Thousands of genes in a genomewide data set are tested against some null hypothesis, for detecting differentially expressed genes in microarray experiments. The expected proportion of false positive genes in a set of genes, called the False Discovery Rate (FDR), has been proposed to measure the statistical significance of this set. Various procedures exist for controlling the FDR. However the threshold  is arbitrary and a specific measure associated with each gene would be worthwhile.p-values is available at .Using process intensity estimation methods, we define and give estimates of the local FDR, which may be considered as the probability for a gene to be a false positive. After a global assessment rule controlling the false positive error, the local FDR is a valuable guideline for deciding wether a gene is differentially expressed. The interest of the method is illustrated on three well known data sets. A R routine for computing local FDR estimates from The local FDR associated with each gene measures the probability that it is a false positive. It gives the opportunity to compute the FDR of any given group of clones (of the same gene) or genes pertaining to the same regulation network or the same chromosomic region. Microarrays are part of a new class of biotechnologies that allow the monitoring of the expression level of thousands of genes simultaneously. Among the applications of microarrays, an important task is the identification of differentially expressed genes, i.e genes whose expressions are associated with the status of the patient (treatment/control for example).The biological question of the identification of differentially expressed genes can be restated as a one (for paired data) or two-sample (for unpaired data) hypothesis testing procedure: is the gene differentially expressed between the two situations? However, when thousands of genes in a microarray data set are evaluated simultaneously by fold changes or significance tests approach, multiple testing problems immediately arise and lead to many false positive genes. In this 'one-by-one gene' approach the probability of detecting false positives rises sharply.p-values that guarantees that the FDR is less than a target level α. The same paper demonstrated that the BH procedure is more powerful than the Bonferroni method that controls the familywise error rate.The False Discovery Rate (FDR), is defined as the expected fraction of false rejections among those hypotheses rejected. In their seminal paper Benjamini & Hochberg  providedThe FDR gives an idea of the expected number of false positive hypotheses that a practitioner can expect if the experiment is done an infinite number of time. As usual with expectation, it gives very little information about the number of false discovery hypotheses in a given experiment.t, is arbitrary. Storey and Tibshirani  ≈ 1, for m0 large enough. This value is a lower bound for V) when m0 is unknown.• Let H1 is generally much smaller than under H0.• The variance of the raw local FDR under  where qj is the q-value of gene j. The q-value may thus be viewed as the mean of the local FDR of the genes with p-values lower than Pj.•  is generally a very variable estimator. Moreover the local FDR should increase with the p-value. This is not the case for the raw local FDR. Therefore it is necessary to use a smoothed estimate.i) isThe smoothed local FDR for j = 1, m, computed at position Pi.where  gives a very valuable guideline for the choice of a threshold. One may consider the curve of the local FDR versus the index of the gene ordered by their p-values: a good candidate for the threshold should be a point with a high second order derivative, which corresponds to an abrupt change in the slope of the curve (see the examples of the following section). The second order derivative of the smoothed local FDR can be computed numerically using finite differences.p-values. The following sections demonstrate how the local FDR can be useful using the data of well known experiments.As an interesting application of the local FDR, it is possible to compute the FDR associated with a class of genes or clones by summing up the local FDR estimate of each clone or gene: one may consider for example clones corresponding to the same gene, genes known involved in a given regulatory network, or gene from the same chromosomic region, and associate a FDR with the whole class. These genes do not need to have consecutive Golub  were intp-values based on random permutations of the ALL/AML labels on Welch t-statistics for each gene, Dudoit . The empirical distribution of the high observed p-values (say above 0.5) is far from the uniform distribution. There are several non-exclusive possibilities to explain this: more than 50% of the genes are differentially expressed, the gene results for non-differentially expressed are correlated or there is a technical problem in the random permutations of the Welch t-statistics.If the distribution of the statistics under p-values, we have computed local FDR estimates. The three genes which have been analysed in detail by Storey and Tibshirani . Furthermore, we will assume, as often, that these p-values are independent. However, the independence of the p-values of differentially expressed genes is not required. Consider a multiple testing situation in which m tests are being performed. Let m0 be the number of non differentially expressed genes. Let I(t) be the set of the genes having a p-value lower than t: I(t) = {i : Pi ≤ t} and R(t) = #I(t), its cardinal. LetLet consider a particular experiment. We observed the differential expression of the genes and compute the associated ordered V(t) = #[I(t) ∩ (i ∈ H0)]andS(t) = #[I(t) ∩ (i ∈ H1)].t, the m genes can be classified according to the following 2 × 2 table Using a threshold The Family Wise Error Rate (FWER) is defined to beP [V(t) ≥ 1].FWER = A classical way to control FWER is given by the Bonferroni inequality. This quantity corresponds to the most direct extension from a test hypothesis procedure but can be very restrictive in a multiple testing procedure.Pi is an unobserved value. It is the same framework as point process (see for example , a natural estimate of FDR(t) isLet FDR and R(t): FDR(t) = E. The expectation of V(t) is m0t and R(t) is observed. Therefore, Storey andS = #[I ∩ (i ∈ H1)].t1, t2) is defined as the expected ratio of V  and R:FDR  = FDR(t). So, the natural estimate of FDR is:It is a generalization of the usual FDR: if t1 does not change the proof, so using the same convexity argument as Storey . If we want to estimate the local FDR around the p-value of the gene i, the question can be restated as how to estimate the ratio of the intensities of two processes around a given point Pi.The local FDR is the FDR(R(t) has to be estimated at each value of t. It is possible to consider small windows of size h, or alternatively, to consider windows of different sizes corresponding to a fixed count for R(t). We have chosen the latter solution, for windows of variable size seem more appealing in the particular context.The intensity of process i) be the local FDR around Pi. To estimate FDR(i) we need to define a neighborhood around Pi. Let Vi = V. Remarking that R = 1, we have FDR(i) = E(Vi). FurthermoreLet FDR(E(Vi) = P(Vi = 1)Vi is a binary variable. Thus FDR(i) provides an unbiased estimation of P(Vi = 1), the probability for gene i to be a false positive.since i is:The raw local FDR estimate for gene H0(i) and H0(i - 1) are true and E = m0. Therefore this estimate is unbiased with mean 1.Assume that Using definition (1), it is direct to obtain:q-value of gene j. The q-value may thus be viewed as the mean of the raw local FDR of the genes with p-values lower than Pj.which equals the H0, it is known that the differences between successive ordered values of independent realizations of the uniform distribution have a Beta distribution with parameters 1 and m0 2 (m0 + 2)] ≈ 1, for m0 large enough.Under the hypothesis  Johnson  Chap. 26H1 is generally much smaller than under H0  is a very variable estimator.The variance of estimates (1) under Pi-1, Pi[ is not symmetric. If we consider the neighborhood interval around Pi defined by t1 = (Pi-1 + Pi)/2, t2 = (Pi+1 + Pi)/2 then we obtain another estimate of the local FDR:This fact is well known in point process literature, . MoreoveNote that (2) is a moving average of order 2 of (1). It is well known that estimates provided by moving average (or kernel estimators) are more stable, see .i) byThis smoothing is generally not enough to obtain usable results and we can consider any kind of smoothing. We propose to estimate FDR for j = 1, m, computed at position Pi.where The smoothing method must be suited to the properties of the raw FDR:p-values corresponding to highly differentially expressed genes• its variance is low for low p-values corresponding to non differentially expressed genes• its variance is very high for p-values and large for p-values corresponding high p-values. The lowess smoothing method has a fixed number of neighbor points. Therefore its window size depends of the density of points around the p-value under concern. The density of points is higher for low p-values which in turn implies a shorter window size, which is a good property. However the adaptation of the window size is not sufficient in some cases such as in the Apo-AI example. Moreover the smoothed FDR should be an increasing function of the p-values, a property which is not satisfied by the lowess smoothing. Therefore we prefer to use an ad hoc moving average smoothing using the following algorithm for computing : let 0 <t1 <t2 <t3 be three pre-definite thresholds and m1 <m2 <m3 <m4 four pre-definite integers.Therefore the window of smoothing should be short for low j≤i  <t1 use a moving average of order min• if maxt1 < maxj≤i  <t2 use a moving average of order min• if t2 < maxj≤i  <t3 use a moving average of order min.• if j≤i  >t3 use a moving average of order min.• if maxt1 = 0.01, t2 = 0.05, t3 = 0.2, m1 = 3, m2 = 5, m3 = 15 and  with the constraint that  is not decreasing. This adaptative moving average method is quite empirical. This topic deserve some more work to build a well assessed smoothing method. This is one of our ongoing research project.We have obtained good empirical results on many data sets with Avner Bar-Hen, Jean-Jacques Daudin and Stephane Robin equally contributed to the statistical work and the redaction task. Julie Aubert coded the R-program and analyzed the three data sets.


Gastrointestinal stromal tumors (GIST) constitute a large majority of mesenchymal tumors of the gastrointestinal (GI) tract, which express the c-kit proto-oncogene protein, a cell membrane receptor with tyrosine kinase activity. GI stromal tumors of the stomach are usually associated with bleeding, abdominal pain or a palpable mass.A 75-year-old male presented with upper abdominal pain and palpable mass. Computed tomographic (CT) scan of the abdomen showed a large mass arising in the posterior aspect of fundus, body, and greater curvature of the stomach. Second day after the admission, there was significant reduction in the size of the tumor, clinically as well as radiologically. Endoscopic biopsy showed large bulge in fundus and corpus of the stomach posteriorly with an opening in the posterior part of the corpus, and biopsy from the edge of the opening reveled GIST. Patient underwent curative resection.Spontaneous ruptured of giant gastric stromal tumor is very rare presentation of stomach GIST. Thorough clinical examination and timely investigation can diagnose rare complication. Gastrointestinal stromal tumors (GIST) are the most common form of mesenchymal tumors arising from the gastrointestinal (GI) wall, mesentery, omentum or retroperitoneum that express the c-kit proto-oncogene protein . This exA 75-year-old diabetic male presented with dull upper abdominal pain of one-week duration. He noticed swelling in left upper abdomen. There was no history of vomiting, fever or gastrointestinal bleeding. He had no significant medical or family history and was non-smoker and non-alcoholic. Physical examination showed a 14 × 10 cm mass palpable in epigastrium and left hypochondrium with minimal intrinsic mobility.Routine biochemical investigations were normal. Ultrasonogram and CT-scan of the abdomen showed large heterogeneous mass of 13 × 10 cm extending from the tail of pancreas to anterior pararenal space, lesser sac to gastrosplenic ligament enveloping the posterior aspect of fundus, body and greater curvature Figure . Fluid aGI stromal tumors express c-kit protein also known as CD 117, and is considered as highly specific marker that differentiates GIST from other mesenchymal tumors such as leiomyomas -10. The Assessment of malignant potential of a primary GIST lesion is difficult in many cases; even as small as less than 2 cm size GIST has certain malignant potentials . Currentet al [Imatinib mesylate, competitive inhibitors of certain tyrosine kinases including the intracellular kinases ABL and BCR-ABL fusion proteins present in some leukemia's, and platelet-derived growth factor receptors , is the et al  had repoet al . The optet al . Role ofSpontaneous ruptured of giant gastric stromal tumor is very rare presentation of stomach GIST. Clinical examination and timely investigations can diagnose this rare complication.The author(s) declare that they have no competing interests.RM, AJ: Preparation of manuscriptNR: performed upper endoscopy and biopsy and helped in preparation of draft manuscript.SOV, SS, PD: surgical management, manuscript revision for scientific contentPD, BV: Revision of manuscript and preparation of final manuscript.All authors read and approved the final version of the manuscript.


Recently, tissue engineering has merged with stem cell technology with interest to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes. ES cells have the unlimited capacity to self-renew and maintain their pluripotency in culture. Upon induction of various signals they will then differentiate into distinctive cell types such as neurons, cardiomyocytes and osteoblasts.1, insulin and ascorbic acid were identified as signals that together with BMP-2 induce a chondrocytic phenotype that is characterized by increased expression of cartilage marker genes in a timely co-ordinated fashion. Expression of collagen type IIB and aggrecan, indicative of a fully mature state, continuously ascend until reaching a peak at day 32 of culture to approximately 80-fold over control values. Sox9 and scleraxis, cartilage specific transcription factors, are highly expressed at very early stages and show decreased expression over the time course of EB differentiation. Some smaller proteoglycans, such as decorin and biglycan, are expressed at earlier stages. Overall, proteoglycan biosynthesis is up-regulated 7-fold in response to the supplements added. BMP-2 induced chondrocytes undergo hypertrophy and begin to alter their expression profile towards osteoblasts. Supplying mineralization factors such as β-glycerophosphate and vitamin D3 with the culture medium can facilitate this process. Moreover, gene expression studies show that adipocytes can also differentiate from BMP-2 treated ES cells.We present here that BMP-2 can drive ES cells to the cartilage, osteoblast or adipogenic fate depending on supplementary co-factors. TGFβUltimately, we have found that ES cells can be successfully triggered to differentiate into chondrocyte-like cells, which can further alter their fate to become hypertrophic, and adipocytes. Compared with previous reports using a brief BMP-2 supplementation early in differentiation, prolonged exposure increased chondrogenic output, while supplementation with insulin and ascorbic acid prevented dedifferentiation. These results provide a foundation for the use of ES cells as a potential therapy in joint injury and disease. Articular cartilage is composed of extracellular matrix (ECM), the matrix-secreting chondrocyte and water, which all account for the tissue's characteristic rigidity as well as its flexibility. These features are necessary in order to warrant life-long survival of the cartilage tissue, especially in the joint where it has to endure pressure forces caused by movement. Chondrocytes arise from a mesenchymal progenitor during development, the same progenitor that gives rise to other mesenchymal cell types including osteoblasts, adipocytes and myocytes. Bone formation can either be endochondral, when chondrocytes mature and calcify to provide a matrix for the invading osteoprogenitors, or intramembraneous involving ossification directly from a mesenchymal ancestor. All these diverse cell types may arise from the same precursor, but are distinguished by specific morphological features and with that, a certain set of characteristic proteins including transcription factors that control their differentiation.Two chondrocyte-specific transcription factors have been identified, Sox9, a member of the SOX-family of transcription factors, and Scleraxis, a member of the basic helix-loop-helix transcription factors ,2. Howev1 and BMPs . Green fluorescing chondrocytes in both cultures appeared either organized in clusters or scattered as seen in figure Genetically manipulated ES cells that express GFP under the control of chondrocyte-specific aggrecan promotor were then used to quantify chondrocyte yield using fluorescence-activated cell sorting. ES cells were differentiated along the chondrocytic lineage using BMP-2 at 2 ng/ml or supplement combination G , insulin [1 μg/ml] and ascorbic acid [50 μg/ml] in various combinations on the differentiation of chondrocytes were examined. Medium was changed every second day.Cells of the mouse ES cell line D3  were kept in permanent culture as described ,18 with 2 containing 0.05% Alcian blue for 48 h. Wash steps in 3% acetic acid, 3% acetic acid/25% ethanol and 3% acetic acid/50% ethanol reduced unspecific binding of the dye.Proteoglycans secreted by ES-cell derived chondrocytes were stained with Alcian blue. Cultures were fixed in 2.5% glutaraldehyde, 25 mM sodium acetate, 0.4 M MgClProteoglycan content of differentiated cultures was determined with the DMMB-assay . Proteog1, insulin and ascorbic acid and fixed on day 32 with ice-cold methanol:aceton (7:3) at -20°C for 10 min. For staining with anti-collagen type II  cultures were digested with pepsin for 15 min at 37°C. Staining with anti-adult proteoglycan (Chemicon MAB 2015) was performed after a 1 h digest with chondroitinase ABC at room temperature. Cells were overlaid with the appropriate dilution of the first antibodies in PBS, 10% FCS at 4°C over night. The corresponding secondary antibody, an AlexaFluor 488 goat anti-mouse IgG (H+L), F(ab')2 fragment  was incubated with the cells for 2 h at room temperature. Cultures were observed in a Leica Fluovert FU fluorescent microscope  with an excitation wavelength of 495 and an emission wavelength of 519 nm.Embryoid bodies were differentiated to the chondrocyte lineage with BMP-2, TGFβ® 7700 Sequence Detector. The accumulation of reaction products during PCR was monitored by measuring the increase in fluorescence caused by the binding of SYBR® Green  to double-stranded DNA. Expression analysis of collagen type II A and B was done in the same PCR run with two differently labelled probes specific for either the A isoform (5'-CGAGATCCCCTTCGGAGAGTGCTGT-3'/VIC) or the B isoform (5'-CCAGGATGCCCGAAAATTAGGGCCAA-3'/FAM) [T-values were standardized against GAPDH expression and induction of expression in treated EBs was normalized to control EBs. Primer sequences for murine GAPDH were 5'-GCACAGTCAAGGCCGAGAAT-3' and 5'-GCCTTCTCCATGGTGGTGAA-3' (Ta = 60°C).Total RNA was isolated from 20 EBs per probe using the RNeasy Midi Kit  according to the manufacturer's instructions with on-column DNase I digestion. The amount of RNA was determined using the RiboGreen™ RNA quantitation reagent and kit (Molecular Probes). 275 ng total EB RNA was used as a template for cDNA synthesis with Superscript II  as described  in a totThe GFP expressing plasmid pEGFP-1 (Clontech) under the control of the Aggrecan promotor (kind gift of John R. Matyas) was stably transfected into D3 ES cells using Invitrogen's Effectene system followed by neomycin selection. Genomic integration of the reporter was analyzed by RT-PCR with specific GFP primers (data not shown). ES cells were differentiated along the chondrocyte lineage, trypsinized into a single cell suspension and subjected to fluorescence-activated cell sorting on day 32 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Ten thousand events were registered per sample and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates.Cells were washed with PBS and without any fixation directly overlaid with Oil-red-O working solution (0.18% Oil-Red-O dye/60% propanol) After a staining period of 15 minutes, ES cell cultures were rinsed in distilled water until desired colour was achieved.Data analysis was performed with ONE-WAY ANOVA on n = 3 experiments using SigmaStat version 2.03 .ADD1 Adipocyte determination- and differentiation-dependent factor 1aP2 Fatty acid-binding proteinBMP Bone Morphogenetic ProteinCbfa1 Core binding factor alpha 1C/EBP CCAAT/enhancer-binding proteinCol CollagenDMMB DimethylmethylenblueECM extracellular matrixES embryonic stemEB embryoid bodyFCS Fetal calf serumFGF Fibroblast growth factorGAPDH Glyceraldehyde-3-phosphate dehydrogenaseGLUT4 Glucose transporter 4IGF Insulin-like growth factorPPAR Peroxisome proliferator-activated receptorSCD Steroyl CoA desaturaseSox Sry-related high mobility group boxTGFβ Transforming Growth Factor beta3 Vitamin D3VDNZN carried out cell culture, biochemical and molecular studies and drafted the manuscript. GK, DER and HJA participated in its design and coordination. All authors read and approved the final manuscript.


A paper published over 20 years ago by Susan Iversen and Mortimer Mishkin on reversal learning continues to inform cognitive neuroscience today One of the hallmarks of human nature is our remarkably flexible behaviour, especially in the social domain, which is perhaps also a major reason for our relative evolutionary success. Our social skills are already being honed in childhood and early adolescence, when we quickly become very adept at forming and breaking alliances within and between groups and spend much of our time engaged in complex social interactions. At best, these interactions enrich our society; at worst, they become ‘Machiavellian’ and exploitative. While science might appear removed from such politics, many scientists would probably agree that science is in fact a social enterprise, sharing many characteristics with other human pursuits, and that any claim to greater scientific truth can only be accorded over decades, even centuries.I have always been fascinated by social intelligence, particularly of the ‘Machiavellian’ kind, and found myself wondering at the start of my doctoral research how one might use neuroimaging to study social intelligence in the human brain. I was also interested by the fact that some of this flexible behaviour is shared with other primates such as chimps, bonobos, and even monkeys, who also spend inordinate amounts of time in social interactions, working out social hierarchies. However, it was not immediately obvious how one might go about designing experiments that would address these somewhat intangible issues of social behaviour.Trawling the scientific literature, I came across the concept of reversal learning. While it is obviously important that we can learn arbitrary associations between stimuli and actions, it is also extremely important that we can relatively easily break these associations and learn others. If we learn that choosing a certain object leads to a reward, it would be rather maladaptive to keep choosing this object when it was no longer associated with a reward but, say, a punishment instead. In order to accommodate complex behaviour, we need to be able to adapt or reverse the learning patterns when things change.For a long time, it was thought that complex behaviour depended crucially on the prefrontal cortex of the brain, but it was not clear which parts were important for reversal learning. This was investigated in a classic paper by the eminent neuroscientists Susan This was not the case for monkeys who had had the medial parts of the orbitofrontal cortex lesioned. These monkeys were not completely unaffected by the lesion, but showed moderate impairment on all but the first of the object discrimination reversals. Furthermore, they had moderate difficulty withholding response between trials on an auditory differentiation task. These results strongly suggested a differential role for the lateral and medial parts of the orbitofrontal cortex.Although the paper was not published in a high-profile journal, this elegant and very significant result has had a huge influence on subsequent research. The paper, like many other great papers, was ahead of its time, and it took almost a decade before the citations started to pick up .There was also some evidence that humans with lesions to the orbitofrontal cortex have problems with reversal learning, but the lesions, caused by neurological insult, were not very clean or focal . In addiThese interesting but nonconclusive results in humans spurred us on to use neuroimaging on a modified version of a probabilistic reversal learning task designed by Julia Hornak and John O'Doherty , whose pWe used functional magnetic resonance imaging to show that dissociable activity in the medial orbitofrontal cortex was correlated with the magnitude of the monetary gains received, while activity in the lateral orbitofrontal cortex was correlated with the monetary losses incurred . This diOne way to solve these problems was to use facial expressions rather than money as the reinforcing stimuli. This made sense, given that the key to social intelligence is the ability to detect subtle changes in communication and act upon these changes rapidly as they occur. Such changes in social behaviour are often based on facial expression and come so naturally to humans (and are in place so early in child development) that some might argue that this functionality is essentially innate. However, our human social behaviour is sufficiently flexible that we can easily learn to adapt our behaviour to most facial expressions. For example, other people's neutral expressions do not normally indicate that our behaviour should change, but it is easy to think of social contexts in which a neutral expression does indeed imply that our current behaviour is inappropriate and should change.I designed a reversal task in which the subject's overall goal was to keep track of the mood of two people presented in a pair and, as much as possible, to select the ‘happy’ person, who would then smile. Over time, the person with the ‘happy’ mood (who would smile when selected), changed his/her mood to ‘angry’. This person thus no longer smiled when selected, but instead changed to a facial expression that signalled that he/she should no longer be selected. In the main reversal task, the facial expression used to cue reversal was an angry expression , while in the second, control, version of the reversal task, a neutral expression was used. By using two different reversal tasks in which different facial expressions signalled that behaviour must change, we were able to determine which brain areas were specific to general reversal learning, rather than just to reversal following a particular expression, such as anger.We used functional magnetic resonance imaging to show that the ability to change behaviour based on facial expression is not reflected in the activity of the fusiform face area , but that general reversal learning is specifically correlated with activity in the lateral orbitofrontal and anterior cingulate/paracingulate cortices  .This result confirmed and extended the results from Iversen and Mishkin's original paper. Further confirmation came from the neuropsychological testing, carried out by Julia Hornak on human patients with surgical lesions to the orbitofrontal cortex, which showed that bilateral  lesions to the lateral orbitofrontal cortex produce significant impairments in reversal learning . Yet, asThe Prince, Niccolò Machiavelli offers a rather pessimistic view on human nature, in which ‘love is held by a chain of obligation which, since men are bad, is broken at every opportunity for personal gain’. It may be that our capacity for rapid reversal learning is sometimes used for less than noble pursuits, both in science and in interpersonal relations in general, but we would be in real trouble if we couldn't learn to change.In his masterpiece,


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders It is a great pity when vested interest and dogma dominate what should be a well-informed and rational debate. The essay by In the United Kingdom, we have had an almost continuous debate since the mid 1980s on topics relating to research on early human embryos. I myself have been involved in some of this debate, especially over the last few years, relating to human embryonic stem cells and nuclear transfer. I will not dwell on the political outcomes of this debate, which are widely known, but I want to stress that it has been one that has been very well informed, with contributions from all sides, including many highly respected moral philosophers and bioethicists. These include notable individuals such as Dame Mary Warnock and bodies such as the Nuffield Bioethics Council, who have been especially valuable because of their independence.So why are the conclusions reached by bioethicists in the UK, who are generally supportive of research involving human embryos, different from those of the President's Council on Bioethics? The same scientific information is available on both sides of the Atlantic. The rules of logic are the same. So it has to be the way the information is interpreted or filtered. This implies bias or vested interest or the input of dogma that is based on belief rather than rational thought. Some examples of this are discussed in the Blackburn and Rowley essay, and they are very worrying. The scare mongering about preimplantation genetic diagnosis is ridiculous—simple mathematics shows that it is implausible to use this technique to screen the usual number of embryos obtained in one round of in vitro fertilisation for more than two or three genetic traits, while we know that intelligence must rely on many more. I am a great fan of science fiction, but I can recognise it as such. I worry that some members of the President's Council seem unable to do this. Many of these daft ideas were already promoted in a book by Francis It is certainly very unfortunate if the input of real science in the Council is to be reduced. The scientific issues are complex. For example, we certainly do not know nearly enough about either adult or embryonic stem cells to say which will be the best for therapies, and of course it is possible that both will turn out to be useful for different problems. Both also offer exciting new ways to explore human disease and the influence of genetics and environment without having to rely on human experimentation. But any committee looking into what is ethically acceptable has to be provided with a balanced view of what will be possible in the near future. There is no point in being too speculative, in part because it is also difficult to predict what will be ethically acceptable in the future. If cures come from the use of human embryonic stem cells, then I suspect that there will be widespread acceptance, as happened with heart transplants and with in vitro fertilisation, both of which were initially greeted with horror by many.It is impossible to have an informed debate without accurate and appropriate information, and there seems little point in having a debate that is not informed. Because of various sensitivities, it seemed to me before the creation of the President's Council on Bioethics that for far too long the issues relating to embryo research had not been considered properly within the United States. The President's Council was therefore an opportunity to redress this situation. But from the evidence I fear it will not succeed. Moreover, it does the general public a disservice to pretend to have a serious committee exploring issues of bioethics when that committee fails to live up to the ideals of impartiality and rationality.


Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem. The precise identification of HLA Class I and Class II alleles is critical for successful hematopoietic progenitor transplants, the development of peptide based viral and cancer vaccines, and investigating immune response ,2. DNA sThere exist two main types of ambiguous typing results obtained with SBT. The first is when a heterozygous sequence can be explained by more than one possible pair of alleles within the region analyzed. The second exists when alleles are defined by a polymorphism outside the region analyzed. In addition to these two situations, a third type of ambiguity arises when an allele has an incomplete sequence in the region analyzed.The prevalence of ambiguities in HLA typing relates to the nature of polymorphisms which exists in the sequence of the major histocompatibility complex (MHC) Class I and Class II genes. The majority of polymorphisms that distinguish one MHC allele from another are oftentimes due to gene conversion, recombination and exon shuffling events. Due to this, polymorphic motifs at given positions are generally shared among several alleles.Sequence-based typing involves PCR amplification and sequencing of specific HLA exons, which are known to be polymorphic, from genomic DNA. For each HLA locus both alleles are amplified and sequenced; therefore, it is not always possible to determine exactly which two alleles were responsible for sequence results. For example two or more different allele combinations can combine to produce identical sequences due to the heterozygous base pair combinations, the first type of ambiguity. More specifically, in the Class I region, HLA-B*070201, 3503 would have the same nucleotide sequence as HLA-B*0724, 3533 in positions 559 and 560 . However, for investigations of genetic inheritance or disease association, the definition of all polymorphisms may be significant.The purpose of this study was to summarize the incidence, nature, and cause of ambiguous HLA SBT results. This represents an important step toward developing strategies to reduce or eliminate this problem.® isolation kit . The DNA was resuspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing.Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE® 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 {ET}.The primary PCR amplification reaction consists of a 1.5 kb reaction encompassing exon 1 through intron 3 of the HLA region. All reagents necessary for primary amplification and sequencing are supplied in the HLA-A or HLA-B AlleleSEQR Sequenced Based Typing Kits . The primary amplification PCR products were purified from excess primers, dNTPs, and genomic DNA using ExoSAP-IT  Each template was sequenced in the forward and reverse sequence orientation for exon 2 and Exon 3 according to protocols supplied with the SBT kit. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide  and analyzed on the ABI PrismSequence based typing analysis of HLA-A and B alleles was performed on a population of 676 normal donors. The racial distribution of the subjects studied was: 615 Caucasian, 13 Asian, 23 African American, 17 Hispanic and 8 Unknown. 672 of the 676 subjects were analyzed for the presence of HLA-A locus ambiguities and 666 were analyzed for HLA-B locus ambiguities. Each allele was counted separately in this analysis in order to determine the total percentage of ambiguous allele combinations. Four new potential alleles were found.At the HLA-A locus a total of 548 ambiguous allele combinations were found. This represented 41% of all HLA-A alleles 548 of 1344)  of the HLA-A ambiguities cannot be resolved with Exon 4 analysis Table .Review of the HLA-B locus results revealed 322 ambiguous allele combinations among the 1332 total HLA-B alleles 24%). Antigens HLA-B*07/08/15/27/35/44, common in Caucasians, produced the largest portion of the ambiguities (279 of 322 or 87%). Each of these ambiguities had an independent reason for occurring. Table %. Antige maintains an updated listing of all ambiguous possibilities [While SBT provides the best available typing of HLA-A and B antigens, it is limited by sequence results that don't allow the precise identification of alleles. We found that 41% of HLA-A alleles and 24% of HLA-B alleles were ambiguously typed. The ambiguities involve some of the most frequent HLA-A and HLA-B antigens: A*01, A*02, A*24, B*07, B*08, B*15, B*27, B*35, and B*44. However, ambiguous allele combinations occur in all loci tested in HLA. The IMGT/HLA Sequence Database The need to initiate additional testing to clarify ambiguous allele combinations must consider whether it is practical to obtain the information and if the information is useful and valuable. The clinical need for the highest resolution HLA typing possible is an important variable that must be considered. When typing is performed for cancer and viral vaccine development studies, high resolution allele data may be necessary to determine if a subject has an HLA type that is appropriate for a study. Utilization of high resolution data may also have implications for hematopoietic progenitor cell transplantation. Transplants involving partially mismatched or unrelated donor-recipient pairs require a higher resolution typing, but those involving HLA identical siblings may not.If it is necessary to resolve an ambiguous typing, a variety of different methods can be used. If the ambiguity is due to an allele that has not been completely sequenced or because the ambiguity is outside the region amplified by the SBT assay, the resolution is dependent on the nature and complexity of the ambiguity. Traditionally, for Class I sequencing purposes most laboratories have performed Exon 2 and Exon 3 analysis alone and for Class II sequencing only Exon 2 analysis. Many of the ambiguities can be resolved by sequencing Exon 4. In fact, in this study the largest portion of the typing ambiguities can be resolved by sequencing exon 4. However, many polymorphisms in exon 4 have no functional significance, so it may not be worthwhile resolving most ambiguities involving exon 4. The requirement for the analysis of Exon 4 to reduce the incidence of typing ambiguity has now been realized by commercial kit manufacturers. Both Celera Diagnostics  and Forensic Analytical/Atria Genetics  now include reagents for analysis of exon 4 in the HLA-A and -B kits. As the use of SBT increases, more data may become available from non-traditional exons in addition to those that have been traditionally sequenced and the number of ambiguities due to unknown sequences will decrease.If the ambiguity is due to an identical heterozygote sequence, as shown in figure Other methods which have been utilized to produce a homologous sequence include cloning, reference strand conformational analysis (RSCA), Pyrosequencing™ and denaturing high-performance liquid chromatography (DHPLC) -11. PyroIn conclusion, although the prevalence of ambiguous allele combinations is high, methods exist to compensate for this problem. As the HLA field continues the discovery of new alleles, alternative approaches to discerning ambiguous allele combinations will need to be investigated in order to reduce the ever-growing number of ambiguities.


If you're an evolutionary biologist, the tired old saw, “You can tell a lot about a person by the company they keep,” represents a fresh new approach to a longstanding problem. Especially if the company in question is a parasite—say, for example, lice—and the problem is tracing the path of human evolution.Homo sapiens interbred with archaic humans. While genetic data have provided insight into recent human evolution, fossils remain the only available evidence for many archaic human species—and the fossil record is notoriously spotty, leaving the data open to multiple interpretations. Two prominent models and a subset of variants have emerged, differing mainly on the question of gene flow: one asserts that modern humans emerged from an archaic ancestor in Africa about 130,000 years ago and then replaced archaic forms in Africa, Asia, and Europe with no gene flow between them; the other proposes gene flow between modern human populations as well as interbreeding between modern and archaic forms in different parts of the world. Both models find support in the available data, but neither can claim a perfect fit with all the data, leaving the possibility of interbreeding an open question.One of the hottest debates in the study of human origins centers around whether modern Faced with a relative paucity of human fossil and genetic data, scientists have been forced to rely on other data sources. Mounting evidence suggests that parasites with an established coevolutionary history with their hosts can serve as a proxy for host evolutionary history, an especially handy tool in the event of insufficient host data. Following this approach in a new study, David Reed and colleagues circumvent the lack of human data by analyzing the next best thing: head lice.As host-specific, obligate parasites—that is, occurring on a single species and not able to survive off that host—lice require direct physical contact between hosts for transmission. As human parasites, lice harbor in their genetic sequence hints of the slings and arrows of evolutionary fortune  that strike their host. Recent studies of the evolutionary history of other human parasites , for example, fall in line with fossil and genetic data that place our origins in Africa.Pediculus humanus) and pubic (Pthirus pubis). Head and body lice obviously occupy different habitats, but are not genetically distinct. Interestingly, P. humanus contains two ancient lineages, offering the opportunity to shed light on this murky period in human evolution. To do this, Reed and colleagues had to reconstruct the evolutionary history of P. humanus, which they did using morphological and genetic analyses of this and other species of lice. They confirmed that P. humanus comprises two lineages—one contains both head and body forms and has worldwide distribution; the other contains only the head louse and is restricted to the New World—but discovered that P. humanus originated long before its H. sapiens host.Two louse species parasitize humans, head/body  of a single host. More likely, the New World louse evolved on an archaic form of humans and then cast its lot with a modern version.H. sapiens and H. neanderthalensis was too recent  to support a concurrent split between the worldwide and New World lice lineages, the split between H. sapiens and H. erectus (about 1.8 million years ago) could. Reed and colleagues propose a scenario in which H. sapiens and H. erectus carried distinct types of lice owing to a million years or so of isolation. As the first waves of modern humans left Africa about 100,000 years ago and modern humans replaced archaic forms, the two forms engaged in enough contact—whether in the form of fighting, swapping clothes, or interbreeding—for archaic lice to make the switch to modern human hosts. Tackling the question of interbreeding, the authors suggest, might best be pursued by studying P. pubis, which requires sexual contact for transmission.While the split between


Caenorhabditis elegans.An mRNA-tagging method was used to selectively isolate mRNA from a small number of cells for subsequent cDNA microarray analysis. The approach was used to identify genes specifically expressed in ciliated sensory neurons of  Caenorhabditis elegans using the mRNA-tagging method, in which poly(A) RNA was co-immunoprecipitated with an epitope-tagged poly(A)-binding protein specifically expressed in sensory neurons. Subsequent cDNA microarray analyses led to the identification of a panel of sensory neuron-expressed genes.It is not always easy to apply microarray technology to small numbers of cells because of the difficulty in selectively isolating mRNA from such cells. We report here the preparation of mRNA from ciliated sensory neurons of  Recent advances in technologies for analyzing whole-genome gene-expression patterns have provided a wealth of information on the complex transcriptional regulatory networks and changes in gene-expression patterns that are related to phenotypic changes caused by environmental stimuli or genetic alterations. Changes in gene expression are also fundamental during development and cellular differentiation, and differences in gene expression lead to different cell fates and eventually determine the structural and functional characteristics of each cell type. Comparative analyses of gene-expression patterns in various cell types will therefore provide a framework for understanding the molecular architecture of these cells as cellular systems.Caenorhabditis elegans is an ideal model organism for investigating development and differentiation at high resolution, because adult hermaphrodites only have 959 somatic nuclei, whose cell lineages are all known. About 19,000 genes were identified by determination of the C. elegans genome sequence ; JN502: Is [acr-5p::flag-pab-1 pRF4]; and JN503: Is [myo-3p::flag-pab-1 pRF4].The initiation codon of a cDNA for of che-2 , acr-5 [2 [acr-5  or myo-35 [myo-3  was insea marker . Stable a marker . Each inin vivo, worms were treated with 1% formaldehyde in M9 for 15 min at 20°C with gentle agitation. The formaldehyde was then inactivated by 125 mM glycine for 5 min at 20°C and washed out by replacing the buffer with four changes of TBS . At this point, worms were dispensed into 0.4 g aliquots, placed in 2-ml microtubes and stored frozen until lysate preparation.To purify poly(A)-RNA/FLAG-PAB-1 complexes from subsets of neurons, we modified a protocol for chromosome immunoprecipitation . Transgeg for 20 min. Five hundred microliters of supernatant, with the protein concentration roughly adjusted to 20 mg/ml, was incubated with 50 μl of anti-FLAG M2 affinity gel beads (Sigma) for 2 h. The affinity beads were sequentially washed three times with lysis buffer supplemented with PMSF, twice with wash buffer  and once with TE . Lysate preparation and purification of RNA-protein complexes were performed at 4°C. Precipitated materials were eluted with 100 μl elution buffer  by incubation for 5 min at 65°C. Elution was repeated and the two supernatant fractions were combined. The eluted RNA/FLAG-PAB-1 complexes were incubated for 6 h at 65°C to reverse the formaldehyde crosslinks. Proteins were digested with proteinase K and removed by phenol-chloroform extraction. Nucleic acid was recovered by ethanol precipitation. Typically, 100 ng nucleic acid was obtained from 0.5 ml cleared lysate of myo-3::PABP. Under the above washing conditions, binding of free poly(A) RNA to PABP was severely impaired (data not shown).Worms were resuspended in 0.45 ml lysis buffer , 1 mM DTT, 0.2 mM PMSF, protease inhibitor cocktail  at the recommended concentration) supplemented with 20 mM ribonucleoside vanadyl complexes  and 1000 U/ml of human placental ribonuclease inhibitor (Takara). Animals were disrupted by vigorous shaking with 2 g acid-washed glass beads (Sigma), and worm debris was removed by centrifugation at 18,000 che-2::PABP worms at L4 to young adult were observed. Cells were identified by their positions and the percentage of stained cells was scored. Responses of the che-2::PABP strain to 1-octanol was assessed as described [For staining of living animals with lipophilic dye, we followed the procedure described before  except tescribed  except teft-3 as a reference, and the results of three independent experiments were averaged. The primers used for the amplification of each gene were: lmn1-52: 5'-CGTTCACCACCCACCAGAA-3' and lmn1-32: 5'-CAAGACGAGCTGATGGGTTATCT-3' for lmn-1; eft3-52: 5'-ATTGCCACACCGCTCACA-3' and eft3-32: 5'-CCGGTACGACGGTCAACCT-3' for eft-3; tax2-54: 5'-GATTAATCCAAGACAAGTTCCTAAATTGAT-3' and tax2-34: 5'-TTCAATTCTTGAACTCCTTTGTTTTC-3' for tax-2; unc8-52: 5'-TCTCAGATTTTGGAGGTAATATTGGA-3', and unc8-32: 5'-GATCTCGCAGAAAAGTTCTGCAA-3' for unc-8; unc54-52: 5'-AACAGAAGTTGAAGACCCAGAAGAA-3', and unc54-32: 5'-TGGTGGGTGAGTTGCTTGTACT-3' for unc-54; snt1-51: 5'-GAGCTGAGGCATTGGATGGA-3' and snt1-31: 5'-CCAAGTGTATGCCATTGAGCAA-3' for snt-1; acr5-52: 5'-AATCGATTTATGGACAGAATTTGGA-3' and acr5-32: 5'-ATGTTGCAAAAGAAGTGGGTCTAGA-3' for acr-5; odr10-51: 5'-TCATTGTGTTTTGCTCATTTCTGTAC-3' and odr10-31: 5'-ATATTGTTCTTCGGAAATCACGAAT-3' for odr-10; del1-51: 5'-TAAACTGCCTCACGACAGAAG-3' and del1-31: 5'-GCCATCAAGTTGAACCAAGAAT-3' for del-1. All primers were designed to include one intron in the PCR product amplified from the genomic DNA for each gene, such that the length and melting point were different from the product amplified from the cDNA. In Figure eft-3 was amplified for 25 cycles, lmn-1, snt-1 and unc-54 for 30 cycles and tax-2, unc-8, odr-10, del-1 and acr-5 for 35 cycles. Amplified DNA was visualized by electrophoresis followed by staining with ethidium bromide.Fifty nanograms of RNA was converted to cDNA using an RNA PCR Kit (AMV) Ver. 2.1 (Takara) according to the manufacturer's protocol. One-tenth of the cDNA from each sample was subjected to a gene-specific PCR reaction in a total volume of 20 μl. Quantification of the PCR products was performed using a FastStart DNA Master SYBR Green I Kit (Roche) with the Light Cycler system (Roche). Serial dilutions of cDNA prepared from poly(A) RNA of wild-type worms were used to generate a standard curve. The ratio of expression levels for each gene was calculated using the amount of Microarrays were prepared using a 16-pin arrayer constructed according to the format of Patrick Brown (Stanford University ) on CMT-A genomic DNA fragment for each gene was amplified by PCR such that it contained an upstream promoter region followed by a partial or full-length predicted coding region. The 3' PCR primers were designed to introduce a restriction site in-frame with GFP in the vectors. The 5' PCR primers were designed to anneal to a sequence 0.6-5.0 kilobases (kb) upstream of the predicted coding region of each gene. The upstream-predicted gene was essentially not included in the promoter fragment. The amplified PCR fragments were cloned into pPD95.70, pPD95.75 (donated by A. Fire) or a Gateway vector (Invitrogen) to create GFP fusions . The resulting reporter plasmid for each gene was introduced into wild-type animals. Transgenic worms at all developmental stages were observed under a differential interference contrast (DIC)-fluorescence microscope. Cells were identified according to their positions  by compaC. elegans genome using BLAT [C. elegans was obtained from the Gene Ontology Consortium [cDNA clones were mapped to the ing BLAT  and BLASing BLAT  and protnsortium .et al. [et al. observed that X -boxes were present about 100 bp upstream of the initiation codons [For Figure et al. . Only ben codons .Caenorhabditis briggsae, whose draft genome sequence has recently been released, was downloaded from WormBase [Drosophila melanogaster, Schizosaccharomyces pombe and Saccharomyces cerevisiae were obtained from NCBI [C. elegans gene.The proteome data set for WormBase . Proteomrom NCBI , and BLA2(che-2::PABP/acr-5::PABP) value for all genes that carried the motif was calculated. To avoid artifactual results due to gene families with close sequence similarities (such as major sperm proteins), groups of genes whose mRNA are expected to cross-hybridize were treated as a single imaginary gene with the average log2(che-2::PABP/acr-5::PABP) value.Protein motifs that preferentially appeared in genes at the higher rank orders were searched for as follows. For all genes represented in our microarrays, the protein motifs contained in each gene product were obtained from the AcePerl server. For each motif, deviation of the average logThe following additional data are available with the online version of this article. Additional data file a table listing the results of the microarray experimentsClick here for additional data fileGenes expressed in sensory neurons, motor neurons, muscles and the intestine, and those with X-boxes shown in Figure 3Click here for additional data fileThe primers and vectors used for reporter constructionsClick here for additional data fileThe legends to the above three tablesClick here for additional data file


Austin Bradford Hill's landmark 1965 paper contains several important lessons for the current conduct of epidemiology. Unfortunately, it is almost exclusively cited as the source of the "Bradford-Hill criteria" for inferring causation when association is observed, despite Hill's explicit statement that cause-effect decisions cannot be based on a set of rules. Overlooked are Hill's important lessons about how to make decisions based on epidemiologic evidence. He advised epidemiologists to avoid over-emphasizing statistical significance testing, given the observation that systematic error is often greater than random error. His compelling and intuitive examples point out the need to consider costs and benefits when making decisions about health-promoting interventions. These lessons, which offer ways to dramatically increase the contribution of health science to decision making, are as needed today as they were when Hill presented them. One of the most cited papers in health research is Austin Bradford Hill's "The Environment and Disease: Association or Causation?" , Hill's Hill outlined a systematic approach for using scientific judgment to infer causation from statistical associations observed in epidemiologic data, listing nine issues to be considered when judging whether an observed association is a causal relationship. Despite widely distributed and clearly elaborated advice to the contrary , Hill's Hill never used the term "criteria" and he explicitly stated that he did not believe any hard-and-fast rules of evidence could be laid down, emphasizing that his nine "viewpoints" (p. 299) Causal criteria are an intriguing subject for the history of science, including the question of why Hill's list seems more popular than others -10 and wThis brief analysis of Hill's "criteria" and what has been made of them can add little new to that topic . Our purpose is to call attention to the seldom-cited last page and a half of the article, which presents lessons that remain overlooked today.t table diverts attention from the inadequacies of the fare" [Hill eloquently warned about overemphasis on statistical significance testing, writing "the glitter of the he fare" (p. 299).he fare" , and litOne implication of Hill's advice is well understood. Emphasis on the p-value  has been soundly denounced for decades ,17. EstiA further inadequacy of the fare is less well appreciated, stemming not from the question of p-values versus confidence intervals, but from systematic errors. No statistical test of random sampling error informs us about the possible impacts of measurement error, confounding, and selection bias. Methods for quantifying such errors  have been developed in epidemiology, particularly over the last five years -25. HillEven as modern epidemiologic analysts become less dazzled by the t-table, replacing significance testing with confidence intervals and introducing quantification of systematic errors, there is still a tendency to completely overlook Hill's other important insight. Hill sought to address the question how to decide whether to take action once causal inferences are made. In his last few paragraphs, he offers an important commentary on the policy recommendations that flow from decisions regarding cause and effect in epidemiology. Since "our object is usually to take action" (p. 300),Even when policy implications are presented explicitly, they are seldom carefully analyzed. Analyzing the implications of a health research finding for decision making is often not terribly difficult, but making recommendations without such analysis can lead to absurd suggestions . One episome health hazard, and act based on the expected gains and losses, with or without statistical certainty. Hill, who was educated in economics, argued that in order to take policy action, we ought to pay attention to the absolute costs and benefits of potential actions. It would clearly be reading too much into the text to suggest that he had a prescient vision of modern probability-weighted cost and benefit based policy analyses and decision theory (those fields were in their early stages at the time of his writing and he never used any of those terms). But, in another memorable phrase, he did make the case for having "differential standards before we convict" (p. 300),Hill points this out (in an example sufficiently ill-chosen that it may have contributed to his important message being ignored): "On relatively slight evidence we might decide to restrict the use of a drug for early-morning sickness in pregnant women. If we are wrong in deducing causation from association no great harm will be done. The good lady and the pharmaceutical industry will doubtless survive (p. 300).Setting aside the impolitic dismissal of women's preferences and the unsupported assertion that there is no great harm at stake  the underlying point might be his most important lesson: Policy actions that appear to create a net benefit  should be taken, even without statistical "proof" of an association, while actions that entail great costs should only be taken with sufficient certainty of substantial benefit.Hill goes on to strengthen his argument: "On fair evidence we might take action on what appears to be an occupational hazard, e.g. we might change from a probably carcinogenic oil to a noncarcinogenic oil in a limited environment and without too much injustice if we are wrong. But we should need very strong evidence before we made people burn a fuel in their homes that they do not like or stop smoking the cigarettes and eating the fats and sugar that they do like (p.300)."Hill clearly stated that the science and data analysis should not be influenced by what is at stake. But health researchers should recognize that the stakes matter, and incorporate a consideration of them into their work. The alternative to carrying out the policy analysis is to leave the weighing of costs and benefits to an unreliable post-science political process.The observation that the costs and benefits matter, despite being rather obvious, is frequently – indeed, typically – overlooked in public health discussions. The popular decongestant phenylpropanolamine was banned on weak evidence without regard to the high cost to consumers ; dietaryMaking a good decision does not depend on having studies with confidence intervals that exclude the null. A best decision can be based on whatever information we have now, and indeed a decision will be made – after all, the decision to maintain the status quo is still a decision ,38. HillThe pursuit of the low p-value  leaves our society postponing apparently useful policy choices while we do more research to try to show what we already believe to be true. It also creates the incentive to use dubious methods  in ordeThis would release us from the trap of letting ignorance trump knowledge. Regulators often fail to act because we have not yet statistically "proven" an association between an exposure and a disease, even when there is enough evidence to strongly suspect a causal relationship. There is a growing movement to escape this mistake by making a similar mistake in the other direction: adopting precautionary principles, which typically call for restrictions until we have "proven" lack of causal association – a decision based on ignorance that merely reverses the default. If we can escape from the false dichotomy of "proven vs. not proven," facilitated by the nonexistant bright line implied by statistical hypothesis testing and by the notion that causality can be definitively inferred from a list of criteria, then we can make decisions based on what we do know rather than what we don't.The uncritical repetition of Hill's "causal criteria" is probably counterproductive in promoting sophisticated understanding of causal inference. But a different list of considerations that can be found in his address is worthy of repeating:• Statistical significance should not be mistaken for evidence of a substantial association.• Association does not prove causation (other evidence must be considered).• Precision should not be mistaken for validity (non-random errors exist).• Evidence (or belief) that there is a causal relationship is not sufficient to suggest action should be taken.• Uncertainty about whether there is a causal relationship (or even an association) is not sufficient to suggest action should not be taken.These points may seem obvious when stated so bluntly, but causal inference and health policy decision making would benefit tremendously if they were considered more carefully and more often. The last point may be the most important unlearned lesson in health decision making.In fairness to those who do not appreciate these points even today, it overinterprets Hill's short paper to claim that he clearly laid out these considerations, or that he was calling for modern decision analysis and uncertainty quantification. But the fundamental concepts were clearly there (and the overinterpretation is not as great as that required to derive a checklist of criteria for determining causation). Several generations of advancement in epidemiology and policy analysis provide much deeper exposition of his points. But Hill still offers timeless insightful analysis about how to interpret our observations. Strangely, these forgotten lessons, which are only slowly and grudgingly being appreciated in modern epidemiology, are hidden in plain sight, in what is possibly the best known paper in the field.1. Interestingly, there are more extreme cases of a scholar's name being immortalized for something contrary to his beliefs. The "Coase Theorem" in economics, from one of the most cited article in the economics and legal literatures  . Cur


Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.The complete sequence of the 1,267,782 bp genome of  Wolbachia provides insights into the origins of mitochondria, as well as the ecology and evolution of endosymbiosisThe genome sequence of  Wolbachia are intracellular gram-negative bacteria that are found in association with a variety of invertebrate species, including insects, mites, spiders, terrestrial crustaceans, and nematodes. Wolbachia are transovarialy transmitted from females to their offspring and are extremely widespread, having been found to infect 20%–75% of invertebrate species sampled , feminization , male-killing , and cytoplasmic incompatibility   .Wolbachia have been hypothesized to play a role in host speciation through the reproductive isolation they generate in infected hosts   . Moreoveult worm .Wolbachia and their invertebrate hosts. This is partly due to the difficulty of working with an obligate intracellular organism that is difficult to culture and hard to obtain in quantity. Here we report the completion and analysis of the genome sequence of Wolbachia pipientiswMel, a strain from the A supergroup that naturally infects Drosophila melanogaster  elements, four likely retrotransposons, and four families without detectible similarity to known elements but with many hallmarks of mobile elements  . Thus, many of these repetitive elements may be useful markers for strain discrimination. In addition, the mobile elements likely contribute to generating the diversity of phenotypically distinct Wolbachia strains  . One of  copies) . However copies) , and the strains ) by alteWolbachia sp. wKue . The other two are closely related to and exhibit extensive gene order conservation with the WO phage described from sp. wKue  .The irregular pattern of GC skew in ith this   and that of many hypothetical and conserved hypothetical proteins.Despite evident genome reduction in  species . Many ofwsp, which is a standard gene for strain identification and phylogenetic reconstruction in Wolbachia (wsp (WD0159), wMel encodes two wsp paralogs (WD0009 and WD0489), which we designate as wspB and