by adding unlabeled MAb. To determine the biodistribution of the MAbs, mice were sacrificed 24 or 72 h after injection. The blood, tumor, and all normal organs were weighed, and the differential radioactivity was measured in a dual channel scintillation counter. The results are expressed as the percentage of the injected dose of radioactivity present per gram of tissue (% ID/g).Two million LS174T cells were grafted s.c. into the right flank of female Swiss nude mice . When the tumors had reached a volume of about 150 mm®-G2 animals were immunized with rhCEA. The XenoMouse®-G2 strain produces both fully human IgG2κ and fully human IgMκ antibodies as part of the normal immune response, as described by Mendez et al. [in vivo tumor targeting purposes. For this reason, (i) hybridoma supernatants were screened by flow cytometry because, by this technique, the selection is made on cell membrane-bound CEA, which is in a conformation as close as possible to that observed in vivo; and (ii) hybridoma supernatants were screened on human granulocytes to eliminate all hybridomas producing anti-NCA antibodies.In order to develop human anti-CEA MAbs, XenoMousez et al. . Fusion H and VL domains sequencing confirmed the VG-IgG2κ and VG-IgM monoclonality and the isotype (data not shown). Flow cytometry analysis demonstrated strong binding of purified MAbs on CEA-expressing cells CO115-5F12  as compared with 10-9 M for MAb X4 , very close to that of 131I-X4 (21.7 ± 7.2% ID/g) . Due to the mouse poly-Ig receptor expression in liver, 125I-VG-IgM was eliminated very rapidly, giving only 3.7 ± 1.0 and 0.3 ± 0.1% ID/g blood at 24 h and 72 h, respectively. This short clearance was responsible for a low tumor uptake . The percentage of injected MAb recovered in the whole mouse at 24 h was only 20.5% for 125I-VG-IgM as compared with 53.7% for 131I-X4.In two other groups of mice, Monoclonal antibodies are now routinely used in the clinic. Whereas the first generation of MAbs were murine or chimeric antibodies, it is now clear that the best clinical results are obtained with humanized or fully human MAbs . Fully hIn some situations, naked MAbs have demonstrated their therapeutic efficacy, particularly, when the target antigen is a receptor implicated in cell proliferation processes ,34. Howein vitro and presented preliminary data on tumor growth inhibition using MKN-45 cells grafted into SCID mice. However, they did not give any results on the biodistribution of their antibodies in radiolabeled form nor indications on how they could be used in RIT. Anti-CEA MAb PR1A3, which exhibits preferential binding to cell-bound CEA, was recently humanized, but to our knowledge, this MAb has not yet been evaluated in experimental or clinical studies [131Iodine or 90Ytrium, in heavily pre-treated patients with metastatic colorectal cancer (MCRC) CT84.66 [Among solid tumors, colorectal cancers represent one of the main causes of death, and CEA is well known as an ideal target antigen for RIT of these cancers . Up to n studies . A few c CT84.66 ,15. One In the present study, the characterization of our fully human anti-CEA MAbs was conducted in comparison with the chimerized anti-CEA MAb X4, which has been shown to be clinically relevant ,24. Furtin vivo use in patients where some circulating CEA can be found. The molecular basis of this observation is not clear. The only possible comparison is with MAb PR1A3, which preferentially binds to cell bound CEA, and to a recombinant chimeric protein containing only the CEA B3 domain [The VG-IgG2κ and VG-IgM affinity constants were found to be similar to that of the control MAb X4 when determined using the BIACORE technology, but the binding of the human MAbs to soluble CEA Figure  was clea3 domain ,38. This3 domain .125I-VG-IgG2κ localized in the tumor up to 26.2 ± 1.7% ID/g as compared with 28.5 ± 2.8% ID/g for 131I-35A7, suggesting a tumor residence time as long as that observed for MAb 35A7 (data not shown). These results could seem contradictory, given the fact that the affinity of VG-IgG2κ for CEA is not as high as that of Mab 35A7. This could be explained by the "affinity barrier" effect described in solid tumors by several authors who demonstrated that very high affinity MAbs localized at the periphery of tumor nodules and that lower affinity MAbs are able to distribute homogenously in these nodules [Using human colon carcinoma bearing nude mice, we obtained high tumor uptakes with VG-IgG2κ, making this antibody a good candidate for future clinical studies Figure . In addi nodules -43.Since, up to now, there are no data available on the biodistribution of any anti-CEA IgM in mice, we decided to analyze the biodistribution of our VG-IgM in LS174T tumor bearing nude mice. The disappointing tumor uptakes  could be attributed to a very short half-life due to poly-Ig receptor expression in the mouse liver which induces a rapid hepatobiliary transport of poly-IgA and IgM ,45. Base2 fragments, or as a bispecific antibody to be used in the affinity enhancement system (AES) approach [In the present study, we described two fully human anti-CEA MAbs. Even though the results obtained in tumor bearing nude mice cannot be extrapolated directly to humans, VG-IgM remains attractive for RIT of CEA-positive peritoneal carcinomatosis in man since humapproach . Furtherapproach , and it approach ,52 and fapproach .No competing interest for VG, FG, EF, MY, MP and AP. RXL and BB are employees of Abgenix, Inc.in vivo experiments. FG and EF participated in the cell culture experiments and antibody characterization assays. MP performed the affinity measurements using BIACORE. MY participated in the design of the study. RXL and BB sequenced the VH and VL domains. AP conceived the study, participated in its design and coordination. All authors read and approved the final manuscript.VG participated in the design of the study, performed all the cell fusions, cell culture experiments, antibody characterization assays and The pre-publication history for this paper can be accessed here: