Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences.For the last decade, our laboratory has been using the sea cucumber actins, and developmental genes, such as Wnt and Hox genes, show interesting expression profiles during regeneration.In the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal regeneration. Here we compared the expression profile of animals at three different intestinal regeneration stages  against the profile from normal (uneviscerated) intestines. The number of differentially expressed probes ranged from 70% at p < 0.05 to 39% at p < 0.001. Clustering analyses show specific profiles of expression for early (first week) and late (second week) regeneration stages. We used semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) to validate the expression profile of fifteen microarray detected differentially expressed genes which resulted in over 86% concordance between both techniques. Most of the differentially expressed ESTs showed no clear similarity to sequences in the databases and might represent novel genes associated with regeneration. However, other ESTs were similar to genes known to be involved in regeneration-related processes, wound healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress response, immune challenge, and neoplastic transformation. Among those that have been validated, cytoskeletal genes, such as Our findings set the base for future studies into the molecular basis of intestinal regeneration. Moreover, it advances the use of echinoderms in regenerative biology, animals that because of their amazing properties and their key evolutionary position, might provide important clues to the genetic basis of regenerative processes. All living organisms exhibit, to some extent, regenerative properties that allow them to deal with environmental events, physical trauma or diseases. Regenerative capacities have been studied in terms of stem cell recruitment, cell dedifferentiation, proliferation and migration, provision of specific regulatory/trophic factors, and expression or re-expression of the developmental program in adult animals . HoweverCentral to regeneration studies is the choice of the model organism, since metazoan species can show large variability in their regenerative capacities. In general, the ability to replace complex body parts decreases as one moves from the basal to the more highly derived taxa. However, even within the same phylum, not all animals are able to regenerate body parts, and not all tissues within a body can be equally repaired ,3. One pHolothuria glaberrima as a model organism for the last decade to dissect the process of how the digestive tract regenerates once it is eliminated by auto-evisceration. We have shown that following evisceration the new intestine regenerates from the free end of the remaining mesentery [Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers (holothurians) are excellent models for studying organ regeneration since they can regenerate many of their organs and appendages. Our laboratory has been using an echinoderm, esentery . Initialesentery -11 as weesentery .We have also used the sea cucumber to explore the role of the genes that allow for such extraordinary regenerative processes . Our appH. glaberrima using microarray technology. For this, microarray slides were made using over 7000 ESTs that we previously identified from normal and regenerating intestine cDNA libraries [We have now overcome this problem by determining the profile of gene activity during intestinal regeneration in ibraries . Here weTechnical analysis showed that the microarrays performed as expected. First, the intensities of the 536 internal controls of the Agilent array perfectly matched the expected ones as shown in the regression model [see additional file H. glaberrima probes in our microarray, 5915 showed similar expression in all groups, including normal (uneviscerated) and 3-, 7- and 14-days post-evisceration (dpe) regenerating intestines.Microarray results showed dramatic differences in gene expression between regenerating and normal intestine and also among animals at different regeneration stages. Of the 14352 Most of the differential expression was observed when normal and regenerating intestines were compared. A large number of probes, 8437 (aprox 58%) showed differential expression at a significance level of p < 0.01 Figure . DiffereIn order to have a graphical representation of the gene expression levels and their significance, we analyzed our results using volcano plots Figure .Results showed not only that a large number of genes are differentially expressed, but a high level of change and significance, mainly during the first week of regeneration. For example, at 3-dpe and 7-dpe the range of odds was significantly larger than at 14-dpe. Comparison with LPS showed a small number of genes being differentially expressed.p-values, we selected the top genes by two criteria: First, those probes with the highest p-value. Second, the fold change of the ratio of the intensities. The sequences were named either with their accession numbers (singlets) or with local identifications for multisequence contigs. All nucleotide sequences have been submitted to NCBI (taxonomy ID: 31192). Genes are shown in order of fold change and probability.The top differentially expressed genes for 3-, 7- and 14-dpe stages of regeneration are shown on Tables cyclophilin, laminin, alpha tubulin, tenascin-R, ferritin. murinoglobulin and NM23. Interestingly, most of the differentially expressed genes showed no significant similarity  to other sequences in publicly available nucleotide or protein databases. Unknown genes, such as Contig 4863-1 and 5300-1, were up-regulated at 3-, 7- and 14-dpe, while the singlet ES728532 showed down-regulation at all regeneration stages. On the other hand, some genes were specific for one stage such as Contig 3933-1 and Contig 4702-1 at 3-dpe. However, these genes were significantly differentially expressed but with higher p-values at other time points.Among the genes in this top 10 list that show known identities, were An alternate strategy to focus on gene groups that are differentially expressed at particular regeneration stages is to compare gene expression among regeneration stages  and not directly to the normal intestine. To do this, we used the expression profile at 7-dpe as reference . Seventeen genes (corresponding to 26 ESTs represented by 55 probes in the microarray) were found to be up-regulated at 3-dpe when compared with 7dpe . Most of these genes have been previously associated with wound-healing, development and regeneration in other animal species. The second group consisted of genes without identity . The validated genes include over-regulated and down-regulated sequences of the microarray.Gene-specific relative RT-PCRs were performed to validate the expression profiles of fifteen genes identified in the microarray as differentially expressed. The sequences of the primers used are shown on Table S1. Validation included genes with putative identity . These genes showed some correlations at particular stages, such as observed for actin 1 and 2 and Hox 12 at 3-dpe. At other stages some of the RT-PCR results although not significant do showed a trend that resembled the microarray results (see actin 3 at 3-dpe). Overall the correlation of RT-PCR and microarray results was 84%, with only 7 of the 45 validated time points not showing the same profile between the microarrays and the RT-PCR. A direct comparison of the RT-PCR and the microarray results is shown on Table The expression profiles of the 15 genes that were validated using RT-PCR confirmed the robustness of the microarray results Figure  and 5. ECluster analysis was performed on the microarray results using hierarchical (not shown) and non-hierarchical methods in order to reveal several distinct profiles of expression Figure .Cluster # A contains probes that were highly over-expressed at 3- and 7-dpe when compared to normal intestine, and whose expression levels are slightly higher in 3- than in 7-dpe and are further decreased at 14-dpe.Cluster # B contains probes that were moderately over-expressed at 3-dpe when compared to normal and whose expression levels decreased at 7-dpe. The expression levels of these probes at 14-dpe were similar to those at 7-dpe.Cluster # C contains probes that were over-expressed at 3-dpe but whose expression peaked at 7-dpe, followed by a decrease in expression levels at 14-dpe.Cluster # D contains probes that showed no significant differences in expression at any stage when compared to normal intestine.Cluster # E contains probes that were slightly under-expressed at 3-dpe when compared to normal. The expression level of these probes was higher at 7- than at 3-dpe and somewhat similar between 7- and 14-dpe.Cluster # F contains probes that were slightly under-expressed at 3- and 7-dpe when compared to normal and whose expression levels increase at 14-dpe.Cluster # G contains probes that were moderately under-expressed at 3-dpe when compared to normal and whose expression levels slightly increase at 7- and 14-dpe.Cluster # H contains probes that were highly under-expressed at 3- and 7-dpe and whose expression levels showed a moderate increase at 14-dpe.S. purpuratus. GO searches  were performed with all the blast results and 1514 unique sequences were found with at least one reported ontology  were "unknowns" that showed no similarity to subjects in the SwissProt database. On the other hand, 41.9% of the sequences (2636) had similarity with database sequences. Of these, 169 showed similarity with hypothetical proteins, most of them from the sea urchin y Figure .In order to determine if particular gene functions could be associated exclusively with specific regeneration stages, diagrams were done with only those genes that showed differential expression between 3-, 7- or 14-dpe and normal. At 3-dpe the GO search showed 1231 results while 1068 were found for 7-dpe Figure  and 7C. In addition to the global view of gene expression provided by microarray analyses, individual gene expression patterns can be determined. We have focused on various genes that have been associated with regenerative processes. For this we only chose those genes whose similarity to genes from other animals was sufficiently high  to be confident that they correspond to holothurian homologues. These genes are classified and presented in three main groups in relation with their function: a) Developmental genes, b) Cytoskeletal-related genes and c) Extracellular matrix-related genes.Many developmental gene pathways have been found to occur or be activated during regenerative processes ,23. Thusp < 0.05. Hox genes stand out among the group of development-associated genes, and our group has previously identified them in the holothurians [Hox 9, 10 and 12 were over-expressed at 3- and 7-dpe and returned to normal levels at 14-dpe. However, while Hox 10 displayed the same levels of over-expression at 3- and 7-dpe, Hox 12 and 9 showed peaks of expression at 3-dpe and 7-dpe, respectively. Hox 5 showed a different pattern of expression altogether being over-expressed only at 14-dpe. Other development-associated genes were also found to be differentially expressed in our microarrays during intestinal regeneration. Once again the expression profile differed among them. For example, Wnt 14 was found to be over-expressed at 3-, 7- and 14-dpe, while BMP-1 was only up-regulated at 7-dpe. In contrast, forkhead box K1 was down regulated at 3-dpe and, similarly, myotrophin was down-regulated at 3- and 7-dpe. Finally, not all development-associated genes showed changes in expression. For example, Kruppel-like factor 2 remained at similar levels of expression during the first two weeks of regeneration.In general, differential expression of these genes mainly occurred at early stages of regeneration. Most of the genes are activated or inhibited at 3-dpe  and 7-dpe . In contrast, at 14-dpe only two developmental genes were differentially expressed, both just at thurians . Four ofHox 12 and Wnt 9 by RT-PCR. The sequences for these genes were obtained from the 7-dpe library and the complete sequence of Hox 12 has been previously characterized by our laboratory [To validate the developmental gene results we chose to study the expression of boratory  RT-PCR sActin 1, Actin 2, Tubulin alfa and alfa-1), others were under-expressed  while others showed no significant changes in their expression (tubulin-beta). Of particular interest were those sequences that showed a specific change at 3-dpe and appeared to only change slightly or not at all at other regeneration stages (tubulin 3 and myosin light 3F chain), since these are more likely to play a role in specific processes occurring during intestinal regeneration.Regeneration induces cellular changes that have been previously reported by our group. Among these are processes of dedifferentiation and migration that are associated with the formation of the new intestinal primordium during early regeneration -9. Such actins 1 and 2 that are over-expressed during early regeneration stages, while actin 3 is down-regulated, was clearly documented in our RT-PCRs.Some of these sequences were validated by RT-PCR. In particular, the differential expression profiles of Echinonectin, Collagen alfa-1, Laminin alpha and Tenascin-R) and three corresponding to matrix metalloproteases (MMPs)   Table . In thisAs mentioned earlier many of the sequences in our microarray slides were unknowns. That is, sequences that showed no similarities to those in databanks. In many cases this was due to the sequence being either too short or being part of the mRNA's UTR. However, in some cases the sequence was long enough to contain a significant ORF that, in principle, should have shown similarities to other proteins when submitted to BLAST searches. These are the most interesting sequences to our research group, particularly those that were differentially expressed during regeneration, since they might correspond to novel holothurian-specific sequences associated with their striking regenerative capacities.One strategy that we explored to study these unknowns was to focus on contigs. That is, those ESTs that shared overlapping sequence identity among them, and that together formed a composed sequence. The number of ESTs from a specific library (stage of regeneration) found to form a contig can be correlated with the expression level for that sequence at the stage from where the cDNA library was made. Therefore, an indirect measurement of the level of gene expression can be inferred from the number of identified ESTs from each library that formed contigs. We identified 596 multi-sequence contigs in our EST databank that had at least two assembled ESTs. Of these, 85.4% had ORFs larger than 100 nt ~33aa), and 46.5% had ORFs larger than 195 nt (~65aa). Only 27 contigs had no ORFs made up of at least 30 nt. We focused on contigs that showed over-expression at 3- and 7-dpe that were composed by at least three different ESTs and where all the EST probes showed similar results in the microarray. Sixteen of these unknown contigs are shown on Table 3aa, and A. japonicus, and 329 sequences from genes of the sea urchin S. purpuratus. Only five of the sea cucumber (Collagen Alpha-1 (CA1); Cytoskeleton actin (cAct); Fibrinogen B precursor (Fib-Bp), Heat Shock protein 90b (HSP-90) and Senescence associated protein (SAP)) showed significant differences in expression in regenerating animals. Two of A. japonicus differentially expressed ESTs were underexpressed during regeneration (Fib-Bp and SAP), while the remaining three were overexpressed. Interestingly, four were similar to the H. glaberrima ESTs and followed the same expression profile (Fib-Bp was the different one). In all cases the probes of these other organisms showed similarity with the C-terminal region of the H. glaberrima sequences. The only sequence from the sea urchin that changed during regeneration was a hypothetical protein (Glean3-07946) showing a decrease in its expression at 3- and 7-dpe. The 24 sequences from zebra fish, human, rat, mice frog and axolotl behaved as expected; none showed any significant labeling or differential expression.Our microarray chip included the sequences of 75 ESTs from another sea cucumber We have now shown by using microarray technology that a large number of genes, represented by EST sequences, are differentially expressed during sea cucumber intestinal regeneration. Nonetheless, microarray results are not fool-proof and must be validated by other methods. An indirect way of validating our results is to compare this microarray data to previous work from our laboratory showing individual gene expression patterns. Two examples of these are the expression of serum amyloid A (SAA)  and of eHox 12, actins 1, 2 and 3), is mainly due to the RT-PCR results not being statistically significant, although in some cases they follow the same trend. It is interesting that these sequences are among the ones known to have closely related sequences in the genome (multiple actin genes and Hox genes), thus suggesting that the lack of correlation at some stages might be due to primers hybridizing with other sequences and increasing the background noise, making the RT-PCR results less sensitive than the microarray.The comparison of RT-PCR results and microarray data obtained for fifteen selected ESTs provides a direct corroboration of their differential expression during intestinal regeneration. These results show that 11/15 of the sequences behave essentially the same whether measured by microarray or RT-PCR. Moreover, even the statistical significance of the result is within the same ballpark figure. The differences between RT-PCR and microarray results of the four remaining sequences  more than a third of the ESTs showed differential expression. This contrasts with other regeneration studies where a smaller number of genes were found. For example, in studies of zebrafish heart regeneration, only about 5% of the assayed genes were found to be differentially regulated  and betwAmbyostoma mexicanum. In this species when the tail, including the spinal cord segment is amputated, 76% of the genes in the microarray were differentially expressed [The reasons for these differences might be due to two factors. First, most of our ESTs were obtained from cDNA libraries of regenerating intestine, thus the original pool of ESTs is probably biased toward those genes that are highly expressed in regenerating tissues. This was confirmed by the results themselves: the set of probes differentially expressed at 3dpe and 7dpe were 81.25% from the 3- and 7-dpe libraries and only 18.75% from the Normal library. In contrast, 42% of the down-regulated sequences come from the Normal library. Second, the process of intestinal regeneration that we are studying is complex and involves organogenesis, not merely wound healing. Furthermore, the intestine is an organ with multiple cell types and embryological origins. The intestine has components of the three embryonic germ layers, the enteric nervous system being an ectodermal derivative, the luminal epithelium an endodermal derivative and the muscular layer and submucosa being mesodermal derivatives. Therefore, when compared to a process such as heart or liver regeneration, where fewer cell types or more limited regeneration is involved, a larger number of genes might be necessary to be modulated to achieve the formation of the new intestine. Moreover, in the study of zebra fish heart, regeneration was limited to the ventricle and only to a portion of the organ since only 20% of the ventricle was removed. This contrasts largely from our studies in the sea cucumber where the new intestine must be completely formed from the remaining mesentery. In fact, a large number of differentially expressed genes was also found in another organism with high regenerative potential, the salamander xpressed . It is iIn view of this difficulty, we have developed certain strategies aimed at singling out those genes that might play important or crucial roles in regeneration. First, is to focus on genes that are differentially expressed at the 3-dpe stage. Previous experiments from our laboratory have shown that the cellular processes associated with intestinal regeneration occur early in intestinal regeneration. By 7-dpe, the blastema-like structure that will be the primordium for the new intestine is already formed . Thus, gThe finding of genes associated with embryonic development being differentially expressed during regeneration is not surprising since developmental gene pathways have been found to occur or be activated during regenerative processes ,23. ThusHox12 had previously been found in our regenerating intestine cDNA library and that there is some evidence for homeobox gene involvement in echinoderm regeneration [Hox 9, 10 and 12) are the ones that showed an increase in expression during intestinal regeneration coincides with findings in the mammalian digestive tract where Hox genes are known to be expressed in a rostral-caudal gradient, and the Hox genes near the 5' end are expressed in the posterior digestive tract, i.e. intestine [One of the most studied groups of genes is the Hox genes, which encode transcription factors that specify the body axis during embryonic development. Hox genes have been implicated in various regeneration processes, including the regeneration of amphibian tails and limbs -33, hydrneration . The finntestine . These antestine ,41.Wnt-9) was overexpressed in the regenerating intestine during the first two weeks of regeneration coincides with published data on this molecular family. Wnt molecules are extracellular molecules that bind to their receptors and activate a signaling pathway that includes other known proteins, such as B-catenin. In recent years, Wnt pathways have been increasingly associated with regenerative phenomena. Wnt was found to be involved in blastema formation of the regenerating limbs of anuran tadpoles [Wnt-9 expression is localized to determine which intestinal cell types are expressing this protein. Wnt pathways have also been involved in other invertebrate regeneration models. In planaria, Wnt is necessary for proper brain pattern formation [Similarly, the finding that a Wnt homologue  has been increasingly associated with regenerative phenomena. In newts, BMPs have been associated with the potential of the ventral iris to regenerate a new lens . And in H. glaberrima from the cellular perspective [In order to understand how changes in gene expression are associated with changes at the tissue and organ levels it is important to have a clear understanding of the cellular events that underlie the regenerative process. For this reason, our laboratory has described the intestinal regeneration process in spective -12. Two in situ hybridization and immunochemical analyses.Previous studies from our laboratory have shown dramatic changes in the cytoskeleton of cells in the regenerating intestine. Many of these changes were associated with de-differentiation of the mesenterial muscle during the initial regeneration stages and with the processes of myogenesis, innervation and luminal epithelial formation in later regenerative stages ,8,10,11.One of the most interesting findings is the under expression of gelsolin in early regenerative stages. Results by Cowin and colleagues  have shoOne of the events that is common to most regenerative processes is the remodeling of the extracellular matrix. These changes, which have been well studied particularly during epithelial wound healing, involve the replacement of the ECM by a transient ECM that facilitates the cellular events associated with the regenerative response . In the what controls organ regeneration? has been highlighted as one of the top scientific issues that deserves to be studied with high priority [First, the regenerative capacity of most vertebrate animals is relatively limited [e.g., intestinal epithelia) and organs  can readily regenerate, but others, like heart and nervous tissues, have little or no regenerative capacity. Second, although development is a process closely akin to regeneration, most animals used extensively to study the molecular aspects of development  lack robust regenerative capacities. Third, those animals with remarkable regenerative capacities, such as coelenterates, flatworms, tunicates, newts and echinoderms have been little studied at the molecular/genetic level [The cellular and molecular processes of organ regeneration have received increasing attention in recent years . In factpriority . Howeverpriority ,64. Firs limited , so popuic level ,20,65.This has started to change and ongoing projects promise a bright future for the identification of regeneration-associated genes. Among the studies/models that need to be highlighted in terms of EST/genomic are those in amphibians ,66,67, zAnalyses of regeneration-associated genes in multiple models will lead to important comparative studies among species with highly developed regenerative capacities or between closely related species where good and poor regenerators are found. The importance of comparative studies using a large range of animal models was highlighted by Sanchez Alvarado and Tsonis  who descDespite the apparent simplicity of the echinoderms, here we show that the organ regeneration is a complex process that involves the up-regulation and down-regulation of thousands of genes. Consistent with previous findings, these genes have been reported as involved in regeneration, development, ECM rearrangement, cytoskeleton reorganization and wound healing such as Wnt-9, BMP-1, Hox12, Tubulin, Tens-R, MMPs and gelsolin respectively. Moreover, a large number of unknown genes were also found to be differentially expressed at different stages of regeneration and might represent novel genes. Finally, here we show that intestine regeneration in the sea cucumber is a novel and important model for studies to identify and characterize the molecular basis of regenerative processes.H. glaberrima arrays. For this we used the "eArray" server from Agilent to design 60-mer probes that were synthesized on the microarray. A design with 15744 spots on each array and 8 arrays per slide was selected. All sequences from the cDNA libraries of normal and regenerating intestines were represented at least twice. Thus, 14352 (91.2%) probes were from 6287 clean sequences from H. glaberrima. We also spotted other sequences. These sequences represented genes associated to development, regeneration, proliferation, wound healing and growth. This pool included 150 probes from the sea cucumber Apostichopus japonicus , 8 from other holothurians that could be found in the NCBI database and 658 from the sea urchin S. purputatus . Finall®  solution for RNA extraction. Uneviscerated  animals were used as controls. One group was injected once with 1 mg lipopolysaccharides (LPS) diluted in 0.5 mL filtered sea water as reported by [Adult sea cucumbers (10~12 cm long) were collected from the north-eastern rocky shores of Puerto Rico. Animals were eviscerated at day 0, by intra-coelomic injection of 0.35 M KCl. They were left undisturbed in the aquaria to allow regeneration for 3, 7 and 14 days. At each time-point, animals were anesthetized by immersion in ice-cold sea water for 45 min. A dorsal incision was made to completely expose the internal cavity and allow the dissection of the intestinal primordium . After dissection, the regenerating intestines were placed in RNAlaterorted by . Intesti®  and the RNAeasy mini kit from Quiagen  The concentration and integrity of RNA were determined using the NanoDrop-1000 Spectrophotometer  and the 2100 Bioanalyzer (Agilent) with an RNA 6000 Nano LabChip® Kit at the Functional Genomics Research center .RNA was extracted using a combination of the  method uRNA samples were amplified and labeled using the Low Input Fluorescent Linear Amplification kit (Agilent). Briefly, cDNA was first synthesized combining 300 ng of RNA with T7 promoter primers, 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP mix, MMLV RT and RNaseOUT. This reaction mix was incubated at 40°C for 2 h. Then, cRNA synthesis and dye incorporation were performed as follows: cDNA of the first reaction was combined with cyanine-3-CTP  or cyanine 5-CTP  and the Transcription Master Mix  at 40°C for 2 h. The fluorescently labeled cRNA products were purified using RNeasy mini spin columns (QIAGEN), subsequently analyzed for yield and dye incorporation using the NanoDrop, and finally stored at -80°C until needed. Only samples that presented a concentration of more than 8 pmol/mg (measurement representing efficiency of dye incorporation) were used for hybridization as recommended by the manufacturer.Cy3 and Cy5-labeled cRNAs were combined, fragmented, and hybridized to an 8 × 15K 60-mer oligo microarray (Agilent) for 17.5 h at 60°C with continuing rotation at 4 rpm. After hybridization, slide arrays were subjected to two successive washes  and dried with the Stabilization and Drying Solution (Agilent). Arrays were immediately scanned in a G250B Microarray Scanner (Agilent) to obtain fluorescence intensities and Cy5/Cy3 ratios for each gene on the array (see below). Scanning parameters were used as recommended by the manufacturer. The resolution settings for scanning were 5 μm using minimum (10%) and maximum (100%) Photo Multiplier Tube detection sensitivities.The experimental design consisted of comparisons between Normal (non-eviscerated) intestinal tissues and 3-, 7-, and 14-dpe regenerating intestines. To determine differences in gene expression between regenerating stages we also compared directly 3- vs. 7-dpe and 7- vs. 14-dpe Figure . CompariTechnically, for each comparison, the microarray experiment was done twice, one using different biological samples, each consisting of a sample pool from 3 animals. Of the two biological replicates one was always performed as a dye-swap in order to correct any dye bias.. The analysis of the microarray data consisted of the following steps: 1) within-array and between-array normalizations; 2) fitting the data to a linear model; and 3) computing differential gene expression. For normalization purposes [2 R/G) was plotted against the overall intensity of each spot /2. Within-array normalization was first applied and M-values were normalized within each array using the Global Loess Normalization method. Aquantile normalization was then applied to the A-values as a method for between-array normalization, to assure that the intensities and log-ratios had similar distributions across arrays. To estimate the average M-value for each gene and assess differential gene expression, a simple linear model was fit to the data, and M-value averages and standard deviations for each gene were obtained. To find genes with significant expression changes between groups, empirical Bayes statistics were applied to the data by moderating the standard errors of the estimated M-values. P-values were obtained from the moderated t-statistic and corrected for multiple testing with the [Microarray data was extracted with Agilent's Feature Extraction software (version 5.1) and analyzed using the Limma package of Bioconductor. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are purposes  MA-plotswith the  method. with the  and non-with the ). under the null hypothesis that there is no differential expression between different stages of regeneration and normal tissues. Significance of the paired t-test was assumed at p < 0.05.The differential expression of the genes of interest was semi-quantitatively determined by gene-specific relative RT-PCR. Primers were designed for optimal performance using the Primer3 and Net primer web-servers [see additional file cDNA: complementary DNA; EST: Expressed sequence tag; Dpe: Days post evisceration; RT-PCR: reverse transcriptase polymerase chain reaction; LPS: lipopolysaccharides; MMP: Matrix metalloproteases; GO: Gene Ontology; ORF: Open Reading Frame; UTR: Untranslated region; mRNA: Messenger RNA; SLS: spindle like structure; ECM: Extra-Cellular Matrix.POP designed and carried out the study, analyzed and interpreted the data, and drafted the manuscript. FRG helped in the design and participated in the molecular experiments. CRC, JRL, ESC, ATMM and KT participated in cDNA library construction, EST characterization and analysis and gene identification and sequencing. JPO, SGD, FSD, JH, and CDA performed most of the validation experiments and participated in the analysis of data. HOZ helped in bioinformatics analysis. JEGA conceived the study, participated in its design, data analyses and helped in the manuscript preparation. All authors have given final approval of the version to be published.Microarray internal controls performance. Numerical and graphical representation of the internal control signals.Click here for fileNormalization procedure. Example of two random selected arrays before and after normalization.Click here for fileDye-swap hybridization. Bottom left corner of one array and the same corner in the corresponding dye-swap microarrayClick here for filePrimers. List of primers used for validation.Click here for file


Depression frequently occurs in the elderly. Its cause is largely unknown, but several studies point to disturbances of biological rhythmicity. In both normal aging, and depression, the functioning of the suprachiasmatic nucleus (SCN) is impaired, as evidenced by an increased prevalence of day-night rhythm perturbations, such as sleeping disorders. Moreover, the inhibitory SCN neurons on the hypothalamus-pituitary adrenocortical axis (HPA-axis) have decreased activity and HPA-activity is enhanced, when compared to non-depressed elderly. Using bright light therapy (BLT) the SCN can be stimulated. In addition, the beneficial effects of BLT on seasonal depression are well accepted. BLT is a potentially safe, nonexpensive and well accepted treatment option. But the current literature on BLT for depression is inconclusive.This study aims to show whether BLT can reduce non-seasonal major depression in elderly patients. Randomized double blind placebo controlled trial in 126 subjects of 60 years and older with a diagnosis of major depressive disorder . Subjects are recruited through referrals of psychiatric outpatient clinics and from case finding from databases of general practitioners and old-people homes in the Amsterdam region. After inclusion subjects are randomly allocated to the active (bright blue light) vs. placebo (dim red light) condition using two Philips Bright Light Energy boxes type HF 3304 per subject, from which the light bulbs have been covered with bright blue- or dim red light- permitting filters. Patients will be stratified by use of antidepressants. Prior to treatment a one-week period without light treatment will be used. At three time points several endocrinological, psychophysiological, psychometrically, neuropsychological measures are performed: just before the start of light therapy, after completion of three weeks therapy period, and three weeks thereafter.If BLT reduces nonseasonal depression in elderly patients, then additional lightning may easily be implemented in the homes of patients to serve as add-on treatment to antidepressants or as a stand-alone treatment in elderly depressed patients. In addition, if our data support the role of a dysfunctional biological clock in depressed elderly subjects, such a finding may guide further development of novel chronobiological oriented treatment strategies.ClinicalTrials.gov identifier: NCT00332670 Depression frequently occurs in the elderly. Prevalence figures of late-life depression of clinical relevance vary between 9% and 18% .The cause of depression is unknown. However, the circadian system and a decreased activity of the suprachiasmatic nucleus (SCN), which is known as the 'biological clock' seems to be involved . The agePatients accept and often prefer non-pharmacological treatments. The few systematic reports of side-effects suggest it is safe with only few counter indications. Light therapy also provides a viable alternative for patients who refuse, resist or cannot tolerate medication.Bright light therapy (BLT) is considered a well and effective treatment of first choice for Seasonal Affective Disorder (SAD) , with anTrials on light therapy in nonseasonal depression have been overviewed by Kripke , and havMartiny and others  investigThe Committee on Chronotherapeutics  delegateMost studies in elderly patients focussed on BLT as a therapeutic means to alleviate disruptive and cognitive symptoms of senile dementia. Recent reviews on the beneficial effects of BLT on sleep and behaviour note that the results of these studies are far from conclusive ,27 Few eThe neurobiological effect of light is mediated through the retina of the eye which is connected to the suprachiasmatic nuclei (SCN) situated just above the chiasmatic crossing of the optic nerve through the retinohypothalamic tract. The SCN is considered the major circadian pacemaker in mammals. Neural connections from the SCN are connected to the pineal gland and by this pathway the melatonin secretion of the pineal gland can be suppressed by light . To dateThere are some indications that certain wavelengths of light are more effective than others in promoting a therapeutic response with the fewest side effects. This could be due to the ability of certain wavelengths of light to more effectively control circadian rhythms . Recent Long-term follow-up of patients with cumulative exposure durations up to 1250 h demonstrated the ophthalmologic safety of light therapy, at least in patients without pre-existing ocular abnormalities .The difficulty of creating a valid placebo condition is one of the main difficulties in light treatment trials. Eastman et al  and GoëlIn SAD, Lewy et al  and TermIn sum, bright light therapy might offer a safe, non-pharmacological alternative for elderly patients with a major depressive disorder. The few systematic reports of side-effects suggest it is safe with only a few counter indications. Light therapy may also provide a viable alternative for patients who refuse, resist or cannot tolerate medication. Finally, it may hasten and potentiate the antidepressant response when used as an adjuvant to conventional antidepressants. Therefore, the aims of the current study were to investigate whether bright light therapy is effective treatment in elderly outpatients suffering from a nonseasonal depressive disorder, when being used as stand-alone or as an adjuvans to antidepressive therapy.The purpose of this study is to investigate the following hypotheses:1. Treatment with bright light improves mood, sleep, concentration and self-sufficiency of elderly depressed subjects. This clinical improvement is accompanied by decreases in cortisol and increase in melatonin concentrations.2. The eventual beneficial effect of bright light treatment can be predicted by the presence of sleep-wake rhythm disturbances as found using muscle activity registration, and by cortisol and melatonin concentrations in saliva and urine over the day and the night.A scheme-diagram of the trial protocol is presented in figure Volunteers are recruited in the Amsterdam region using four strategies: (1) referrals from several outpatient psychiatry secondary care settings specialized in elderly care in the Amsterdam region, (2) referrals from general practitioners, (3) advertisements in local newspapers, folders from 25 apothecaries and waiting rooms of general practitioners, (4) an active case-finding procedure by screening elderly populations from 16 General Practitioners Offices using the 15-item version of the Geriatric Depression Scale . SubjecThe inclusion criteria are 60 years or more of age, a primary diagnosis of Major Depressive Disorder according to DSM-IV criteria, as assessed with the Structured Clinical Interview  by a traCandidates are not admitted to the study if any of the following criteria were present: (1) insufficient fluency in Dutch, (2) living outside the Amsterdam region, insufficient address for follow-up, or unwillingness to return for follow-up, (3) light treatment in the past, (4) history of light-indicated migraine or epilepsy, (5) presence of physical illnesses that require urgent specialized treatment  for which surgery is scheduled in near future), (6) the presence of other major axis-I disorders like bipolar disorder, dementia, delirium, all psychotic disorders, PTSD, a recent history of a suicide attempt, the presence of the DSM-IV Seasonal Pattern specifier, (7) diseases that could interfere with HPA-axis functioning, or (8) diseases or medication that could elevate the risk of light-therapy associated side effects , (9) current drug or alcohol abuse less than 3 months before the study, (10) any drugs  known to interfere with endocrine function. Also excluded were all subjects who were taking (11) SSRIs or SNRIs shorter than 2 months.We chose to use dim red light as a placebo, reasoning that because the red part of the spectrum is biologically relatively inactive, there would be no substantial effect. Another advantage of red light as placebo is that it induces very strong positive expectations quite comparable to those of bright white light ,30.® boxes type HF 3304). Mistblue filters (Leevellen Lee 061) are built in to arrange the active bright-light condition. Blood-red filters (Leevellen Lee 789) are built in for the placebo dim-red light condition. On the outside of the boxes no difference is observable between the two conditions. Emitted light spectra and intensities are quantified using a lux meter at 40 cm from two light boxes. Bright light consists of approximately 10.000 lux. Dim red light consists of less than 50 lux. Environmental light in the subject's home at the designated sitting place of light therapy is measured at the start of the light therapy using a lux-meter.Patients receive either early morning bright blue light or early morning dim red light. Portable light boxes are used with a fixed (non-tuneable) light-intensity . Participants will be asked to note their compliance in their trial-diaries.Martiny et al.  found a Randomisation was prepared by an independent researcher (BU) using a computer generated table, blocking in subsets of 10, transcribed to closed envelope messages. Stratification is applied for use antidepressant medication . After inclusion the researcher sends an email with name, contact information and condition i.e. AD+ or AD-) to one of our two specially trained protocol-blinded instructors. As a check the instructor writes the allocated condition onto a paper which is kept in a special envelope in the same box with the closed envelopes.The study is presented to the patients to study whether light therapy would be a beneficial treatment modality for major depressive disorder. The blue light and red light conditions are presented to study whether a specific part of the light spectrum would be more effective than another.Light treatment is blinded to the investigators as the lamps are delivered at the patient's homes by a specially trained protocol blinded instructor, so the instructors are led to believe that the blue and red light conditions are to study which part of the spectrum worked better than the other.The patients are asked not to refer any details of their condition to the investigators as this would harm the study. When patients however do reveal their condition, the researcher will be replaced immediately by another researcher to perform psychometrical interviewing and ratings.To evaluate the blinding procedure, and in order to assess participants' general expectations for light therapy and their specific expectations in respect to their allocated light boxes, we measure expectations before the light box was installed using a four item expectations questionnaire. Briefly, participants rate, on a 7-points scale , how they thought light therapy would improve their symptoms. We use four questions to be rated: (1) To what extent do you expect your problems will improve without treatment? (2) To what extent do you expect your problems will improve with light treatment? (3) To what extent do you expect your problems will improve with the blue light treatment? (4) To what extent do you expect your problems will improve with the red light treatment?Data will be analyzed using SPSS 16.0. Analyses are performed using intention-to-treat principles. Therefore, drop-outs are distinguished into 'drop-outs before T1 assessment' and 'drop-outs after T1 assessment'. When only a randomisation assessment (T0) is available, subjects are considered lost to follow-up. For 'drop-outs after T1 assessment' the principle of LOCF (last observation carried forward) will be used. For sensitivity analysis, the more conservative LOCF variant will be calculated where T0-data will be carried forward, and a completers analysis will be performed.2-statistics.Baseline characteristics across the treatment groups are compared using analysis of variance (ANOVA) and χAs the primary analysis efficacy of treatment is tested by performing a repeated measures analysis of variance (RMANOVA) on HADRS-17 scores at T0, T1 and T2. To control for possible difference in baseline severity scores baseline values for HADRS-17 are used as covariates. Repeated contrasts are applied to compare effects in the treatment period with effects after the treatment period. As secondary analyses efficacy of treatment is tested by performing RMANOVA on the other depression symptom scales . As tertiary analyses depression outcome measures are dichotomised to responders and non-responders, where response is defined as a decrease of ≥ 50% on the HADRS-17, HAM-D6, SIGH-SAD and MADRS scales. Responder rates will also be presented as odd's ratios and NNT's.The current study is executed in accordance with the principles laid down in the Helsinki Declaration . ParticiDepression severity is assessed by a trained research physician and qualified research psychologists  using the Montgomery-Åsberg Depression Rating Scale (MADRS) , the comSecondary outcome measures are clinical response (i.e. 50% reduction in depressive symptoms) on the 29-items SIGH-SAD, the Atypical-8 subset from the SIGH-SAD, the HAMD-6 depression core features subscore, and the MADRS. Specially trained clinical research staff conducted the interviews.At each visit and 4 additional times, subjects are systematically asked about possible side effects by the blind raters: early morning awakening, headache, agitation, drowsiness, irritability, tight muscles, nervousness, anxiety, tremor, dizziness, fatigue nausea, diarrhoea, dry mouth, anorexia, dyspepsia, constipation, excessive sweating, rash, asthenia, viral infection, upper respiratory infection, flulike syndrome, nasal congestion and hot flushes . Each it1. Activity of the HPA-axis was assessed by free cortisol assessment using 8 saliva samples per respondent per time. As a measure of a natural "stress" response of the HPA-axis, the morning cortisol awakening response (CAR) is assessed by taking saliva samples at T0, T1 and T2 , and the cortisol evening curve . Saliva samples are collected using cotton swaps . Subjects are instructed to be seated during the last 15 minutes before saliva collection. During the entire sampling period, subjects can watch TV or perform leisure activities. Eating and drinking , and smoking will be permitted. Saliva containers are kept in the patient's refrigerators and are to be collected by the psychometrist the next day after psychometry, which are delivered to the clinical labwhere it is spinned down and stored at -85°C. Cortisol will be assayed on an Elecsys using a standard cortisol kit (Roche). Saliva samples are frozen in order to do all measurements at once.2. To determine a patients circadian phase position the Dim Light Melatonin Onset  will be calculated after measuring the melatonin evening curve using measurements in saliva samples  at T0, T1 and T2.3. Total cortisol excretion at T0, T1 and T2 will be determined using 24-hour urine collections. Collections are carried in the participants' own homes, following procedures provided through detailed written and verbal instructions. Urine is collected for 24 hours after the first voided urine following awakening, and included the first voided urine on the following day. Three liter polyethylene collection bottles were used. The completeness of data collection is ascertained by measurements of the 24-hr urine volume and creatinine excretion. Only participants demonstrating good compliance, using this quality control step, will be included in the analyses. The urinary free cortisol (UFC) will be determined by radioimmunoassay using a commercially available , Los Angeles USA) antibody specific for cortisol and I-125-labeled antigen. Urinary measurements are performed by the laboratory at arrival.Before, during treatment and three weeks after treatment circadian rest-activity will be measured using actography. The small (57 × 46 × 22 mm) and light-weight (70 g) wrist-worn actigraph, which senses movement-induced accelerations, as described elsewhere .Estimates of sleep parameters are obtained using the validated Sleepwatch Analysis Software 201 . The software automatically calculates Sleepstart and Sleepend, which are limited to occur at any time between the habitual Bed Time and Get Up Time as read from the sleep logs and entered into the software; Assumed sleep (the difference between Sleep End and Sleep Start), Actual Sleep time  gives an indication of of the stability of the 24-hour rest activity pattern over days. The intradaily variability (IV) gives an indication of the fragmentation of the rhythm, by quantifying the number and strength of transitions between periods of rest and activity. L5 quantifies the activity level during the core sleep period. M10 describes the daytime activity level. AMP is an absolute amplitude measure and calculated as the difference between M10 and L5. The relative amplitude (RA) is calculated by dividing AMP by the sum of L5 and M10 ,49.Subjective sleep quality is scored using the Pittsburgh Sleep Quality Inventory (PSQI) at T0, T1 and T2 . The PSQAn abbreviated version of the Social Rhythm Metric SRM-17 , the SRMAt T0, T1 and T2 participants will do neuropsychological tests addressing working memory, using the digit-span forward and the digit span backward, learning and memory using the Rivermead stories immediate and delayed recall, and verbal fluency using animals, insects and occupations.Details about the social support is measured using Social Support Questionnaire (SSQ) , at T0, In order to measure self-sufficiency we chose to use the MOS-short form General Health Survey  . De SF-2To additionally measure both the ADL and instrumental ADL disability with respect to the aid and services provided by professional home help and district nursing agencies we will use the Groningen Activiteiten Restriction Scale .The general self-efficacy was measured with the Dutch version of the General Self-Efficacy Scale (GSES) the Algemene Competentie Schaal (ALCOS) . The ALCA Dutch version of the Philadelphia Geriatric Center Morale Scale  is used A subset of consecutively included participants is invited for 1,5 Tesla MR-scanning at T0 and after therapy at T1. Scan protocols consist of structural MPRAGE/T1- and T2 weighted magnetic resonance scans of the brain, functional-MRI scans using encoding, facial recognition task and working memory tasks (N-back). In addition, adrenal volumetry is performed using abdominal MPRAGE of adrenals.AD: Antidepressant; BLT: Bright light therapy; CAR: Cortisol awakening response; DLMO: Dim Light Melatonin Onset; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders 4th Edition: GDS-15: Geriatric Depression Scale 15item version; HAMD-6: 6-core-item subscore from HDRS-17; HDRS-17: Hamilton Depression Rating Scale 17 item version; HPA-axis: Hypothalamus-pituitary adrenocortical axis; MADRS: Montgomery-Åsberg Depression Rating Scale; MDD: Major depressive disorder; MPRAGE: Magnetization Prepared RApid Gradient Echo; SAD: Seasonal affective disorder; SCID-I: Structured Clinical Interview for DSM-IV; SCN: Suprachiasmatic nucleus, SIGHSAD: Structured Interview Guide for the Hamilton Depression Rating Scale – Seasonal Affective Disorder Version; UFC: Urinary Free Cortisol.The authors declare that they have no competing interests.RL is the principal investigator, participated in the study design, patient recruitment and trial coordination, drafted the manuscript, MMAN is the postdoc, participated in the study design, patient recruitment and participated in the trial coordination, DJV participated in the neuroimaging design, BU is a neurologist-epidemiologist and participated in the study design, set up the randomisation system, EJWS is psychophysiologist, conceived of the study, and participated in the study design, conceived the fabrication of the trial conditions, and supervisor of actometry, JHS is the trial methodologist and chief research of the academic department of psychiatry, participated in recruitment strategies, in methodology and interpretation issues, WJGH is the study director, conceived of the study, obtained funding, participated in design and coordination and helped on the manuscript. All authors read and approved the final manuscript.


Material wear testing is an important technique in the development and evaluation of materials for use in implant for total knee arthroplasty. Since a knee joint induces a complex rolling-gliding movement, standardised material wear testing devices such as Pin-on-Disc or Ring-on-Disc testers are suitable to only a limited extent because they generate pure gliding motion only.A rolling-gliding wear simulator was thus designed, constructed and implemented, which simulates and reproduces the rolling-gliding movement and loading of the knee joint on specimens of simplified geometry. The technical concept was to run a base-plate, representing the tibia plateau, against a pivoted cylindrical counter-body, representing one femur condyle under an axial load. A rolling movement occurs as a result of the friction and pure gliding is induced by limiting the rotation of the cylindrical counter-body. The set up also enables simplified specimens handling and removal for gravimetrical wear measurements. Long-term wear tests and gravimetrical wear measurements were carried out on the well known material pairings: cobalt chrome-polyethylene, ceramic-polyethylene and ceramic-ceramic, over three million motion cycles to allow material comparisons to be made.The observed differences in wear rates between cobalt-chrome on polyethylene and ceramic on polyethylene pairings were similar to the differences of published data for existing material-pairings. Test results on ceramic-ceramic pairings of different frontal-plane geometry and surface roughness displayed low wear rates and no fracture failures.The presented set up is able to simulate the rolling-gliding movement of the knee joint, is easy to use, and requires a minimum of user intervention or monitoring. It is suitable for long-term testing, and therefore a useful tool for the investigation of new and promising materials which are of interest for application in knee joint replacement implants. During physiologic flexion and extension of the knee joint, a combined rolling and gliding motion occurs. This complex motion pattern and the incongruence of the corresponding femur and tibia condyles make the design of total knee prostheses one of the most challenging tasks in implant development. In particular, the properties of the materials responsible for the tribological pairing are of essential importance. Such materials must be biocompatible and bear mechanical load. Furthermore, they should be durable as well as exhibit very low friction and low wear rates. Wear is one of the primary factors limiting the lifetime of current knee prostheses, either directly due to material failure as a result of wear, or by aseptic loosening resulting from the accumulation of wear debris and the ensuring biological reaction ,2. For tin vivo as realistically as possible in order to predict the wear behaviour of the device in clinical application. On the other hand, prosthesis geometry and design represent confounding factors contributing to wear, which cannot be isolated from the contributions of the material and surface properties alone. Moreover, complete knee-implant samples are associated with high production and acquisition costs. Thus, a TKA knee wear simulator is less suited as a materials testing advice than for product evaluation, with the aim of performing comparisons in the context of the product certification process.Wear testing of complete total knee arthroplasty (TKA) devices for purposes of certification or product comparisons is typically carried out with commercially available knee wear simulators according to international standards such as ISO 14243. These standards seek to simulate the boundary conditions applied to a prosthetic device To avoid the complexity and confounding effects of a knee wear simulator, as well as the over-simplification of Ring- and Pin-on-Disk approaches for purposes of materials testing, a device was constructed which includes the essential knee-like rolling-gliding motion of the knee-joint. The primary objective was to improve standardized material wear testing for applications in TKA, whereby the intended application of the device was the investigation of the effect of manufacturing processes such as machining and polishing on the surface quality, geometric tolerances, and in turn wear characteristics of biocermaic materials. Further important goals in conceiving the device were that it should be relatively inexpensive, easily transportable, capable of applying millions of loading cycles while requiring minimal user intervention, and allow wear to be measured according to standardized gravimetric methods .Herein, we describe the technical specifications, design and implementation of the device, as well as first results obtained. Data obtained from tests of the tribological pairings cobalt chrome against polyethylene (MoP), ceramic against polyethylene (CoP), and ceramic against ceramic (CoC) are reported as example applications. We hypothesized that the different wear behaviour measured with the new simulator for these well characterized material-pairings would be similar to reported values. Furthermore, tests were carried out on three further CoC tribological parings, each with a different surface roughness, and one CoC paring with convex/concave shaped running surfaces.The physiological rolling-gliding motion of the human knee joint is represented in many typical prosthesis designs by motion occurring between the femur component and the polyethylene tibia plateau. The normalised gait cycle (ISO 14243-1) accordingly permits the following types of motion: gliding, rolling with slip, and rolling between the femur component and tibia plateau, depending on the range of motion of the knee prostheses design ,6.Our aim was to induce these three different types of motion  on simplified specimen geometries. A concept was adopted in which a base-plate was horizontally moved relative to a rotating cylindrical counter-body under axial loading Figure. . In thisThe "Rolling-Gliding Wear Simulator" created based on this concept consists of a horizontally oscillating stage onto which the base-plate is mounted, as well as a cylindrical counter-body which is statically vertically loaded Figures. , 2 and 3The material samples can be removed from the device for gravimetric wear measurement according to ASTM F1715 and ISO 14243-2, or alternatively for analysis of the wear surface profile using topological methods such as scanning electron microscopy or others .Material specimens are held in place in their two respective mounting points by a keyway and rectangular key design, with the addition of a small clamping mechanism to prevent lateral motion or dropping out of the cylindrical counter-body Figures. , 4. ThisAn adjustable eccentric driven by a servo motor  supplies the horizontal motion of the translation stage. A fluid tray, equipped with a heat-exchanger, is mounted on the translation-stage Figures.  and 4 inAn electronic control module and user interface generated using LabView  are used to manage the testing device. The software allows the user to adjust the number of testing cycles, the rotation speed of the servo motor and the amount of distilled-water added per hour. The software records the input data coming from sensors which read the linear and rotational movements, and the relative amounts of rolling, rolling and gliding, and pure gliding are graphically displayed Figure.  top. A hThe wear tests are conducted under the application of a constant compressive axial load of 714 N (700 N load bars + 14 N dead load of structure) to the cylindrical counter-body. Since a single condyle is being modelled in this wear test, 714 N reflect about a half of an average normal load during stance phase of gait (0% - 66% of gait cycle) . The geo®delta, CeramTec AG). The CoCr and PE specimens were tested as provided by the manufacturer; the ceramic specimens were tested as provided, or finished by the authors. The materials were tribologically paired as follows: a UHMWPE plate against a polished CoCr counter-body (TP1), a UHMWPE plate against a ceramic counter-body (TP2), and a ceramic plate against a ceramic counter-body (TP3. and TP4). Furthermore, three ceramic-ceramic tribological pairings with surfaces finished to different degree of roughness (Ra) were tested: ground to Ra = 825 nm (TP5), polished to Ra = 382 nm (TP6), and highly polished to Ra = 52 nm (TP7). Finally, another ceramic-ceramic pairing was carried out with convex and concave shaped surfaces of the base-plate and the counter-body (TP8), whereby the base-plate had a convex radius of 36.48 mm and the counter-body a concave radius of 37.45 mm, which represents a difference in radius of 0.97 mm. For every test series performed, a reference material sample was included, which was unloaded.Altogether eight tribological pairings were tested, consisting of three different materials: Ultra High Molecular Weight Polyethylene , Cobalt Chrome , and a zirconia toughened alumina bioceramic material , which is a typical pairing in knee prostheses, had an overall volumetric PE wear of 2.540 mm3/(106 cycles) calculated based on density of 0.943 mg/mm³ (Table. TP2) using ceramic instead of CoCr shows a lower volumetric wear rate of 0.406 mm3/(106 cycles) for the soft base-plate. The wear of the hard ceramic counter-body was not detectable.The investigated hard-soft combination of MoP TP, which i40 mm3/10 cycles cTP3, TP4) show a low volumetric wear for both the base-plate, 0.192 ± 0.002 mm3/(106 cycles) and the counter-body, 0.195 ± 0.063 mm3/(106 cycles) calculated with a density of 4.37 mg/mm3 for the bioceramic.The average result of two hard-hard CoC material pairings of (TP5-TP7) are below 0.161 mm3/(106 cycles), (Table. TP8) show a low wear rate of less than 0.067 mm3/(106 cycles). The gravimetric-wear rate of the base-plate and counter-body of CoC material pairings displays typical patterns, whereby the base-plate exhibits an initial higher slope with a following nearly linear behaviour over three million cycles of motion , as well as for CoC material pairings with different frontal plane geometries and surface roughness. Typical wear patterns of a CoC material pairing were also described.3/(106 cycles) which was over one order of magnitude lower than the MoP pairing at about 2.5 mm3/(106 cycles). The relative reduction of wear in the CoC parings compared to MoP we observed is similar to the reduction seen in total hip arthroplasty. Values reported there range from 8-35 mm3/(106 cycles) for MoP [3/(106 cycles) for CoC pairing [3/(106 cycles) [3/(106 cycles) to a CoP (Alumina-PE) pairing at 0.7 mm3/(106 cycles) [We observed a volumetric wear rate of the CoC pairings of less than 0.20 mm for MoP ,13, and  pairing ,14-16. S cycles) ,18. The Regarding the wear patterns of the CoC pairings investigated, reproducible patterns were observed on the cylindrical counter-body and base-plate. The ceramic base-plate Figure. , bottom The investigation of CoC tribological pairings under the knee-like loadings and kinematics is especially interesting in the context of the realisation of low-wear CoC knee prostheses. Two interesting aspects related to the application of the CoC material pairing in knee-prostheses are the influence of geometric tolerances and surface quality on wear . GeometrThe results of preliminary experiments on the effect of surface roughness on wear Table.  suggest As we have shown, the wear we observe with our device is comparable in magnitude and trend to wear-tester results of complete prosthetic devices. The device thus seems to be suitable for making comparisons between different material pairings. It should however be remembered, that the simplified boundary conditions applied  and the simplified geometry of the specimens cannot, and are not intended to represent the effect of complex design elements of knee prostheses, such as fixed or mobile inlays and or guiding or stabilizing structures. A further important consideration and possible limitation of our device is that it does not produce cross sheering motions, which are believed to be of importance in PE wear ,24. The The concept of rolling and gliding a cylindrical counter-body against a plate has also been employed in a further "wheel-on-flat" apparatus in order to analyse the wear behaviour of polyethylene material in response to increasing traction forces . The resThe uncomplicated use of the testing device and the simple design of the specimens give the user the possibility of studying a wide range of technical or even natural materials with minimal user intervention over multiple million cycles of motion. The device thus allows different and new materials and or surfaces to be studied efficiently under the rolling-gliding motions similar to those to which the knee prosthesis materials are subjected to in use. The contact force and the rate of rolling and gliding can easily be changed or adjusted. By this means the behaviour of different materials and surfaces can be studied before manufacturing complete knee prosthesis components. The testing device presented herein will provide basic-research regarding issues important to the design and manufacture of ceramic-ceramic material pairings for application in TKA.The authors declare that they have no competing interests.BIR carried out the tests series, implemented the evaluation, analysed the data and prepared the manuscript. CH participated in the design of the study, carried out the design and construction, performed the data-analysis and helped to draft the manuscript. AT and BD supplied and prepared the specimens and analysed the geometric and surface quality. SO advised in the conception and the design of the device. All authors read and approved the final manuscript.


