DNA repair genes encode proteins that protect organisms against genetic damage generated by environmental agents and by-products of cell metabolism. The importance of these genes in life maintenance is supported by their high conservation, and the presence of duplications of such genes may be easily traced, especially in prokaryotic genomes.Xanthomonas species were used as the basis for phylogenetic analyses of genes related to DNA repair that were found duplicated. Although 16S rRNA phylogenetic analyses confirm their classification at the basis of the gamma proteobacteria subdivision, differences were found in the origin of the various genes investigated. Except for lexA, detected as a recent duplication, most of the genes in more than one copy are represented by two highly divergent orthologs. Basically, one of such duplications is frequently positioned close to other gamma proteobacteria, but the second is often positioned close to unrelated bacteria. These orthologs may have occurred from old duplication events, followed by extensive gene loss, or were originated from lateral gene transfer (LGT), as is the case of the uvrD homolog.The genome sequences of two Xanthomonas, seem to have arisen from old events and possibly enlarge both functional and evolutionary genome potentiality.Duplications of DNA repair related genes may result in redundancy and also improve the organisms' responses to environmental challenges. Most of such duplications, in  The availability of complete genome sequences from different organisms makes it possible to identify, by similarity, potential homologs of genes that have been experimentally tested in other living beings, resulting in the recognition of putative functions for the proteins encoded by them. This research concept represents a revolutionary tool in modern biology, as the data generated allow for recognition of the presence or absence of genes, giving indications of the metabolic pathways present in such organisms and revealing possible particularities of the individuals in their natural habitat. Moreover, the possibility of obtaining gene sequences from organisms that have long diverged makes it feasible to use these data to trace their evolutionary origin . ComputeAll known forms of life present efficient systems to maintain the integrity of their genetic material. As DNA is under constant attack by different environmental agents and metabolic by-products, evolution has provided organisms with several DNA repair pathways to remove or to tolerate lesions in their genetic material. In fact, these pathways have at least two important contrasting roles in evolution, safeguarding the genome, and allowing for a certain level of mutations in the course of evolution. The critical balance of these two activities is probably the best reason for the high levels of conservation observed in DNA repair related proteins, even across the three kingdoms, Bacteria, Archaea and Eukarya. Detailed studies of many of the different DNA repair genes and protein domains have been described previously ,7, confiXanthomonas axonopodis, X. campestris and Xylella fastidiosa, we found that certain DNA repair genes present duplications in both Xanthomonas species. The great relevance of gene duplication for expanding gene families, as well as for gene innovation, is a consensus among researchers. New genes can facilitate survival in new environments or make possible the use of new metabolites. We investigated the phylogeny of such duplicated genes, in search of evidence on the mechanisms by which cells evolve their DNA repair machinery. Although most DNA repair genes follow the conserved vertical transmission found for 16S rRNA phylogeny, the data clearly show that duplications may have arisen by different means. Examples of a recent duplication, as well as of other old or LGT events, generating paralogs, are presented. These results also help in tracing the origins of LGT events that lead to redundancy and also to functional diversification, especially of Xanthomonas bacteria.In this work, we investigated the DNA repair genes in the recently described genomes from the Xanthomonadales group ,9 followX. fastidiosa, when compared to both Xanthomonas species: ada-alkA (fusion), tag, phr, dinB and ligB. For evolution purposes, however, the presence of several duplications, especially in the Xanthomonas genomes, will be focused here.DNA repair-related genes are normally highly conserved, and grant good genetic information for investigating the evolution of organisms. Orthologs of known bacterial DNA repair proteins were identified in the plant pathogen bacteria of the Xanthomonadales group and are listed in Table Neisseria and Ralstonia).The RecA protein is almost universal among bacteria and is a clear example of phylogeny that follows 16S rRNA gene evolution. In fact, this gene is always unique in all genomes analyzed and has been proposed as an alternative molecule to be used in systematic studies of Bacteria . Figure E. coli, the RecA protein has been shown to participate in recombinational repair, as well as in the control of a set of physiological changes to DNA damage, known as SOS response. The induction of this regulon stems from the co-protease activity of the RecA protein, which cleaves a repressor protein, denominated LexA, allowing the expression of the set of SOS genes. The presence of lexA and recA homologs in the Xanthomonadales indicates that these bacteria also present a SOS regulon. However, in Xanthomonas genomes, there are two copies of the lexA gene. The phylogeny of LexA proteins is presented in Figure lexA gene is not found in all groups of bacteria. The products of the two copies of the Xanthomonas lexA paralogs are positioned close together in the same branch of Xanthomonadales, indicating a recent duplication of such a gene. The fact that they had branched before Xylella's single lexA divergence indicates that this bacterium may have lost the duplication. Gene losses are expected to be extensive in Xylella, in agreement with this bacterium having a more specialized parasitic way of life [lexA in Xanthomonas points to a highly controlled SOS regulon, that may be important for fine-tuning the bacterial responses to stress induced in environmental changes. Indeed, functional characterization of lexA1 in X. campestris has been performed by gene disruption, and the data indicate this protein controls the expression of the recA gene, as expected for the SOS regulatory circuit [lexA gene in Deinococcus radiodurans does not change the level of RecA expression, suggesting that LexA protein may be related to functions other than controlling the SOS regulon in that organism [In  of life . The dup circuit . The funorganism .uvrB gene from X. campestris has been cloned and it was shown to participate in the resistance of these bacteria after UV irradiation [Methanothermobacter thermautotrophicus, Methanosarcina sp and Halobacterium sp. NRC-1 [The nucleotide excision repair (NER) pathway is one of the most important, versatile and conserved systems of DNA damage removal in bacteria. It recognizes damages, which cause significant helix distortions in the DNA molecule, these being excised as an oligonucleotide by several enzymes that act as helicases and endonucleases. The main proteins that participate in such a pathway are known as UvrA, UvrB and UvrC, which, in sequential steps, interact with one another, recognize the damaged strand (UvrA), open the double helix (UvrB), and cleave DNA (UvrC) at both sides, few nucleotides away from the lesion. Subsequently, the oligonucleotide containing the damage is removed by a DNA helicase known as UvrD. The adiation . The NERp. NRC-1 . Other ap. NRC-1 .uvrA, uvrC and uvrD are duplicated in Xanthomonas and the phylogenies of the proteins encoded by these orthologs, together with the single uvrB, are presented in figure Xanthomonas being positioned in the same branch of the Xylella's ortholog, close to gamma proteobacteria as expected for a vertical descent. However, for the duplications, the patterns are different and more complex. The second ortholog of uvrA in X. axonopodis is phylogenetically closer to the also duplicated genes found in several other bacteria (figure D. radiodurans) points to an origin due to horizontal gene transfer from other bacteria. The functions of both uvrA orthologs are not necessarily related to DNA repair, since this activity can be provided by either one of them. It is relevant to mention that for the duplication found in D. radiodurans, White et al [The genes a figure , thus inte et al  have prote et al .X. axonopodis is close to the uvrC orthologs from other gamma proteobacteria, following a vertical transmission. However, a protein with considerable similarity to the N-terminal of UvrC is found in both Xanthomonas species. These orthologs form an independent branch of the phylogenetic tree of UvrC, close to a heterogeneous group of gram-positive bacteria. The role of this second UvrC homolog of E. coli in DNA repair (named Cho protein, for UvrC homolog) has recently been investigated in detail, and was shown to have an endonuclease activity in damaged DNA [in vitro [Azotobacter vinelandii, Escherichia coli, Salmonella typhimurium, Shiguella flexneri, and Xanthomonas. Curiously, in Beta proteobacteria, Ralstonia metallidurans and Chromobacterium violaceum, an endonuclease domain similar to Cho appears as a C-terminal fusion with a putative 3'-exonuclease, corresponding to the epsilon subunit of DNA polymerase III. A similar fusion protein was found in Mycobacterium tuberculosis, and an interesting coordinated action of these two activities (endonuclease and exonuclease) was proposed as a new mechanism of DNA repair [cho in these bacteria, but, again, one would have to concede extensive gene loss in other species. Once more, horizontal transfer events, involving part of the uvrC gene  to some gamma proteobacteria, could explain the limited occurrence of cho only in these bacteria. Moreover, some organisms present a duplication of the complete uvrC gene (Clostridium acetobutylicum and Listeria). The proteins encoded by these paralogs are closely positioned in the tree, thus indicating a recent origin for them, and, possibly, functional redundancy.A similar pattern of evolution was observed for the phylogeny of UvrC protein figure . One of aged DNA . This prin vitro . Cho mayin vitro . The oriA repair . A formeuvrD gene is part of a DNA helicase family, which includes the pcrA and rep genes [uvrD gene participates in the removal of damaged DNA strand, after the incision steps of NER or DNA mismatch repair have occurred. The evolution tree of these proteins is presented in figure uvrA and uvrC, there are two orthologs for these genes in the X. axonopodis genome. One of the orthologs encoded by the uvrD gene is closer to the gamma proteobacteria, branching with X. fastidiosa, following a typical vertical inheritance, while the second is similar to proteins from other prokaryotes, including Archaea and Bacteria, mainly from the firmicutes and alpha proteobacteria groups. The second ortholog of this gene from Sinorhizobium meliloti is found in plasmid DNA for which an alien origin is suggested, due to its lower GC content [uvrD duplication in the X. axonopodis genome indicates that it is located close to transposon-related genes  in both Xanthomonas is another remarkable feature of BER in this group. Phylogenetic trees generated for this protein family show these orthologs located in different branches, indicating that the duplication is not a recent event. However, a miscellaneous branching pattern of the tree obtained for the different bacterial groups, results difficult to track the evolution of these genes (data not shown).Base excision repair (BER) protects genetic material from a wide range of DNA damaging agents . The plaNA genes . This duet al [ligA gene, encoding the NAD-dependent DNA ligase, is found in the Xanthomonas and Xylella genomes. The phylogeny for these proteins is presented in figure Xanthomonas genomes present extra copies of putative ATP-dependent DNA ligases (two in X. axonopodis and three in X. campestris). The phylogenetic tree of ATP-dependent DNA ligases is shown in figure Mycobacterium tuberculosis and Streptomyces coelicolor. The other bacterial orthologs branch independently, wherein alpha proteobacteria predominate. A curious observation is the high and variable number of ATP DNA-ligases within the genomes of plant symbiontes, especially in the alpha proteobacteria (six in Agrobacterium tumefasciens, nine in Sinorhizobium meliloti and eleven in Mesorhizobium loti). It is possible that these X. axonopodis genes may have arisen from recent duplications, soon after gene introduction in alpha proteobacteria, probably by means of LGT from Archaea. The fact that these bacteria inhabit a common niche in plants could facilitate mutual gene exchange. However, the function of such ligases in these bacteria is unknown. As the NAD (+)-dependent ligase is present in all bacterial genomes, these Archaea-related ligase orthologs, present in certain bacteria, seem to be redundant, their roles in cell metabolism remaining a puzzle. Since NAD+-dependent DNA ligases are typically eubacterial, and cannot be replaced [+-dependent DNA ligases appear to perform more accurate ligation reactions, with few mispaired nucleotides being allowed in the DNA extremities [+-dependent DNA ligase from Thermus species exhibit enhanced mismatched ligations under certain conditions but catalyze reactions with 1–2 orders of magnitude more discriminative towards correct nucleotide matches than the ATP-dependent DNA ligase from T4-phage [Thermococcus kodakaraensis, Nakatani et al [The DNA ligases catalyze the joining of breaks in the phosphodiester backbone of the DNA molecule and, thus, play an essential role in several processes of DNA repair, replication and recombination. These enzymes are evolutionary related, although two distinct families of DNA ligases are found: one that is typical for Bacteria, using NAD+ as cofactor, and a second that is typical of Eukaryotes and Archaea, but using ATP as cofactor . As forreplaced , the preremities . NAD+-deT4-phage . Workingni et al  found thAgrobacterium sp T-DNA [Rhizobium species amplicons present in pNGR234 plasmid [Xanthomonas the presence of the ATP-dependent DNA ligase could also be linked to the elevated number of transposable elements [Therefore, the presence of ATP-dependent DNA ligases in certain bacteria could be connected to the ligation of DNA breaks under several contexts that would generate genetic variability, with possible evolutionary advantages. In the alpha-proteobacteria group, a non homologous end joining appears to be important for the integration of inserting elements in the genome of host plants, as occurs with sp T-DNA  or for t plasmid . In Xantelements . Howeverphr) in X. fastidiosa, while present in both Xanthomonas, may be related to its limited habitat within the plant xylem or inside the insect vector [X. campestris bears the umuC and umuD genes, which encode the DNA polymerase V (UmuD'2C), related to translesion synthesis [X. axonopodis uvrD2 gene.Some other features of the DNA repair genes in Xanthomonadales are interesting to mention Table . The abst vector . It is aynthesis . In factynthesis , suggestXanthomonas (5.1 Mbp) are much larger than that of X. fastidiosa (2.7 Mbp), it was not a surprise to find more duplicated DNA repair genes in the former bacteria. Mechanisms for the protection of genetic information may reflect how the organism deals with stress and a hostile environment. Thus, the increased number of DNA repair genes in Xanthomonas may be due to the fact that these bacteria have a more variable habitat when compared to Xylella, which lives most of the time inside its hosts as a parasite. In this work, DNA repair genes, which appeared duplicated in the genomes, were analyzed focusing on their evolution, although it should be pointed out that their functions in vivo remain to be investigated. The duplicated genes found in Xanthomonas have close orthologs in other bacteria. As Xanthomonas are very closely related to Xylella, it is thus possible that, in these cases, duplications arose before the split Xanthomonas-Xylella, and were lost in the latter.Since the genomes of lexA paralogs of Xanthomonas, but this seems to be an unusual example. The possibility that gene duplications would have occurred in an early common ancestor, followed by gene loss in most other bacteria, cannot be the only explanation for many of the genes analyzed. Therefore, horizontal gene transfer among different bacteria possibly originated some of these paralogs. A clear example of recent LGT is the uvrD duplication, which is often found associated with DNA mobile elements. An origin from other life kingdoms may also have occurred, as for the ATP dependent DNA ligase, a common ligase in Archaea, which may have been acquired and were established in certain bacterial genomes, including Xanthomonas. The LGT, more than any other genetic process, makes possible faster ecological changes with the immediate incorporation of a gene or group of genes [For the genes investigated, evolution patterns indicate that duplicated genes may result mainly from relatively ancient origins. Phylogenetic errors of construction, such as long branch attraction effect, cannot be completely excluded, although the presence of close orthologs reinforce the trees generated. Moreover, the duplications are normally positioned near to orthologs also found in the genomes of some distantly related bacteria. A clear exception is the most likely recent duplication found for the of genes . Eventuaof genes ,33. In fof genes .uvrC homolog (cho), which has been found to have a complementary function in E. coli [The new genes acquired by lateral gene exchange are expected to be maintained when they provide a selective advantage to the recipient cell . For the E. coli -20. The Mycobacterium tuberculosis some DNA repair genes are induced by DNA damage, independently of the RecA protein [uvrA, uvrD and ligB, which are duplicated in Xanthomonas. This novel mechanism present in M. tuberculosis may also occur in Xanthomonas and support the idea that these duplications are also required protecting the genome against damage. Curiously, the duplicated genes found in Xanthomonas do not replace the orthologs that present vertical inheritance, similar to 16S rRNA. This reinforces the idea that they may complement the known DNA repair mechanisms with other different functions. The search for such novel functions for these genes may not only improve our knowledge on how cells protect their genomes against DNA damage, but also about how DNA repair processes evolve in bacteria.In  protein . Among tXanthomonas was performed comparing the two different genomes of the species X. axonopodis and X. campestris. As most of their genes are very similar, only X. axonopodis homologs are shown, differences being described in the text. Protein sequences from complete genomes (Bacteria and Archaea) were aligned using the ClustalX multiple sequence alignment program [Sequences of DNA repair-related proteins were obtained at the National Center of Biotechnology Information GenBank database (35). The list of organisms, with abbreviations used and proteins analyzed, is shown in Table  program  with man program . Parsimo program . Bootstr program . The samXanthomonas sp. All authors read and approved the final manuscript.MM-P carried out the phylogenetic analyses and, together with RSG and CFMM, designed and conceived the ideas and the writing of the manuscript. CL gave substantial contribution on the possible involvement of ATP-dependent DNA ligases, when present in bacterial genomes. KML-B and KAA participated in the sequencing and DNA repair genes annotation in Accession numbers for the genes indicated in the figures. Contains all accession numbers of the genes used in the phylogenetic analyses.Click here for file


We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an  Platelet activation and aggregation play an important role in the development of cardiovascular disease, which is the leading cause of death in the United States. Over the last several years, enormous advances have been made in understanding the molecular mechanisms that regulate platelet function. A general paradigm has emerged that endothelial cells synthesize and release substances that inhibit platelet activation (e.g. nitric oxide and prostacyclin) except near a site of vascular injury, while platelets synthesize and release substances that promote further platelet activation. In contrast to this paradigm, several recent studies have suggested that, in the presence of certain agonists (IGF-1, adenosine diphosphate, insulin, and isoproterenol), platelets will produce nitric oxide (NO), a potent inhibitor of platelet activation . Althoug2+ mobilization [Platelet-derived NO is synthesized by membrane-bound endothelial-type nitric oxide synthase (eNOS). A common signaling mechanism shared by many eNOS agonists is binding to a surface receptor, followed by activation of phosphatidylinositol-3-kinase (PI3K), which results in phosphorylation of eNOS at serine 1179 through Akt -8. The alization . The con-17 mole NO/platelet (determined by microelectrode in PRP for 2 minutes following addition of 5 μM ADP), is sufficient to alter platelet function [As a small, hydrophilic free radical NO is highly diffusible in the aqueous environment of the blood. However, it is also highly reactive with a very short half-life estimated to be only on the order of a few seconds  in the bThe purpose of our study was to determine if platelet-derived NO plays a role in thrombus formation in the presence of shear stress. We examined the effect of several external factors on platelet thrombus formation, including insulin, the β-adrenoceptor agonist isoproterenol, and shear stress. Previous studies showed that insulin and isoproterenol both induced NO formation by platelets ,4. Shear-1 are shown in Figure Platelets from whole blood adhered avidly to the collagen-coated surface in the presence of physiologically relevant levels of fluid mechanical shear stress. Very little, if any, platelets were detected on the albumin-coated portion of the slide. Detectable levels of platelet adhesion were evident within 20–30 seconds of the initiation of blood flow over the surface. Platelet adherence occurred predominantly at the interface between albumin and collagen and increased as a function of time similar to results that have been obtained by others using a similar system -16 Repre-1 resulted in increasing inhibition of platelet accumulation on the surface. Higher concentrations of either insulin or isoproterenol had no additional affect on the extent of platelet accumulation on the surface (data not shown). Inhibition of thrombus formation was significant (p < 0.05) up to 6 minutes with 1000 pM insulin and up to 3 minutes with 1000 μM isoproterenol. Significance was determined with SPSS using the Post Hoc test as described in Methods.The effect of insulin and isoproterenol on mural thrombus formation was studied and the results are presented in Figures -1. In the presence of L-NAME as well as either insulin or isoproterenol, thrombus formation is increased, returning to levels seen in the absence of added agonist. This suggests a role for nitric oxide in mediating the agonist induced reduction in platelet accumulation on the surface. Platelet thrombus formation on the surface at lower (100 s-1) and higher (2000 s-1) levels of shear rate are shown in Figures -1) platelet accumulation on the surface is not significantly altered by either insulin (500 pM) or isoproterenol (100 μM). Higher concentrations of either agonist had no affect as well (data not shown). In addition, L-NAME by itself or in combination with either agonist did not significantly affect platelet deposition on the surface  as well as L-NAME, a specific inhibitor of nitric oxide production. The results are presented in Figures  Figures . At the  Figures , platele-1 we observed a curious result when the blood was treated with L-NAME but in the absence of any agonist. The results are presented in Figure -1 even in the absence of insulin or isoproterenol.At the high shear rate of 2000 s-1 and in the presence of insulin was slightly increased by addition of ODQ. No significant effect of ODQ on thrombus formation was observed with isoproterenol  that we tested, platelet derived NO did not appear to play a role in modulating platelet accumulation on the surface. This result was not surprising due to the many types of adhesion receptors on platelets that function well in a low shear environment. The primary platelet receptors for collagen III on a surface are GPVI and α2β1 [2β1 and αIIbβ3 into a high affinity state through a process termed inside-out signaling. In this high affinity state, these integrins are capable of supporting platelet-platelet interactions and promoting thrombus growth. Therefore, platelet adhesion at low levels of shear rate can be mediated by a number of different adhesion molecules, some of which do not require platelet activation. Our results are consistent with this model since we found that platelet derived nitric oxide did not inhibit the rate of thrombus growth at these low, venous levels of shear rate.At the low, venous level of shear (100 sand α2β1 ,24. Addiand α2β1 . Because-1), integrins α2β1 and αIIbβ3 play a more significant role in platelet adhesion and recruitment. These platelet integrins are essential for thrombus formation at intermediate to high shear [At intermediate shear  and in the presence of whole blood. Platelet-derived NO did not affect platelet adhesion at low shear but had a significant effect at intermediate and high shear. Production of NO in platelets was dominated by receptor-mediated interactions at intermediate shear and mechano-transduction at high shear. This study and future work may lead to a better understanding of platelet-derived NO and its role in healthy and diabetic vascular wound healing.Low molecular weight heparin, HEPES, NaCl, collagen III from calf skin, bovine serum albumin (BSA), L-nitro-amine-methyl-ester (L-NAME), insulin from porcine pancreas, and isoproterenol were obtained from Sigma. Recombinant hirudin (r-hirudin) was obtained from Pentapharm and 1H-,4oxadiaz2O and 10 mL 95% ethanol. After washing was complete, the cover slips were then placed in a 95% ethanol bath for at least 12 hours. Before use, each cover slip was rinsed with an additional 20 mL 95% ethanol and allowed to air-dry for 15 minutes.Each cover slip was washed with 20 mL nanopure HCollagen III solution was prepared at 0.8 mg/mL in 17 mM acetic acid (pH 2.6) at least 24 hours prior to use. BSA solution was prepared at 0.1% in 10 mM HEPES/115 mM NaCl buffer (pH 7.4). Half of each cover slip was coated with collagen and allowed to incubate for 4 hours in a humidified environment (80–90%) at room temperature (~24°C). After incubation, each slide was rinsed with 10–15 mL of HEPES/NaCl buffer solution to remove excess collagen and coated with 0.1% BSA solution for at least 1 hour.Venous blood collected from healthy donors (30–60 mL, depending on shear rate and length of experiment) was anticoagulated with low molecular weight heparin to a final concentration of 20 U/mL or r-hirudin to a final concentration of 40 anti-thrombin units/mL (ATU/mL). The fluorescent dye mepacrine was then added to a final concentration of 10 μM and allowed to incubate for 10–15 minutes. All donors gave informed consent to participate in our study according to methods approved by the University of Oklahoma Institutional Review Board.in vitro parallel plate flow chamber similar to those previously characterized [-1, 0.013 × .8 cm for shear rate of 500 s-1, and 0.013 × .25 cm for shear rates of 1,000 and 2,000 s-1.The arterial flow environment was modeled with an cterized ,33. The In some studies, 500 pM insulin or 100 μM isoproterenol was added to the anti-coagulated blood 10 minutes before the start of an experiment. In other studies, L-NAME, an inhibitor of platelet NO synthesis was added at a concentration of 200 μM for 20 minutes before the start of an experiment. In additional studies, ODQ, an inhibitor of guanylate cyclase was added at a concentration of 100 μM for 20 minutes before the start of an experiment ,19,34. IIn all studies, blood was used in experiments within two hours of collection. The order of experiments was varied randomly to insure that time-dependent platelet phenotype was not skewing the results. Additionally, flow cytometric studies of platelets showed no significant change in platelet activation, as measured by P-selectin expression, over the course of 4 hours (data not shown).® image analysis software by Inovision. A background image was acquired after blood began flowing over the surface but before adhesion of any platelets. This image was subtracted from subsequent images. Platelets were identified based on size and intensity using adjustable parameters. Images were acquired and analyzed every 10–30 seconds over the course of each experiment, which lasted from 5–10 minutes.A syringe pump provided the flow through the flow chamber. The flow chamber was placed on the stage of a Nikon Diaphot 300 inverted microscope. The illumination was provided by a 75W Xenon light source passing through a 480 nm excitation filter and a 40 × fluorite objective lens. The fluorescent emission from adherent platelets was passed through a 514 nm filter and converted to analog signal by an image intensifier and CCD camera. The image was recorded on VHS tape for subsequent analysis. The extent of mural thrombus formation on the surface was quantified in terms of the percent coverage of a representative area of the collagen-coated surface at the interface between the collagen and albumin. The analyzed area was approximately 100 μm wide by 150 μm in the direction of flow. Digital image analysis was performed on an SGI Indy workstation running the ISEEStatistical significance of the results was assessed with the aid of the software package SPSS (version 11.5 for Windows). The statistical tests used were the Post Hoc test for multiple comparisons and the student's t-test. Results were deemed significant if p < 0.05.BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; eNOS, endothelial nitric oxide synthase, FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; L-NAME, L-nitro-amine-methyl-ester; NO, nitric oxide; ODQ, ,2,4oxadiThe authors declare that they have no competing interests.RW performed all of the experiments and MN conceived of the project and coordinated the data analysis. All authors read and approved the final manuscript.


Human endogenous retroviruses (HERVs) comprise a large class of repetitive retroelements. Most HERVs are ancient and invaded our genome at least 25 million years ago, except for the evolutionary young HERV-K group. The far majority of the encoded genes are degenerate due to mutational decay and only a few non-HERV-K loci are known to retain intact reading frames. Additional intact HERV genes may exist, since retroviral reading frames have not been systematically annotated on a genome-wide scale.gammaretroviral types. One encodes a protein similar to a recently discovered zebrafish retrovirus (ZFERV) while another shows partial, C-terminal, homology to Syncytin (HERV-W/FRD).By clustering of hits from multiple BLAST searches using known retroviral sequences we have mapped 1.1% of the human genome as retrovirus related. The coding potential of all identified HERV regions were analyzed by annotating viral open reading frames (vORFs) and we report 7836 loci as verified by protein homology criteria. Among 59 intact or almost-intact viral polyproteins scattered around the human genome we have found 29 envelope genes including two novel .This compilation of HERV sequences and their coding potential provide a useful tool for pursuing functional analysis such as RNA expression profiling and effects of viral proteins, which may, in turn, reveal a role for HERVs in human health and disease. All data are publicly available through a database at  This stands in stark contrast to various types of repetitive DNA, and it has been estimated that transposable elements alone take up almost half of our genome [pol) [env) [Retroviridae most of the seven genera are represented by endogenous members, and HERVs are divided into class I, II and III depending on sequence relatedness to gammaretroviruses, betaretroviruses or spumaviruses, respectively. Many HERVs are named according to tRNA usage (i.e. HERV-K has a primer binding site that matches a lysine tRNA), while others have been more or less provisionally named by their discoverer. It seems increasingly clear that the nomenclature for endogenous retroviruses (ERVs) needs to be revised to accommodate such wide diversity. Furthermore, it is evident that many more ERVs are yet to be discovered as retroviral elements are present in most, if not all, vertebrates and even in some invertebrates [Phylogenetic analysis of the retroviral