wspC, respectively. While these paralogs are highly divergent from wsp  and do not amplify using the standard wsp PCR primers  is actually an ortholog of wspB  and not strains .Buchnera, Rickettsia, Chlamydia, and Wigglesworthia, that all have very low levels of repetitive DNA and mobile elements. The recently sequenced genomes of the intracellular pathogen Coxiella burnetti , we propose that the acquisition and maintenance of these repetitive and mobile elements by wMel have played a key role in shaping the evolution of Wolbachia.We propose that the mobile DNA in layi see . The lacchia DNA , 1998 orWolbachia  has been suggested previously for intracellular bacteria, based in part on observations that these bacteria have higher evolutionary rates than free-living bacteria  in wMel is particularly relevant since this pathway is one of the key steps in regulating mutation rates in other species. In fact, wMel is the first bacterial species to be found with two mutL homologs. Overall, examination of the predicted DNA repair capabilities of bacteria . Given wMel involve population-size related factors, such as genetic drift and the occurrence of population bottlenecks. Such factors have also been shown to likely explain the high evolutionary rates in other intracellular species  the free-living α-Proteobacteria, and (iv) mitochondria within α-Proteobacteria. Since amino acid content bias was very severe in these datasets, protein LogDet analyses, which can correct for the bias, were also performed. In LogDet analyses of the concatenated protein alignment, both including and excluding highly biased positions, mitochondria usually branched basal to the Wolbachia–Rickettsia clade, but never specifically with Rickettsia  do not even branch within the α-Proteobacteria, although these genes almost certainly were encoded in the ancestral mitochondrial genome  and two conserved hypothetical proteins . The α-Proteobacterial origin of these genes suggests that at least some of the genes of the de novo nucleotide synthesis pathway in eukaryotes might have been laterally acquired from bacteria via the mitochondria. The presence of such genes in other Proteobacteria suggests that their absence from Rickettsia is due to gene loss . We used the ene loss . This fiWolbachia are excellent candidates for such transfer events since they live inside the germ cells, which would allow lateral transfers to the host to be transmitted to subsequent host generations. Consistent with this, a recent study has shown some evidence for the presence of Wolbachia-like genes in a beetle genome , riboflavin biosynthesis, pyrimidine metabolism , and chelated iron uptake (using a single ABC transporter). The two Rickettsia species have a relatively large complement of predicted transporters for osmoprotectants, such as proline and glycine betaine, whereas wMel possesses only two of these systems.Many of the predicted metabolic properties of avengers . This liwMel genome is predicted to encode few proteins for regulatory responses. Three genes encoding two-component system subunits are present: two sensor histidine kinases (WD1216 and WD1284) and one response regulator (WD0221). Only six strong candidates for transcription regulators were identified: a homolog of arginine repressors (WD0453), two members of the TenA family of transcription activator proteins (WD0139 and WD0140), a homolog of ctrA, a transcription regulator for two component systems in other α-Proteobacteria (WD0732), and two σ factors (RpoH/WD1064 and RpoD/WD1298). There are also seven members of one paralogous family of proteins that are distantly related to phage repressors (see above), although if they have any role in transcription, it is likely only for phage genes. Such a limited repertoire of regulatory systems has also been reported in other endosymbionts and has been explained by the apparent highly predictable and stable environment in which these species live . The second operon contains virB3, virB4, and virB6 as well as four additional non-vir CDSs, including three putative membrane-spanning proteins, that form part of a 15.7 kb operon (WD0859–WD0853). Examination of the Rickettsia conorii genome shows a similar orga-nization  is also present in the wMel genome. WD0818 is quite divergent from virB8 and, as such, does not appear to have resulted from a recent gene duplication event. RT-PCR experiments have failed to show expression of this CDS in wMel-infected Drosophila (data not shown). PCR primers were designed to all CDSs of the wMel Type IV secretion system and used to successfully amplify orthologs from the divergent Wolbachia strains wRi and wAlbB (data not shown). We were able to detect orthologs to all of the wMel Type IV secretion system components as well as most of the adjacent non-vir CDSs, suggesting that this system is conserved across a range of A- and B-group Wolbachia. An increasing body of evidence has highlighted the importance of Type IV secretion systems for the successful infection, invasion, and persistence of intracellular bacteria within their hosts , it is not likely to have been acquired from other species. However, it is possible that these genes can be transferred among Wolbachia strains via the phage, which in turn could lead to some variation in host–symbiont interactions between Wolbachia strains.Genes involved in pathogenicity in bacteria have been found to be frequently associated with regions of anomalous nucleotide composition, possibly owing to transfer from other species or insertion into the genome from plasmids or phage. In the four such regions in wMel has 23 ankyrin repeat-containing genes, the most currently described for a prokaryote, with C. burnetti being next with 13. This is particularly striking given wMel's relatively small genome size. The functions of the ankyrin repeat-containing proteins in wMel are difficult to predict since most have no sequence similarity outside the ankyrin domains to any proteins of known function. Many lines of evidence suggest that the wMel ankyrin domain proteins are involved in regulating host cell-cycle or cell division or interacting with the host cytoskeleton: (i) many ankyrin-containing proteins in eukaryotes are thought to be involved in linking membrane proteins to the cytoskeleton  angulation ; (iii) s repeats ; and  has provided insights into the biology of Wolbachia, helped identify genes that likely play roles in the unusual effects Wolbachia have on their host, and revealed many new details about the evolution of Wolbachia and mitochondria. Perhaps most importantly, future studies of Wolbachia will benefit both from this genome sequence and from the ability to study host–symbiont interactions in a host (D. melanogaster) well-suited for experimental studies.Analysis of the wMel DNA was obtained from D. melanogaster yw67c23 flies that naturally carry the wMel infection. wMel was purified from young adult flies on pulsed-field gels as described previously  after prerinsing with 70% ethanol followed by TE buffer and, after concentration, rinsed with TE. The retentate was incubated with proteinase K at 56°C for 2 h and then stored at 4°C. wMel DNA for gap closure was prepared from approximately 1,000 Drosophila adults using the Holmes–Bonner urea/phenol:chloroform protocol  showed that 3,912 of the 10,642 contigs were likely contaminants from the Drosophila genome. To aid in closure, the assemblies were rerun with all sequences of likely host origin excluded. Closure, which was made very difficult by the presence of a large amount of repetitive DNA (see below), was done using a mix of primer walking, generation, and sequencing of transposon-tagged libraries of large insert clones and multiplex PCR  and wMel (1.2 Mb).The complete genome sequence was determined using the whole-genome shotgun method . For theplex PCR . The finWolbachia and their hosts may undergo lateral gene transfer events  to analyze the phylogenetic profile , and (iii) to determine the presence of homologs in different species. Orthologs between the wMel genome and that of the two Rickettsia species were identified by requiring mutual best-hit relationships among all possible pairwise BLASTP comparisons, with some manual correction. Those genes present in both Rickettsia genomes as well as other bacterial species, but not wMel, were considered to have been lost in the wMel branch  toanch see . Genes panch see . For theMarchantia polymorpha and Reclinomonas americana , leaving 32 genes for investigation: atp6, atp9, atpA, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad9, rpl16, rpl2, rpl5, rpl6, rps1, rps11, rps12, rps13, rps14, rps19, rps2, rps3, rps4, rps7, rps8, yejR, and yejU. Using FASTA with the mitochondrial proteins as a query, homologs were identified from the genomes of seven α-Proteobacteria: two intracellular symbionts (W. pipientis wMel and Rickettsia prowazekii) and five free-living forms . Escherichia coli and Neisseria meningitidis were used as outgroups. Caulobacter crescentus was excluded from analysis because homologs of some of the 32 genes were not found in the current annotation. In the event that more than one homolog was identified per genome, the one with the greatest sequence identity to the mitochondrial query was retrieved. Proteins were aligned using CLUSTALW . PCR products were cloned into pGem-T vector  as previously described . The four partial wsp sequences were aligned using CLUSTALV  and cDNA synthesized using SuperScript III RT (Invitrogen) using primers wspBR, WD0817R, WD0853R, and WD0852R. RNA isolation and RT were done according to manufacturer's protocols, with the exception that suggested initial incubation of RNA template and primers at 65°C for 5 min and final heat denaturation of RT-enzyme at 70°C for 15 min were not done. PCR was done using rTaq , and several primer sets were used to amplify regions spanning adjacent CDSs for most of the two operons. For operon virB3-WD0853, the following primers were used: (virB3-virB4)F, (virB3-virB4)R, (virB6-WD0856)F, (virB6-WD0856)R, (WD0856-WD0855)F, (WD0856-WD0855)R, (WD0854-WD0853)F, (WD0854-WD0853)R. For operon virB8-wspB, the following primers were used: (virB8-virB9)F, (virB8-virB9)R, (virB9-virB11)F, (virB9-virB11)R, (virB11-virD4)F, (virB11-virD4)R, (virD4-wspB)F, and (virD4-wspB)R. The coexpression of virB4 and virB6, as well as WD0855 and WD0854, was confirmed within the putative virB3-WD0853 operon using nested PCR with the following primers: (virB4-virB6)F1, (virB4-virB6)R1, (virB4-virB6)F2, (virB4-virB6)R2, (WD0855-WD0854)F1, (WD0855-WD0854)R1, (WD0855-WD0854)F2, and (WD0855-WD0854)R2. All ORFs within the putative virB8-wspB operon were shown to be coexpressed and are thus considered to be a genuine operon. All products were amplified only from RT-positive reactions .Click here for additional data file.Figure S2wMel codon usage. Multivariate analysis of codon usage was performed using the CODONW package (available from http://www.molbiol.ox.ac.uk/cu/codonW.html).Proteins with fewer than 100 residues are excluded from this analysis because their effective number of codon (ENc) values are unreliable. The curve shows the expected ENc values if codon usage bias is caused by GC variation alone. Colors: yellow, hypothetical; purple, mobile element; blue, others. Most of the variation in codon bias can be traced to variation in GC, indicating that the mutation forces dominate the (289 KB PDF).Click here for additional data file.Figure S3wMel. Transporters are grouped by predicted substrate specificity: inorganic cations (green), inorganic anions (pink), carbohydrates (yellow), and amino acids/peptides/amines/purines and pyrimidines (red). Transporters in the drug-efflux family (labeled as “drugs”) and those of unknown specificity are colored black. Arrows indicate the direction of transport. Energy-coupling mechanisms are also shown: solutes transported by channel proteins (double-headed arrow); secondary transporters ; ATP-driven transporters (ATP hydrolysis reaction); unknown energy-coupling mechanism (single arrow). Transporter predictions are based upon a phylogenetic classification of transporter proteins (Overview of the predicted metabolism (energy production and organic compounds) and transport in proteins .(167 KB PDF).Click here for additional data file.Table S1(649 KB DOC).Click here for additional data file.Table S2(147 KB DOC).Click here for additional data file.Table S3(718 KB XLS).Click here for additional data file.Table S4(116 KB DOC).Click here for additional data file.Table S5(24 KB DOC).Click here for additional data file.Table S6(26 KB DOC).Click here for additional data file.Table S7(34 KB DOC).Click here for additional data file.Table S8(117 KB DOC).Click here for additional data file.Table S9(47 KB DOC).Click here for additional data file.wMel has been given GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession ID number AE017196 and is available through the TIGR Comprehensive Microbial Resourceat http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=dmgThe complete sequence for Wolbachia sp. wAlbB outer surface protein precursor wsp gene), AF020070 (Wolbachia sp. wRi outer surface protein precursor wsp gene), AJ252062 (Wolbachia endosymbiont of D. immitis sp. gene for surface protein), AJ580921 , AJ580922 , and AJ580923 .The GenBank accession numbers for other sequences discussed in this paper are AF020059 (


Dysphagia and feeding intolerance are common in neurologically handicapped children. The aim is to determine the etiologies of feeding intolerance in neurologically handicapped children who are intolerant of tube feedings.Eighteen neurologically handicapped children, followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin who were intolerant of gastrostomy feedings. The charts of these 18 patients were reviewed. Past medical history, diagnoses, history of fundoplication and results of various tests of gastrointestinal function including barium contrast radiography, endoscopy and antroduodenal manometry were documented.Five of 11 children had abnormal barium upper gastrointestinal series. Seven of 14 had abnormal liquid phase gastric emptying tests. Two of 16 had esophagitis on endoscopy. All 18 children had abnormal antroduodenal motility.In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Oral pharyngeal dysphagia due to disordered swallowing has become increasingly recognized in children with cerebral palsy and other neurodevelopmental disorders. This has led to the increasing use of enteral tube feedings either for full or supplemental nutritional support. Symptoms of foregut dysmotility, such as vomiting, retching gagging and bloating, are often associated with tube feeding in neurologically handicapped children -4. PreviIn order to elucidate the mechanisms behind these symptoms we reviewed the charts of a group of children followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin with neurological dysfunction, who were intolerant of tube feedings and who had undergone antroduodenal motility studies as part of their evaluations.2 b1receptor antagonists are summarized in Table The charts of 18 neurologically handicapped children  with dysphagia and symptoms of foregut motility were reviewed. The symptoms, underlying disorders, feeding route, the presence of fundoplication, and the use of prokinetic agents and HFollowing an overnight fast, antroduodenal motility studies were performed using a multilumen catheter with 8 recording ports spaced 2.5–5 cm apart, passed through the gastrostomy either under fluoroscopic guidance or endoscopically, connected to a low compliance, pneumohydraulic capillary infusion system  and a computerized motility system . Fasting activity was recorded for 3–4 hours. Erythromycin (1 mg/kg) was given intravenously over 10 minutes and the recording continued for another hour. Octreotide (0.5 mcg/kg) was then given intravenously over 5 minutes. 45 minutes later a liquid meal was given followed by an additional 2–3 hour recording period. The meal varied and consisted of the usual formula and volume given at home. Patients receiving jejunal feedings or TPN were given a bolus gastrostomy feeding. In the one patient receiving TPN, the TPN was discontinued during the study. Prokinetics were stopped at least 48 hours prior to study.Phase 1 of the MMC was defined as motor quiescence. Phase 2 was defined as the time between Phases 1 and 3 and is characterized by random contractions of varied amplitude and frequency. Phase 3 of the MMC is characterized by an aborally propagating cluster of repetitive contractions with a frequency of 11–13/minute in the duodenum and 3/minute in the antrum with a duration of 3–10 minutes. The tracings were analyzed by visual inspection.This study was approved by the Research and Publications Committee/Human Rights Review Board of the Children's Hospital of Wisconsin and the Institutional Review Board of the Medical College of Wisconsin.Eleven children had had recent upper GI series. Of these 5 were normal, 3 had gastroesophageal reflux and 3 had paraesophageal hernias , 1 had a bezoar and 1 had esophageal dysmotility. Fourteen patients had liquid phase gastric emptying studies. Of these 7 were normal, 6 had delayed emptying and 1 had rapid emptying. Two of 16 patients who had had recent endoscopies had esophagitis, 14 were normal. The diagnoses and clinical histories of the patients are summarized in Table No patients had a normal antroduodenal motility study Figures ,2,3. In Following erythromycin eight had a normal response consisting of antral contractions with a frequency of 3/min followed by phase 3 like activity in the duodenum. Three patients had no response and seen had abnormal responses consisting of abnormal clusters in three, no antral response in three and no duodenal response in three.Fourteen patients had a normal response to octreotide consisting of cessation of antral activity and the development of phase 3 activity in the duodenum. Two patients had continued antral contractions and two did not develop phase 3 duodenal activity. These patients had non-propagating duodenal clusters.Fourteen patients had normal postprandial phase 2-like activity. Eight developed premature phase 3 activity within 30 minutes following the meal. Seven patients failed to develop phase 3 activity during the 2–3 hour postprandial monitoring period and seven had no antral contractions in the postprandial monitoring period. One patient had a retrograde MMC.Three patients developed severe pain or irritability associated with antral or duodenal contractions following erythromycin  or octreForegut dysmotility is common in children with neurodevelopmental disorders such as cerebral palsy -4. Up toRavelli and Milla have shown that gastric electrical activity as measured by the electrogastrogram (EGG) was abnormal in 31/50 neurologically handicapped children . Eleven In a previous study we compared gastric electrical activity as measured by EGG in a group of neurologically handicapped children who were tolerant of their tube feedings to a group that were intolerant or symptomatic during tube feedings . The perThere have been few reports of antroduodenal motility, which have focused on neurologically handicapped children with feeding intolerance. DiLorenzo and colleagues reported that 25/28 children who remained symptomatic following fundoplication had abnormal antroduodenal motility .Due to the nature of our patients there are some limitations in the design and interpretation of our study. Because many of our patients had had multiple formula changes, we decided to use the formula, which the child was receiving at the time of the study, so that any symptoms occurring during the studies could not be attributed to a formula change. Bolus feeds were given to all patients during the study even those who had been receiving drip feeds. The fasting state could not be recorded for 4 hours in all patients, thus it is possible that absence of phase 3 of the MMC in these patients might not be abnormal. Since normal manometry data have not been published for children, non propagating clusters may or may not be normal in our patients.Since only 12 of our 18 patients had undergone fundoplication, we agree with DiLorenzo et al  that theIn this study we have confirmed that the incidence of foregut dysmotility is very high in neurologically handicapped children with feeding intolerance. Prokinetics and acid suppression did not resolve the symptoms in our patients. Twelve of our 18 patients had had fundoplications and two had undergone two fundoplications in unsuccessful attempts to control what had been thought to be reflux symptoms. While there is no way to know how much abnormal antroduodenal motility contributed to our patient's feeding disorders, following antroduodenal manometry a number of our patients were treated successfully with jejunal feeding, suggesting that while they had foregut dysmotility, midgut motility is normal.In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Thus in this patient population generalized foregut dysmotility may mimic reflux and the decision to perform a fundoplication should be made very cautiously and only after a complete evaluation of foregut motility particularly in children with gagging retching and forceful vomiting.None declared.The pre-publication history for this paper can be accessed here: