One important application of microarray experiments is to identify differentially expressed genes. Often, small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value before a statistical test is carried out. Then, there are two types of data: truncated values and original observations. The truncated values are not just another point on the continuum of possible values and, therefore, it is appropriate to combine two statistical tests in a two-part model rather than using standard statistical methods. A similar situation occurs when DNA methylation data are investigated. In that case, there are null values (undetectable methylation) and observed positive values. For these data, we propose a two-part permutation test.p-values in comparison to the original two-part test. We found this for both DNA methylation data and microarray data. With a simulation study we confirmed this result and could show that the two-part permutation test is, on average, more powerful. The new test also reduces, without any loss of power, to a standard test when there are no null or truncated values.The proposed permutation test leads to smaller The two-part permutation test can be used in routine analyses since it reduces to a standard test when there are positive values only. Further advantages of the new test are that it opens the possibility to use other test statistics to construct the two-part test and that it avoids the use of any asymptotic distribution. The latter advantage is particularly important for the analysis of microarrays since sample sizes are usually small. The addition of a methyl group at the carbon-5 position of cytosine is a modification of DNA called DNA methylation. In mammalian cells, DNA methylation is essential for proper development . The met2 with two degrees of freedom (df = 2).DNA methylation data can be obtained using the MethyLight technology . When thIn microarray data it is relatively common that small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value. Recent examples used different cutoff values: 1, 20, and 50, respectively -11. AsidDue to the truncation there are two different types of data: truncated values and original observations. Since the truncated values are not just another point on the continuum of possible values, it would be inappropriate to use a standard statistical method that would treat all values equally . The twop-value being the probability of the permutations giving a value of the test statistic as supportive or more supportive of the alternative than the observed value X2 = B2 + W2. Under the null hypothesis of no difference between the groups, X2 is asymptotically χ2-distributed with df = 2 [X2. This test, called two-part permutation test here, is a permutation test based on the sum statistic X2. It is carried out by permuting the group labels for the whole sample. Thus, all observations, truncated and non-truncated values  are reallocated to the groups. When performing this two-part permutation test the exact permutation distribution of X2 is determined. This distribution, computed by generating all possible permutations or, for the p-values given below in Table p-value. Since it is a permutation test based on the sum X2, it is neither necessary to determine the permutation distributions of the summands B2 and W2 nor to calculate the p-values of the univariate tests related to B2 and W2.The test statistic for the two-part test is h df = 2 ,7. Alter. Siegmund et al. [We use DNA methylation data from 7 regions and 87 lung cancer cell lines, 41 lines are from small cell lung cancer and 46 lines from non-small cell lung cancer ,4. The pp-values of the two tests. For most regions the two-part permutation test gives a smaller p-value than the original two-part test. The only exception is the region APC. However, the original two-part test's p-value for this region is 0.1684 and, for a p-value of this size, a small change in the value is usually of no importance.Table . The sample size in this microarray experiment is 10 per group. As mentioned above, expression levels below 50 were set to 50. This data truncation occurred in 2,215 (28%) out of 7,902 genes. First, we consider these 2,215 genes. Figure Tschentscher et al.  performep-values. Often, the two-part permutation test gives a smaller p-value than the original two-part test. For instance, for 19 genes the p-value is ≤ 0.001 when the latter test is applied. The permutation test gives a p-value ≤ 0.001 for these 19 and for 39 additional genes. As usual, the majority of genes do not show any indication of being differentially expressed. For these genes a slight change in the p-value is of no importance. Thus, in Table p-value of the original two-part test is ≤ 0.1. Out of the 2,215 genes 514 remain. As shown in Table p-values of the two-part permutation test are, on average, distinctly smaller than those of the original two-part test.Table W2. Of course, one has to define a priori whether the two-part test or the Wilcoxon test will be used to analyze the data. If the original two-sided (asymptotic) Wilcoxon test were chosen and applied, one could compare W2 with critical values from a χ2 distribution with one degree of freedom (df). If the two-part were chosen there is df = 2 and, in case of no truncated values, power is lost compared to the original Wilcoxon test. For instance, the 95% percentile of the χ2 distribution with df = 1 is 3.84, but it is 5.99 for df = 2. The permutation test using the sum statistic X2 does not suffer from this power loss: When there are no truncated values there is, of course, no difference in the proportions of truncated values, and the test statistic X2 is, for every permutation, the sum 0 + W2. Thus, the two-part permutation test reduces to the exact two-sided Wilcoxon rank sum test when there are no truncated values. Consequently, this permutation test does not only give smaller p-values, but it is also applicable in routine use whether or not truncated values are present.For a large proportion of genes (72% in this data set) there are no truncated values. In that case, the two-part statistic reduces to the sum 0 plus the squared standardized rank sum p1 and p2, and the remaining observations were generated according to a lognormal distribution (with median 1 and σ = 1). Then, the values of one group were shifted if applicable. In some other configurations all observations were generated according to the lognormal distribution and, in one group, shifted. Then, values smaller than a cutoff value were truncated.The two different tests, the original two-part test and the two-part permutation test, were compared in a Monte Carlo simulation study performed using SAS version 8.2, 5,000 simulation runs were generated for each configuration. The sample size of 10 per group was chosen as in the microarray experiment presented above. In some configurations some randomly chosen values were set to 0 according to binomial distributions with the probabilities p1 = p2 = 0.3 and a significance level of α = 0.05 the simulated type I error rates were 0.049 for both the original two-part test and the two-part permutation test.The type I error rates of the two tests are very similar. With e.g. p-value ≤ 0.1 of the original two-part test. In all considered configurations the median of the difference between the p-values of the original two-part test and the two-part permutation test is positive. The finding that the p-values of the permutation test are smaller corresponds to a higher power of this test. The power is given in Table Table B2 and W2 by the exact permutation distributions. If so, one would compute two exact p-values although the aim of a two-part test is to receive one p-value that combines information from both parts. Therefore, the two-part permutation test uses the exact permutation distribution of the sum statistic X2. That this permutation distribution of the sum is generated rather than to simply replace the asymptotic distributions of the summands by their exact permutation distributions may be the reason why the permutation test is more powerful.Previous research demonstrated that a two-part test is appropriate and powerful in the presence of a clump of zero observations . In this paper we propose a permutation test for such situations with two types of data. Usually, nonparametric tests can be performed based on an asymptotic distribution or based on a permutation null distribution. The two approaches often give similar results, especially when the sample sizes are large. However, in the case of a two-part test one cannot simply replace the asymptotic distributions of A disadvantage of a permutation test is that it can be computer-intensive. However, this issue is less relevant now due to faster algorithms [, chap. 1p-values of the tests from individual genes [In microarray experiments it is common to investigate thousands of genes simultaneously. The approach presented here for the identification of differentially expressed genes is to consider a univariate testing problem for each gene. A correction for the multiplicity of genes is a subsequent step, that is, like the previous step of normalizing the data, outside the scope of this paper. A common approach to the multiplicity problem is to consider a procedure for testing the genes simultaneously for differential expression with the test on an individual gene being implied in the simultaneous test. For such a procedure different proposals have been made recently. For instance, there are methods based on the al genes -23. In aAside from the shown improvement in power, the proposed two-part permutation test has three important advantages. First, it avoids the use of any asymptotic distribution and, therefore, can safely be applied in case of small sample sizes that are common in microarray experiments. Second, it reduces without any loss of power to the exact Wilcoxon test if there were no truncated (or zero) values. Thus, it can be used in routine analyses. Third, the permutation test opens the possibility to use other tests to construct the two-part test. Thus, tests with unknown or non-standard null distributions can be used. For instance, one could replace the Wilcoxon test by the Baumgartner-Weiß-Schindler test  that wasMN performed the statistical analyses and drafted the manuscript. TB prepared the microarray data. TB and KHJ participated in the design of the simulation study and helped to draft the manuscript. All authors read and approved the final manuscript.