Antimitochondrial antibodies (AMAs) are the classic serologic            marker in primary biliary cirrhosis (PBC). However, there have been only             limited attempts to study changes in titer or isotype analysis of such AMAs in            patients followed for long periods of time		We took advantage of stored sera from well-characterized patients with            PBC followed for a period of 7-28 years (mean duration of 13.5 years). Immunoblot            and enzyme-linked immunosorbant assays were performed against PDC-E2,            BCOADC-E2 and OGDC-E2 as well as isotype analysis of antigen-specific IgG,            IgA and IgM antibodies against each of these mitochondrial autoantigens. Sera            were analyzed for total IgG, IgA and IgM by radial immunodiffusion. The sera titer            of AMAs was significantly higher in younger patients with PBC. Indeed, age of onset            of clinical PBC was a significant predictor for the highest values of sera AMAs. In            contrast, the AMA titer did not significantly change over time in this prolonged            longitudinal study. The total sera levels of the individual immunoglobulins did not            show a time-dependent change, when based on age of onset of the disease. Higher             titers of AMAs were noted in the younger patients. Furthermore, despite this long             follow-up, there was no evidence for a significant change in AMA levels; also, levels            were not influenced by drug therapy used during the period of observation.


Oncorhynchus kisutch) returning to the Chehalis River in British Columbia, Canada, were crossed to create pure hatchery, pure wild, and hybrid offspring. A proportion of the progeny from each cross was reared in a traditional hatchery environment, whereas the remaining fry were reared naturally in a contained side channel. The resulting phenotypic differences between replicates, between rearing environments, and between cross types were compared. While there were few phenotypic differences noted between genetic groups reared in the same habitat, rearing environment played a significant role in smolt size, survival, swimming endurance, predator avoidance and migratory behaviour. The lack of any observed genetic differences between wild- and hatchery-born salmon may be due to the long-term mixing of these genotypes from hatchery introgression into wild populations, or conversely, due to strong selection in nature—capable of maintaining highly fit genotypes whether or not fish have experienced part of their life history under cultured conditions.With the current trends in climate and fisheries, well-designed mitigative strategies for conserving fish stocks may become increasingly necessary. The poor post-release survival of hatchery-reared Pacific salmon indicates that salmon enhancement programs require assessment. The objective of this study was to determine the relative roles that genotype and rearing environment play in the phenotypic expression of young salmon, including their survival, growth, physiology, swimming endurance, predator avoidance and migratory behaviour. Wild- and hatchery-born coho salmon adults ( Climate change, over-fishing and habitat alteration are suspected to be contributing to declines in Pacific salmon stocks to the point that some populations are now seriously threatened Oncorhynchus kisutch) were supplanting wild coho Concern that hatchery-reared coho salmon  have been shown to evolve reproductive isolation in fewer than thirteen generations O. mykiss) in just one or two generations Salmon are capable of a high degree of phenotypic plasticity, which means that they can physiologically and behaviourally adapt to their environment The relative effects of rearing-environment and genotype on salmon phenotype, behaviour and survival are generally unknown and need to be assessed All work involving live fish reported in this paper was annually reviewed and pre-approved as meeting or exceeding the standards laid out by the Canadian Council on Animal Care. The project guidelines were approved by The University of British Columbia's Committee on Animal Care at Suite 102, 6190 Agronomy Road, Vancouver, BC (permit A06-0153).−3), survival, colour, fin quality, disease presence, gill Na+/K+−ATPase activity level, microarray gene expression profile, predator avoidance ability, swimming endurance and migratory behaviour—including speed, timing and habitat use. See Physiology for further details on the methods used.Returning wild- and hatchery-born coho adults were crossed at the Chehalis Hatchery  during tEighty crosses of returning coho adults were carried out at the Chehalis Hatchery on 1 February 2007 . Blood samples were taken and stored in 0.01 N sodium hydroxide for later genetic identification. The eggs of each female parent were divided in two equal groups so that one half could be fertilized by a wild male and the other by a hatchery male to test for maternal effects . Each crAs there was a small percentage (∼10%) of the returning adults of early 2007 that were released from the hatchery unclipped, the parental otoliths were analysed to confirm which of the unclipped fish were wild-born, and which had actually been hatchery-reared. One year later (January 2008) otoliths were collected from 20 clipped and 20 unclipped returning adults from the 2004 broodstock for analysis. All of the hatchery fish from the 2004 broodstock had been adipose-clipped prior to release; therefore their otoliths could be used as a control to compare with the parental otoliths. Many of the otoliths from hatchery-origin fish were too crystallized to be able to see growth rings, so the degree of crystallization was used as an indicator of origin O. clarkii) were trapped in the natural habitat areas and released into the wild. Following this removal, there were still mammalian, avian and aquatic predators observed in both of the natural habitat areas. Further detail on the natural rearing environment can be found in (Three months post-fertilization (early May 2007), the unfed fry from each full-sibling group were divided into four rearing groups and released into the four separate habitats . The two hatchery rearing groups had 100 individuals from each full-sibling group, as they had a much greater expected survival rate than those reared naturally. The natural-rearing groups had one-half of the remaining individuals from each sibling group. A total of 3,600 fry were released into each of the hatchery troughs, and 23,000 fry were released into each of the natural habitats. Whereas the coho fry in the side-channel rearing areas were left to feed on naturally-occurring food sources, the hatchery-reared fry were fed daily on a typical hatchery diet . To reduce the predation pressure in the natural habitats and ensure that some fry survived the year, 135 resident coho smolts and 2 cutthroat trout . Thus, with 3,600 fry in each, the hatchery-rearing density was 4,167 fry per m3. Two months prior to their release, the pre-smolts in the hatchery were moved to a larger trough with a gate at one end connecting to the Chehalis River.The starting density in the natural rearing area was 46,000 fish in 3,230 mSize sampling was conducted monthly in the hatchery from April 2007 (fry ponding) to June 2008 (smolt release). The naturally-reared fish were trapped using baited minnow traps and sampled during February, April, May and June 2008. All of the fish were returned to their habitat following measurement. During May and June 2008, DNA samples were taken non-lethally (adipose clip) from the naturally- and hatchery-reared pre-smolts  to identify genetic cross groups. Microsatellite analysis was conducted on eight loci  according to methods in −3) +/K+−ATPase activity assays could be conducted At the time of the smolt release during late June 2008, a physical assessment was conducted on 30 hatchery- and 30 naturally-reared fish. The smolts were euthanised prior to sampling in buffered tricaine methanesulphonate . Mass and fork length was measured and condition factors calculated  and hatchery-reared (n = 10) coho smolts was assessed in a swim tunnel on 25 and 26 June 2008. Each smolt was given 5 min to acclimate at 0.1 m · sDuring 25 and 26 June 2008 two groups of naturally-reared (n = 20 in each) and two groups of hatchery-reared coho smolts (n = 20 in each) were placed in identical partially-covered troughs and allowed to acclimate for two days. The time to eat an egg dropped 30 cm from the edge of the covered area, was recorded five times for each of the four groups. After the fourth group was finished, a predatory attack was simulated on the first group by moving a plastic heron up and down in the tank quickly with its beak penetrating the water. The beak was kept just under the surface of the water as an egg was dropped into the tank. The time taken for the egg to be eaten was recorded. The heron's beak was removed after 90 s, and the trial was stopped after four minutes if the egg was still uneaten. Five repetitions were done per group per trial with a minimum of one hour between trials. Two trials were done per day, for a total of four trials.2. The fish were released 24 hours later on the 23 and 24 June at nightfall (2130 h) from the hatchery together with 4,000 coho smolts. Manual tracking (VEMCO VR100) in the Chehalis River hatchery pool  were tagged with 7 mm-diameter tags  according to methods in pool C1,  was carrAcoustic receiver arrays (VEMCO VR2s and VR3s) were moored along potential migratory routes to monitor the smolts' behaviour. Approximately 2 km downstream of the release site, one stationary receiver was located in the Chehalis River C2, . Arrays The wild and hatchery parent groups sampled in January 2007 did not differ in size when compared by origin and gender . However2, and 0.1 individuals per m3. The hatchery rearing density was 4,167 individuals per m3.Growth comparisons between fish in the two natural and two hatchery rearing areas during February and April 2008 found no difference between replicates ; however, hatchery-reared fish were significantly larger than their naturally-reared siblings  for analysis of body-size differences within each rearing environment. None of the genetic cross groups were significantly