polymerase gene (pol)  and envel) [env)  have idel) [env) ,5. Withitebrates ,7.With a single exception (HERV-K) all HERV groups are ancient (i.e. entered the genome prior to human speciation) and entered our genome at least 25 million years ago ,8,9 presi) replication and (random) insertion of new proviral structures, ii) effect on adjacent cellular genes, iii) long range genomic effects and iv) expression of viral proteins (or RNA). Since the majority of HERVs are highly defective no de novo insertions have been observed and presumably HERV mobilization very rarely results in spontaneous genetic disorders or gene knock-outs as seen with other active retrotransposons such as L1 elements [gag, (pro), pol and env lead to expression of 3 viral polyproteins (Gag, Gag-Pol and Env) that are processed by a viral or host protease into the active structural and enzymatic subunits. Although most HERV genes are no longer intact, a small fraction has escaped mutational decay. For a subgroup of HERV-K (HDTV) all proteins can apparently be expressed and particle formation has been detected in teratocarcinoma cell lines [env genes from HERV-H [The presence of endogenous retroviral sequences in our genome has several possible implications: elements . Howeverelements . Moreoveelements . Such efelements . The frell lines . Furtherll lines ,22. Locim HERV-H , HERV-W m HERV-H  and HERVm HERV-H . Conservm HERV-H ,27. Recem HERV-H  was idenm HERV-H .i) the possible association of Rec (HERV-K) with germ-cell tumors [ii) the immunosuppressive abilities of HERV-H Env in a murine cancer model resulting in disturbed tumor clearance [iii) the possible superantigenic (SAg) properties of envelopes from HERV-K and HERV-W [In the literature, expression of HERVs has frequently been linked with human disease including various cancers and a number of autoimmune disorders . While cl tumors , ii) thelearance  and iii)d HERV-W ,34 and td HERV-W , schizopd HERV-W  and typed HERV-W . SAg expd HERV-W ,38. One Among Env-associated effects the mechanism of SAg-like activity is believed to involve true epitope-independent stimulation of T-cells, while the mechanism of action of the immunosuppressive CKS-17-like domain is still unknown. This immunosuppressive peptide region maps to the envelope gene  and may pol gene [env gene [env genes by nucleotide similarity [et al. [env genes in a recent survey by de Parseval et al [Computer-assisted identification of HERV loci has previously been reported. These include searching conserved amino-acid motifs within the pol gene ,40 and eenv gene , detectimilarity  and compmilarity ,42. Curr [et al. ,42 provi [et al. . Howeveral et al  and a deIn order to screen the human genome for HERV-related sequences we have performed multiple nucleotide BLAST searches and subsequently clustered neighboring hits into larger regions up to about 10 kb in size Figure . The que Our data include i) chromosomal coordinates and sequence information of the 7836 HERV regions, ii) annotation of ~38000 retroviral ORFs within these regions and iii) graphical visualization of individual HERV regions . Table gag genes (HERV ID 26658–9. W). We also find other examples of closely situated HERV loci as for instance HERV ID 44313 that is composed of two proviruses of distinct origin (HERV-K and a γ-retrovirus-like sequence) both severely degenerated.The 7836 HERV regions (~2.7 per Mb) are not uniformly distributed among the 22+2 chromosomes (χ2 test, P < 10-300), and only 13% of our 7836 HERV regions are situated inside a gene, despite that 33% of the genome is spanned by genes  for intragenic HERVs to be orientated antisense relative to the gene  genes are significantly reduced as compared to a random distribution  are short . Our method only detects a single non-betaretroviral Gag ORF above 500 codons , while two long Pol ORFs are both present in full-length HERV-Fc and HERV-H elements (HERV ID 1178 and 10816) that also harbour intact env genes [Of the ~38000 retroviral ORFs 25% are classified as Gag, 7% as Pro, 55% as Pol and 13% as Env proteins. This correlates well with the expected size of the nv genes ,45.If one extends the search criteria and scans the human genome for retroviral genes where a single mutation (one nucleotide insertion, deletion or substitution) either removes premature termination or restores the correct reading frame, the number of long Gag, Pol and Env proteins increases two-fold to 27, 23 and 43, respectively  shows similarity to a recently reported full-length endogenous retrovirus from Zebrafish (Danio rerio), dubbed ZFERV [gammaretroviruses [gammaretroviruses (weak similarity to Feline leukemia virus). Also, two loci (HERV ID 44200–1 and 44204–5) harbor novel Env-like ORFs that C-terminally show homology to Env from HERV-W/syncytin-1 [pol and env reading frames. The same deletion is present in the HERV-K18 Env locus that has been reported to have SAg-like activity [Our method detects 29 Env ORFs (stop to stop) above 500 codons Table , which ced ZFERV . A phylooviruses . An analncytin-1 ,27 and Hncytin-1 , while tactivity .env genes four of 6 ESTs are also derived from placental tissues.We mapped 265 ESTs to one of the 42 HERV regions that encode a long Gag, Pol or Env ORF Figure . The ESTWe report a mapping of 7836 loci in the human genome that show nucleotide sequence similarity to retroviral genomes and importantly, we provide a detailed analysis of their coding potential by annotation of all viral ORFs (stop-codon to stop-codon fragments longer than 62). This compilation of HERV regions and their corresponding viral ORFs is available as a searchable database . A graphgag, pol or env genes can be restored by a single point mutation or a single insertion-deletion event.The vast majority of the mapped HERV regions contain several frame-shift mutations or in-frame stop codons that truncate the viral ORFs and thus testify to their old association with the human genome. In fact, we detect only 42 proviruses that have retained Gag, Pol or Env ORFs in the size range that approach full-length proteins  in several of these provirus loci strongly suggests that selection has preserved the env genes. In case of syncytin-1 and -2 (HERV-W and HERV-FRD members, respectively) evolutionary conservation can be understood in functional terms, since the encoded envelope proteins have been suggested to play an essential role in placental development by causing trophoblast syncytia formation [Within our list of intact or almost-intact viral ORFs in the human genome, we detect only a single al Table . The fraormation ,48. Compormation ,49,50.env gene. Potential cellular roles for envelope genes that may drive purifying selection include i) protection from infection by related retroviruses by receptor interference as demonstrated for the murine fv4 locus [ii) mediator of organized cell-cell fusion like the syncytin genes [iii) a hypothesized role in preventing the immune response against the developing embryo by means of the immunosuppressive domain [Concerning other ancient loci such as HERV-R (erv3) no evidence for a physiological role has yet been established despite a remarkable conservation and expression of the v4 locus , ii) medin genes -28 and ie domain .env genes not detected in the recent survey of intact human envelope genes [gammaretroviral genera. Whether this gene is still active or whether the encoded protein still maintains function and/or plays a cellular role is yet to be established. Although we were unable to detect any unambiguous EST matches to this gene . BLAST ne Table , RT-PCR ta Table . We havebetaretroviral env genes five turned out to carry a specific 292 bp deletion that fuses the pol and env reading frames. This deletion variant of the HERV-K (HML-2) group is indicative of the type 1 genomes [Among the long  genomes  that des genomes ,54. It i genomes , and a senv gene reported by us was only detected due to inclusion of the ZFERV sequence [Although our analysis is extensive it is most likely not exhaustive. The sensitivity is obviously limited by our query sequences, and some ancient HERVs may have suffered from the mutational decay to a degree which makes is impossible to detect them by homology. For instance, the ZFERV-related sequence , and altsequence  also poisequence . Thus, isequence . Finallysequence ), and lii) there is an unequal distribution of HERVs between chromosomes and along the genome. In particular the Y chromosome stands out with a five-fold excess of our vORF positive  HERV sequences (Table ii) HERVs are underrepresented within genes and iii) HERVs found in introns are predominantly orientated in the antisense direction  and the presence of flanking direct repeat sequences (putative LTRs). Subsequently, ORFs were categorized based on a library of known retroviral proteins and non-retroviral proteins.), with default parameters except for W = 8, E = 0.001, V = 1000000, B = 1000000. Search results were stored in a MySql database and mapped to chromosomal positions using Ensembl Bioperl packages [In order to cover as many different HERV families as possible we compiled a query set of 237 publicly available sequences from Genbank, published papers and Repbase sequences . These spackages .Overlapping BLAST hits were clustered into putative HERV regions allowing a gap of 500 nucleotides between hits. A region-score was calculated based on the sum of e-value weighted hitlengths divided by region length. Only regions longer than 300 nucleotides and a region-score > 3.0  were kept, resulting in 45658 putative HERV region.Detection of direct flanking repeats (putative LTRs) were done by comparing a window before and after the HERV region.pro and pol genes . Therefore the predicted proteins in particular for gag and env genes may contain incorrect N-terminal regions that must be removed by looking for appropriate start codons. ORF lengths below 63 aa were discarded as the probability of finding ORFs less than 63 aa in a random sequence increases to more than 0.05 .For the 45658 putative HERV loci, we scanned the DNA sequence (including 1000 bases flanking the locus) for forward open reading frames (stop-codon to stop-codon) of lengths > 62 aminoacids (aa). Stop-codon-to-stop-codon fragments were chosen to accommodate the use of non-conventional translational initiation by retroviruses at the internal All ORFs were then assigned to a category by FASTA searching against a library of known retroviral proteins (RV) and known non-retroviral proteins (NON_RV). RV proteins were downloaded from NCBI and categorized into either GAG, POL, PRO, ENV, ACC (accessory protein) or UNWANTED (for unwanted or unknown proteins). NON_RV proteins consists of all human SwissProt proteins of length 400–700 aa not including the words "endogenous, virus, envelope, env-, env, gag-, gag, pol-, pol, reverse". The final library consisted of 6260 records (3454 RV proteins + 2806 NON_RV proteins). ORF was assigned to the same category as the highest scoring hit. All loci with a significant RV ORF (vORF) were flagged as HERVs (E < 0.0005) – this data set consists of 7836 loci. Manual inspection of long ORF above 400 codons revealed that two envelope ORFs (ORF ID 86185 and 312172) were (mis)categorized as non-significant (NonS) due to low sequence similarity to our retroviral protein library.In order to match the human ESTs to the vORF positive HERV regions we first performed an all against all search using NCBI MegaBLAST . The outERV Endogenous retrovirusEST Expressed sequence tagHERV Human endogenous retrovirusLTR Long terminal repeatvORF Viral open reading frameThe authors declare that they have no competing interests., while LAA prepared query sequences, detailed analysis of the results and drafted the manuscript and PV and CW performed the statistical analysis. All authors read and approved the final manuscript.The study was conceived by LAA and FSP; PV and LAA participated in designing and coordinating the study; PV carried out all programming and compilation of Table 1. Genes with one or more vORF HERVs inside. Genes were selected from Ensembl (Current Release 21.34d.1) and compared with all HERV regions containing a retroviral ORF. 813 genes (642 with descriptions) contained 1182 HERVs (969) overlapping the gene chromosomal coordinates (exons + introns). The HERV score is a measure of the density of retroviral blast hits in the region.Click here for fileTable 2. ESTs matching HERVs containing a long viral ORF. ESTs were compared to HERVs using megaBLAST. Only ESTs that best matched the target HERV were kept. Finally, ESTs mapping conclusively to the same genomic regions as the target HERV were kept. EST library information (organ and tissue) was parsed from Genbank. The positions are in NCBI35 coordinates due to overly stringent settings of EST mappings in the NCBI34 mapping at UCSC. HERV positions were lifted to NCBI35 coordinates using the "lift genome annotations" tool at Click here for file