Microarray-based comparative genome hybridization experiments generate data that can be mapped onto the genome. These data are interpreted more easily when represented graphically in a genomic context.We have developed Caryoscope, which is an open source Java application for visualizing microarray data from array comparative genome hybridization experiments in a genomic context. Caryoscope can read General Feature Format files (GFF files), as well as comma- and tab-delimited files, that define the genomic positions of the microarray reporters for which data are obtained. The microarray data can be browsed using an interactive, zoomable interface, which helps users identify regions of chromosomal deletion or amplification. The graphical representation of the data can be exported in a number of graphic formats, including publication-quality formats such as PostScript.Caryoscope is a useful tool that can aid in the visualization, exploration and interpretation of microarray data in a genomic context. Eisen he data . Howeve.g., see ), the ge.g., see ,4).In addition to being able to view and browse arrayCGH data, it is also important that the data be readily connected to annotation sources, such that a user can easily determine the identity and attributes of the gene represented by a reporter that was present on a microarray, which for instance may show evidence of amplification or deletion. For example, in arrayCGH experiments using tumor cells as the DNA source, there is an obvious value in rapidly determining whether a deleted region contains a tumor suppressor gene.Finally, researchers frequently need to create figures, for publications, communication with co-workers, supplemental websites, or presentations. Thus researchers should be able to produce the visual representation of their data in a variety of graphic formats.Caryoscope was originally implemented as a Web form, generating either a bitmap or a clickable PDF output. When this became an important day-to-day tool for our users -9), we c, we c9])et al.  or a taOnce the user opens a file, Caryoscope automatically displays one of the datasets contained therein Figure . CaryoscView modes toolbar  that appear immediately when mousing over the features, and can navigate to related URLs by clicking on each feature. Typically, users at Stanford University (our primary source of testers and users) link GenBank accessions, associated with the cDNA clones that are on their microarrays, to SOURCE Gene Pages , and a user can compute a moving average of the values. Both these computations can be controlled from the Export dialog. Specifically, Caryoscope supports vector  output for scalable publication-quality results, and raster  output for ease of viewing, posting on supplemental websites, and inclusion in presentations.To prepare diagrams, the user can export the Caryoscope display to a variety of graphics formats via the A user may export graphics from Caryoscope via the command line mode, without having to invoke the interactive user interface. For example, to export a view of a dataset as a PDF file, the user could invoke Caryoscope as follows:     java -jar caryoscope-run.jar            -inputFile 3395-2004-04-04.csv           -visibleDataset RAT2_MEAN           -outputFile 3395.pdf           -outputFormat pdf-listFormats option provides a list of available graphics output formats, and the -help option prints a brief summary of the options.All settings may be modified via the command line. The A comparison of the features of Caryoscope with other, similar software is presented in Table In addition to immediately executable copies, the complete source code for Caryoscope is available without limitations from our website , and isWe update Caryoscope frequently (approximately once every three weeks) and post news items on the website. We also send e-mail announcements to people who have requested them.Caryoscope is useful for viewing both arrayCGH and expression data in the context of genomic position. It helps a biologist gain insight by providing a high-level view of a large amount of data at once, where patterns can be perceived at a glance.A biologist studying amplifications or deletions in tumor cells may create and export graphics representing arrayCGH and expression data for the same cells using Caryoscope, and visually compare the two side-by-side. For instance, co-located regions that are amplified at the DNA level and over-expressed at the RNA level would provide excellent confirmation of the results. Using the zooming and panning features of Caryoscope, the biologist could focus on specific regions of interest.Minimum feature width of, say, 2 pixels , one can use the n Figure  settingsWe intend Caryoscope to be a bench-top visualization tool that biologists can use immediately to get day-to-day work done, with a very low "cost of entry" for getting started. This led us to a number of design choices.Caryoscope can be launched from our web site without a prior installation step. Since it is Open Source, anyone, including any for-profit organization, can use it without restrictions and without having to obtain a license or register for access.The input file formats and output graphical formats we chose are all in common usage. In particular, the TXT and CSV input formats can be generated using any popular spreadsheet or database software, or even with a plain text editor, without having to do any programming.i.e., the tooltips and hyperlinks).Finally, we built Caryoscope to be content-neutral, with no hard-coded specificity to any research field. Thus, users of Caryoscope may control how annotations are treated as numerical data, and can "program" data-driven interactive behavior of the display  of that pixel.Modern computer graphics systems use a technique called "anti-aliasing"  and Figu to rendeWe will develop our display methods further to ensure that we can provide an easy-to-read display while retaining the subtle variations in the data. Following the spirit of medical diagnostic imaging , wherebOne solution, suggested by , is to dWe are particularly concerned about the use of Caryoscope (or similar) graphics in vector formats  that are subsequently rendered on diverse display devices and printers. Since we have no control over the rendering at the destination, it is likely that the same vector output could look very different on different devices. Once we have studied this problem in more detail, we intend to provide practical usage guidelines for researchers.Our experience with the application, and how it is used, leads us to believe that the current zooming paradigm should be revisited. While the model of a continuously zoomable 2D space provides users with the features they need, it can lead to displays whereby the data in specific regions of adjacent chromosomes are juxtaposed, even though their being next to each other is not intrinsically meaningful .A common user request is to display two or more arrayCGH and/or expression datasets side by side, either to determine regions of recurrent deletion or amplification, or to discern visually the impact that changes in chromosome copy number may have on transcript levels . AnotheCaryoscope currently provides a flexible method to visualize, explore and create images of microarray data in a genomic context. With such a tool, microarray researchers will be able to answer questions about how genome copy number or genome position plays a role in biological processes or human diseases.Caryoscope is a useful, flexible Java application for the visualization of microarray data in a genomic context. It is available as Open Source under the permissive MIT License, allowing anyone to use or modify it.Project name: CaryoscopeProject home page: Operating system(s): Platform independentProgramming language: JavaOther requirements: Java 1.4 or higherLicense: MIT LicenseAny restrictions to use by non-academics: NoneIABA compiled the functional requirements for the Java version of Caryoscope and designed and implemented the software. CAR conceived and wrote the original Perl version of Caryoscope. THB aided and participated in the design of the study, dealt with user support and worked with users to get feedback and suggestions for further development. CAB aided and participated in the design and evaluation of the study and provided feedback and suggestions for future development. GS supervised and participated in the design of the study. All authors read and approved the final version of the manuscript.


Temperature is a frequently used parameter to describe the predicted size of lesions computed by computational models. In many cases, however, temperature correlates poorly with lesion size. Although many studies have been conducted to characterize the relationship between time-temperature exposure of tissue heating to cell damage, to date these relationships have not been employed in a finite element model.We present an axisymmetric two-dimensional finite element model that calculates cell damage in tissues and compare lesion sizes using common tissue damage and iso-temperature contour definitions. The model accounts for both temperature-dependent changes in the electrical conductivity of tissue as well as tissue damage-dependent changes in local tissue perfusion. The data is validated using excised porcine liver tissues.The data demonstrate the size of thermal lesions is grossly overestimated when calculated using traditional temperature isocontours of 42°C and 47°C. The computational model results predicted lesion dimensions that were within 5% of the experimental measurements.When modeling radiofrequency ablation problems, temperature isotherms may not be representative of actual tissue damage patterns. The mitigation of primary and metastatic tumors by radiofrequency ablation is a developing research area. The goal of ablation is to necrose treatment volumes by raising the temperature of targeted tissues. Ablation probes are inserted percutaneously, laparoscopically, or during surgery into cancerous tumors. Once positioned, high frequency alternating current (450–550 kHz) is delivered through an uninsulated electrode into the surrounding tissues to a dispersive ground pad that is applied to the patient. The electromagnetic energy is converted to heat by resistive heating.While the usage of radiofrequency ablation devices is well established, efforts to optimize treatment strategies are ongoing. An important consideration in optimizing ablation is determining what treatment volumes are necessary and acceptable. In liver ablation, for example, treatment volumes generally extend a centimeter beyond the dimensions of a tumor -3. Sincein vitro and in vivo in animal models show wide variations, since many of the key parameters (i.e. tissue perfusion) cannot be controlled  = 0     (Eq.1)∇·}     (Eq.4)- 6.584 × 10whereN) = N [10.394-2.3776 N + 0.68258 N2 - 9.13538 N3 + 1.0086 × 10-2 N4]σ . The thermal properties of liver used in the model were acquired from Tungjitkusolmun et al.