different in mass or length within a rearing environment . However+/K+−ATPase activitiy was higher in the hatchery-reared than the naturally-reared fish .The hatchery-reared pre-smolts were lighter (more yellow) in colour than their naturally-reared siblings (green/brown with more pronounced parr marks). In the higher-density hatchery groups, the incidence of scale loss and fin damage was greater. The hatchery fish also had elevated amounts of adipose tissue and longer gill-rakers, whereas the naturally-reared fish had short gill-rakers and almost no body fat. Fifteen percent of the hatchery fish had eye damage, compared to only one percent of the naturally-reared fish. Skin parasites were observed in 37% of the naturally-reared fish and none of the hatchery-reared fish. Some of the naturally-reared fish also had parasites in their gills (7%) and body cavity (13%). No BKD or IHN was detected in either group. Gill NaThe mRNA levels of IGF-I and -II were similar between rearing groups, but the mRNA levels of GHR were significantly lower in the hatchery-reared smolts than in their naturally-reared siblings . Many ge−1) by the fish did not differ significantly between the naturally- and hatchery-reared groups . However, the naturally-reared fish took longer to fatigue (903±894 s) than the hatchery fish . The naturally-reared fish were also more agitated in the tunnel, scaring easily when their tails touched the down-flow grate. Conversely, the hatchery fish seemed to prop themselves up against the back grate and side of the tunnel with their tails intermittently.The swimming endurance of the naturally-reared fish was significantly greater than those reared in the hatchery troughs, even though they were smaller. The mean relative velocity swum . The naturally-reared fish also took longer to recover after the “predator attack” . Of the 60 fish tagged, 83% were detected by moored receivers. All of the hatchery-reared smolts except two departed from the vicinity of the hatchery C1,  the nighThe four genetic cross types reared in the hatchery  had similar migratory behaviour and body size. All left the hatchery pool the night after release and travelled quickly downstream, arriving at C2 the same night. They arrived at H2 approximately three days post-release and spent very little time near the H2 array. Three of the smolts  were detected leaving the Harrison River. The two hybrids travelled quickly from H2 to H3, whereas the pure hatchery smolt spent two days in the shallow area of the Harrison River before travelling downstream to F1 and F2.Most of the tagged fish stopped in the wide shallow area in the lower Harrison River (between H2 and H3). In total, only 8 of the 60 tagged fish (13%) were detected downstream of the Harrison River. Five of those eight were naturally-reared. The eight fish were similar in that they all travelled quickly downstream, spending 0–3 d between H2 and H3. One of the hatchery-reared fish was detected in the Fraser River at F1 and F2, five and seven days after its last detection at H3. Two of the naturally-reared fish were detected in the northern Strait of Georgia during July and August 2008 NSOG,  and two Only one smolt (hatchery-reared) moved upstream and was detected at H1. This individual was recorded at H2 five days post-release, presumably heading downstream to the lower Harrison River. Two nights later, however, the fish was recorded again at H2, then at H1 the same night. It was never detected again and may have continued further upstream into Harrison Lake.In the continuing debate of nature versus nurture, this study has found that the effects of rearing environment on phenotype and behaviour far outweighed the effects of any genetic differences existing between second-generation hatchery- and wild-born coho salmon in this system. Coho reared in a natural environment had more normal-type otoliths, greater swimming endurance and predator-avoidance abilities, and longer migrations than their hatchery-reared siblings. There were no observable differences in growth, survival or migratory behaviour between pure-wild- and pure-hatchery-genotype groups reared in the same environment. These results indicate that few, if any, genetic differences are present between the hatchery-reared and wild-born coho salmon studied. To what extent the wild-born fish are populated by previous generations of hatchery fish, however, is unknown.The fact that there was little difference observed between the offspring of wild- and hatchery-born coho reared in the same environment suggests that there may not be a strong genetic difference between the two groups. While some studies have observed genetic influences on phenotypic differences between wild and hatchery populations Studies examining the long-term captive breeding effects on coho populations in Washington and Oregon have found that the genotypes of many wild populations have transformed to more closely resemble the genotypes of hatchery-reared fish Early rearing conditions seem to affect coho reproduction investment, which can directly influence overall fitness. Studies have found that hatchery-reared fish had greater ovary mass Balancing the risk of predation with the benefit of feeding is important for maximizing individual fitness, and is expected to be influenced by both genotype (e.g. inherent growth rate) and rearing environment (experience with predators in nature) Feeding on natural sources as a pre-smolt likely increases a smolt's ability to find good quality food in the wild. Releases of hatchery-reared fish that had been hand-fed pellets were more surface-oriented and more likely to approach moving objects than were naturally-reared fish Naturally-reared coho smolts were more likely to be detected downstream of the Harrison River than their hatchery-reared siblings. Of the fish detected entering the Fraser River, 63% were naturally-reared, despite the fact that the naturally-reared fish made up only 32% of the total releases. These long migrants travelled quickly through the Harrison system and its shallow, predator-rich feeding areas. Many factors could have influenced this behaviour. The normal-type otoliths and better physical condition of naturally-reared smolts may improve their orientation, balance and swimming ability, allowing them to migrate further and faster. The foraging and predator-avoidance abilities of the naturally-reared smolts may have also increased their chances of survival.The incidence of coho residualisation has not been fully investigated in the Fraser River system. This study has provided some evidence that coho may be residualising in the Harrison River and Harrison Lake. Mortalities or tag losses may have accounted for some of the fish that were not detected beyond the Harrison River. The possibility of some tagged fish passing by receiver arrays undetected also exists. Further quantification of coho residualisation in this system could be accomplished through the use of PIT tags and seining.Distinguishing between hatchery-reared and naturally-reared salmon using only otoliths usually involves an analysis of early growth rings The microarray analysis demonstrated that the metabolic rates of naturally- and hatchery-reared siblings differed. For example, the mRNA levels of cytosolic PEPCK gene were lower in the hatchery-reared smolts. PEPCK is a rate-limiting enzyme of gluconeogenesis +/K+−ATPase in the naturally-reared fish was inconsistent with the elevated levels of GHR mRNA in the same fish. The stimulatory effect of GH on smolting and ATPase would suggest that the elevated GHR mRNA reflects a more active role of GH in the naturally-reared fish—which should lead to more ATPase activity. Earlier work showed higher levels of Na+/K+−ATPase in wild than hatchery coho smolts from the Quinsam watershed The lower levels of Na+/K+−ATPase activity levels, and survival As the goal of salmon enhancement programs is to improve the conservation status and productivity of wild salmon stocks, smolt quality and fitness are of high priority. Behavioural deficiencies due to artificial rearing environments have been considered a primary cause of lower hatchery survival rates In the system studied, hatchery-reared fish are capable of returning to and spawning in the wild, and as such, wild-born fish may be comprised of a mixture of fish that have some hatchery ancestry and some ancestry from lineages that have been wild for many generations. Since the relative proportions of these ancestries in the population are unknown, it is not possible to determine whether the lack of observable genetic effects on offspring phenotype was due to 1) a significant mixture of their genotypes through interbreeding, 2) hatchery practices that maintain wild genetic diversity, or 3) strong selection for fish with appropriate “wild” genotypes  that acts to canalize genetic variance for non-neutral loci between hatchery and wild-derived salmon in the population. However, if the Chehalis River hatchery is selecting for genotypes distinct from wild fish, and hatchery fish are contributing significantly to wild populations by breeding in the wild, then the data from the present study indicate that selection for wild genotypes in nature is occurring rapidly such that returning hatchery fish are not distinguishable phenotypically from those born of wild parents. Further studies tracking the contribution of hatchery genotypes to wild populations will be required to resolve these important questions.File S1Habitat assessment and carrying capacity.(0.02 MB DOC)Click here for additional data file.Figure S1A scale diagram of the natural rearing area near the Chehalis River Hatchery. The water flow begins at the circle on the right, next to the Chehalis Hatchery, flowing downstream to the left. The first habitat runs from the water source to the first fence. The second habitat is considerably shorter in length, from the first fence to the second fence. The numbers in pink indicate the width of channel in meters, and significant features are labeled.(3.94 MB TIF)Click here for additional data file.Table S1Phenotypic characteristics of naturally- and hatchery-reared coho salmon. Statistical differences were established at P<0.05 with the Mann-Whitney U test.(0.09 MB DOC)Click here for additional data file.