Parallel computing is frequently used to speed up computationally expensive tasks in Bioinformatics.a) pair-wise sequence alignments that are used as a first step to multiple alignment account for most of the CPU time in DIALIGN. Since alignments of different sequence pairs are completely independent of each other, they can be distributed to multiple processors without any effect on the resulting output alignments. (b) For alignments of large genomic sequences, we use a heuristics by splitting up sequences into sub-sequences based on a previously introduced anchored alignment procedure. For our test sequences, this combined approach reduces the program running time of DIALIGN by up to 97%.Herein, a parallel version of the multi-alignment program DIALIGN is introduced. We propose two ways of dividing the program into independent sub-routines that can be run on different processors: (By distributing sub-routines to multiple processors, the running time of DIALIGN can be crucially improved. With these improvements, it is possible to apply the program in large-scale genomics and proteomics projects that were previously beyond its scope. Multiple sequence alignment continues to be an active field of research in Computational Biology and a number of novel approaches have been developed during the last years, see  for an oet al. evaluated the capability of alignment programs to detect conserved non-coding sites in genomic sequences. These authors conclude that DIALIGN can produce alignments with high coverage and sensitivity, as well as specificity to detect constrained sites [DIALIGN  is a vered sites .anchored alignment option [Though DIALIGN produces alignments of high quality, it is slower than alternative multi-alignment programs. Especially if large genomic sequences are to be aligned DIALIGN is far more time-consuming than the above mentioned specialized programs for genomic alignment. A recently introduced t option  can be ut option -21. Herechain of local fragment alignments with maximum total weight score is identified. A fragment or fragment alignment is defined as an un-gapped local pair-wise alignment, and the weight score of such a fragment is calculated based on a P-value i.e. on the probability of its random occurrence, see [For multiple alignment, the DIALIGN algorithm works as follows: in a first step, all respective optimal pair-wise alignments are carried out. This means that, for each pair of input sequences, a nce, see  for a dence, see .N input sequences, N × (N - 1)/2 pair-wise alignments are to be calculated; fragments contained in these pair-wise alignments are then used to build up a multiple alignment in greedy fashion. If the maximum sequence length is bounded by some constant, the time-complexity of this algorithm as a function of the number N of sequences is as follows: Performing all pair-wise alignments takes O(N2) time. During the greedy procedure, O(N) independent fragments can be included into the multiple alignment; additional fragments would be either inconsistent or already contained in the existing multiple alignment. Accepting a single fragment takes O(N2) time since for each accepted fragment, so-called consistency frontiers are to be updated. These frontiers are used to decide if subsequent fragments are consistent with previously accepted fragments. Thus, the worst-case time complexity of our multi-alignment algorithm is O(N3). Test runs with real data show, however, that the real time-complexity is something between quadratic and cubic, see [For a set of bic, see  for a derelative proportion of CPU time used for pair-wise alignments decreases with the number of input sequences, as can be expected from the above theoretical considerations.The current version of DIALIGN uses an efficient algorithm to update the consistency frontiers. This means that, although performing all pair-wise alignments has a lower theoretical time complexity than processing the fragments from these alignments in the greedy algorithm, for realistic data sets most of the CPU time is spent on the pair-wise alignments. For example, for a set of 20 protein sequences with an average length of 367 amino acid residues, as much as 97.4 % of the CPU time is used to perform the 20 × 19/2 = 190 pair-wise alignments. However, the N × (N - 1)/2 pair-wise alignments to the available processors in order to balance the work load. Here, we are using a greedy algorithm to find a satisfactory work-load distribution in reasonable time: we first assign a processor to the pair-wise alignment with the longest expected running time, then assign a processor to the second-largest pairwise alignment etc.The pair-wise alignments that are calculated as the first step of the multi-alignment procedure are completely independent of each other. Thus, it is obvious that the total program running time can be crucially reduced by running these procedures on parallel processors. Here, an important point is to distribute the work load evenly to the different processors in order to minimize the total program running time. To this end, our algorithm first estimates the running time for each pair-wise alignment as a function of the sequence length. As outlined in , the runanchored alignment procedure has recently been implemented [CHAOS to identify a chain of high-scoring local alignments for each pair of input sequences . To select a consistent sub-set of these local alignments, a greedy algorithm is used, see [The quadratic time complexity of the original DI ALIGN algorithm for pair-wise alignment is clearly not efficient enough to align large genomic sequences. To improve the running time of DIALIGN for long sequences, an lemented ,24. For sed, see  for detatrie data structure to identify pairs of segments with a user-defined upper bound on the number of mismatches per segment pair. It defines a local alignment as a chain of such gap-free local alignments that are located within a certain distance from each other. Finally, the algorithm returns an optimal chain of such local alignments. After the consistency check, these alignments are used by our algorithm to define anchor points in order to narrow down the search space for the final pair-wise alignment procedure that is performed by DIALIGN as explained in [CHAOS uses a ained in .x is assigned to a residue y through one of the anchor points, this means that x is the only residue that can be aligned with y in the final output alignment. Whether or not x and y will be aligned depends on the degree of local sequence similarity that DIALIGN detects. Moreover, all residues to the left of x can be aligned only with residues to the left of y and vice versa, see Figure non-anchored alignment, then the result of the anchored alignment procedure will necessarily be the same as for the non-anchored procedure. In particular, this is the case if all anchor points are part of the optimal non-anchored alignment.To be precise, an anchor point is a pair of segments, one segment from each of the two input sequences; this way each position in the first segment is assigned to the respective position in the second segment. If a residue cutting positions to split the input sequences into smaller sub-sequences, and we reduce the program running time for DIALIGN by aligning these sub-sequences independently on multiple processors. This procedure is related to Stoye's well-known divide-and-conquer approach to multiple alignment [In the present study, we use selected anchor points as lignment  and to tlignment .non-additive weighting function w for segment pairs (fragments). If a large fragment f is split into two smaller sub-fragments f1, f2, the sum w(f1) + w(f2) is, in general, lower than the original weight w(f), see [It should be mentioned that, unlike the above outlined anchoring procedure, distributing sub-alignments to multiple processors may well affect the resulting output alignments, even if the selected cut positions are consistent with the optimal alignment. No matter how well our anchoring positions are chosen, we can generally not expect the optimal alignments of our sub-sequences to coincide with the optimal alignment of the original input sequences The reason for this behaviour is that DIALIGN uses a (f), see . As demoTo reduce the total running time of the alignment procedure as far as possible, our algorithm distributes the sub-sequence alignments evenly to the available processors. At the same time, it minimizes the loss of alignment quality that may be caused by splitting the sequences into too many subsequences. To this end, we first identify a chain of anchor points using CHAOS. The program then divides the sequences at every anchor point, thereby producing a large set S1 of pairs of relatively small sub-sequences. The running time for each of the corresponding sub-alignments is estimated as described above. Using these estimates, the sub-sequences in S1 are concatenated in such a way that a set S2 of larger sub-sequences is obtained that can still be evenly distributed to the available processors. Here again, we use a greedy approach to assign processors to sub-alignments. As a result, our algorithm minimizes the program running time by balancing the work load among the processors while it maximizes the length of the aligned sub-sequences, thereby reducing the possible loss of alignment quality.) that is a Linux-SMP cluster with two Pentium III processors (650 MHz) and 1 GB main memory per node. The nodes are connected by a Myrinet 1.28 GBit/s switched LAN. The software was also compiled and tested on a Sun fire with 8 processors and on an ordinary Linux-based workstation pool that generally exists in every institute.We decided to use the message-passing interface (MPI) ,28 for oat al. [genomic sequences has recently been investigated by Pollard et al. [existing approach, we do not evaluate the quality of the produced output alignments; the ability of our software to produce biologically meaningful alignments under various conditions has been evaluated in the above cited papers. Herein, we compare the running time of our parallel software to the original serial version of the program.The performance of existing multi-alignment software has been evaluated in detail. All programs have been extensively tested by their authors; in addition several independent studies have been carried out using numerous sets of real and artificial benchmark data. The quality of multiple-protein-alignment programs including DIALIGN has been systematically studied by Thompson at al.  and by Lat al. . The abid et al. . Since tp of processors. Using 64 processors, the running time for these data sets can be reduced by 94.82%, 90.80% and 75.90%, respectively, compared to the serial running time. The observed differences in the relative speed-up are due to the different proportion of CPU time that is spent on the pairwise alignments, see [As a first test example, we aligned sets of proteins with 20, 55, and 100 sequences, respectively. The program distributed the pair-wise alignments to different processors as described in section. Table nts, see . The larnts, see ). Furthents, see .bp (note that, for each sequence pair, a chain of n anchor points divides the sequences into n + 1 pairs of sub-sequences). These pairs of subsequences were concatenated to obtain a set S2 of larger sub-sequences that can be evenly distributed to the available processors.Next, we looked at the improvement of program running time that can be achieved for large genomic sequences using the algorithm described in section. As a test example, we used a set of three syntenic genomic sequences from mouse, rat and human. Each of these sequences is around 1 MB in length. The program CHAOS identified a total of 15,818 anchor points; 4,294 for human/mouse, 4,072 for human/rat and 7,452 for mouse/rat. Most of these anchor points were consistent with each other, only 121 out of the 15,818 anchor points had to be discarded because of consistency problems [Using CHAOS anchor points, the serial version of our program took 267,574 s = 74 h 19 m 34 s to compute the multiple alignment of our input sequences on a single processor of our cluster. We estimate that, without the anchoring option, the original DIALIGN program would have taken around three weeks to align these sequences. By contrast, the parallel version of the program with 64 processors took only 6,583 s = 1 h 49 m 43 s to align the same sequence set, corresponding to a running time improvement by 97.5 %. With a speed-up from more than three days to less than two hours, DIALIGN can now compute long-range multiple alignments of genomic sequences that were, until recently, far beyond its scope.Göttingen Bioinformatics Compute Server (GO-BICS) at . The program code is available on request.The program will be available online through MS parallelized DIALIGN, installed it on the Kepler cluster, performed the test runs and wrote parts of the manuscript. KN and MK participated in the design of the project and manuscript preparation. BM supervised the project and wrote most of the manuscript. All authors read and approved the final manuscript.


Miscarriage is a common event but is remarkably difficult to measure in epidemiological studies. Few large-scale population-based studies have been conducted in the UK.This was a population-based two-stage postal survey of reproductive histories of adult women living in the United Kingdom in 2001, sampled from the electronic electoral roll. In Stage 1 a short "screening" questionnaire was sent to over 60,000 randomly selected women in order to identify those aged 55 and under who had ever been pregnant or ever attempted to achieve a pregnancy, from whom a brief reproductive history was requested. Stage 2 involved a more lengthy questionnaire requesting detailed information on every pregnancy (and fertility problems), and questions relating to socio-demographic, behavioural and other factors for the most recent pregnancy in order to examine risk factors for miscarriage. Data on stillbirth, multiple birth and maternal age are compared to national data in order to assess response bias.The response rate was 49% for Stage 1 and 73% for the more targeted Stage 2. A total of 26,050 questionnaires were returned in Stage 1. Of the 17,748 women who were eligible on the grounds of age, 27% reported that they had never been pregnant and had never attempted to conceive a child. The remaining 13,035 women reported a total of 30,661 pregnancies. Comparison of key reproductive indicators  with national statistics showed that the data look remarkably similar to the general population.This study has enabled the assembly of a large population-based dataset of women's reproductive histories which appears unbiased compared to the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility. Despite improvements in obstetric care in the UK over the past fifty years, it is estimated that around one in five pregnancies will end in miscarriage  ,2. The pAlthough many large-scale population-based studies of miscarriage risk have been conducted elsewhere -10, relaStudies of miscarriage have tended to be clinical-based, and are thus subject to selection bias. For example, gestations are later among miscarriages reaching hospital-based clinics. Many miscarriages are managed at home, and some are not reported to a clinician. Not only is miscarriage hard to measure, and different clinical sources rarely see the full range of cases, but reported risks of miscarriage tend to be pregnancy-rather than woman-based: estimates of risk tend to relate to the proportion of pregnancies ending in miscarriage, and there are very few studies examining the risk of experiencing one, two or more miscarriages, or the chances of conceiving following a miscarriage . Large pAn increasing number of couples are also seeking help for problems achieving a pregnancy. Although it is estimated that up to 15% couples experience such problems , few popWe now report on a large UK population-based survey of reproductive health, the National Women's Health Study. The study design was developed from several other large epidemiological surveys of reproductive outcome which showed that a postal method could be used to obtain full reproductive histories from large study populations ,14,17,18Eurodirect [This was a population-based cross-sectional postal survey of reproductive histories of adult women living in the United Kingdom in 2001, designed to enable the construction of a retrospective population-based reproductive cohort and a case-control study of risk factors for miscarriage. A sample of women was randomly selected from electronic electoral registers for England, Wales, Scotland and Northern Ireland held by the company rodirect . All UK Eurodirect based on forename, whereby the sampling frame was restricted to women thought likely to be aged 55 and under on the basis of their name. This process was based on empirical data relating to birth certificates going back to the beginning of the 20th century, from which it could be calculated that, for example, those named "Elsie" are likely to be aged over 55, and those named "Kylie" under 55 years. Predictions are further refined by examination of combinations of names within a household (a "Jane" married to or living with an Alfred likely to be older than a "Jane" married to or living with a "Darren") and length of residency (e.g. someone registered to vote at the same address for 12 years has to be over 30). We requested a random sample of 61,000 women likely to be aged 55 and under . After removing those known to be under age 18 at study , the final sample consisted of 60,814 women.In order to reduce possible biases associated with memory, we aimed for a sample aged 55 years and under at survey. Date of birth is not, however, routinely recorded on the electoral register. To avoid unnecessary mailing and expense, we therefore made use of a probabilistic process offered by The study received approval from the Trent Multi-Centre Research Ethics Committee and the Ethics Committee of the London School of Hygiene & Tropical Medicine.The postal survey had two stages. Stage one consisted of a single-page "screening" questionnaire which asked for details of all pregnancies experienced by study participants, as well as periods of infertility and infertility treatment. This form was sent to the whole sample and included "opt-out" boxes to be ticked if the recipient had never been pregnant and had never attempted to have children, and/or was over age 55, and/or did not wish to take part. The second stage of the study consisted of a longer postal questionnaire which was sent to all those responding to Stage 1 who had ever been pregnant or who reported ever attempting to conceive and who agreed to be re-contacted. Excluded from this second stage were women who had had one or more termination for non-medical reasons (i.e. for reasons other than that a defect had been identified in the fetus or that continuing the pregnancy would put the mother at risk) and no other pregnancies. The Stage 2 questionnaire requested more general detail about the women ; detailed information on all pregnancies (including whether the pregnancy was the planned, the result of infertility treatment, father's date of birth and whether father had remained the same); plus socio-demographic and behavioural details relating to the most recent pregnancy. These details included questions relating to weight at start of pregnancy, nausea, smoking, coffee and alcohol consumption, diet, vitamin intake, ill health, air travel, sexual intercourse, occupation and stress levels. The most recent pregnancy was selected to minimise biases related to recall, and since it could be at the start, middle or end of the reproductive careers of these women whose ages at survey ranged from 18 to 55 years potential biases relating to ending reproductive careers on a "success" were not expected to be large. For those whose most recent pregnancy had ended in miscarriage , brief information relating to clinical management of miscarriage and the advice given was also requested. Permission to access clinical notes relating to outcomes reported in the questionnaire, and to contact the women for further study if needed, was also requested. In order to increase the number of cases for the case-control analysis of risk factors for miscarriage, women who had had a miscarriage recently (since 1995) but whose last pregnancy was not a miscarriage were sent a third questionnaire. This was a shortened version of the Stage 2 questionnaire, containing only those questions relating to biological, socio-demographic and behavioural details of the most recent pregnancy, but now requesting these details in relation to the most recent miscarriage. Such women then had two pregnancies in case-control analyses and standard errors were computed using a robust method based on the "sandwich estimate" to account for this statistically.A free telephone helpline was run throughout the study, to answer queries and refer on to other organizations for professional help, if appropriate, and this was well used.all first registered births, if live. No formal statistical comparisons of maternal age were made, partly because the numbers were so large that slight, non-meaningful, nuances in the data would give a statististically significant result, and render the comparison meaningless, and partly because the average ages in the general population, though comparable, were expected to be similar but slightly older in the general population data owing to the fact that the data related to births within marriage only. Births where the date of birth or maternal age were not known were excluded from all comparisons with population data.All analyses in this paper were performed using Stata statistical software . To inveThe response to the first stage of the study is summarised in Table 12,695 women aged under 55 at survey had been pregnant at least once. These 12,695 women, whose average age at survey was 40.5 years, had started their reproductive careers from 1963 to 2002, 75% having their first pregnancy in 1980 or later (Table 11,424 (88%) women ever attempting to have children (successfully or unsuccessfully) agreed to participate in the second stage of the study. Of these 596 (5%) were not sent a Stage 2 questionnaire, 212 because they had only ever had one or more termination of pregnancy for non-medical reasons, and 384 because their Stage 1 form arrived back after mailing had ended. A total of 10,828 women were thus sent a second stage questionnaire. The response to this second stage was high (73%), though 2% of women had decided that they no longer wished to participate  birth was remarkably similar to national data for both Stage 1 and Stage 2 data Table . ExactlyUsing a novel method, the National Women's Health Study has enabled a large UK population-based dataset to be assembled, comprising full reproductive histories, including any history of infertility, for 13,035 women, 12,695 of whom had conceived 30,661 pregnancies. We have obtained further detailed information for 7,702 of these women (18,391 pregnancies), including fertility diagnoses for both male and female partner (if appropriate), and lifestyle and behavioural risk factors for the most recent pregnancy. Seventy-five percent of these women consented to their medical notes being accessed in relation to information reported in the questionnaire, and 90% agreed to be contacted again, thus providing the means to carry out a population-based cohort study of these women at some time in the future.UK population-based data, collected at government level by England & Wales, Scotland and Northern Ireland, relate to registered births (live and still) and terminations of pregnancy, with Scotland also routinely collecting maternity data on hospital deliveries at any gestation. The National Women's Health Study goes one step further than this, providing the whole reproductive picture. Rather than being a pregnancy-based, cross-sectional survey, the data collected for each woman covers the complete spectrum of reproductive outcomes from infertility problems through miscarriage, ectopic pregnancies and terminations , to live and stillbirths, and does not rely on legal definitions for inclusion in the dataset. Furthermore, unlike most epidemiological studies of adverse reproductive outcome such as miscarriage, the data source is not clinical , but relates to women selected randomly from the UK electoral register. And for outcomes such as infertility no other data currently exist to enable estimation of how many pregnancies in the population as a whole result from fertility treatment.The study does rely on maternal recall and this could be a source of bias. Studies of self-reported reproductive history and exposures relating to reproductive events have, however, found maternal recall to have acceptably high reliability, and to be little affected by time from event -26.In terms of the key reproductive indicators of stillbirth, multiple delivery rates and maternal age at first birth, the data look remarkably similar to the general population. We therefore feel confident that response was unlikely to be related to adverse reproductive outcome. Indeed, the average age at survey of around 40 years, coupled with average ages at first birth which are exactly as would be expected from general population data, could be seen to indicate that non-responders to the survey tended to concentrate among younger women who had not yet tested their fertility. In addition, we feel confident that those responding to the more detailed Stage 2 questionnaire are an unbiased sample of those responding to Stage 1. Both Stage 1 and Stage 2 data can thus can be considered unbiased with respect to reproduction, and representative of patterns among all women in the UK population who have ever tried to have children, hence prevalence estimates might be taken as unbiased estimates of hard-to-measure outcomes such as miscarriage and pregnancies conceived through assisted reproduction techniques. Such data will be invaluable as population-based reference data for epidemiological studies of reproduction.In addition to both pregnancy-and woman-based population prevalence estimates, further papers to follow include reports of case-control analyses of behavioural and lifestyle risk factors for miscarriage.In summary, we have assembled a large population-based dataset of women's reproductive histories which appears representative of the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility.None declaredNM and PD initiated the research and participated in protocol design, data collection, analysis and writing the paper. SP participated in data collection and analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Echocardiography has the ability to noninvasively explore hemodynamic variables during pharmacologic or exercise stress test in patients with heart failure. In this review, we detail some important potential applications of stress echocardiography in patients with heart failure. In patients with coronary artery disease and chronic LV dysfunction, dobutamine stress echocardiography is able to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Exercise testing, in combination with echocardiographic monitoring, is a method of obtaining accurate information in the assessment of functional capacity and prognosis. Functional mitral regurgitation is a common finding in patients with dilated and ischaemic cardiomyopathy and stress echocardiography in the form of exercise or pharmacologic protocols can be useful to evaluate the behaviour of mitral regurgitation. It is clinical useful to search the presence of contractile reserve in non ischemic dilated cardiomyopathy such as to screen or monitor the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents. Moreover, in patients with suspected diastolic heart failure and normal systolic function, exercise echocardiography could be able to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Finally, in the aortic stenosis dobutamine echocardiography can distinguish severe from non-severe stenosis in patients with low transvalvular gradients and depressed left ventricular function. The identification of viable hibernating myocardium in patients with coronary artery disease and chronic left ventricular (LV) dysfunction is, up to today, the most common use of stress echocardiography in patients with heart failure. However, to search viable myocardium or the presence of contractile reserve is only one of plugs of the physiopathologic puzzle in a failing heart Figure  and 2. ITherefore, the present review will detail some important potential applications of stress echocardiography in patients with heart failure in the evaluation of the different clinical and physiopathologic aspects of heart failure syndrome.The most common cause of heart failure in the Western world is coronary artery disease, accounting for up to 60% of cases . In patiAfter brief episodes of coronary occlusion and reflow a reversible global LV dysfunction can occur. This phenomenon was called myocardial stunning . It is cHibernating myocardium" was first termed by Rahimtoola to indicate the state of reversible dysfunctional myocardium, which was considered to be the result of a state of persistently impaired myocardial function at rest, caused by reduced coronary blood flow, and which could be partially or completely restored to normal either by improving blood flow or reducing oxygen demand [The term "n demand .Echocardiography can detect viable myocardium during infusion of drugs which have ability to elicit an enhanced contractile response by recruiting contractile proteins. At least two drugs have these proprieties: the dobutamine, a synthetic β1 agonist with additional α1- and β2-stimulating properties and the enoximone that inhibits cyclic adenosine monophosphate-specific phosphosdiesterase ,7. RoutiAn initial evaluation of end diastolic wall thickness of akinetic segments with resting echocardiography can be used as an initial screening technique for assessment of viability. Indeed, akinetic regions with an end diastolic wall thickness <6 mm do not contain viable myocardium and do not improve in function after revascularization . HoweverDuring stress echocardiography is possible to observe four response patterns based on regional wall function: normal, ischemic, viable and necrotic. In the normal response, a segment is normokinetic at rest and normal or hyperkinetic during stress. In the ischemic response, a segment worsens its function during stress from normokinesis to dyssynergy. In the necrotic response, a segment akinesia remains akinetic during stress. In the viability response, a segment with resting dysfunction improves during stress. During pharmacologic stress, a viable response at low dose can be followed by ischemic response at high dose (biphasic response). This biphasic response is suggestive of viability and ischemia, with jeopardized myocardium fed by a critically stenosed coronary artery . A restiregional function after revascularization was 84% and 81% respectively [The overall sensitivity and specificity of dobutamine echocardiography for predicting recovery of ectively . In a stectively . On the Sensitivity of dobutamine echocardiography may be affected by several factors such as the severe reduction of myocardial blood flow that can preclude the contractile response, the premature interruption of dobutamine infusion, resting tachycardia that may renders the myocardium ischemic and dobutamine can only augment ischemia . On the global myocardial function after revascularization. At this purpose there is a relation between improvement in left ventricular ejection fraction (LVEF) and the number of segments with contractile reserve, indicating that extent of jeopardized but viable myocardium determine the magnitude of improvement of LV function after revascularization. Usually a level of ≥ 4 viable segments, which corresponds an improvement in wall motion score index >0.25 (about 20% of left ventricle), is advised as a cutoff value to predict improvement of LVEF [The main clinical issue to search the myocardial viability is that patients with evidence of hibernating myocardium who do not undergo revascularization have poor prognosis with high incidence of cardiac events . In cont of LVEF . Thus, p of LVEF   to calculate the regugitant volume and the effective regurgitant orifice area (EROA), the LV volumes to assess the myocardial contractility and the tricuspid regurgitant jet velocity to measure the sPAP that is an useful index of the haemodynamic burden of MR.First, in symptomatic patients with LV dysfunction and a clinical picture suspicious for new or worsening MR, but not evident at resting echo examination, exercise echocardiography can demonstrate a worsening of MR which helps to correlate this scenario with the patient's symptoms  unmask patients at high risk of poor outcome. The cardiac mortality rate of medically treated patients with dynamic MR during exercise is 39% at 20 months which represents excess mortality in patients in functional class II or III . While in the ischemic cardiomyopathy the search of myocardial viability is focused to find the presence of reversible segmental myocardial dysfunction and its possible effect on global systolic LV recovery after revascularization, in DCM the primary end point is to evaluate the presence of residual global contractile reserve. Both dobutamine and exercise testing have been used in the study patients with DCM, but there is a clear predominance for the use of dobutamine test. The doses of dobutamine utilized vary from investigators, but safety in its use in this population has been documented in doses as high as 40 μg/kg per minute. In the interpretation of results both wall motion score index and the LV volume to derive LVEF must be calculated.LV systolic function at the time of diagnosis has been proposed to be the strongest predictor of survival in DCM, but now the presence of contractile reserve recognised by dobutamine echocardiography seems to be the best marker of good prognosis in patients with severe LV dysfunction at rest ,38. PatiStress echocardiography may also help in the identification of patients in the initial phase of cardiomyopathy overt normal resting echocardiographic parameters. Both dobutamine and exercise have to be used to screen for the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents .The prevalence of diastolic heart failure in the community is now to be at least as high as that reported in previous studies of hospitalised patients; almost half of all patients with heart failure have diastolic heart failure . The terResting echocardiography is most useful in the assessment of LV size, LVEF and the use of Doppler-derived indices of diastolic function has impact on the identification of diastolic dysfunction. However, to determine whether an abnormality of diastolic function is the cause of the patient's symptoms, we need to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Therefore, the stress echocardiography, in particular exercise echocardiography could be useful in dyspnoeic patients with apparently normal LV function to unmask the presence of diastolic dysfunction (signs of elevated LV filling pressure) during exercise as cause of symptoms.Patients with diastolic heart failure, as well as those with diastolic dysfunction and little or no congestion, exhibit exercise intolerance for several reasons. First, an elevated LV diastolic and pulmonary venous pressure during exercise causes reduction in lung compliance, which increases work of breathing and evokes the symptom of dyspnoea . Second,All these aspects can be assessed during exercise echocardiography Table . In partStress echocardiography with dobutamine infusion is particularly useful in clinical decision making in patients with aortic stenosis with LV dysfunction and low transvalvular gradients. In this group of patients, an important clinical question rises: is the low gradient a consequence of low cardiac output due to a severe aortic stenosis which has led to LV dysfunction or is the low gradient a consequence of LV dysfunction is unrelated to aortic stenosis and the aortic stenosis is an incidental finding?It is well known that the transvalvular gradients are flow-depentent parameters so that they are influenced by LV function. The aortic valve area can be accurately determined by Doppler echocardiography with continuity equation and that correlate well with Gorlin formula . HoweverTherefore, the assessment of valve area does not solve the diagnostic dilemma in these patients, because we cannot distinguish between severe fixed from flow-dependent (relative) aortic stenosis. Thus, it is important to perform pharmacological manoeuvres to increase cardiac output so that valve area can be calculated at higher flow rate. Dobutamine stress echocardiography until 20 γ/kg/min with concomitant evaluation of cardiac output, aortic valve area and gradients, is a useful and reliable test to distinguish between severe fixed from relative aortic stenosis in presence of low gradient and LV dysfunction. On the basis on test results, it is possible to distinguish 3 groups of patients  Figure : 1. Pati2 rather than an increase of ≥ 0.3 cm2 from baseline alone [When interpreting the results of a dobutamine study in these patients to rule out or confirm definitively the presence of a severe fixed aortic stenosis, it is advisable to use an absolute cut-off value of the aortic valve area at peak of dobutamine >1 cmne alone ,50.Beyond the identification of viable hibernating myocardium, stress echocardiography is particular useful in patients with systolic and diastolic heart failure to assess the different physiopathologic component of heart failure syndrome and can aid to an appropriate clinical decision making.LV: left ventricularMR: mitral regurgitationLVEF: left ventricular ejection fractionDCM: dilated cardiomyopathyTAPSE: Tricuspid annular plane systolic excursionsPAP: Systolic pulmonary artery pressureThe manuscript is not under consideration elsewhere and the data presented have not been previously published. All authors have read and approved the manuscript. No financial support was received for this study. The content of this manuscript is not associated with any financial interest or other relations that could lead to a conflict of interest.Concerning the authorship, the listed authors have contributed as follows to the manuscript:EA and MP: 1) conception, design, analysis and interpretation of data, 2) drafting of the manuscript and 3) final approval of the manuscriptMO and AM: 1) critical revision of the manuscript for important intellectual content, and 3) final approval of the manuscript.


A couple of years ago, a paper was published in a high-profile journal that challenged a long-established model of cell signaling. While researchers in the field mostly greeted the results with skepticism, some went into the lab to investigate the discrepancy. Many elements of this pathway, called the Wnt pathway, have been well characterized. The standard model of Wnt signaling holds that when the Wnt protein binds to its receptor, it initiates a labyrinthine signaling cascade that sends a protein called β-catenin into the cell's nucleus where, together with a protein complex, it initiates transcription. In the absence of this signal, β-catenin binds to an inactivating complex in the cytoplasm and is targeted for degradation. The paper that disputed this view suggested that β-catenin can effect gene expression without entering the nucleus and that it can activate the Wnt pathway while tethered to the cell membrane.Before that paper was published, Nicholas Tolwinski and Eric Wieschaus had shown that β-catenin, also known as Armadillo (Arm) in the fruitfly, is sent into the nucleus in response to Wnt signaling. Upon entering the nucleus, Arm interacts with a second protein complex to activate transcription. Now Tolwinski and Wieschaus have reexamined the function of Arm in the fruitfly and have demonstrated that the pathway “in fact does depend on the nuclear localization of β-catenin.” While their paper was in the final stages of acceptance, the dissenting paper was retracted, after it was learned that the results had been fabricated. Tolwinski and Wieschaus' findings confirm what had already been known about Arm's role in Wnt signaling and also fill in important details about how it works.Multicellular organisms rely on elaborate communication networks of signaling proteins and enzymes to exchange information between cells. The Wnt signaling pathway regulates the expression of a host of different genes during embryogenesis to control body patterning and cell differentiation in organisms from fruitflies to mammals. Miscommunications in this tightly regulated pathway contribute to a variety of developmental defects and cancers.In the developing fruitfly, Wnt signaling is normally restricted to the front of each larval segment, where it produces a smooth surface; the rear of the segments, where Wnt signaling is absent, is hairy. If Wnt signaling is overexpressed, it produces fruitfly larvae with only smooth segments; lack of Wnt signaling produces hairy segments. Using the smooth phenotype as a measure of Wnt signaling, Tolwinski and Wieschaus delved deeper into the role of Arm in this signaling process.These experiments are complicated because Arm functions not just in Wnt signaling, but also in cell adhesion. The trick is to make the endogenous Arm (the version encoded by the fly genome) defective for signaling, while leaving the cell adhesion functions fairly normal. Set against this “background,” an additional Arm protein is expressed that is tethered to the membrane; it still retains the protein domains required for signaling, but it's stuck on the inside of the membrane and can't move into the nucleus. In this background, any signaling in response to Wnt must mean that Arm can signal to the nucleus without actually having to get inside. Tolwinski and Wieschaus prove—again—that this is not the case. They do this by showing that the weak, medium, and strong endogenous Arm mutants—these classifications reflect the severity of the mutations' effects—have different effects on signaling in the presence of the membrane-tethered Arm.It's clear, they argue, that tethered Arm cannot signal on its own and must somehow be helping the weaker mutants signal. To further investigate how the tethered Arm activates the endogenous mutants, Tolwinski and Wieschaus developed two new and “cleaner” Arm mutants that impair Arm's signaling ability but have no effect on its cell adhesion function. The tethered Arm could not produce a completely smooth phenotype with these nonsignaling endogenous mutants. These experiments, the authors conclude, indicate that the tethered form of Arm produces its transcriptional effects by activating the endogenous Arm protein. Normal activation of the pathway liberates Arm proteins from the inactivating complex, which allows them to enter the nucleus and activate transcription. Tethered Arm appears to accomplish this by sequestering the inactivating complex at the cell membrane, preventing it from interfering with endogenous Arm.Even though Tolwinski and Wieschaus started these experiments based on what turned out to be fabricated results, their investigations produced valuable contributions. They not only reaffirm the standard model of Wnt signaling, but reveal important new insights into the workings of a major player in the pathway.


Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.The HOLA assay, developed for detection of the kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.The method is capable of detecting both the East and West African  Anopheles gambiae, the major vector of malaria in sub-Saharan Africa, threatens to compromise the successful use of insecticide-treated materials [An. gambiae sensu stricto in West Africa [The successful trials of pyrethroid insecticide-treated nets for malaria control in various endemic settings has led to the Roll Back Malaria initiative adopting the approach as one of the cornerstones of its malaria control programmes -3. Howevaterials . Resistat Africa  and has t Africa . Whilst t Africa , there it Africa .kdr allele. This allele encodes a modified voltage-gated sodium channel that has reduced sensitivity to DDT and pyrethroids. Molecular studies identified a single point mutation in the kdr allele that causes an amino acid substitution in domain II of the protein [An. gambiae; the first causes a leucine to phenylalanine amino acid change and has been found in several West African countries [Anopheles mosquitoes is not yet fully understood. However, monitoring its frequency, as a rapid indicator of the development of resistance, should be an integral component of insecticide resistance management programmes.One allele commonly associated with resistance to permethrin is the knock-down resistance or  protein . Two difountries -11, whilountries ,12. The kdr mutations involves a multiplexed PCR technique. Single Nucleotide Polymorphism (SNP) detection is problematic with simple PCR approaches, requiring the use of highly toxic reagents [kdr resistance allele frequencies in An. gambiae, it is broadly applicable where target-site insensitivity is an important mechanism of resistance to insecticides and to chemotherapeutics.The most commonly used method for identifying the reagents  or prohikdr mutation), Kisumu (a susceptible line from Kenya, established in 1953), and Odumasi . Adult females were stored at -20°C before extraction. Field caught specimens were collected using resting catches from Asembo in western Kenya in May 2004, and by pyrethrum spray collections in Odumasi, Ghana in June 2003. Samples were dried over silica gel for later analysis.Specimens were obtained from laboratory colonies of RSP , 0.3 μM each primer (Qiagen), Taq DNA polymerase 0.034 U/μl (Qiagen 201203). Reaction conditions were 94°C for 4 min, 25 cycles of 94°C for 25 sec, 56°C for 20 sec, 72°C for 8 sec; and a final extension step of 72°C for 10 min  was set up for each oligo pair. Four reactions were set up for each PCR sample to test for the East and West resistant alleles and the susceptible allele . The reaction conditions were 95°C for 5 min, 25 cycles of 94°C for 1 min, 58°C for West African kdr detection or 60°C for East African kdr detection for 2 min; with a final hold at 4°C. Ligated products were kept at 4°C in the dark and used as soon as possible for SNP analysis.3 μl of PCR product from the above reaction was used in a hot ligation with Detector and Reporter oligonucleotides (MWG Biotech) Table . Aliquot96-well plates (VWR 402 200 402) were prepared using 100 μl of 5 μg/ml streptavidin (Sigma S4762) per well. The plate was left to dry overnight and then washed 4 times in 250 μl of 1 x PBS with 0.1% v/v Tween 20. Buffer was removed by tapping the plate upside down and 200 μl of blocking solution (1x PBS, 0.1%v/v Tween 20, 2%w/v BSA) added for 1 hour. Four more washes of 250 μl with PBS were carried out before plates were covered with a plastic seal and stored at 4°C for up to one week.20 μl of TNE (10 mM Tris-HCl pH7.5, 1 mM EDTA pH 8.0, 0.2 M NaCl) was added to the hot ligation reaction and then all 40 μl was placed in a well of the streptavidin plate and allowed to incubate at room temperature for 30 min in the dark. The ligation reaction was carefully removed with a multichannel pipette and the plate washed twice in 250 μl of freshly prepared wash buffer 1 (10 mM NaOH, 0.05%v/v Tween 20) and then twice in 250 μl of wash buffer 2 (0.1 M Tris-HCl pH7.5, 0.15 M NaCl, 0.05%v/v Tween 20).40 μl of 75 mU/ml HSP-conjugated antifluorescein Ab (Roche 1 426 346) solution in 1% w/v BSA solution was placed in each well and incubated at room temperature for 30 min. The plate was then washed three times in 250 μl of wash buffer 2. All buffer traces were removed by tapping the plate upside down on a paper towel and 100 μl of room-temperature TMB solution (Roche BM Blue Pod Substrate 1 484 281) added. At least 5 min were allowed for the colour to develop before plates were scored. Plates were read at 680 nm in a Molecular Devices Versa Max plate reader to provide a quantitative method of scoring which could be compared to the visual method of scoring to check reliability.kdr allele. Heterozygotes are easily distinguishable. The protocol presented here for kdr detection is reliable and gives unambiguous results (Table An. gambiae from East Africa compared to the commonly used PCR multiplex approach. The genotype was unambiguously determined by the HOLA technique, whereas the PCR results were more difficult to interpret and often required a repeat reaction (Table kdr allele is rare in the Kenyan population [A schematic of the HOLA approach is given in Figure ts Table . Visual on Table . There won Table . It is bpulation  and so wThe HOLA method allows for over 40 samples to be screened on a single microtitre plate. As shown in Figure An. gambiae mosquitoes to be characterized for the East and West African kdr mutations. Homozygotes and heterozygotes can be easily distinguished using low cost equipment and a simple methodology which makes this technique suitable for use in resource-poor countries. In our hands the method is more reliable than the current multiplex PCR approach, less ambiguous and may be more sensitive for the detection of heterozygotes.The HOLA method allows fresh and stored DNA – Deoxyribonucleic acid.ELISA – Enzyme-linked immunosorbent assayHOLA – Heated oligonucleotide ligation assay.Kdr – Knock down resistance.PCR – Polymerase chain reaction.SNP – Single nucleotide polymorphism.kdr mutation and drafted the manuscript. HR conceived of the study and participated in its design. NPR carried out the multiplex PCR. PJM and EDW helped draft the manuscript. WCB developed the HOLA technique. MJD participated in the design of the study and substantially helped draft the manuscript.AL developed the HOLA method for the


The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting.We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes.Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers. In the post genome sequence era, repetitive sequences, erstwhile considered junk and devoid of function, are increasingly being implicated in many cellular functions, genome organization and diseases -8. Alu rAlus have been shown to harbor a number of regulatory sites like hormone response element (HRE), and a couple of ligand activated transcription factor binding sites -24. ThesAlus originally demonstrated to have non uniform distribution on the chromosomes through banding studies ,34 have Identification and analysis of various permutations and combinations of these regulatory elements in otherwise conserved repetitive Alus are mostly excluded from genetic analysis. Since, Alus occupy a tenth of the human genome, it is imperative to identify those, which might assume function in the proper context. Our primary aim in this analysis is to find out if any bias exists in the distribution of transcriptional regulatory sites in Alus of various evolutionary ages and their distribution with respect to the functional classes of genes.As a first step toward examining the role of these regulatory sites, we mapped their most probable positions on Alus, using in house developed algorithms Figure . This waNearly all the analyzed regulatory sites for RNA polymerase II (RNA pol II) are distributed in the region between A- Box and B-Box with more clustering near the B-Box region Figure . There iin vitro as well as in vivo studies in the 'B' Box that 'G' and 'T' residues at the 1st and 3rd positions respectively are very critical for it's functioning [th position in this case is mutated to "T" in the older families. The Yb8 family that has been reported to be transcriptionally and retropositionally active amongst the younger subfamilies, retains the B'-Box element in a significant fraction. This suggests that even though retropositionally competent younger Alus are hypothesized to be transcriptionally active, only a minority retains consensus B'-Box. It is possible that the enhancing activity of the A Box is sufficient to drive transcription from the weaker B'- Box in the younger subfamilies. Our findings corroborates well with an earlier study in which presence of all subfamilies in the RNA polymerase III driven Alu transcript pool was reported [Majority of Alu retroposition has ceased at least 30 million years ago and only a few Alu subfamilies are still active ,17,41. Tctioning . Our anactioning  for the reported . Additioreported . Additioreported ,49, tranAlus have been demonstrated to exert effects at transcription, post-transcription as well as at the translation level. In an earlier study on complete chromosomes 21 and 22, we have demonstrated that the Alu elements are clustered in genes of signaling, metabolic and transport proteins and rarely present in the structural and information proteins . This clGene inversions, duplications and formation of pseudogenes have been extensively reported to be mediated both through retrotransposition as well as recombination of Alus. This, in many cases, has also been associated with aberrant gene expression. For instance, presence of AML sites in an Alu upstream of MPO gene, has been first demonstrated to be associated with Acute Myelocytic Leukemia . This isAnalysis of regulatory sites within Alus suggests that a polymorphic Alu has the potential to transpose and recombine which allows it to integrate at random sites in the genome. They also harbour potential regulatory sites, which could evolve to become accessory sites for RNA pol II transcription as revealed by their clustering in older subfamilies. Further, the Alu sequence due to acquisition of novel functions could form a part of the transcription repertoire involved in the regulation of the downstream /associated genes and create novel regulatory networks Figure . These rComparison of sequences in the regulatory regions of many homologous genes in human have shown accumulation of Alus, not only post divergence from non-human primates but also during primate evolution . PerhapsCurrently, Alus are repeat-masked in all studies pertaining to identification of predisposition markers in complex disorders. With such wide spectrum of nuclear receptors, which play a major role in maintaining normal physiological state and affect as diverse processes as development, reproduction, general metabolism, residing in Alus, it therefore becomes imperative to screen for variations in these sites. This might have important consequences in the candidate genes for those complex diseases that are triggered in response to hormonal imbalances as well as other environmental cues.126 polymorphic Alu sequences cited in literature ,40 were Information about the regulatory sites and their sequences was collected from various literature sources Table . CharactTwo different programs were written in order to locate the most probable biologically significant regions. A local alignment based program, Xalign, was implemented in C++, Red Hat 7.3 based Linux. This program finds the probable sites by aligning the consensus of regulatory site with the query sequence. Multiple queries with a size upto 600 nucleotides can be taken at a time. Another program, Promotif, was implemented in C++, Red Hat 7.3 based Linux, using the probabilistic modeling approach. It uses the position weight matrix, normalization of the positions with conservation index , and inter-nucleotide dependence in terms of transition matrix to find out the sites. Position weight matrices were generated using Gibbs Motif Sampler, for every site included in the program. The sequences for position weight matrix generation were carefully selected based on the sequence and length reported for each binding site. The final length for search was fixed at the lowest length observed. This provides element specific matrix with lesser chance for the selection on non-RE regions. For the sites analyzed, it had an in built transition matrix, position weight matrix and conservation index. Batch analysis of over a thousand Alu sequences can be performed with this program.Using the annotated sequences from literature as well as from NCBI web page, training set for the probabilistic model was created. Training was done for approximately 70% sequences and rest of the sequences were taken as test set. Details of the program along with the equations used are available on request.About 126 recently integrated Alus from younger subfamilies were searched in the human genome using BLASTn at NCBI server and regulatory sites were mapped in these regions using the programs discussed above.Alus in the promoter regions and intronic regions of functionally classified genes  of chromRS developed the algorithms and programs for identifying regulatory and significant regions, carried out the analysis of distribution of these sites in Alu subfamilies, association analysis and drafted the manuscript. DG was involved in chromosome 22 analyses. SKB participated in the design of the study. MM conceived of the study, participated in its design, analysis, coordination and manuscript preparation. All authors read and approved the final manuscript.The analysis over the promoter and intronic regions has been performed through the data given in the supplementary table file, supplementary table 3_ravishankar et al. Format: .xls. For human chromosome 22, the data contains the accession number, associated Alu family, the respective positions, functional class of the region and further details, for each associated regulatory element found within the Alu repeats in the 5' flanking promoter and intronic regions. The zipped file name is supplementary 1.zip. Details about programs used are on request for academic users.Click here for file


Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies.We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency—a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation—and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets.These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity. State-of-the art analysis of patients' response to melanoma vaccines yields lessons about cancer vaccines and rationale vaccine design in general The immunotherapy of cancer holds promise in harnessing the host immune response to specifically target tumor cells without harming normal tissues. Strategies involve adoptive cellular therapy or active immune induction (commonly referred to as “cancer vaccination”). Cancer vaccines may consist of whole tumor cells or tumor lysates, but identification of tumor-associated antigens (TAAs) over the past decade has made possible the use of specific proteins or peptides as cancer vaccines. The anti-tumor potential of TAA-specific CD8+ T cells has been illustrated by the demonstrated capacity of adoptive T cell therapy to reduce tumor size . While eVarious reasons for the paradoxical coexistence of cancer cells and TAA-specific T cells within patients have been proposed ,19. One While isolated T cell clones with low RE have indeed been generated from melanoma patients following heteroclitic peptide vaccination, the proportion of vaccine-elicited T cell responses these cells represent in vivo is not clear. If predominantly high-RE, tumor-cytolytic T cells are generated, then a small fraction of low-RE T cells generated would be of little consequence. However, if predominantly low-RE T cells are generated, then this low proportion of high-RE T cells may be an important factor in the observed lack of clinical effectiveness of current cancer vaccination strategies. To address this important issue, we undertook a systematic examination of the complexity of T cell responses induced by heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses which develop in some patients. Typically, responses to vaccination are examined following in vitro expansion from patient samples, which may alter the composition of cells and consequently not reveal the proportion of cells in vivo having sufficiently high RE to lyse tumor targets. Although staining with peptide–MHC tetramers provides a direct estimate for the number of TAA-specific T cells present in vivo, and intensity of tetramer staining has been employed as a parameter for isolation of high-RE, tumor-lytic T cells , staininTo analyze and compare T cell responses in melanoma patients on a single-cell level, we generated and examined a large number of cytotoxic T lymphocyte (CTL) clones derived from post-vaccination or endogenous anti-tumor T cell responses. Each clone was analyzed for T cell receptor (TCR) variable chain beta (VB) expression, RE, and ability to lyse melanoma targets. Importantly, these clones were generated directly ex vivo through tetramer-guided sorting, which minimizes the selection bias that could be introduced by prior in vitro expansion. Therefore, data from these clones could be taken to estimate the complexity of the responses in vivo.9/l or more, hemoglobin of 90 g/l or more, and total white blood cell count of 3.0 × 109/l or greater. Tests for human immunodeficiency virus, hepatitis C antibody (Ab), and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 antigen positive by a microcytotoxicity assay. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute  and the Los Angeles County/University of Southern California Institutional Review Board. Analysis of the patient samples was approved by Stanford University's Institutional Review Board. Peripheral blood mononuclear cell (PBMC) samples were isolated from patients after vaccination with the heteroclitic peptides MART 26–35 (27L) (ELAGIGILTV) and gp100 209–217 (210M) (IMDQVPSFV) at the University of Southern California Norris Cancer Center . Clinical-grade peptides used were provided by the Cancer Therapy Evaluation Program of the NCI under an Investigational New Drug application BB 6123 held by the NCI. Immunizations (1 mg of each peptide emulsified with incomplete Freund's adjuvant) were administered every 2 wk for 8 wk, then every 4 wk for 12 wk, and then once 8 wk later. PBMC samples were collected 4 wk after the final immunization and stored at −130 °C. Samples were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration in a solution of 90% Iscove's Modified Dulbecco's Medium (IMDM) and 10% fetal bovine serum (FBS).All patients had resected stage III or IV melanoma, as determined by the 1988 modified American Joint Commission on Cancer staging system. They were required to have a magnetic resonance imaging or computed tomographic scan of the head and computed tomographic scans of the chest, abdomen, and pelvis showing no indication of disease within 4 wk of therapy to verify that they were clinically free of melanoma. Eligibility criteria included age 18 y or older, creatinine of less than 180 μmol/l, bilirubin of less than 110 μmol/l, platelet count of 100 × 10For isolation and detection of peptide-specific T cells, patient PBMC samples were stained and analyzed by fluorescence-activated cell sorting (FACS) as previously described . BrieflyThe HLA-A*0201-positive melanoma lines Malme-3M and A375 and the T2 cell line were purchased fromATCC (Manassas, Virginia, United States) and maintained according to instructions provided by theATCC. The HLA-A*0201-positive melanoma line mel526 was obtained from the Surgery Branch of the NCI. While Malme-3M and mel526 express both MART and gp100, A375 does not express MART or gp100 and served as a negative control. Expression (or lack thereof) of these antigens by each cell line was further confirmed by immunohistochemical staining. Cells were trypsinized using Trypsin/EDTA solution  before use. T2 cells were HLA-A2.1+ and were pulsed prior to assays with peptides indicated in the text.7/ml) in CTL medium.Effector cells, which include clones, cell line, and PBMC samples, were frozen and analyzed in batches. The cells were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration  in 100% EtOH, 100 μl of target cells, 100 μl of effector cells, and 1 μl of CD107-APC Abs. The cells were mixed well using a multichannel pippetor. The plate was centrifuged at 300g for 1 min to pellet cells, then placed into an incubator at 37 °C for 4 h. After the incubation, the plates were centrifuged to 500g to pellet cells, and the supernatant was removed. Cell–cell conjugates were disrupted by washing the cells with PBS supplemented with 0.02% azide and 0.5 mM EDTA, and mixed vigorously, then stained with additional Abs.All assays were done at least twice, with duplicates for each condition. The effector to target (E:T) ratio used was generally 1:2, with 2 × 102 to allow for growth. Potential clones became visible around day 14 and were then transferred to 24-well plates containing 1 ml of CTL medium with 100 units/ml IL-2. Wells were selected based on cell confluency for expansion and further analysis. Clones confirmed to be tetramer-positive were expanded in T-25 flasks containing irradiated JY cells and fresh PBMCs in 25 ml of CTL medium containing PHA. IL-2 was added to a final concentration of 50 units/ml on day 1 and then every 2 d thereafter for 2 wk.CD8+ T cell clones were derived by FACSorting individual tetramer-positive cells from PBMC samples prepared for flow cytometry as described above. CD8+ tetramer-positive T cells were sorted under sterile conditions into 96-well plates, one cell per well, using a FACS Vantage (Becton Dickinson). Wells contained 100 μl of CTL IMDM, with 10% FBS, 2% human AB sera, and penicillin, streptomycin, and L-glutamine, supplemented with 100 units/ml IL-2. Sorted cells were expanded in vitro using standard protocols. Briefly, irradiated feeder cells (JY cells and fresh PBMCs) were added to wells containing the sorted T cells, and the 96-well plates were incubated at 37 °C with 7% CO51Chromium, washed, and resuspended to 105 cells/ml. One hundred microliters of target cells were incubated with 100 μl CTL clones at 10:1 E:T ratio for 4 h. Percent specific release of 51Chromium from target cells was calculated from 40-μl cell-free supernatants.Target cells were as described above under CD107 Mobilization Assays, and were labeled overnight with −7 M and decreasing by log steps to 10−13 M. T cell clones were incubated with T2 targets at 10:1 E:T ratios for 4 h, then chromium release was measured and percentage cytotoxicity calculated by standard methods. Prior to each cytotoxicity assay, clones underwent ficoll-hypaque centrifugation to remove dead feeder cells and were determined to be greater than 80% CD8+ tetramer-positive T cells by FACS. The E:T ratio was based upon live T and target cells. For each T cell clone, percent cytotoxicity was plotted against peptide concentration. The peptide concentration at which the curve crossed 40% cytotoxicity was defined as the RE of that clone [Chromium-labeled T2 targets were pulsed with a range of peptide concentrations, generally starting at 10at clone .g, and incubated 4 h at 37 °C. Percent specific release of 51Chromium was calculated from 40 μl of cell-free supernatant.Cells were isolated directly from PBMCs from patient 422 by FACS as described above. Cells were collected in microfuge tubes containing 1 ml of ice-cold 90% IMDM with 10% FBS. Collected cells were washed and resuspended to 83,300 cells/ml in 90% IMDM with 10% FBS. Targets were prepared as described above and resuspended to 8,300 cells/ml in 90% IMDM with 10% FBS. A total of 2,500 sorted cells (30 μl) and 250 target cells (30 μl) were transferred to a microcentrifuge tube , centrifuged 1 min at 200RNA was extracted from clones and tetramer-positive cells using TRIzol  and reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). PCR was performed using 34 different 5′ primers that specifically amplify all functional TCR VB genes. Most of the 5′ primers used have been previously described . These pA standard software package  was used to provide descriptive statistical plots. Barcharts were provided with standard errors on them. Linear plots were provided with standard errors computed at each point. A linear regression (using least squares) of percent specific lysis on recognition efficiency is shown in To address the complexity of T cell responses against melanoma in vivo, patients with vaccine-induced or endogenous TAA-specific responses were selected. In recent cancer vaccine trials ,4,5, manPatient 422 had the largest detectable TAA-specific CD8+ T cell response (2.5% G209–2M-tetramer-positive) and thus sufficient numbers for examination of lytic function immediately following isolation. To test whether peptide-vaccine-induced T cell responses were functionally active directly ex vivo, T cells isolated by G209–2M-tetramer-guided cell sorting from patient 422 were tested for lysis of peptide-pulsed and melanoma target cells in microcytotoxic assays B. The diTo assess the functional status of the smaller TAA-specific CD8+ T cell responses in the other five patients—which were too small for direct cytotoxicity assays after sorting—we utilized a novel FACS assay for degranulation based on CD107 mobilization . All sixTo substantiate the generality of these findings, we analyzed four additional patients with vaccine-elicited responses. One subject responded to G209–2M only (patient 722), one to M26 only (patient 713), and two to both G209–2M and M26 (patients 721 and 735). Similar to the first four vaccine-elicited patients, these four additional patients  exhibited variable reactivity to melanoma targets, ranging from 13% to 49.6% .To confirm and further investigate the differences in tumor reactivity between endogenous and vaccine-elicited responses, we reasoned that analysis of a set of clonal CTL lines that represented the tetramer-positive population would provide an accurate estimate of the complexity of the TAA-specific T cell response in each patient. A large number of clonal CTL lines (more than 200) were generated by FACS of individual G209–2M- and M26-tetramer-positive cells directly from PBMC samples . Up to 8Peptide specificity and CD8 expression of each clone was confirmed by staining with G209–2M- and M26-tetramers and anti-CD8 monoclonal Ab (data not shown). To obtain an accurate reflection of the total T cell population detected with tetramer in each patient, we decided to rigorously examine at least one representative clone for each subpopulation expressing a different TCR VB . MultiplTo determine the effectiveness of tumor lysis by the different TAA-specific T cell clones that were propagated, clones were analyzed for their ability to lyse melanoma cell lines mel526 and Malme-3M. A375 cells served as a control for antigen-specific killing. In addition, each CTL clone was examined for antigen-specific lysis of T2 cells pulsed with high levels (1μg/ml) of G209–2M or M26 peptides. “Efficient lysis” in these experiments was defined as 40% or greater specific release of radiolabel from the target cells; 10% or less specific release was categorized as “low or no lysis,” and 10% to 40% was termed “intermediate lysis.” All but two of the CTL clones elicited from endogenous anti-tumor responses (from patients 132 and 461) exhibited “efficient lysis” of both the mel526 and Malme-3M melanoma cell lines . In contWe hypothesized that CTL clones that did not efficiently lyse melanoma targets may be incapable of recognizing the relatively low surface densities of native peptide present on tumor cells. CTL clones selected for analysis of tumor lysis were also assessed for RE for the native and heteroclitic peptides via a ten-log range of dilutions. This is illustrated with clones 132.1 and 476.105 A. There 10 value of the peptide concentration required for 40% specific lysis at an E:T ratio of 10:1. For clones 132.1 and 476.105, these scores were 11.1 and 8.3 for assays with G209n peptide  to isolate such cells. CTL clones were selected directly from patient PBMC samples without enrichment in culture to closely reflect the composition of the antigen-specific T cell response in vivo at the time of isolation.3-fold higher concentration of peptide for similar levels of lysis of targets compared to T cell clones that were tumor-lytic. Side-by-side comparison of endogenous responses and vaccine-induced responses suggests that low RE TAA-specific T cell responses may be preferentially driven by heteroclitic peptide vaccination. Thus, high doses of peptide and/or the higher levels of expression of heteroclitic peptide on APCs may induce and actively propagate predominantly T cells with RE too low for recognition of physiological levels of the native peptide present on tumor targets. These data suggest an inverse relationship between antigen density and the RE of T cells elicited. This would be an important consideration in design of future vaccine strategies.Our data revealed that T cell populations induced by vaccination were significantly different from endogenous responses: while some CTLs elicited by vaccination could kill melanoma targets, most were inefficient in tumor cell lysis. In contrast, nearly all clones from endogenous responses were efficient at melanoma cell lysis. This difference was related to RE for the native peptide. Clones that did not lyse tumor cells required up to 10Differential recognition of native and heteroclitic peptides by many T cells may also account for the induction of non-tumor-lytic clones by heteroclitic peptide vaccines, which has been suggested previously ,32. HoweAnother implication of this study is that the number of cells measured by current methods, including ELISPOT or staining with MHC tetramers, may not correlate directly with the RE or tumor reactivity of T cell responses to vaccination. For example, of the nine clones analyzed from patient 517, none were efficient in tumor cell lysis, yet these cells were detectable by MHC tetramer staining. T cells with low RE for native TAA do not efficiently lyse tumor, and therefore are unlikely to have an impact on clinical outcome. Furthermore, it may be possible that low-RE TAA-specific T cells may interfere with elicitation of high-RE T cells, either by direct competition for antigen on APC surface ,34 or doOur data support the notion that not only quantity, but quality, of the T cell response elicited by vaccination may be important for clinical efficacy. There are a number of strategies to increase the magnitude of T cell responses to peptide vaccines. These include using various adjuvants, such as incomplete Freund's adjuvant and immunomodulatory agents, such as IL-12 , GM-CSF In summary, we have demonstrated that vaccination with heteroclitic peptide at high concentrations may drive T cell responses of variable tumor-cytolytic potential in cancer patients—and that the ability to lyse tumor cells correlates with the T cell's RE for native peptides. This represents an important—but not sole—factor in explaining the lack of correlation between immunological and clinical responses after vaccination for cancer. Importantly, the situation is different in endogenous responses, in which cells are predominantly of high RE. This suggests that the manner in which T cells are elicited in vivo are different in these two settings and may underlie their differences in biology.Our immune system protects us against infectious diseases. It can also recognize and destroy early cancer cells before they form tumors. Researchers have been trying to find a way to boost the anti-cancer function of the immune system so that it can kill even established tumors. This is the idea behind developing vaccines for treating cancer—the vaccine alerts and boosts the patient's immune system and so helps to fight the cancer. The idea of enlisting the immune system against cancer has been around for a long time. There have been some spectacular successes, but it has proven difficult to find vaccines that work in more than just a few patients. And we don't yet understand why vaccines seem to work in some patients but not in others.Peter Lee and colleagues are trying to find out why some patients respond to vaccines and others don't by looking at the immune response in vaccinated patients. In this study, using state-of-the art technology, they examined patients who received different vaccines against the skin cancer melanoma. They concentrated on the so-called killer T cells (cytotoxic T cells), which directly attack and kill tumor cells, and analyzed them in great detail.Most cytotoxic T cells produced by patients after vaccination—including vaccination with so-called heteroclitic peptides that had been specifically designed to provoke a very strong immune response—did not kill tumor cells very well, but a few of them did. These results provide some explanation as to why cancer vaccines haven't been as successful as many had hoped, but also suggest that if it were possible to get more of the potent T cells or to expand the ones that are already produced with the current vaccines, there would be a stronger anti-tumor response.How to get effective cancer vaccines remains an open question. But at least technologies such as those used in this study now exist that allow researchers to analyze how the immune systems of different patients react to vaccination and hence can guide the development of better vaccines.http://www.cancerresearch.org/The Cancer Research Institute: http://www.cancerresearch.org/US Food and Drug Administration page on cancer vaccines: http://www.cancer.med.umich.edu/learn/cancervaccines.htmUniversity of Michigan page on cancer vaccines:


This study reinvestigated the recent finding that females – but not males – die in traffic accidents on Friday the 13th more often than on other Fridays (Näyhä S: Traffic deaths and superstition on Friday the 13th. Am J Psychiatry 2002, 159: 2110–2111). The current study used matched setting and injury accident data base that is more numerous than fatality data. If such an effect would be caused by impaired psychic and psychomotor functioning due to more frequent anxiety among women, it should also appear in injury crashes.We used the national Finnish road accident database for 1989–2002. To control seasonal variation, 21 Fridays the 13th were compared in a matched design to previous and following Fridays, excluding all holidays, on number of accidents, male/female responsibility for accidents, and the number of dead, injured and overall number of active participants (drivers, pedestrians and bicyclists) as a consequence of the accident.There were no significant differences in any examined aspect of road injury accidents among the three Fridays, either in females or males. Women were not overrepresented in crashes that occurred on Fridays 13th.There is no consistent evidence for females having more road traffic crashes on Fridays the 13th, based on deaths or road accident statistics. However, this does not imply a non-existent effect of superstition related anxiety on accident risk as no exposure-to-risk data are available. People who are anxious of "Black Friday" may stay home, or at least avoid driving a car. One widely spread superstition is that Friday the 13th brings bad luck. However, the few studies published on human behaviour and its consequences on that day show inconsistent results, whether they be on economic behaviour -6. A recThis interpretation is not without problems. First, although the author repeatedly refers to driving, it should be noted that he also included water and air traffic accidents. Secondly, as the author pointed out himself, his data included passengers killed in accidents, who typically have no control on the task. Impaired psychic and psychomotor functioning due to anxiety, which could indeed be more frequent in females due to their higher neuroticism rate , superstIn spite of Näyhä's fairly conservative conclusion, his results have been widely publicised as evidence that superstitious female drivers die on Fridays the 13th [We reinvestigated the case using the national Finnish road accident data base of injury accidents  for 1989There were 24 Fridays the 13th during the study period. However, three of them were excluded because two were Good Fridays and one followed a Thursday holiday. To control seasonal variation in traffic and weather-type, the remaining 21 Fridays the 13th were compared with the previous Fridays the 6th and the following Fridays the 20th on the number of accidents, male/female responsibility for accidents (police officer judgment), the number of dead, injured and overall number of active participants as a consequence of accident, separately for women and men. Active participants included drivers, bicyclists and pedestrians who actively controlled their motion in traffic and may get involved in crashes. Motor vehicle passengers were excluded. Nine holidays or otherwise unusual control Fridays were replaced by the mean values of the accident variables (e.g. number of accidents, number of injured) from the previous and following years' closest Fridays. For example, Friday the 20th in June 1997 fell on Midsummer holiday eve, and was replaced by mean values gathered from Fridays June 14th, 1996 and June 12th, 1998. This was done to preserve size of the sample. To avoid violating parametric assumptions, the Friedman analysis of variance by ranks  was usedTables 2 = 3.534, df = 2, p = 0.171); in active participants, for females (χ2 = 0.025, df = 2, p = 0.987) or males (χ2 = 0.173, df = 2, p = 0.917); in injured active participants, for females (χ2 = 1.162, df = 2, p = 0.559) or males (χ2 = 0.532, df = 2, p = 0.767); and in dead active participants, for females (χ2 = 2.735, df = 2, p = 0.255) or males (χ2 = 0.448, df = 2, p = 0.799) among three Fridays. To test the Gender × Day interaction, we also computed female/male ratios of active participants for each Friday of each triplet, and applied the Friedman test to check whether this ratio is systematically higher on Fridays 13th, as expected from Näyhä's [2 = 0.400, df = 2, p = 0.819) indicating that, with respect to men, women are not overrepresented in crashes that occur on Fridays 13th. The odds for women being involved in an injury tend to be even somewhat smaller on Friday 13th. There was no such overrepresentation in injured (χ2 = 1.615, df = 2, p = 0.446) or dead (χ2 = 2.1, df = 2, p = 0.350) women among active participants.Comparisons of 21 triplets showed no significant difference in injury accidents (Friedman χ2 = 0.514, df = 2, p = 0.774).Finally, we similarly checked a possible Gender × Day effect in legally responsible crashes (responsibility drawn from police reports), computing female/male ratios of guilty participants for each day and triplet, but did not found any effect(Friedman test, χThis study could not find any indication of overrepresentation of women in injury crashes on Friday the 13th. This is inconsistent with Näyhä's  results It is to be noted that both Näyhä's study  and thisHowever, our data only implies that, in comparison to men, women are not overrepresented in injury road accidents on Fridays 13th in Finland for 1989–2002. We do not and we cannot conclude anything about women's performance in traffic on Fridays 13th, or about their accident risk (given certain exposure to risk), or about the effect of superstition on those risks. For such conclusions, disaggregated individual level data is needed with detailed information of exposure to risk and respective accident outcome. People themselves adjust their exposure to risk at several levels, while making trip decisions, choosing transport mode, or selecting routes to the destination (see the "multiple sieve model" of accident output ). TherefWe conclude that, in the Finnish traffic accident statistics for 1989–2002, females have not incurred more injury  road traffic accidents on Fridays the 13th than expected, as a driver, bicyclist or pedestrian. We suggest that Näyhä's contradicting result on fatalities is due to different sampling, non-optimal setting and chance in a fairly small data. However, this does not imply a non-existent effect on accident risk as no exposure-to-risk data  are avaiThe authors declare that they have no competing interests.Both authors participated in each stage of research and manuscript preparation.The pre-publication history for this paper can be accessed here:


A single, fertilized egg divides into apparently identical daughter cells. As these twins divide again and again, differences emerge among their progeny, establishing segments that will distinguish back from front and head from tail in the growing embryo. Development of segments—and, later, distinct tissues—requires a carefully coordinated square dance of gene expression machinery. Proteins coded by special genes called transcription factors call out the steps by binding to DNA to block or encourage expression of specific genes. With to-the-minute timing, transcription factors call other genes into action to produce the proteins that will determine cell fate. Developing organisms express different transcription factors at specific times and locations to coordinate the changes that make some cells head, others tail; one, a neuron, another, muscle.Drosophila melanogaster. Misregulation of Hairy and related factors is associated with cancer and developmental defects across species. Mutations in the gene that codes Hairy lead to overexpression of genes involved in development, but it is not clear whether Hairy normally represses those genes directly, or through intermediaries. To find the answer, Susan Parkhurst and colleagues at the Fred Hutchinson Cancer Research Center in Seattle, Washington, set out to identify Hairy's direct targets.A transcription factor called Hairy is one of the first activated during segment development in the fruitfly Drosophila cells called Kc cells and in embryos collected at the peak of Hairy expression during Drosophila segmentation. Because they searched approximately half of the expected Drosophila genome, the researchers estimate that they identified roughly half of all Hairy target genes. The list included genes known to act during segmentation, as expected, as well as many others with roles in cell division, growth, and shape.The researchers found a total of 59 genes bound directly by the Hairy protein in cultured Drosophila C-terminal binding protein (dCtBP), and Drosophila Sir2 (dSir2)—may help to set the tempo and timing of Hairy's repression of genes.Of the 59 Hairy targets identified, only one appeared in both Kc cells and embryos. The lack of overlap may reflect a difference in developmental stage between the Kc cells, which are thought to be precursors to neurons, and the relatively undifferentiated embryos, and suggests that Hairy's role changes with context, such as the stage of development or tissue type. But Hairy doesn't act alone. Like most transcription factors, Hairy requires assistant proteins, called cofactors, to do its job. The availability of Hairy's known cofactors—Groucho, Groucho was thought to be Hairy's main assistant, but Parkhurst and colleagues found that only one of the identified Hairy target genes bound Groucho in Kc cells. The majority of Hairy targets overlapped with those of dCtBP, most often in combination with dSir2. All of the cofactors also bound to non-Hairy targets, suggesting that they assist other transcription factors as well.Drosophila genes has grown 2-fold to include almost all of the expected genome. Repetition of these experiments with the expanded gene set could identify most or all of Hairy's target genes.Next, Parkhurst's lab plans to explore the biological functions of the 59 Hairy target genes, to see how they help Hairy coordinate development. Meanwhile, the list of known The current results suggest that Hairy plays roles in segmentation, cell division, and tissue formation that evolve as an organism develops. Differences in cofactor involvement could help regulate Hairy's repression of genes. This paper demonstrates a powerful technique to explore how developing embryos keep gene expression in step.