[;N is the normality of an electrically equivalent sodium chloride solution, un et al. and Duckun et al..o) is applied to the conducting tip of the ablation probe. The outer surface of the model serves as an electrical ground return (V = 0). An electrically insulating boundary condition is applied to the non-conducting portions of the probe such that n·(σ∇ V) = 0; where n is the unit vector normal to the surface, σ is the electrical conductivity, and V is the voltage at the insulating surface. A thermal boundary condition of T = Tamb is applied to the outer surfaces of the model to simulate ambient temperature. Since the thermal mass of the probe is small compared to the surrounding tissue, we assumed that heat conduction into the probe itself was minimal. Thus, all other surfaces of the ablation probe are considered to have a thermally insulating boundary condition such that n·(k ∇ T) = 0.A source voltage  and Matlab  to calculate temperature and tissue damage. While conventional finite element models effectively solve field solutions using a nonuniform geometrical mesh, tissue exposure calculations are integrated at each point in the model over the course of ablation and are more easily calculated using uniform rectilinear grids. As shown in Figure -3 cubic meters of blood/ cubic meter of tissue/ second) [Given the axial symmetry of the problem, we used a 2D-axisymmetric mesh consisting of 13,641 nodes and 26,880 elements. The Femlab 'Fldaspk' ordinary differential equation solver was used to achieve convergence. This is a robust variant of the traditional ODE15s stiff differential equation solver used in solving finite element problems in Matlab. Ablations were simulated at source voltages of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and 30 volts. For each of the source voltages, we varied the initial level of tissue perfusion at 0%, 20%, 40%, 60%, 80%, and 100% normal tissue perfusion (6.4 × 10 second) . AmbientTo validate the computational model, experimental measurements were made in 6 freshly excised porcine liver sections. A single needle ablation probe with a 2 cm uninsulated tip was inserted 3 cm into each liver tissue. Since commercial RF ablation generators operate using either constant temperature or constant power feedback algorithms, an experimental constant voltage RF generator 500 kHz) was used 00 kHz wa,59. ThisComputational model calculations were made at 20, 25, and 30 volts following the same experimental protocol. Ambient temperature for these calculations was 22°C instead of the 37°C temperature used in the main simulations. The calculated lesion sizes were directly compared with the measurements in tissue.Table -3 mb3/ mt3/s), the electrical conductivity changes as much as 260% using a 30 volt source. The electrical conductivity is indirectly a function of tissue perfusion since tissue perfusion is zero in the necrosed treatment volume. Tissue perfusion lowers the tissue temperature outside the treatment volume which helps to conduct heat away from temperatures within the ablated area..Table 2, where σ is the electrical conductivity, ρ is the tissue density, and |E| is the magnitude of the electric field. The data shows that the SAR is highest with increasing source voltage with no tissue perfusion. Initially, this seems counterintuitive as one would expect a higher maximum SAR for perfused flows, where a greater amount of power is needed to compensate for the convective heat loss. This observation can be explained by the large changes in the electrical conductivity (Table Table 2), whereas cell damage exhibits an S-shaped curve. Figure Figure Figure Figure A comparison of lesion volumes with no tissue perfusion computed using 63% and 100% iso-damage threshold contours and 42°C, 47°C, 60°C, and 90°C isothermal contours is presented for the cases of no tissue perfusion Table . The senTable To validate the computational model, ablation experiments were performed at room temperature (22°C) in excised porcine liver tissue using 20, 25, and 30 volt constant voltage radiofrequency sources (500 kHz). Ablations were made for a 15 minute exposure time. Figure To date, several computational studies have been performed to described the rate of lesion growth in radiofrequency ablation applications. In many cases, these studies use surrogate endpoints such as temperature isotherms and thermal dosing to calculate equivalent expressions for lesion size. While many models exists that account for far-more elaborate parameters such as tissue perfusion through large blood vessels, the interpretation of such models is difficult since most do not account for transient changes in tissue properties and often report tissue temperature only ,56,57,60In this study, we created a computational simulation that tested some of the basic assumptions made in modeling lesion growth problems. We developed a model where tissue perfusion and the electrical conductivity are allowed to vary at each time step and spatial position as a function of tissue damage and temperature. These simulations are significantly more time-consuming since gross simplifications to heating mechanisms are not made. Although our model geometry is simpler than others that appear in the literature, we chose to ignore large vessels since their position and impact are highly variable. We chose a simpler geometry so that the impact of damage-dependent tissue perfusion and temperature-dependent electrical conductivity could be assessed more directly.The damage-dependent tissue perfusion accounts for physiological observations of tissue coagulation and local cessation of blood flow. Unlike thermal dosing, where thermal injury is calculated globally over the entire duration of an ablation, tissue damage is calculated at every time step. The intermediate tissue damage that results at every timestep influences the local tissue perfusion and creates a moving boundary condition which changes the local heat sink properties. Ignoring the intermediate timesteps causes tissue perfusion to remain constant throughout the entire ablation, which results in an underestimation of the true lesion size. The use of temperature-dependent electrical conductivity greatly affects modeling results, as the electrical conductivity has been shown to increase dramatically over the course of tissue heating . When coAn important outcome of this study is the demonstration that, temperature isotherms and tissue damage patterns are not synonymous. Traditional use of temperature isotherms that are used to define lesion size rely on coagulation temperature for protein (42–47°C) and grossly overestimate lesion dimensions. Our studies show that temperature decrease is gradual, while tissue damage decreases rapidly as a function of distance. It is this sharp decrease in tissue damage that causes lesion boundaries to appear fuzzy, as predicted by our model. The results also demonstrate that ablation lesions continue to grow after the applied power is terminated. Lesions continue to grow while temperature envelopes collapse after ablation since sufficiently high temperature are present to accrue tissue damage. In nearly all cases, lesions continued to grow several minutes following the ablation. A comparison of the resulting lesion dimensions between fully perfused and non-perfused tissues show that the lesion width decreases 38–46% and the lesion depth decreases 18–20% when tissue perfusion is accounted for in the model. Previous studies have shown that tissue perfusion can account for as much as 50% change in the size of the lesions generated during ablation .An important observation in this study is the resemblance of the 60°C isocontour to lesion size. While the 42°C and the 47°C isotemperature contours are poor indicators of lesion size, 60°C is highly correlated with the lesion volumes. Seemingly, this would suggest that time-intensive tissue damage calculations need not be made since a critical temperature of 60°C can be used to identify lesion size. However, this is only true if the calculated temperature is a function of both transient changes in tissue perfusion and the electrical conductivity. In the absence of either of these phenomena, lesion sizes calculated at 60°C would underestimate lesion size.The validation data demonstrate that the model accurately accounts for the behavior of lesion growth in tissue. There are, however, a few limitations to this model. First, it is well established that temperature elevation of tissues results in the denaturing of proteins, which may drastically change the electrical conductivity of tissue in a nonlinear fashion ,57. PrelA second limitation in our model is that it is only valid for temperatures below 100°C. At temperatures above 100°C, tissues begin to boil and generate gas. When this occurs, some of the energy that contributes to temperature increase is used to change the water content of tissues into gas. At substantially higher temperatures, the composition of gas may be highly complex as tissue begins to burn and break down. Although gas generation is commonly seen in clinical use of radiofrequency ablation, impedance rises due to tissue charring limit the progressive rise in temperature. The complexity of multi-phasic ablation was beyond the scope of this study.The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.


Health and nutrition inequality is a result of a complex web of factors that include socio-economic inequalities. Various socio-economic indicators exist however some do not accurately predict inequalities in children. Others are not intervention feasible.To examine the association of four socio-economic indicators namely: mothers' education, fathers' education, household asset index, and land ownership with growth stunting, which is used as a proxy for health and nutrition inequalities among infants and young children.This was a cross-sectional survey conducted in the rural district of Hoima, Uganda. Two-stage cluster sampling design was used to obtain 720 child/mother pairs. Information on indicators of household socio-economic status and child anthropometry was gathered by administering a structured questionnaire to mothers in their home settings. Regression modelling was used to determine the association of socio-economic indicators with stunting.One hundred seventy two (25%) of the studied children were stunted, of which 105 (61%) were boys (p < 0.001). Bivariate analysis indicated a higher prevalence of stunting among children of: non-educated mothers compared to mothers educated above primary school (odds ratio (OR) 2.5, 95% confidence interval (CI) 1.4–4.4); non-educated fathers compared to fathers educated above secondary school ; households belonging in the "poorest" quintile for the asset index compared to the "least poor" quintile ; Land ownership exhibited no differentials with stunting. Simultaneously adjusting all socio-economic indicators in conditional regression analysis left mothers' education as the only independent predictor of stunting with children of non-educated mothers significantly more likely to be stunted compared to those of mothers educated above primary school . More boys than girls were significantly stunted in poorer than wealthier socio-economic strata.Of four socio-economic indicators, mothers' education is the best predictor for health and nutrition inequalities among infants and young children in rural Uganda. This suggests a need for appropriate formal education of the girl child aimed at promoting child health and nutrition. The finding that boys are adversely affected by poverty more than their female counterparts corroborates evidence from previous studies. Health outcomes are a result of a multi-layered conceptual framework with proximal and distal determinants . ProximaIn recent decades, development agencies and governments have emphasized efficacious interventions to improve child survival and healthy development. However, most of these interventions have traditionally addressed proximal determinants with limited effort on distal determinants since many of them lie outside the health sector. Recent calls were made for interventions to address the more distal determinants of health outcomes ,3. To miTo assess inequalities of child health and nutrition, the World Health Organisation recommends use of linear growth retardation (stunting) -6. In thStunting varies systematically with socio-economic status with the poorest most affected by it. This relationship is observed in a number of studies in the African region -18. UnfoThe present study was conducted in a rural setting, in Uganda. It examines the independent influence of four different components of socio-economic status: mothers' education, fathers' education, household asset index, and land ownership on growth stunting, a proxy for health inequalities. Results of this study should provide policy makers and programme managers with useful information in child health and nutrition planning for rural areas.The study was carried out in Hoima district, which is located in western Uganda and shares a border with the Democratic Republic of Congo along Lake Albert. The district has a population density of 80 per square kilometre with a total population of approximately 370,000, comprised of mainly peasant farmers of the Banyoro tribe . The disThe present survey utilised a two stage probability proportional to size cluster design . Data waA structured questionnaire constructed in English and translated into Runyoro, the local language, was pilot tested and updated in survey similar settings and administered face-to-face to mothers/caregivers in their home settings. Trained non-medical university students gathered data in September and October 2002. To minimise bias, training of interviewers emphasized proper and random identification of respondents, eligible children, and questionnaire administration. A village leader followed data collectors through the village, and traditional village protocol was observed.Information about the educational level of parents, household durable assets, materials of the dwelling structure and land ownership was collected as proxy makers of household socio-economic status. Child's age was obtained through birth certificates, health cards, or recall using a calendar of local events while taking precautions to minimize field errors . RecumbeThe dependent variable (stunting) was constructed using child length, age and sex, and was defined as length less than minus 2 standard deviations (<-2 SD) from the median of the NCHS/WHO reference population .The education level of fathers and mothers was assessed on scales ranging from (1) never went to school, to (7) college/university. Two categorical variables were constructed yielding four categories for mothers: (1) no formal education, (2) stopped in primary, (3) completed primary (minimum 7 years of school) and (4) stopped above primary. Five categories were yielded for fathers because they were more educated: (1) no formal education, (2) stopped in primary, (3) completed primary, (4) stopped in secondary, and (5) completed secondary 4 or above (minimum 11 years of school).Household durable assets  were assessed as (1) available/ in working condition and (2) not available/ not in working condition. Four components of the dwelling structure were assessed including number of rooms, roof – (1) thatched, (2) corrugated iron sheets or tiles; floor – (1) mud, (2) cement; wall – (1) thatched, (2) mud and pole, (3) unburned bricks, (4) burnt bricks built with mud, (5) burnt bricks built with cement and (6) cement/concrete blocks. A household wealth (asset) index was constructed from 15 variables (household durable assets and dwelling structure) using principal components analysis . The indLand is usually given special socio-economic status in many subsistence agricultural communities in Uganda . In ordeChild age in months was assed as a continuous variable and a categorical variable was constructed yielding four categories: (1) 0–5 months, (2) 6–11 months, (3) 12–17 months, and (4) 18–23 months. Child sex was ordinal (1) male, (2) female.Data was entered in Epidata software , and wasApproval of the study was granted by Makerere University Faculty of Medicine Ethics and Research committee, the Uganda National Council for Science and Technology and the Regional Committee for Medical Research Ethics, West Norway (REK vest).Of the children studied 366 51%) were females. The median age (inter-quartile range) was 10 (5–16) months for females and 11 (5–16) months for males respectively. The mean age difference between boys and girls was not statistically significant (p = 0.34). A total of 148 (21%) mothers and 59 (9%) fathers never attained any formal education  had a graded pattern with stunting across strata with the least educated or most poor being more likely to have stunted children. The corresponding p-values for test of trend was <0.001, 0.03 and 0.01 for mothers' education, fathers' education and household wealth index, respectively (Table The extent of association between socio-economic indicators was examined. The correlations (Spearman's rho) between mothers' and fathers' education was 0.50; mothers' education and household wealth index = 0.37; mothers' education and land ownership = 0.12; fathers' education and household wealth index = 0.39; fathers' education and land ownership = 0.14; and household wealth index and land ownership = 0.18. Their colinearity was sufficiently low to allow them into the same model.The four socio-economic indicators , child age and sex were simultaneously entered in a backward conditional logistic regression. Only mothers' education, child age and sex appeared in the last step model. Children belonging to mother with no formal education or to mothers who stopped in primary school were significantly more likely to be stunted compared to their counterparts with mothers who were educated beyond primary school OR = 2.1, Table , Table 1Stunting differentials for both sexes within and across socio-economic indicators did not favour male children. There was a graded relationship of stunting among males unlike females with almost all socio-economic groups except land ownership Table . More maThis study examined how socio-economic indicators namely mothers' education, fathers' education, household asset index and land ownership relate with inequalities in child health and nutrition using growth stunting as the proxy for the inequalities. Findings showed that mothers' education was a robust predictor for inequalities of child health and nutrition. Secondly, boys were more affected by low socio-economic status than girls. Indirectly the study also indicates that it may be unsuitable to bunch together socio-economic indicators as they could have low covariance or could conceptually not be interchangeable and thus unable to serve as adequate proxies for one another.While interpreting findings of this study one should consider the fact that some of the fixed factors known to influence linear growth such as birth weight ,8 and maThe socio-economic predictor variables used in the study were assumed to represent distinct dimensions reflecting different domains of influences from the society. The relatively low colinearity between the indicators used supports this assumption. This favours discussions on choice of socio-economic indicators where researchers are urged to use a number of socio-economic indicators rather bunching them together as each could have its unique contribution ,32.Our results regarding the relationship of socio-economic status with stunting are similar to findings in other studies -18. As oThis emphasizes the need for promoting the education of the girl child with a principal aim of improving child health, which is in line with recommendations by UNICEF . HoweverAdditionally, mothers' education could be used as a suitable indicator to guide effective targeting especially with regard to identifying the most vulnerable families in rural subsistence agricultural communities. This is in appreciation of the fact that achieving universal coverage for child-health interventions presently lies far beyond the capacity of many health systems in low-income countries ,35. It iIt was surprising that land ownership and access, perceived by many in Uganda as the ultimate form of security or socio-economic status , showed Concordant with findings in other studies ,36, age This study highlights the importance of maternal education in child well being. From our data any increment in maternal education is likely to have a positive influence on child growth. Governments of low-income countries need to ensure that the girl child receives appropriate formal education. For effective targeting of families with children in greatest need or at highest risk of health and nutrition hazards, policy makers and programme managers could be guided by mothers' education. Findings that males appear to be more adversely affected by poverty than their female counterparts corroborate evidence from previous research.All authors participated in the design of the study, the interpretation of findings and write-up of the manuscript. HW coordinated and supervised field data collection and performed the statistical analysis. All authors read and approved the manuscript.The authors declare that they have no competing interests.


An increasing number of microbial genomes are being sequenced and deposited in public databases. In addition, several closely related strains are also being sequenced in order to understand the genetic basis of diversity and mechanisms that lead to the acquisition of new genetic traits. These exercises have necessitated the requirement for visualizing microbial genomes and performing genome comparisons on a finer scale. We have developed GenomeViz to enable rapid visualization and subsequent comparisons of several microbial genomes in an interactive environment.Here we describe a program that allows visualization of both qualitative and quantitative information from complete and partially sequenced microbial genomes. Using GenomeViz, data deriving from studies on genomic islands, gene/protein classifications, GC content, GC skew, whole genome alignments, microarrays and proteomics may be plotted. Several genomes can be visualized interactively at the same time from a comparative genomic perspective and publication quality circular genome plots can be created.GenomeViz should allow researchers to perform visualization and comparative analysis of up to eight different microbial genomes simultaneously. Escherichia, Bacillus, Helicobacter, Mycobacterium, Streptococcus, Staphylococcus and Listeria are available. Genomic data too, is diverse, ranging from COG functional classification data [Current efforts in genome sequencing have led to a rapid increase in the number of microbial genome sequences. A total of 522 ongoing microbial genome projects are listed in the GOLD database  and whilion data ,4, expreMicrobial Genomes Viewer [GenoMap [GenomeAtlas [GenomePlot [BugView [Genome2D [Several genome visualization tools have been developed in the last few years. The s Viewer  offers a[GenoMap  can be uomeAtlas  providesnomePlot  also pro[BugView  is anothGenome2D  offers uGenome2D  and ACT Genome2D  are convGenomeViz has been programmed in ActiveTcl. ActiveTcl  is availGenomeViz uses the concept of "tags" which may be applied to groups of genes for classification-type data. A tag file is tab-delimited text file of three columns. It has the "tags", their colors, and their brief descriptions. A pre-prepared tag file for the COG functional categories is available for immediate use. The map file has all the information required to create the plot . It is also a tab-delimited text file. Both file formats (tag and map) are easy to manipulate in a spreadsheet application like Microsoft Excel. However, care must be taken while manipulating data in such applications since errors may creep in the data as demonstrated by Zeeberg et al. . The mapIn order to minimize initial difficulty that users may encounter in creating their own map files, we provide pre-prepared map files for over a hundred genomes. Of these nearly seventy genomes are loaded with the COG classification scheme and may be used immediately. The program also performs checks on the input map file for possible formatting errors and attempts to indicate location of errors (if any) before creating the plot. Several types of plots may be created; on either single or double strands and color gradients and line-graphs are available for numerical data. Once the plots are done, mouse-over on any gene immediately displays associated information from the map file in a display area.Using GenomeViz, it is also possible to search, highlight and retrieve genes of interest. Each loaded genome may be queried separately. Regular expression searches are fully supported and results are highlighted in the genome. For instance, the simple expression ribosomal|ribosome will mark in color all ribosomal proteins in any genome and retrieve all the information for these genes from the map file. The "|" operator is the standard OR operator in Tcl expressions. Genes involved in iron metabolism/regulation which are usually annotated with keywords like ferrous, ferric or iron may be retrieved with the expression "ferric|ferrous|iron". The results can be saved as a text file. Users can also use their own annotations to visualize and query their genome of interest provided these annotations are available in the map file format. It is also possible to display, in different colors, the results of different queries on the same genome by changing the search color before performing a search. This will enable visualization of, for instance, the distribution of genes/operons involved in iron and zinc metabolism/regulation separately.A 'Select COGs' option enables one to retrieve all genes from a particular COG category, e.g. "Cell division and Chromosome partitioning" or "Transcription". Each loaded genome can thus be queried separately. Usually, this is more useful when using a special tag file  that colors all genes as "grey", so that a neutral background is available for highlighting the distribution of genes of a particular COG category over the entire genome. Categories of interest can be highlighted in different colors simply by changing the selection color before selecting the category. Results of each query are also displayed in a text box from where they may be saved as a text file. Thus, GenomeViz allows a rapid overview of the similarities in distribution of various functional categories in closely related genomes Figure . It is aSeveral options are available for printing the circular plot. The graphics can be directly sent to the printer or saved to a PostScript file and read by standard graphics programs. A number of page size options are available and extra large plots spanning many pages may also be printed. A detailed program manual is available with notes on installation, usage and examples.We describe a rapid and convenient application GenomeViz for simultaneous visualization and comparison of varied genomic data from several microbial genomes. Future updates for software and data will be available from the project home page.Project name: GenomeViz• Project home page: • Operating system(s): Linux, Solaris, Unix• Programming language: Tcl/Tk• Other requirements: ActiveTcl, PERL• License: Free for academic use• Any restrictions on use by non-academics: Contact corresponding author for a license.• COG: Clusters of Orthologous GroupsSIGI: Score-based Identification of Genomic IslandsRG conceived the program, wrote and tested it, prepared the manuals and the website. TC oversaw the entire development process. TH offered suggestions on program features. RG and TC prepared the manuscript. All authors read and approved of the final manuscript.


Coordination of [Ca2+]i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca2+]i activity in individual cells and a robust coordination of [Ca2+]i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca Two-photon laser scanning microscopy was used to image calcium concentration changes in astrocytes in the cerebral cortex of anesthetized rats Astrocytes are nonneuronal cells of the brain with some known and hypothesized functions  in the cytosol undergoes large changes spontaneously or in response to various physiological and pharmacological manipulations, such as mechanical stimulation, membrane potential depolarization, and activation of metabotropic glutamate receptors  and/or metabotropic glutamate receptors  ester in juvenile rats in vivo. We find that [Ca2+]i dynamics in astrocytes is rather quiescent during baseline anesthesia. However, increased population bursting, brought about by attenuating γ-aminobutyric acid (GABAA) receptor-mediated neurotransmission, leads to increased magnitude [Ca2+]i surges, and the [Ca2+]i changes become more strongly coordinated in neighboring astrocytes.We have used two-photon laser scanning microscopy (2-PLSM) to monitor cytosolic CaTo examine the depth of penetration of the Fluo-4 AM, coronal brain slices (300 μm thick) were acutely prepared after the residual dye was washed off from the craniotomy. A large number of cells below the craniotomy showed fluorescence labeling . On the 2+]i transients , cells with astrocytic appearance (n = 185) were selected for long-term (10–20 min) monitoring. For quantitative studies, three states of [Ca2+]i activity were distinguished: (a) quiescent state with very slow (less than 0.025 Hz) oscillations of baseline fluorescence level, (b) [Ca2+]i spikes , and (c) [Ca2+]i plateau potentials . [Ca2+]i spikes and [Ca2+]i plateau potentials were automatically detected. In the control (baseline) condition, 11% of astrocytes had at least one spike event, and 52% had at least one plateau event in 10 min. The mean frequency of [Ca2+]i spikes among the cells that had at least one [Ca2+]i spike was 0.121 ± 0.098 per minute  and the mean frequency of [Ca2+]i plateau was 0.118 ± 0.058 per minute (mean duration: 160.4 ± 114.9 s).In our initial experiments, we made a large number of line scans (sampling rate ∼200 Hz) of dye-loaded cells to examine whether some of them were neurons. We never observed short-lasting [Ca2+]i dynamics were affected by increasing neuronal activity, we induced regularly occurring population bursts by local application of bicuculline (n = 7 rats). Large amplitude (0.69 ± 0.26 mV) synchronous field events (approximately 100 ms) occurred at relatively regular frequency (0.15 ± 0.06 Hz), associated with multiple unit discharges. No significant difference was observed in average heartbeat frequency between the control sessions and bicuculline sessions .To investigate whether the baseline values of [Ca2+]i in astrocytes (n = 214 cells). First, the incidence of [Ca2+]i spikes and plateau events was counted in the absence and presence of bicuculline-induced population bursts. Under bicuculline condition significantly more astrocytes had [Ca2+]i spikes , whereas the probability (52% versus 54%) of plateau potentials did not differ significantly. The mean duration of plateau potentials, however, was significantly longer  after bicuculline treatment. Among the cells that exhibited at least one spike or plateau event, there was not a significant difference in frequency of the event occurrences . Thus, the major difference between control and bicuculline conditions was the higher proportion of active astrocytes under bicuculline.We used two methods to evaluate the effect of neuronal activity on [Ca2+]i changes in the frequency domain. The 0ΔF/F trace was considered as a continuous process, and the power spectrum estimate was calculated with a multi-taper method for each astrocyte and averaged across cells. There was a general increase of power at all frequencies in bicuculline-treated animal. The most consistent significant increase (p < 0.05) of power appeared in the frequency range of 0.10–0.24Hz, reflecting the increased incidence of [Ca2+]i spikes. Short-term cross-correlation of neuronal field bursts and [Ca2+]i signals (± 10 s) did not show a significant time-locked relationship  and distant cell pairs (greater than 50 μm). In control conditions, the temporal correlation of [Ca2+]i signals in neighboring pairs was somewhat larger than in distant pairs, but this difference was not significant . Nevertheless, [Ca2+]i signals in astrocytes were not completely random, since the cross-correlograms had wide central peaks at the 10–100 s scale  was similar to those in the control condition. However, synchrony between local pairs  increased several-fold relative to both distant pairs under the same condition  and to local pairs in the baseline condition .In individual experiments, propagation of synchronous activity could be observed visually  but the  s scale B. In conuculline C. Correln = 1,512 cell pairs, r = 0.019, p = 0.46). In contrast, a significant negative correlation was found between the synchrony of [Ca2+]i signals in the bicuculline condition .Using a different approach, the magnitude of the zero-timelag correlation coefficient for each cell pair was plotted against distance between the cell pairs. Under control condition, no notable relationship was observed between these variables  and NMDA receptors  in close contact with small brain vessels . It has ompounds . Furtherompounds . These fMale and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in 3, 1.1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol.Fluo-4 AM  was mixed with 2 μl of Pluronic  and 5 μl of dymethyl sulfoxide  for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF)  was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons  were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method (NW = 4). For the calcium event detection, 0ΔF/F signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of 0ΔF/F signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of 0ΔF/F (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50% 0ΔF/F from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the 0ΔF/F signals to unity  so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted.Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change 2PO4, 155.17 mM NaCl, 2.96 mM NaHPO4, pH approximately 7.4), the slices were treated with S100B antibody  in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody  in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity  because this antibody stains the somatic region of astrocytes as well as its processes . Followiactivity .Video S1The primary somatosensory cortex (P15) was stained with Fluo-4 AM in vivo and subsequently imaged in vitro. Acute slices (approximately 300 μm thickness) were cut in cold ACSF after the cells were loaded in vivo. .(49 MB AVI).Click here for additional data file.Video S2Same slice as shown in (48 MB AVI).Click here for additional data file.Video S3Detailed imaging of in vivo-loaded acute slice preparation of the primary somatosensory cortex . Z step = 1 μm; scale bar = 20 μm.(39 MB AVI).Click here for additional data file.Video S4Empty circles in layers II/III, presumed unloaded neurons (note their absence in layer I). The loaded cells have typical glial morphological appearance. Z step = 1 μm; scale bar = 50 μm.(50 MB AVI).Click here for additional data file.Video S5Acute slices (300 μm thickness) were prepared from the in vivo Fluo-4 AM-loaded somatosensory cortex. The slices were subsequently incubated in EDAC containing saline followed by formalin fixation. The loaded astrocytes were identified by examination at various depths and numbered (left). Next, the slices were processed for immunocytochemistry with astrocyte marker S100B. Depth scans (1 μm between the frames) were taken again to determine immunoreactivity of cells with S100B (right movie). An overlapping set of the cells was identified to be S100B-immunoreactive, indicating that nearly all Fluo-4 AM-loaded cells were astrocytes.(5 MB AVI).Click here for additional data file.Video S6Movie taken from a P14 rat. Image was taken with 2 Hz sampling rate for 10 min and compressed to 36 s for display purposes. Note spatial- and light-emission-stability of the recorded cells. Note also that at frames approximately 9 s and 15 s, two of the astrocytes in the middle display transient increased fluorescence. Scale bar 50 micro μ.(55 MB AVI).Click here for additional data file.


Adenoid cystic carcinoma is a rare malignant parotid tumor. Metastasis can occur even a decade or more after initial treatment of the primary.We report a 60 year old female patient who presented with adenoid cystic carcinoma of the parotid gland. She underwent a total conservative parotidectomy followed by adjuvant radiotherapy. While on follow up, patient developed multiple liver metastases which manifested three years later. Patient lived for another two years before she died of her disease.Although distant metastases of adenoid cystic carcinoma develop frequently, isolated metastasis to liver is unusual. Even after manifestation of distant metastasis, patients can be expected to live for a number of years. Palliative chemotherapy can be considered in symptomatic cases while the usefulness of metastatectomy is controversial. Adenoid cystic carcinoma (ACC) is a rare malignant neoplasm of the salivary gland. Salivary gland neoplasms constitute 3% of cancers of all sites, of which, 10–15% are malignant . Though A 60 year old woman presented with a small swelling beneath the right ear lobe of 4 months duration. The swelling measured 2 × 1 cm placed in the superficial part of the parotid and was not fixed. There was no facial nerve palsy or palpable cervical nodes. A fine needle aspiration cytology (FNAC) was carried out which showed the lesion to be ACC [Although ACC is the second most common malignant salivary gland neoplasm and constitutes approximately one third of all salivary gland malignancies it constitutes only 2% of parotid neoplasms . As ACC Radiotherapy has been used as a primary modality for patients with surgical contraindications and in those with unresectable neoplasms. Though no improvement in survival is reported, the use of adjuvant radiation improves locoregional control and disease free survival. This patient received adjuvant radiation and did not have any locoregional recurrence.Regional metastasis is less common occurring in about 17% while systemic failure occurs in 33 to 50% of the patients ,8. ThougACC is a rare malignant tumor of parotid gland. Metastasis can manifest very late and hence a long term follow-up and a high index of suspicion is necessary to diagnose them early. An annual ultrasound study of abdomen would be desirable on follow-up. Unlike metastasis from other malignancies, these grow indolently and long term survival can be expected even with multiple metastases as also evidenced in the present case. Chemotherapy could be considered in selected patients as a therapeutic option in metastatic disease.ACC: Adenoid cystic carcinomaFNAC: Fine needle aspiration cytologyGy: GrayNone declaredKH was the principal clinician who planned the evaluation and procedure, in addition to conceptualizing and drafting the article.SRMG was the pathologist.Both the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The aim of this study was to explore the ethical challenges in surgery from the surgeons' point of view and their experience of being in ethically difficult situations.Five male and five female surgeons at a university hospital in Norway were interviewed as part of a comprehensive investigation into the narratives of nurses and physicians about being in such situations. The transcribed interview texts were subjected to a phenomenological-hermeneutic interpretation.No differences in ethical reasoning between male and female surgeons were found. They reasoned in both action and relational ethical perspectives. Surgeons focused on their relationships with patients and colleagues and their moral self in descriptions of the ethical challenges in their work. Dialogue and personal involvement were important in their relationships with patients. The surgeons emphasized the importance of open dialogue, professional recognition, and an inclusive and accepting environment between colleagues.The surgeons are personally challenged by the existential realities of human life in their relationships with patients. They realized that ethical challenges are an inherent part of performing surgery and of life itself, and say that they have to learn to "live with" these challenges in a way that is confirmed both socially and by their inner moral self. This means accepting their personal and professional limitations, being uncertain, being fallible, and being humble. Living with the ethical challenges of surgery seems to contribute to the surgeons' confidence and vulnerability in their professional identity. It is important for surgeons to be and to act in a right and good way towards patients, relatives, and colleagues. Studies have shown, however, that physicians often are in doubt about the best and correct actions to take for the patients in specific situations -3. This A relational ethical perspective means reflecting on the challenges we encounter in our relationships with others and how to fulfil our social roles and obligations in a good way – as a human being, a surgeon, and a colleague. It tries to answer questions such as "How can I adequately meet the challenges that confront me in the relationships in which I am involved in this situation?" . The quaAction and relational ethical perspectives are not interchangeable as surgeons have a dual responsibility for their actions in specific situations as well as their way of being in their relationships ,7. BeingMany authors argue that the respect and trust in the physician-patient relationship have eroded in recent years in spite of the physicians' increased therapeutic capabilities ,12. ShorMedical problems are always existential problems too because suffering, anxiety, life, death, and cure involve the core of human existence. Physicians are working with emotionally intense issues and have to accept the possibility of failures, continuing suffering, and death on the part of their patients . MeetingAlthough most clinicians are aware of the uncertainty and the limitations of medicine and their responsibility to try to reduce the likelihood of error, ,20 the bSurgeons' relationships with their colleagues have become more important as surgery has changed from relying heavily on the performance of individual surgeons to relying on a team of providers ,25. SeveThe vulnerability of patients is emphasized in the literature of medical ethics. Less is written about the vulnerability of the physicians in their relationships with patients, relatives, and colleagues. Being involved may engender feelings of helplessness and vulnerability in the physician. MacLeod  argues tStudies show that physicians seem to experience uncertainty and fallibility in different ways. Less experienced female physicians in paediatric care put on an air of certainty while the more experienced gained a kind of security by allowing themselves to feel uncertain . Female Few empirical studies have been found that explore the ethical challenges of surgery from the surgeons' point of view and their experience of being in ethically difficult situations. The present study is part of a comprehensive investigation of ethical reasoning among male and female physicians and nurses within surgical units. The results of this interview study with male and female surgeons will be presented in two articles. The present study describes the surgeons' experiences of being in ethically difficult situations from a relational ethics perspective. The other paper describes the ethical dilemmas as experienced by the surgeons from an action ethical perspective. The results from the interviews with the registered nurses (RNs) working in surgical units are in progress and will be addressed in a third paper.The aim of this study is to explore the meaning of being in ethically difficult situations in surgery as narrated by male and female surgeons.th Regional Ethics Committee in Norway.Five male and five female surgeons working on surgical units at a university hospital in Norway participated in the study. All were experienced and had been working in health care from 9 to 31 years (median = 21.5), and in surgery between 5 to 21 years (median = 13). The surgeons worked full time and were on duty when the interviews were conducted. No individual characteristics will be disclosed in order to guarantee confidentiality. The surgeons gave their informed consent to participate in the study, which was also approved by the 5The interviews were conducted by the first author and lasted from 35 to 75 minutes (median = 55). They were tape recorded and subsequently transcribed verbatim. The interviewees were asked to tell about one or more ethically difficult care situations that they had experienced in their work as surgeons. What constituted an ethically difficult situation was not defined, allowing the interviewees to determine what they considered ethically difficult themselves. The aim of the interviews was to obtain as many rich narratives as possible without interrupting the surgeons' narrative flow and reflection. If the surgeons did not spontaneously reflect on the events they talked about, their reflections were sought. Questions were asked when the interviewer wanted the interviewees to elaborate on their stories or had difficulty understanding the narration. These questions referred to the interviewees' thoughts, feelings, and actions . Field nThe method of interpretation used was inspired by the French philosopher Paul Ricoeur's phenomenological hermeneutics , and devnaïve reading was made of all the transcribed interviews as a whole to gain a first impression of the surgeons' experiences of being in ethically difficult situations during their clinical work. The repeated naïve reading was made as open-minded as possible, without any deliberate analysis of the text. The naïve reading shows the direction the structural analysis may take. Second, a structural analysis was performed in order to validate or refute the initial understanding obtained from the naïve reading and to explain what the text was saying. The interviews were divided into meaningful parts and patterns, i.e. one sentence, parts of a sentence, or a whole paragraph with a related meaning content. The meaning units were condensed and discussed among all the authors, and themes and subthemes were identified, and presented in 'Results'. Third, a comprehensive understanding was developed, taking into account the authors' pre-understanding, the naïve reading, and the structural analysis (results). The text was read as a whole and interpreted in relation to relevant theories of ethics and results from previous investigations into the meaning of being in ethically difficult care situations  what the situation is. I never try to conceal anything. That will only torment them".Talking openly with patients is also important for the surgeons in situations when they are in doubt about the right thing to do. They experienced