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 [Since, in microarray experiments, the sample sizes, i.e. the numbers of replications, are usually very small ,16, the ed value ,18. Thusp-values. In addition, the permutation test reduces, without any loss of power, to a single test if no truncated (or null) values were present. Thus, the proposed test is applicable in routine use whether or not truncated (or null) values occur. After the definition of the tests in the following section, we present our findings for DNA methylation data and microarray data. We then confirm the results using simulations.We found that, in the case of a two-part model, the permutation test is not only a way to avoid the use of an asymptotic distribution, but also is a more powerful test, i.e. a test that produces, on average, smaller n1 and n2 be the numbers of independent observations regarding one gene (or one region in case of methylation data, respectively), for two groups to be compared. The observed numbers of truncated values  in the two groups are denoted by m1 and m2. To compare these numbers m1 and m2 Lachenbruch [As briefly mentioned above a two-part test statistic is the sum of two squared statistics, one comparing the proportions of truncated values and one comparing the positive values. Let henbruch  used the, , and . Under the null hypothesis the proportions of truncated values are not different between the two groups, and B2 is asymptotically χ2-distributed with df = 1. B2 is always well defined, unless there are only truncated values in both groups or no truncated values at all. For these two extreme cases we set B2 = 0.where t test, and the Kolmogorov-Smirnov test. The use of the latter test in a two-part model, however, was too liberal, the type I error rate was close to 0.065 (for a significance level of α = 0.05 [For the second part in Lachenbruch's two-part model one can use different tests, Lachenbruch -8 considα = 0.05 ,8). Bothα = 0.05 ,11. Nonpα = 0.05 .The standardized rank sum statistic based on the non-truncated  values is defined asRS is the rank sum, i.e. the sum of the ranks in group 1. When there are ties within the non-truncated values the denominator slightly changes [[W = 0.where changes [, p. 109]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 (e.g., PostScript and PDF) output for scalable publication-quality results, and raster (e.g., JPEG and PNG) 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 (DICOM, ), wherebOne solution, suggested by , is to dWe are particularly concerned about the use of Caryoscope (or similar) graphics in vector formats (such as PostScript, PDF and SVG) 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 (.g., see ). 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 [The growth of ablation lesions remains a central issue in the development of radiofrequency ablation devices. Knowing the expected shape of lesions is essential for treatment planning and procedure optimization. To date, many approaches have been attempted to characterize lesion size. The results have varied widely. Ablation lesions generated ntrolled ,5. In adMany computational studies have been reported in the literature to predict the growth of lesion size during ablation -19. HoweA(s-1), and the activation energy ΔE (J mol-1). Arrhenius parameters have been determined in skin [A and ΔE determine the amount of cell damage incurred for a specific tissue type. In combination with computational modeling techniques, it is then possible to calculate the distribution of cell damage surrounding ablation probes.Several investigators have demonstrated that tissue damage is a function of both temperature and time -30. As t in skin -35, arte in skin ,37, bloo in skin -40, panc in skin ,42, hear in skin , cornea  in skin -46, musc in skin , prostat in skin , ovary [ in skin , kidney  in skin -52, and  in skin . For a sIn this study, we compare the temperature distribution and tissue necrosis patterns for a hepatic ablation probe at body temperatures. At each time step, the specific absorption rate (SAR), temperature, and the tissue damage are calculated. The level of tissue perfusion is varied for the models to determine the maximum variation in lesion size resulting from a typical ablation. These data are validated experimentally using an ablation probe in liver tissue.Radiofrequency ablation probes operate between 460–550 kHz. At these frequencies, the wavelength of the electromagnetic energy is several orders of magnitude larger than the size of the ablation electrodes. Thus, the primary mode of energy transfer is through electrical conduction and can be modeled as a coupled quasistatic electrical conduction and heat conduction problem. The electric field is solved by using Laplace's equation,σ(T)∇V] = 0     (Eq.1)∇·[where ∇ is the gradient operator, σ (T) is the temperature-dependent conductivity (Siemens/meter), and V is the electric potential (Volts). Temperature is solved by using a modified Pennes bioheat equation ,3 [C is the heat capacity of tissues, 3600 J/kg-K [k is the heat conduction coefficient, 0.502 W/K-m [b is the density of blood, 1000 kg/m3 [b is the heat capacity of blood, 4180 J/kg-K [-3 sec-1 [amb is the ambient body temperature, 37°C; and Qm is the metabolic heat source term. For all cases, we assumed that the metabolic heat source was insignificant. The tissue state coefficient (α) ranges from 0–1 depending on the local level of tissue damagewhere ρ is the density, 1060 kg/m3 ; C is th0 J/kg-K ; k is th02 W/K-m ; ρb is t00 kg/m3 ; Cb is t0 J/kg-K ;α is a t-3 sec-1 ;Tamb is At each time step, the cumulative damage integral is computed using the well established Arrhenius equationc(t) is the concentration of living cells, R is the universal gas constant, A is a "frequency" factor for the kinetic expression (s-1), and ΔE is the activation energy for the irreversible damage reaction (J-mol-1) [A and ΔE are dependent on the type of tissue and have been characterized for liver tissues by Jacques et. al. (A = 7.39 × 1039 s-1 and ΔE = 2.577 × 105 J-mol-1) [where Ω(t) is the degree of tissue injury, J-mol-1) . The kinJ-mol-1) . The parJ-mol-1) . In the J-mol-1) . Once tiFigure N) = σ(25,N) {1.000-1.962 × 10-2Δ + 8.08 × 10-5Δ2σ(T,NΔ [3.020 × 10-5 + 3.922 × 10-5Δ + N(1.721 × 10-5Δ- -6Δ)]}     (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 (VA hybrid finite element model was developed using Femlab (Comsol, Burlington MA, USA) and Matlab (Mathworks, Natick MA, USA) 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 (OR 1.7, 95% CI 0.8–3.5); households belonging in the "poorest" quintile for the asset index compared to the "least poor" quintile (OR 2.1, 95% CI 1.2–3.7); 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 (OR 2.1, 95% CI 1.1–3.9). 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 (cupboard, hurricane lamp, radio, bicycle, fuel, boat, telephone, refrigerator, motorcycle and car) 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.