relief if the patient had an answer to their doubts. They said that patients who are seriously ill usually have thought about life and death issues and have a conception of their condition, and that the question of withholding or withdrawing treatment seldom comes as a surprise to them. Some patients say that they have lived a good life and do not want an operation. Others strongly want an operation even though their prognoses are poor. "We often reach an agreement about ending a treatment that either does not lead to a meaningful life afterwards or leads to a life that would be experienced as a heavy burden".The surgeons said that they become personally involved with their patients, focusing on patients as persons and their quality of life as much as on their medical treatment. Being involved and knowing the patients' background and what they really want in life makes difficult ethical decisions easier to handle. The surgeons explained that caring for the patient can be felt as a personal, emotional burden. "It's not easy when people you have established a relationship with die. The only way to run away from it is to relinquish your responsibility. But that means disassociating yourself from or rejecting the patient. So you have to care, to be involved and to be a human being".The surgeons are involved in many patients' lives and destinies and said that keeping a certain distance protects their feelings and is a way of caring for themselves. They said that keeping a certain distance is necessary in order to give the patient neutral advice and the most suitable medical treatment. The surgeons feel a responsibility to care in situations where they find it difficult, for instance when they dislike the patients' personality, behaviour, or values. They said they work hard to get involved and care for demanding and non-compliant patients.Ethical challenges are discussed in both formal and informal social arenas among the surgeons. All new patients are presented at the daily morning meeting between all the surgeons, including what had been done to them and why. Only questions and short objections to the patients' diagnostics and treatments are raised at these meetings or shortly after. The surgeons arrange separate meetings to discuss problematic cases. "We assemble the nurses, the anaesthesiologists, the surgeons, and even other specialists like the nephrologists when we have patients who reside a long time in the intensive care unit. In a way we create a meeting-place for the case and discuss whether we should withdraw a life-sustaining treatment or start additional treatment for a kidney failure for instance." The informal running dialogues during the day were equally important for the surgeons. "You have to make the decision yourself, but we always discuss the problem together before difficult decisions are made. The discussions do help and are experienced as mutual support". The surgeons expressed confidence in the consensus that usually is achieved in these discussions. "I know that I would be content to receive the treatment we agree upon myself".The surgeons emphasized the importance of having a caring relationship with their colleagues. They said that talking together and giving and receiving collegial support is necessary in order to live with the personal responsibility of being in ethically difficult situations and with the emotional burden of decision making. "People say that surgeons are a bit tough and do not talk about feelings, and that may be true. But in my experience we really care for each other. Perhaps we do not go all mushy about our feelings, but we understand when someone is in difficulty. I have experienced receiving good support in such situations. Colleagues contact you and say: "Ok, listen, a couple of years back the same thing happened to me", or: "You must not take this too hard, it could have happened to anybody". That helps".Personal and emotional support is informally and silently shared among trusted colleagues and great value is attached to it. After having presented a difficult decision about withholding treatment at a morning meeting, one of the surgeons commented: "That same afternoon, four or five of the most experienced surgeons came to me independently, and told me that they thought that what I had done was great. They said that most surgeons were not able to do what I had done. I remember it well because I think it was so well said".The surgeons emphasized the importance of "playing with an open hand" and that openness and honesty in the relationships with colleagues presupposes a trusting atmosphere that allows everybody to feel free to voice their opinion and be listened to. They feel that it is important that everyone who is involved in the treatment and care of the patient should have an opportunity to express their opinion and to be heard before any final decision is made. Openness and honesty are particularly important when medical errors occur or when something has gone wrong during an operation. Talking about medical errors or mistakes is considered an opportunity for learning and for improving surgical routines. "You have to have an including and accepting environment that allows you to say that you could have chosen a different solution. If there is no room for you saying something like that, then there will be a tendency to conceal it. We all make mistakes and we all make wrong deliberations and sometimes choose bad solutions. We have to live with that. Therefore it's important that we try to learn from the cases where something [erratic] happens".The surgeons focused on the necessity of dialogue and cooperation with their colleagues. "We are used to working close together in a team and it makes these difficult situations easier to handle". They found it satisfactory to work in a hospital because "there is always someone you can ask for advice when in doubt". They said they find it easier to talk to patients about difficult treatment options when the question has been discussed with experienced colleagues or senior surgeons first. This is especially important for less experienced physicians. The surgeons also make a contrast between the importance of cooperation with colleagues and the burden of being alone and being responsible decision makers.The surgeons said that they experience ethically difficult situations as an important part of their everyday activities that cannot be separated from the rest. "These situations are a part of our profession that are not necessarily experienced as difficult, but are sometimes unpleasant to be in". They said they have to experience these situations personally and be involved in order to understand them and to learn to live with them.Some ethically difficult situations are experienced as dramatic and tragic, especially when the surgeons feel the personal responsibility for saving the lives of trauma patients after major accidents. "If you are not able to cope with being in this situation and be responsible, and be the leader of the trauma team trying to save peoples' lives after a serious accident, if you cannot do that but go on wondering if you have done something wrong, then I think you will find something else to do".The surgeons told about situations where they are alone on duty and responsible for rapid deliberations and decisions in acute and emergency situations as especially challenging. "There are no other times in this job when you feel as lonely as you do in those situations". The decision whether to continue or withdraw treatment from traumatized young patients and children is experienced as especially challenging to make alone. "It's not easy [being alone]. It's the kind of decisions you often ponder about for several days afterwards, also at home. It's the sort of decisions you really try to closely think through, and it often troubles you even after you have made a decision".The surgeons said they have to learn to live with the unpredictable consequences of their decisions. "The practice of surgery is very specific and you feel more responsible in a way than in other areas of medicine. You have the feeling of being the direct cause when things go wrong, e.g. that you operated at the wrong time, you operated incorrectly or that you should have found the right diagnosis earlier".The surgeons spoke about "living with" the inherent uncertainty of surgery and emphasized that they can never be completely sure of the right thing to do in ethically difficult situations. They have to live and work with the uncertainty of the course of the disease, the patients' chances of survival, the risk of serious and fatal complications and the uncertainty of the patients' quality of life after extensive operations. The surgeons said they have to learn to live with the uncertainty of their deliberations and operations as there can be no right answer to ethical challenges, and no criteria to guide them when they make their decisions.Living with uncertainty is experienced as both frightening and satisfying. The surgeons said that not knowing what to do in an uncertain situation, finding a way, and succeeding in their attempt to restore health or save life is a satisfying aspect of their work. They also commented that they have to live with their doubts and fears of being too active and the risk of knowingly imposing severe complications and a poor quality of life on their patients.The surgeons said that their aim is to make all patients better. Accepting the limitation of surgery and not being able to cure a particular patient or alleviate his or her suffering is not an easy task for surgeons. They commented that it feels difficult not to be able to or not to be allowed to help patients. Sometimes they feel guilty if the treatment does not succeed, but said that they have to accept the possibility of making mistakes as an essential part of the profession. "You have to face the reality of how things are. What we are dealing with are human beings in marginal situations where things can go wrong. Everybody who is dealing with these things makes errors of deliberation and judgment. It's a part of the game that you have to live with when the margins are so tight. It would not be better if you quit. Your dearly purchased experience will be of use to nobody then".The surgeons said that they are focused on healing and curing and that they try to operate on the patients in most situations. "I believe that surgeons feel that it's good to do something, curing, and saving lives. That is what we have learned to do. We are in a profession that can do many useful things and that is the gratifying part of practicing surgery, – that you can identify a problem and do something about it".The clinical experience of deliberating and choosing and finding workable solutions in clinical and ethical difficult situations makes the surgeons confident. "Most surgeons are action-oriented. If not, it's almost impossible to practice surgery, because you have to makes decisions all the time and be accountable for your decisions". Receiving social confirmation from patients, relatives, and colleagues when they succeed contributed to the surgeons' experience of confidence in their own decisions and actions. The surgeons also said that having the courage of your convictions and a set of personal ethical values is equally important in order to do a "proper and conscientious job". This means acting according to the patients' best interests and the standards of their profession. Their conscience helps them to decide which action is morally wrong, to make controversial decisions, and to voice personal, professional, and moral opinions to colleagues and patients.The surgeons emphasized that although they deal with existential issues of life and death, they do not rule over these human conditions. "The essence is that we are not almighty. We do not save lives, we just postpone death. The only thing that is certain is that we are all going to die. You are just doing repairs and trying to help the patients to live a bit longer. " "Fortunately it's not we who rule over life and death. What we can do means less than you think and that is how it should be. I think it's important that we all acknowledge this and everyone else too".All the surgeons said they are against active euthanasia. They also commented that a humble attitude in their work helps them acknowledge their personal and professional limitations and to recognize what they cannot and ought not do. "There is a dimension in surgery of being right about having a good reason to operate and being considerate about the benefits of carrying out an operation".The surgeons in this study reasoned in a relational ethics perspective, focusing on dialogue, openness, and involvement in their relationships with patients and colleagues. While the surgeons in the interviews described their experience of being in ethically difficult situations, it seems at the same time that identity was central to their experience of ethics and their enactment of moral agency. The surgeons identified themselves by telling about their ethical experience, expressing the way they are living their identity. By narrating our lived experience, we give meaning to our experiences. We understand ourselves through the stories we tell and live, as well as those told about us and by interpreting them. Personal identity is said to be constructed through the stories we tell about our lives, stories which are in turn shaped by more general institutional, cultural, or national meta-narratives that live within the culture, of surgery, medicine, and society -42. ThusBeing open and honest about all aspects of patients' treatment and care is experienced as important for the surgeons. This is even true in situations when they have to disclose bad news and tell the patient that they are not able to or did not succeed in their efforts to restore health. Openness of speech is one of the spontaneous expressions of life, designated as "utterances of life" according to the writings of Løgstrup . That opHuman life means expressing oneself with the expectation of being met by others according to Løgstrup . OpennesAlthough disclosing bad news to the patient is experienced as an emotional challenge by the surgeons, concealing the truth does not seem to be an option they consider. Concealing the truth from the patient is experienced as difficult. It seems that surgeons need to share their experiences by speaking openly to patients so they can live with their demanding professional life in a satisfactory way. Therefore, honesty and openness are important because the relationships with patients are important for the surgeons' lives.The surgeons said that it is important not to hide their uncertainty and doubts from themselves or from their patients. They accepted that uncertainty is an inherent part of their profession and realized that they often have to decide in spite of lack of scientific knowledge. They also deal with problems that may have no desirable solutions. Previous studies of surgeons , female The surgeons said that they sometimes feel guilty when they have to disclose the bad news to patients that the treatment did not succeed. We feel guilty when we fail to do what is required of us in a situation according to Løgstrup , either Feeling guilty is our fundamental ethical condition as human beings according to Pahuus  because The surgeons said that they experience a relief when the patients give answers to their own uncertainty and doubts about the right or good thing to do. It seems as if the surgeons are relieved when patients want to decide for themselves and take responsibility for their lives, appreciating that they do not hand over this responsibility to the surgeons. The surgeons in this study do not fit into the traditional and stereotypical picture of the paternalistic and authoritarian physicians . On the The surgeons emphasized the importance of being personally involved with the patients by focusing on patients as persons and their quality of life as much as their medical treatment. However, they experienced the challenge of finding the balance between involvement and distance in the relationships, between caring for the patients and themselves, and in giving professional and neutral information. Distance based on knowledge, skills, and experience with previous cases and situations is necessary in order to help the patients in a professional manner ,46. PersIn their narratives, the surgeons focused on cooperation and the relationships with their colleagues. They experienced being listened to and together they seem to create and recreate a collegial environment where they can express their doubts and admit their errors of deliberation and decisions.The surgeons said that they experience taking difficult decisions alone as a burden. Working in a hospital is experienced as satisfying because they appreciate working together. They seemed to accept that they are mutually dependent on each other and appreciated the support they receive in dialogue with colleagues. Interdependence and exchange is a fundamental way of human lives according to the ethics of Løgstrup . He callTo be seen and listened to by colleagues, be taken seriously, and comforted when something goes wrong is an answer to fundamental human needs. Openness about personal doubts and failures also make the surgeons vulnerable. The interdependency among surgeons in this study is not experienced as a burden, but as mutual support. The surgeons' acceptance and appreciation of their collegial dependency may be an answer to why surgeons are able to stand in ethical difficult situations and live with them in a satisfying way. The dialogue with colleagues reduces the surgeons' doubts, although their doubts cannot and do not disappear.The surgeons receive social confirmation for being good surgeons through dialogues with patients, relatives, and colleagues. Their experience of being of use to society as a whole also confirms their identity as good surgeons. To be confirmed is to be seen, listened to, and accepted by other persons. A person's identity is dependent on other persons in good or bad relationships according to Ricoeur , as wellThe surgeons in this study emphasized the importance of dialogue with patients and colleagues. All identities are socially bestowed, socially sustained, and socially transformed and people make meanings of their actions by participating in communicative contexts. We all need recognition and social confirmation from others in order to construct our identities ,48. ThusThere is a close relationship between identity management and values in a society since human beings usually want to be something they find valuable. What constitutes morality is created, recreated and communicated in social relations ,49. NarrThe modern self is described as multi-voiced and dialogical and moral action is determined through a process of inner dialogue. To open up to the different voices within and enter into a dialogue, one can either reach consensus with oneself or experience conflict and dissatisfaction with oneself ,49. The The modernization and secularization of western societies emphasize the ideology of individual autonomy and freedom of choice. At the same time, the way in which social structures control and restrict individual performance and freedom becomes less visible. It is not in every person's or profession's power to choose, in other words, act as his/her conscience directs . The surAccording to the surgeons, their choices of actions and ways of being in relationships with patients and colleagues have to be accepted and confirmed by their inner moral selves. They speak about "having the courage of your own conviction", "taking a stand", "having a personal set of ethical values" as important in order to "live with" the ethical challenges of surgery. The surgeons in this study revealed their identities through their narratives about being in ethically difficult situations.Being a surgeon means having the necessary specialized knowledge, skills, and experience to perform complicated, extensive, and technically advanced operations in principally uncertain and unpredictable situations. This identity is socially sustained through dialogues with patients, relatives, and colleagues. To have an identity as a surgeon is also to understand that one is vulnerable and exposed in these relationships and to know what is at stake in one's own life as well as in the lives of the patients and colleagues.In this study uncertainty, fallibility, and humility seem to be equally important in the surgeons' identities as are their responsibility and confidence. The social importance and benefit of their work to patients and society is confirmed both socially and by their moral selves. This seems to outweigh their personal uncertainty and vulnerability. Thus, surgeons seem to appreciate their work and have learned to live with, and to a certain extent even appreciate, its most difficult dimensions. The meaning of being a surgeon is to be able to live with the tensions of contradictions, ambivalence, dilemmas, and paradoxes in their practice and to find workable solutions in these situations.The author(s) declare that they have no competing interests.KT participated in the design of the study, carried out the interviews, participated in the analysis and completed the manuscript.AN read the interviews, participated in the analysis and helped to draft the manuscript.VS participated in the design of the study, read the interviews, participated in the analysis and helped to draft the manuscript.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here: