The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2  linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances.6000 long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths.Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cRWe obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate. It is also the only bird species for which the genome has been studied in detail and a lot is expected from its use in comparative genome analyses. This will help to detect sequences conserved between species, which should correspond to unknown exons and to regulatory or other functional regions. Such analyses will therefore be essential for the annotation of other genomes, including that of human ,2. Chick,4. Thank,6. Never. TherefoRH panels are available for several domestic animals: cow , pig 9]9], horseWe present here the radiation hybrid map of chicken chromosome 2, built by using markers chosen from the GGA2 genetic map and a substantial number of additional markers developed from chicken EST data. Markers from the genetic map are essential to anchor the RH map onto GGA2. Markers developed from EST data were chosen on the basis of existing comparative mapping information data, indicating conservation of synteny with HSA1, HSA3, HSA6, HSA7, HSA8, HSA9, HSA10, HSA18 and HSA22. They were used primarily to saturate the map in markers and also to increase the precision of comparative maps. The GGA2 RH map obtained was aligned with the genomic sequence assembly to detect eventual discordances.Eighty eight microsatellite markers and 10 genes were selected from available data in the literature or the databases. In addition to this, 219 markers were developed from EST data to saturate the map. At the time this study was initiated, comparative mapping data suggested conservation of synteny of GGA2 with 9 human chromosomes and therefore genes were selected from regions of HSA1 (9 markers), HSA3 (28 markers), HSA6 (22 markers), HSA7 (10 markers), HSA8 (84 markers), HSA9 (19 markers), HSA10 (7 markers), HSA18 (35 markers), HSA22 (5 markers). Primers were developed using the Iccare web server ,19,20.LOC51059 and ALDH5A1) should have been integrated into the GGA2 RH map, given their location within the sequence assembly. One explanation, concerning the lack of these 2 markers, could be that the retention frequency, that is unusually low for these two markers and indicative of PCR problems, did not allow attaining a LOD score value significant for linkage. The other 70 markers map either to other chromosomes or to unknown regions. They correspond to the external boundaries of the regions of conserved synteny with human, from which EST were chosen for marker development.Altogether, genotyping data were obtained for a total of 270 markers. Two-point analysis with CarthaGene using a LOD threshold of 4 enabled the construction of 5 GGA2 linkage groups containing a total of 198 markers, including all the microsatellite and gene markers from the genetic map. Out of the remaining markers, two , the 3 markers (MCW157, MCW189, and MCW0073 / HSF1) were in the same order in the linkage group, but its orientation is different from the genetic map. These markers are mapped on the comprehensive RH map (less significant than the framework one), so the orientation given in the genetic map may be the right one.The final RH map of chicken chromosome 2 is 2794 cRThe corresponding comprehensive map comprises a total of 198 markers  could be localized on the GGA2 RH map. As this localisation could be questioned, the identity of the fragment was confirmed after sequencing of the PCR product.Previous data suggested that the  on GGA2 . We haveAs already observed for other chicken chromosomes -17,24-26). The sequence coverage is 6.6X. For GGA2, 147.590.765 bp were sequenced, 166 known genes were identified, and a total of 1432 genes were defined altogether when including results from prediction programs.A first draft chicken genome assembly was recently deposited into public databases by a team led by R. Wilson and W. Warren, from the Washington University School of Medicine in St. Louis  and ABR0336 is found on the GGA27 sequence. The RH two point LOD scores between CENTG3, CSPG5, ABR0336 and their neighbours on GGA2 (MCW082, ACVR2B or PAXIP1L) are higher than 6. In addition, the genetic map confirms the localization of ABR0336 on chicken chromosome 2. In the same region of the RH map, MCW071 (a microsatellite in the EN2 gene) belongs to a genomic region with no sequence available (no blast hit); the genetic map confirms the RH position of this marker. These results suggest possible improvements to be made in the sequence assembly for this region of chromosome 2.In the upper part of the chromosome, several markers absent in the sequence assembly could be localized on the RH map: sequences corresponding to TSG gene, at position 116.4 Mb, is close to EYA1 and STAU2, whereas on the RH map TSG maps at 1660 cR, close to PTPN2 and LEI0147, located at positions 96.4 and 97.3 Mb respectively. At the same position as TSG on the RH map, the VAPA gene corresponds to a sequence fragment of unknown location. Moreover, the RH map in this region is in agreement with comparative mapping, so we suspect there may be problems in the sequence assembly in the TSG region.On the sequence assembly, the PRKDC and MAPRE2, and the second SDCBP and ADL114. In both cases, EST markers mapped close to these genes are missing in the GGA2 sequence assembly: CEBPD near PRKDC and ASPH near SDCBP are in the fraction of unassigned sequence. However, in the case of the PRKDC-MAPRE2 region, the sequence assembly is in agreement with the gene order on human chromosome 8. The order of the markers in the RH map is supported in both cases by a difference of LOD greater than 8, when compared to the alternate order.Around position 2100 cR, two local inversions between the RH map and the sequence orders are observed. The first concerns HEY gene at position 2330 cR is localized on GGA26 in the assembly, whereas the two-point LOD scores with its neighbours on the GGA2 RH map are trust-worthy (11.5 with IMPA1, 8.8 with PKIA). The MATN2 and NCALD genes are in the unknown fraction of genomic sequence, whereas the 2-point LOD scores with their neighbours, around 2550 cR, are higher than 6. At the end of the chromosome (position 2690 cR), we observed an inversion between the RH map and the sequence concerning the NOV gene and the microsatellite LEI228. The order in the RH map is supported by a difference of LOD greater than 4 when compared to the order in the sequence assembly.In the lower part of the chromosome, several markers localized on the RH map are absent from the GGA2 sequence assembly: the Altogether, out of 198 markers localized on GGA2 through RH mapping, only one, labelled "no hit" in figure  also used for RH genotyping data collection.The GGA2 RH map is available on the ChickRH web server We have built a high resolution radiation hybrid map of chicken chromosome 2 using the chickRH6 panel. Our goal was to provide jointly a source of potential polymorphic markers and of candidate genes for QTL mapping on this chromosome. In the course of our work, the first draft chicken genome assembly was released and we aligned it to our GGA2 RH map. Although the sequence assembly is globally in good agreement with our data, a limited number of discrepancies and the mapping of sequence fragments of unknown location or of markers not present in the genomic sequence, show that RH mapping it still useful.Future developments of the chicken RH map will now be based on the genomic sequence, using it for choosing STS markers in selected regions to develop RH framework maps and thereby detect eventual problems in the genomic sequence assembly. This is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study.The generation of the 6000 rads chicken RH panel has already been described . This pa and the ChickAce database .Seventy-seven microsatellite markers well distributed along chicken chromosome 2 were selected from the published chicken genetic map. In addition, 11 other microsatellite markers and 10 genes were selected from other published data . Primer . All publicly available chicken EST (> 420,000) were collected in a local database and compared to the human genome sequence. EST selection targeted towards GGA2 was based on available comparative mapping data with the human genome. Long introns, whose positions could be predicted in chicken on the basis of their position in the human genes, were avoided and primers were chosen in the most divergent regions of the chicken/human sequence alignments, to avoid cross-amplification of the hamster DNA present in the hybrids. Information on primers for EST markers is given in the additional table.Primers for 219 EST were designed using the Iccare web server , . All p2 (Life technologies: Carlsblad, CA, USA), 0.3 U Taq DNA polymerase (Life technologies), 1X buffer (Life technologies), 200 μM of each dNTPs (Life technologies), 0.25 μM of each primer, and 1X loading buffer (350 mM sucrose and 0.2 mM cresol). After a 5 min denaturation step, 30 to 35 cycles (depending on the primers) of 30 sec at 94°C, 30 sec at annealing temperature, 30 sec at 72°C, were performed, followed by a final elongation step of 5 min at 72°C. Chicken DNA was used as positive control; hamster DNA and TE were used as negative controls.PCR were carried out in 15 μl reactions containing 25 ng hybrid DNA, 2 mM MgClPCR amplification results were scored on 2% agarose gels. Each marker was genotyped twice.. First, linkage groups were constituted by using a two-point LOD threshold of 4, after which a 1000:1 framework map  for each group was built under a haploid model. Then, the framework maps obtained were aligned on the genetic map and the orientation and distance between each group were estimated using the "printbestmap" option in the Carthagene program. Finally, after this final validation, all distances between markers were re-evaluated under a diploid model.The CarthaGene program  was usedMarkers not included in the framework map, but displaying two point Lod scores higher than 4 with framework markers, were mapped relative to the framework map by using the CarthaGene "buildfw" option which calculates the most likely position of a marker and gives a confidence interval.The map was drawn using the MapChart software . browser.Data on the location of the chicken genes in the sequence assembly was obtained by BLASTN searches using the Ensembl Data on human gene order were obtained from the Iccare web server.SL, MD, SB, FV and KF carried out the molecular study. SL built the RH map, and drafted the manuscript with FP. MM made the ChickRH6 panel. AV coordinated the project and finalized the manuscript.Description of the markers. The GCTXXXX markers were developed using the Iccare web server. The EXTXXXX markers were chosen from published information.Click here for file


In this report we re-examine some recent experiments with digital organisms to test some predictions of quasispecies theory. These experiments revealed that under high mutation rates populations of less fit organisms previously adapted to such high mutation rates were able to outcompete organisms with higher average fitness but adapted to low mutation rates.We have verified that these results do hold in the original conditions and, by extending the set of initial parameters, we have also detected that the critical mutation rate was independent of population size, a result that we have found to be dependent on a different, contingent factor, the initial fitness vector. Furthermore, in all but one case, the critical mutation rate is higher than the error threshold, a key parameter in quasispecies theory, which prevents its extrapolation to natural viral populations.From these results we conclude that digital organisms are useful tools for investigating evolutionary patterns and processes including some predictions from the quasispecies theory. RNA viruses are among the most infective pathogens affecting plants, animals and humans. Several of their features such as their reduced genomes, high genetic heterogeneity, large population sizes, short generation times and fast evolutionary rates place them among the best models for evolutionary and population genetic studies ,2. TheseAs a consequence, selection, that translates in competition with the host and among viral variants, usually results in the persistence of the most infective, pathogenic or more persistent variants. The molecular bases for this genetic variability are three mechanisms differentially used by each kind of virus: mutation, homologous and non-homologous recombination and genome rearrangement .10individuals in short times), high mutation rates (in the order of 0,1–1 mutations per genome and replication round (m/g/r) derived from lack of proof-read correction in the polymerase), and small genome sizes (ranging from 3 to 30 kilobases).Attempts to model the evolutionary dynamics of RNA viruses incorporate their most relevant features, such as large population sizes .- Genetic drift has no relevant effects: their tiny genomes, large population sizes and high mutation rates allow the exploration of all the neutral space around the master sequence.- The average consensus sequence remains stable along quasispecies evolution.This model contrasts sharply with conventional population genetic models in which the existence of a large number of neutral mutations would lead to genetic drift of the population and the individual is the unit of selection rather than a cloud of related variants . This laThe difficulty in experimentally testing some predictions of quasispecies theory has led to the search of alternative systems. In this work we have used digital organisms as an approximation to the population dynamics of RNA viruses. Digital organisms are self-replicating entities and compete for access to resources, in this case CPU cycles, as implemented in the AVIDA platform ,15.Avidians are programs (genomes) composed by arrays of logical instructions (genes) that allow them obtaining CPU cycles. There are 28 possible instructions. The number of instructions in a digital organism is equivalent to the genome size in a biological organism ,17.Many studies have been done using the AVIDA platform. The possibility that genome sizes change during the course of evolution and the rewards that they can obtain by the combination of functions have allowed investigations about the evolution of genomic complexity . Furthersurvival of the flattest and not survival of the fittest.In this work we have focused in a recent study by Wilke et al.  with digHere we have extended the original experimental conditions in order to study the effect and interaction between three of the key factors in the quasispecies model: population and genome sizes and mutation rates. Our results indicate that chance events in the form of historical contingency play an important role in the evolution of these populations. Moreover we have established a new, corrected mutation rate necessary for quasispecies formation with a higher value than the original one. The implications of this correction are discussed.We considered three factors affecting the critical mutation rate in our digital organisms: genome size, population size and the influence of the initial fitness vector. This is a vector of randomly assigned priorities for the first time evaluation of each organism fitness, incorporated to prevent the system from collapsing if all organisms simultaneously try to enter the CPU, and can be interpreted as a historical, contingent factor in evolution. The range of genome sizes studied varied from 54 to 272 instructions Table . Our expTable As expected from our preliminary results, the use of an initial random vector for each experiment and not for each organism resulted in clear differences with the results encountered by Wilke et al. . These dNevertheless, despite these differences between organisms we cannot conclude that there is a globally significant effect of population size on critical mutation rate. Using Bonferroni's correction for multiple comparisons we obtained a new significance level α' = 0.0045. Therefore only organism C148 has a significant, positive correlation (r = 0.932, P = 0.002). But the differences in the use of a random or fixed initial fitness vector are clear and can be observed by comparing Figures Table The quasispecies model requires a series of conditions to be fulfilled. These requirements are related to four key factors: population size, mutation rate, genome size and neutrality ,23. In o1) The use of different initial fitness vectors for otherwise identical experiments results in unpredictable effects on critical mutation rates for different population sizes. These effects were not detected in the original experiments by Wilke et al.  and can 2) There is no significant correlation between genome size and critical mutation rate.3) The originally calculated critical mutation rates underestimate their real values.Despite the lack of a general correlation between critical mutation rate and population size, the comparison of Figures Another key factor in the quasispecies model is genome size. The establishment of the quasispecies is easier in populations with small genomes, as in these the number of neutral sites is reduced and therefore the neutral space is also smaller. However the relationship between critical mutation rate and genomic size to be expected is somewhat contradictory. On the one hand, larger genomes need higher mutation rates because the neutral and adaptive landscapes are larger. On the other hand, it is well known that large genomes require a stability not supplied by high mutation rates, hence the existence of an error threshold that will be discussed later. In fact Eigen  proposedOur correction to the critical mutation rates estimated in the original paper relates directly to the limits imposed by the mutation rate. In most cases Wilke et al.  obtainedgag region in isolates from different patients, an indication of the limited adaptive solutions able to produce escape mutants to the immune response of cytotoxic T lymphocytes [In conclusion, although some predictions from quasispecies theory are not fulfilled in our experiments, we do have observed the principal prediction that lower fitness competitors can win the competition to high fitness ones, but only under very high mutation rates. Recently, several papers ,23,30 haphocytes . Furtherphocytes .Analogies are very useful in science, but they have to be used cautiously. Similar features and dynamics between digital organisms and RNA viruses are tempting and usually lead to conclude that both kinds of entities are governed by the same laws. This is not necessarily the case, as practitioners of the comparative method know. In any case, digital organisms are an extraordinary system to experiment with controlled, repeatable evolution conditions and further work with them is necessary to ascertain which evolution features are of their own and which are of common application to other evolving entities.The project was started with the twelve pairs of organisms generated in a previous experiment  that hadWith these twelve pairs we followed the same experimental procedure designed by Wilke et al. . BasicalIn AVIDA organisms occupy the limiting environmental resource, the computer CPU, depending on their "fitness". In order to prevent the collapse of the system when all the competing organisms simultaneously try to use the CPU, there exists one feature designed to prevent the simultaneous replication of all organisms at the start of the competition, when all the organisms might be equally fit since they have not been tested yet in the environment. This is achieved by asynchronously introducing organisms in the competition system by assigning an initial fitness to each organism that introduces a small time lag in the accession to the CPU. For this, Wilke et al.  generateDuring exploratory experiments we noticed that this vector could play a decisive influence in the result of the competition. In consequence, we divided the study into two parts. In the first one we kept the vector assigned by Wilke et al.  to each The critical mutation rate is "the midpoint between the highest rate at where A prevailed and the lowest rate where B prevailed" . It reprIt is necessary to clarify the conceptual difference between the critical mutation rate and the error threshold. The first one is the rate at which the prediction of quasispecies theory that organisms with lower fitness can win the competition is fulfilled. However the error threshold is the genomic mutation rate beyond which the information in the molecules that compose the quasispecies loses sense due to mutational degeneracy . In pracWe used versions 1.4 and 1.6 of the AVIDA program. Basically, digital organisms are chains of instructions that act over the CPU with the objective of reproducing as fast as possible. In this manner the CPU time becomes the limiting resource in their evolution. During replication their genomes can mutate and, as a result, a system with variation and therefore with selection and evolution is obtained. Genome sizes of the twelve pairs of digital organisms varied between 54 and 272 instructions . Generally 50 generations were enough for the fixation of the A or B organism in the population. Configuration files used in these experiments are available from the authors web site .Each mutation rate-population size combination was replicated six times. For the verification of the relation between the population size and the critical mutation rate we used Pearson's correlation coefficient and a significance level of 5% using Bonferroni's correction . The samIC performed all the simulations, made the statistical analyses and wrote the first draft of the manuscript. AM contributed to the design of the experiments and the discussion of the results. FGC designed and supervised the experiments, discussed the results and their analyses and wrote the final version of the manuscript. All the authors read and approved the final manuscript.


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Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity.c-myc, FGF-2 and HTLV-1). The tricistronic recombinant amphotropic viruses containing the IRES from EMCV, FGF-2 or HTLV-1 were equally efficient in inducing the expression of an exogenous gene in the transduced murine or human cells, without displaying any cell type specificity. The simultaneous presence of several IRESes on the same mRNA, however, can induce the differential expression of the various cistrons. Here we show that the IRESes of HTLV-1 and EMCV interfere with the translation induced by other IRESes in mouse melanoma cells. The IRES from FGF-2 did however induce the expression of exogenous cDNA in human melanoma cells without any positive or negative regulation from the other IRESs present within the vectors. Tumor cells that were genetically modified with the tricistronic retroviral vectors, were able to induce an in vivo anti-tumor immune response in murine models.We have constructed and compared different retroviral vectors containing two co-stimulatory molecules (CD70, CD80) and selectable marker genes linked to different IRES sequences (IRES from EMCV, Translation of the exogenous gene is directed by the IRES and its high level of expression not only depends on the type of cell that is transduced but also on the presence of other genetic elements within the vector. Gene therapy protocols would strongly benefit from the development of a one step technique that would allow cells to be genetically modified through the introduction of several therapeutic genes. In order to induce the translation and expression of exogenous cDNAs, carried by a single vector, researchers have cloned internal ribosomal entry sites (IRES) upstream from these exogenous cDNAs. This approach should lead to the translation of three cistrons from an unique mRNA and therefore to the consequent expression of the three encoded proteins -6.in vitro vector translation efficiency had been established [in vivo expression of heterologous proteins, they do, however, give little information as to the potential in vivo interactions that occur between different IRESs cloned into the same MuLV-based retroviral vector.In most cases, the IRES from EMCV is cloned into polycistronic vectors as it induces high levels of DNA translation ,7,8. Theablished -14. ThesEMCV), the IRES of the c-myc proto-oncogene (IRESC-MYC), the IRES of FGF-2 (IRESFGF-2) and the IRES of the HTLV-1 lentivirus (IRESHTLV-1) [EMCV and the second cistron was translated from the IRESEMCV, IRESC-MYC,, IRESFGF-2 or IRESHTLV-1.We cloned tricistronic vectors encoding three different cDNAs. This involved using at least two IRESes to translate the second and third cistrons. Using the same IRES twice in a single vector could, however, induce recombination events and the loss of the second IRES and cistron. In the same way, using the same cistron twice could lead to a competition between the two IRESs for the binding to cell type specific translation factors. For these reasons, we chose to clone and compare the efficiency of different IRESes cloned into the same vector. We chose the IRES of EMCV (IRESSHTLV-1) ,15-22. Tin vivo anti-tumor immune response, regardless of the percentage of co-stimulatory molecules expressed by the transduced cells.Recently, it has been shown that retroviral vectors derived from MuLV contain an additional IRES on the 5' gag sequence ,23. The Methods. Expression of the first open reading frame (cDNA encoding a co-stimulatory molecule) occurs from one IRES (MuLV) located at the 5' extremity of the mRNA. The second and third open reading frames are translated from two identical or two different IRESes. TFGEMCVNEO or TFGEMCVZEO were constructed so that the translation of the selection gene and the second co-stimulatory molecule could be induced from two identical IRESEMCV  from one promoter  Figure . The conV Figure . TFGHTLVV Figure .EMCVNEO, TFGEMCVZEO, TFGHTLV-1ZEO, TFGHTLV-1NEO, TFGFGF-2ZEO or TFGFGF-2NEO but none after transfection with TFGcMYCZEO or TFGcMYCNEO. We tested the supernatants from ψCRIP or 293T transfected cell suspensions for the presence of replication competent retroviruses (RCR) through the dosage of reverse transcriptase activity. The supernatants from all the transfected packaging cells were free of reverse transcriptase activity. The titers of the different retroviruses produced different results, depending on the experiments, varying from 104 to 106 particles/ml. We used 105 particles of each type of retrovirus that was produced (TFGFGF-2ZEO, TFGHTLV-1ZEO or TFGEMCVZEO) or the 48 hour supernatants from transiently transfected TFGcMYCZEO ψCRIP or 293T cells to transduce different types of mammalian cells. The murine cells used were either NIH-3T3 fibroblasts or B16.F10 melanoma cells. To establish a human model, we cultured melanoma cells from biopsies as described in the Methods. During the course of this study we obtained 23 melanoma biopsies. Fifteen cell cultures were obtained from these biopsies which represents a yield of 65 %. The quality and characteristics of the melanoma cells were determined by immunohistochemistry, on cytospin cells, using anti-cytokeratin (KL-1) (negative control), anti-S100 protein, anti-melanA/MART1 and anti-HMB-45/gp100 antibody staining as described in the Methods. The 15 human cell cultures obtained all stemmed from melanoma cells (data not shown).We generated retroviral vectors using the different plasmids described in Figure CMYCZEO transduced cells, we obtained roughly the same number of zeocin or G418 resistant clones with the different retroviral constructs. Both murine and human cells could be successfully genetically modified using the engineered trigenic retroviral vectors. No vector was statistically more efficient in obtaining a higher yield of resistant clones. However, the TFGEMCVZEO, TFGFGF-2ZEO and TFGHTLV-1ZEO cells were long lasting, viable and could be expanded, whereas neither murine nor human TFGcMYCZEO transfected cells displayed long-term viability.The transduced cells were selected for their resistance to G418 or zeocin. Fifteen days after the transduction with each retroviral vector, we selected G418 or zeocin-resistant cells. Apart from the TFGCells were stably transduced with MFG derived vectors encoding CD70, CD80 and an antibiotic resistance gene. After selection with the appropriate antibiotics (G418 or Zeocin), the stably transduced cells were analyzed for the expression of the different mRNAs, by RT-PCR using specific primers. The aim of the RT-PCR analysis was to show, through the expression of the full length RNA, that successful transcription of the construct had been achieved. We were uncertain whether the transcription of the ectopic DNA was complete. RT-PCR analysis was performed using cells that were resistant to Zeocin. We are convinced that the mRNA transcribed from the ectopic DNA contains the IRES zeocin cassettes. As we can noticed in figure These RT-PCR results suggest that there was no downregulation of CD70 or CD80 expression at the transcriptional and post-transcriptional levels.FGF-2ZEO. Figure FGF2ZEO or TFGHTLV-1ZEO retroviral vectors  and cells that expressed the two molecules.In murine cells (either fibroblasts or melanoma cells), a high percentage of cells only expressed CD70. This was not due to the expression of the CD70 molecule, in itself. Indeed, when we used constructs with CD80 as the first cistron, we obtained a high percentage of cells that only expressed CD80. This percentage could be attributed to a negative regulation of the translation of the third cistron.HTLV-1ZEO, TFGFGF-2ZEO or TFGEMCVZEO transduced murine NIH-3T3 cells, we respectively found 35%, 31% or 44% of cells that expressed undetectable levels of CD70 or CD80. Expression levels were assessed regardless of the resistance gene that was used (NEO or ZEO).Even after the selection of transfected cells, we obtained cells which did not express detectable levels of co-stimulatory molecules. Indeed, in TFGWe tested the expression of the two co-stimulatory molecules immediately after transduction or after selection. No correlation could be established between the time of analysis and the percentage of cells expressing both molecules. One clone could be composed of a majority of cells (60%) expressing both molecules at day 5 post selection, only have a small percentage of these cells at day 15 and up to 95 % on day 25. Indeed, even within a selected population of cells, the percentage expressing both co-stimulatory molecules could vary from 5 up to 95 % of the total cell number.After transduction with a single vector, 20 clones were selected. Within the same clone, some cells expressed only one co-stimulatory molecule whereas others expressed both molecules. These observations are reflected by the high SD of the percentage of cells expressing co-stimulatory molecules (Table 1 - see None of the vectors used were found to be adequate for the transfection of murine cells.HTLV-1ZEO (CD80 first) or TFGHTLV-1ZEO2 (CD70 first) we obtained cells that expressed only the first cistron. We obtained a better yield of cells expressing the two co-stimulatory molecules when using the TFGFGF-2ZEO construct (70% of cells expressing the two molecules in a stable manner). Again, even within a selected population of cells, the percentage of cells expressing both co-stimulatory molecules could vary considerably .For human cells, we chose to use a pool of transduced cells, rather than clones, to stay as close as possible to the reality of human clinical protocols. When we used TFGFGF-2 induced the translation of the second cistron, and IRESEMCV the translation of the third cistron, led to a high percentage of cells expressing the three cistrons.In human melanoma cells, the use of tricistronic vectors in which IRESin vitro splenocyte proliferation [HTLV-1ZEO (52 % CD70, 21 % both CD70 and CD80), TFGFGF-2ZEO (5 % CD70, 33 % both CD70 and CD80), TFGEMCVZEO (27 % CD70, 16 % both CD70 and CD80) or double-transfected with DFG CD70 and DFG CD80 (>65 % both CD70 and CD80), we showed that different percentages of cells expressing both co-stimulatory molecules (even as little as 20 %) could induce a proliferative response in splenocytes. A substantial increase in spleen cell proliferation was observed when the two molecules were expressed following genetic modification with all the viral vectors. This increase was similar to that induced by the use of double transfected cells (data not shown).Among selected cells, the percentage expressing both co-stimulatory molecules varied. We have previously shown that melanoma cells expressing high levels of CD70 alone or in combination with CD80 induced 5 cells, from each type of tumor, into the flanks of C57BL/6 mice  and double transfected cell tumors (p < 0.001) on day 20  expressing both co-stimulatory molecules to observe an immune response.To determine whether the local expression of CD70 and CD80 could affect tumor establishment, we sub-cutaneously injected 10e Figure . These c0 Figure . This dein vivo immune response, we transduced TS/A adenocarcinoma cells using the same retroviral vectors, as previously described. We pooled the zeocin resistant cells and chose two pools. In the first pool of cells transduced with TFGEMCVZEO, 34 % only expressed CD70 and 23 % expressed both CD70 and CD80. In the second pool of cells transduced with TFGFGF-2ZEO, 73 % only expressed CD70 and 20 % expressed both CD70 and CD80. These two pools were injected sub-cutaneously into syngeneic immunocompetent BALB/c mice. These injections led, in all cases, to the development of tumors. However, when the cells were double-transfected with two independent vectors and more than 70 % of cells expressed both co-stimulatory molecules, most of the palpable tumors spontaneously decreased within 10 days. In contrast, only a delay in the appearance of the tumors derived from transduced cells could be observed. This delay was more or less pronounced depending on the percentage of cells expressing both molecules. There was no delay in the appearance of tumors derived from the pool containing 34 % of cells expressing only CD70 and 23 % of cells expressing CD70 and CD80. A delay was observed in the appearance of tumors derived from the pool containing 73 % of cells expressing only CD70 and 20 % of cells expressing CD70 and CD80  to design polycistronic vectors that could allow the expression of several proteins from the same mRNA. Several authors have reported the construction of different vectors using the IRESnous gene,10,11,28m HTLV-1 ,18,20,21c-myc or HTLV-1.As we have generated retroviral vectors derived from the well known MFG vector (MuLV vector), we already had one additional IRES upstream from the first ATG codon (initiation codon of the first exogenous cDNA). This IRES has been described by Vagner and coll . The firc-myc proto-oncogene was used, we never obtained long-lasting zeocin or G418 resistant murine or human cells, whether these were tumor cells or fibroblasts. So far, most of the cellular mRNAs that contain IRESes and code for proteins involved in the control of cell proliferation and differentiation, require stringent regulation like for example the c-myc mRNAs [c-myc is used, translation is strongly downregulated [The choice of IRES that was used to express the cDNA encoding the G418 or zeocin-resistance genes was unimportant as we obtained resistant cells in all the cell lines tested: murine tumor cells (B16.F10, TS/A), murine fibroblasts (NIH3T3), packaging cell line (ψCRIP), human melanoma and lung adenocarcinoma cells (A549). However, when the IRES from the yc mRNAs ,15,29,30yc mRNAs ,19. Thisyc mRNAs . Such obWe obtained long-lasting viable resistant cells when we used the IRESes from EMCV, FGF-2 or HTLV-1. The number of clones obtained after transduction or transfection was roughly the same depending on the experiments and the cell lines tested. This indicated that only a few (if any) recombination events occurred when we used the same IRES (EMCV) twice in the same retroviral vector.trans-acting factors, the La antigen and PTB have been found to bind to picornavirus IRES elements and to be essential for their internal initiation of translation [trans-acting factors that are dependent on a specific IRES may be required. It could be that the MuLV IRES (first exon) binds those trans-acting factors with a higher affinity than the IRES from EMCV (third exon). Anthony and Merrick suggested that translation factors, that have a higher affinity for the cap structure than for the IRES element, could be sequestered at the m7GpppN cap structure and would therefore be unavailable for the internal initiation of translation at saturating concentrations of capped bicistronic RNA [The first and third IRESes are responsible for inducing the translation of the two co-stimulatory molecules. These IRESes competed to induce the expression of the two exogenous cDNAs. Indeed within the same population of selected cells, whatever the retroviral vector used, we were able to obtain cells that only expressed the first exon, or only the third exon or both exons. The percentage of cells that expressed both co-stimulatory molecules varied with the cell passage and from one selected clone to another. This is true regardless of the cell line tested: murine tumor cells (B16.F10, TS/A), murine fibroblasts (NIH3T3), murine packaging cell line (ψCRIP), human melanoma cells and human lung adenocarcinoma cells A549). There is a difference in the IRES-dependent mechanism that occurs in cellular and viral internal initiation. Currently, two cellular nslation ,31,32. Hnslation ,33. Borm. There inslation . The actnslation . It is ponic RNA . This woin vivo anti-tumor response in murine models. We first showed that B16.F10 cells that were genetically modified by the tricistronic vectors, could induce in vitro proliferation of spleen cells. These B16.F10 cells were then injected into syngeneic animals and tumor growth was monitored. We observed that in this murine model (C57BL/6) or in the BALB/c murine model (breast adenocarcinoma TS/A cells), co-expression of the two co-stimulatory molecules by at least 50 % of cells was necessary to induce an anti-tumor response. CD70 expression, alone or in association with a low level of CD80 expression, was not sufficient to induce anti-tumor immunity. These findings show that at least 50 % of the genetically modified cells must express the CD70 and CD80 co-stimulatory molecules before starting an immunotherapy protocol. Establishing cultures of human melanoma cells derived from biopsies was very difficult, we obtained a low yield of 63 %. We could infect human melanoma cells with the tricistronic retroviral vector with an efficiency of 50 %. However, within the 50 % of cells that were genetically modified we could only obtain a high percentage of cells expressing both co-stimulatory molecules when we used the tricistronic recombinant amphotropic viruses obtained with the IRES FGF-2.We have previously shown that two co-stimulatory molecules (CD70 and CD80), expressed on the surface of tumor cells could induce an anti-tumor immune response when the cells were injected into a syngeneic animal . In tumoin vitro and in vivo, to a variety of dividing cell types has been applied to research and gene therapy for the past 10 years. Our work shows that it is difficult to chose which IRES must be inserted into a polycistronic gene therapy vector, when the aim is to ensure a high level of translation of the exogenous gene. This level of expression will depend on the type of cell that is transduced but also on the presence of other genetic elements within the vector.The ability of retroviral vectors carrying IRESes to deliver genes, The murine melanoma B16.F10 cell line and the mouse mammary adenocarcinoma TS/A cell line, previously described, were cultured in RPMI 1640 supplemented with 2 mM glutamine and 10% fetal calf serum (FCS) (GIBCO-BRL, Cergy-pontoise, France). NIH-3T3 cells and the cysteine-rich intestinal protein (ψCRIP) and 293T packaging cell lines, were purchased from the American Type Culture Collection (Rockville, Md, USA). These three cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % FCS . All celMelanoma tumor biopsies were dissected into small explants and then enzymatically digested with collagenase (5 mg/ml) and hyaluronidase (3 mg/ml)  for 1 hour at 37°C under agitation. The cells were then centrifuged (5 minutes at 1200 rpm) and transferred to a culture flask and left to proliferate for 10 days. These cells were cultured in RPMI 1640 supplemented with 2 mM glutamine, 10% FCS, non-essential amino acids and vitamins (SIGMA, France). During the first 4 days after the first passage, cells were cultured with 0.1 mg/ml of G418 (GIBCO-BRL, Cergy-pontoise, France) to remove fibroblasts as described by Mouriaux and coll. The quaenv gene of the MFG vector.The pMDgag/pol and pBA-GALV plasmids were obtained from the Genethon (Evry, France). The tricistronic vectors are MFG-based retroviral vectors. These vectors were derived from the DFG-human-CD80 (hCD80) and DFG-human-CD70 (hCD70) vectors that have previously been described . The firEMCV. We digested the Blue-script plasmid (PKs) (Stratagene, La jolla, CA, USA) with Xho1 and EcoRV to insert the cDNA encoding IRESEMCV (636 bp), obtained from PCR amplification of pIRES-EGFP , and obtained a PKs IRESEMCV vector. This vector was digested by Nco1 and EcoR1 and ligated with the cDNA encoding either CD70 or CD80, obtained from the PCR amplification of DFG-CD70 or DFG-CD80 respectively. The PKs IRESEMCV CD70 or PKs IRESEMCV CD80 vectors were digested by BamH1. These inserts have been cloned into either DFG CD80 NEO or ZEO by partial digestion using BamH1, generating the (T) tricistronic vectors TFGEMCVNEO or TFGEMCVZEO with CD80 as first cistron and CD70 as third cistron or in DFG CD70 NEO or ZEO generating the same type of tricistronic vectors but where CD70 is the first cistron and CD80 is the third cistron.In these vectors, the translation of the genes encoding the two co-stimulatory molecules and the cDNA encoding the G418 or zeocin resistance genes are under the control of the IRESc-myc oncogene (cMYC)(602 bp) and the IRES of the HTLV-1 lentivirus (249 bp) upstream from the cDNA encoding the G418 or zeocin resistance genes in the PKs plasmid. We generated different combinations of cDNAs with BamH1 sites on both the 5' and 3' ends: IRESHTLV-1-NEO, IRESHTLV-1-ZEO, IRESFGF-2-NEO, IRESFGF-2-ZEO, IREScMYC-NEO and IREScMYC-ZEO. These constructs were inserted downstream from the gene encoding the first co-stimulatory molecule on the BamH1 site. We generated the TFGFGF-2NEO or ZEO, or TFGc-MYCNEO or ZEO and TFGHTLV-1NEO or ZEO tricistronic vectors  (547 bp), the IRES of the s Figure . All the4 viral particles/ml/48 hours/106 CRIP cells. 293T cells were triple-transfected with the different plasmids: TFG, pMDgag/pol and pBA-GALV. Estimation of the viral titer in transiently transfected 293T cells showed the presence of 106 viral particles/ml/48 hours/106 293T cells. Fibroblasts or tumor cells (B16.F10, TS/A or human melanoma) were transduced with the retroviral vectors using polybrene (8 μg/ml) (SIGMA, France). The stably transduced cells were selected using G418 or Zeocin depending on the viral particles used, and the resistant cells were either cloned or pooled and used in subsequent experiments.CRIP packaging cells were transfected with the different plasmids using the lipofectamine technique followed or not by selection with G418 (1 mg/ml) (from Life Technologies, GIBCO-BRL, France) or Zeocin (0.2 mg/ml) (CAYLA, France). The resistant CRIP clones were expanded and screened for viral titers which were of approximately 102 culture-flasks for 72 h until they reached sub-confluence. Total RNA was isolated from a suspension, as described by Choczynski and Sacchi (1987) [6 cells using TRIZOL™ reagent following the manufacturer's recommendations (Life Technologies, Cergy Pontoise, F). RNA solutions were treated with 5 units of DNAse 1 (Roche Diagnostics, Mannheim, D) to remove any contaminating genomic DNA. mRNA was transcribed into cDNA using the Ready-to-go™ kit (Amersham Pharmacia Biotech. Inc. Piscataway, NJ, USA) and random primers were purchased from Life Technologies. Amplification of cDNA was carried out using 1U/100 μl of Taq DNA polymerase (Roche Diagnostics, Mannheim, D) in a PTC-100™ Programmable Thermal Controller (MJ Research, Watertown, Mass, USA) after 33 temperature cycles consisting of denaturation at 94°C (60 s), annealing at 65°C (60 s) and elongation at 72°C (120 s). The following primers were used: MFG sense primer (Trigen 1): 5'-TGTAAAACGACGGCCAGTCACGTGAAGGCTGCCGACC-3', ZEO anti-sense primer (trigen 5): 5'-CAGGAAACAGCTATGACCCACCGGAACGGCACTGGTC-3', ZEO sense primer (trigen 4): 5'-TGTAAAACGACGGCCAGTGACCAGTGCCGTTCCGGTG-3' and MFG anti-sense primer (trigen 10): 5'-CAGGAAACAGCTATGACCGCCTGGACCACTGATATCCTGTC-3'. PCR products and the lambda/hindIII molecular weight marker (Promega, Lyon, F) were separated by electrophoresis on 0,8% agarose (Roche, Mannheim, D), Tris Borate EDTA (Interchim, Monluçon, F) gels and visualized by staining with ethidium bromide.Cells were cultured in T-75 cmi (1987) , of 5 × Transduced cells were stained for membrane expression of the two co-stimulatory molecules using PE-conjugated mouse anti-human CD70 mAb and FITC-conjugated mouse anti-human CD80 mAb (Pharmingen, Hamburg, D) as previously described [Female C57BL/6 (H-2b), or BALB/c (H-2d) mice were obtained from CERJ Janvier . All ear-tagged mice were kept in the special pathogen-free animal facility in our institution and were used for experiments between the age of 6 to 8 weeks. To establish subcutaneous (s.c.) tumors, 105 cells  were suspended in 0.1 ml of PBS and injected s.c. into the flank. Animals were examined daily until the tumor became palpable, the diameter of the tumor, in two dimensions, was then measured twice a week. The animals were sacrificed when the tumor size reached 2.5 × 2.5 cm in the control group. The statistical significance of the data was established using the two sample student's t-test. A p-value of less than 0.01 was considered to be statistically significant.in vivo studies, SB the synthesis of the retroviral vectors (molecular biology and virology). PR performed the characterization of the melanoma biopsies. LC provided all the IRES sequences. BC and ACP are responsible for conceiving this work. BC participated in the design, coordination and drafting of this manuscript. GF is the director of the laboratory. All authors read and approved the final manuscript.VDE carried out the cellular and Click here for file


The interpretation of data obtained in a movement analysis laboratory is a crucial issue in clinical contexts. Collection of such data in large databases might encourage the use of modern techniques of data mining to discover additional knowledge with automated methods. In order to maximise the size of the database, simple and low-cost experimental set-ups are preferable. The aim of this study was to extract knowledge inherent in the sit-to-stand task as performed by healthy adults, by searching relationships among measured and estimated biomechanical quantities. An automated method was applied to a large amount of data stored in a database. The sit-to-stand motor task was already shown to be adequate for determining the level of individual motor ability.The technique of search for association rules was chosen to discover patterns as part of a Knowledge Discovery in Databases (KDD) process applied to a sit-to-stand motor task observed with a simple experimental set-up and analysed by means of a minimum measured input model. Selected parameters and variables of a database containing data from 110 healthy adults, of both genders and of a large range of age, performing the task were considered in the analysis.A set of rules and definitions were found characterising the patterns shared by the investigated subjects. Time events of the task turned out to be highly interdependent at least in their average values, showing a high level of repeatability of the timing of the performance of the task.The distinctive patterns of the sit-to-stand task found in this study, associated to those that could be found in similar studies focusing on subjects with pathologies, could be used as a reference for the functional evaluation of specific subjects performing the sit-to-stand motor task. In the last decade quantitative movement analysis has been increasingly used in clinical contexts . This anModern complex instrumentation and models, such as stereophotogrammetric systems and multi-segment models of the human body, provide a thorough and faithful description of the subject's movement at a local level (e.g. joints kinematics), to be used at its best as a support to the functional assessment of subsystems of the locomotor apparatus (e.g. joint function) . HoweverHowever, even simplified experimental setups and models may provide a large amount of biomechanical data that requires considerable human efforts to be interpreted . In factdata mining techniques ) lend themselves to be effectively used in this context since they may reveal meaningful patterns and data structures from massive databases [data mining technique was applied to the data yielded by the analysis of sit-to-stand (STS) trials performed by healthy adults and carried out using the above-mentioned MMIM approach. The STS motor task was chosen because it has been shown to be adequate for determining the level of subject-specific motor ability [The aim of this study was to extract knowledge regarding the execution of a specific motor task. The term "knowledge" refers here to any relationship among attributes associated with the phenomenon under analysis. These relationships can be intended as causal and, therefore, suitable for the interpretation endeavours, or at least as tools for evidencing the presence of a repeatable pattern of variables. The declared goal was pursued by searching relationships among large amounts of biomechanical quantities by using an automatic method. Some atabases ,11. A sp ability . In addiIn order to properly frame this study, a summary description of the MMIM approach and an overview of the KDD process are reported.A MMIM is a model of a portion of the musculoskeletal system that includes the invariant aspects of both the modelled mechanical system and the motor task being performed. Therefore, a MMIM requires a minimum amount of measurements and provides a physiology-related description of the motor task . In analThe KDD process was introduced in order to provide a framework in which data-miners could work in a logical and sequential way, considering all the research aspects from the data acquisition to the information extraction ,14. An iInitially, the domain understanding, the parameter selection, and the goal definition need to be set. A subset of interest of the stored dataset can then be isolated. Pre-processing is performed to reduce noise and fill possible gaps in the target dataset. Elimination of outliers, corrections of wrong elements in the database and reduction of dimensionality are crucial transformations to reach an adequate level of suitability of the database.Data mining "is a well-defined procedure that takes data as input and produces output in the form of models or patterns" [atterns"  and is tatterns" , Descripatterns" , Predictatterns" , Retrievatterns"  and Discatterns" .Innovative techniques for the data mining have been introduced to be used either in conjunction with or in alternative to traditional statistical methods for two main reasons. First, while classical statistics is applied to data collected according to a specific goal of the analyst, data mining methods are applied to data already collected and aim at finding unknown relationships among them. Secondly, data mining allows to infer general rules with adequate approximation, even if the amount of data available is not as large as that generally required by inferential statistics .data mining technique is based on the specific analysis. Prediction, clustering, classification and research of association rules are the most common tasks and each of them may be accomplished with various algorithms. Finally, data interpretation helps the user in managing and understanding the results: visualisations (clustering) or extraction of symbolic rules are common ways of evaluating the discovered knowledge.The selection of the T, each experimental trial is a record of D and is made of a set X of literals called items. An item represents a specific value of an attribute of a table of D, and a record can be represented as an attribute (i.e. an output variable or a model parameter) together with its value [I = {i1, i2,...., im} a set of items of D therefore, T can be seen as a group of items such that T I. An association rule can be defined as a logical implication:The technique of research of association rules, which aims at finding the most recurrent patterns in a database, was selected for the data mining. Given a database D of experimental trials ts value . The proX YX I is the antecedent of the rule, Y I is the consequent and X ∩ Y = . A rule X Y, over a set of trials T, has a confidence c if c % of the trials in T containing X, also include Y. The same rule in the same context has a support s if s % of the trials in T contain X Y. The confidence of a rule X Y can be calculated from the support of the antecedent X and the support of the union of the antecedent X and the consequent Y:where X and Y in the sense that the presence of a pattern X in a trial implies, with a high probability, the presence of Y in the same trial. Given a set of trials T, finding "interesting" association rules in T is the problem of generating all the rules whose both support and confidence are greater than a set threshold (minimum support and minimum confidence).Confidence is an index of the validity of a rule. A high confidence means that there is a strong relationship between The extracted rules were reported in the following format:A → B [c % ]where the first item was the antecedent of the rule, the item which followed was the consequent, while the value indicated in square brackets was the confidence. The implication was intended to be valid only one way, from the left to the right. In case of validity of both directions, "definitions" were obtained:A ←→ B [c-min %]c-min was the lowest of the two c values associated with the one way rules. The search for association rules followed the path illustrated in Figure composed by two rules having the two items both as antecedent and consequent whose confidence A, generated a variable number n of partitions . The first partition 1_n Aincluded the lowest values of A and the last partition n_n Aincluded highest values of A. Items  similar to the qualitative items could thus be generated  were used to cluster the values of the attributes. SOMs are widely known as a powerful clustering tool  and coulhniques. . The lathniques.  from thehniques. . Values minimum support were found. The set of itemsets that appeared frequently in the transactions of the database was then identified. This step of the process was the most demanding in terms of processing time and computer memory occupation.The search of frequent itemsets was performed on the selected dataset. Itemsets with support greater than the The search for association rules was accomplished by using the APRIORI algorithm  which waCk of itemsets, counting their occurrences;• building of a candidate set Lk on the basis of support constraints.• defining "large itemsets" In figure minimum confidence reached the following phase. The selected association rules represented the knowledge extracted from the database expressed in a quasi natural language that the user could interpret. Efforts were made toward a clustered representation of the set of rules to increase readability and interpretability of information.Each frequent itemset generated a set of rules and each rule was scored by its confidence. Only rules whose confidence was higher than the A software project for the data mining phase was purposely designed and implemented as follows: the software received as input all data from the database and returned a text file containing a list of the discovered association rules and all the possible unified rules and definitions derived from the entire dataset. Support and confidence thresholds were set to 35% and 85%, respectively. Considering that in a rule more than one consequent can be found , a maximHealthy adult volunteers (N = 110), both males and females between 22 and 87, participated in the study, executing a total of more than 1100 trials. They were initially asked to sit on a seat. The height of the seat was set at a value equal to the subject's tibial plateau height . Subject® software . First, force platform signals were digitally low-pass filtered (second order Butterworth filter 15 Hz cut-off frequency). Data were then fed into the TIP model, which yielded the kinematic and kinetic time functions (displacement, velocity, force/couple and power) of the LA and SA. FA variables were not analysed since their contribution to the motor strategy was considered negligible, given the sagittal symmetry of the STS motor task. From these functions a subset of kinematic and kinetic variables (KK-set) was extracted including time events of the task , positioned under both the seat and the subject's feet. Data were collected at a sampling rate of 100 Hz and pre-processed with an internally developed Labview3_3TMaxSADispASO) as a consequent of the following kinematic and kinetic items:Various rules and definitions were found. Among them, some referred to obvious relationships such as those related to symmetry between right and left coordinates, others related a single item of a temporal parameter  3_6SAVelSo, at seat-off;b) 3,4_6MaxLADispASO, 4_5MaxLAForceASO, 3_6 MaxSADispASOand 2,3_5MaxSAVelASO, after seat-off;c) and of the following time events:1_6Duration, 3_6TMaxSADispBSO, 3_6TMaxSAVelBSO, 2_6TMaxSACoupleBSO, 2_6TMaxSAPowerBSO, 5_6 TMaxLADispASOand 3_5TMaxSAPowerASO.TMaxSADispASO was the attribute with the lowest number of partitions (three partitions) and its last partition included about 90% of the observations.The attribute The discovered definitions and rules that could not be easily predicted are illustrated in Figure The definitions related exclusively time instant items:3_6 TMaxSADispBSO←→ so3_6 t[95 %],• 3_5 TMaxSAAngVelASO←→ 3_5 TMaxSAPowerASO[95 %],• tSo (partition 3 of 6). The second definition associated the time instant of maximum sagittal velocity to that of maximum power, both after the seat-off. Moreover, meaningful rules were found that involved as consequent the item 1_6MaxSAPowerBSO. This item showed relationships, with a value of confidence varying between 86% and 96%, with the following kinematic and kinetic items:The first definitions related the 'average' time of occurrence of maximum sagittal displacement during BSO (partition 3 of 6) to 'average' values of 2_6MaxSACoupleBSO, 3_6MaxSADispBSO, 4_5MaxLAForceASO, and 2_5MaxSAVelASO; and the following temporal items:2_6TMaxSACoupleBSO, 1_6TMaxSAPowerBSO, 3_5 TMaxSAPowerASOand 3_6SAVelSo.The partitions corresponding to the attributes involved in both rules and definitions, their support and their range of variability expressed in the relevant units of measurement (UoM), are reported in Table The items reported in Table 3_3 TMaxSADispASOto various temporal, kinematic and kinetic items needs a further analysis to be interpreted. In fact, the attribute TMaxSADispASO was mapped in only three partitions and most of its observations were concentrated in the last partition. This circumstance rendered highly probable the presence of rules relating the item 3_3 TMaxSADispASOto those items of the various attributes with support higher than 35%. Therefore, these rules were used to highlight items involved with a considerable support and therefore the usefulness of such rules was deemed limited. In general, when interpreting a rule/definition found, the analyst should be aware not only of both its confidence and the support of the items forming it, but also of the number of partitions in which the attributes involved in the rule/definition were divided. The fewer are the partitions used for a quantitative attribute, the higher is the probability of finding rules/definitions unsuitable for drawing specific patterns. This is particularly true when most of the observations fall in a single partition of the attribute. Conversely, some of the rules and definitions discovered by the data mining analysis highlighted relationships that could not be easily predicted otherwise. The two definitions reported in the results section, that related time instant items, indicated that specific 'average' timings  of the sit-stand task were closely related. This finding is consistent with those present in the literature [The data mining analysis allowed for the discovery of both definitions and rules relating various items obtained from a MMIM analysis of the STS motor task. The most obvious and/or expected relationships, such as those related to the symmetry between right and left coordinates, also noticeable by a visual examination of the task as performed by the investigated subject, were included in the set of discovered rules and definitions. The finding of such relationships provided elements to confirm the validity of the data mining analysis. The set of rules found that related the temporal item terature ,33. In p1_6 MaxSAPowerBSOwith several KK-set items showed the importance of the SA in the execution of the task. In fact, almost all rules relating KK-set items to the 1_6 MaxSAPowerBSOregarded the sagittal actuator. When the maximum SA power during BSO occurred early in the task, its value was among the lowest (1_6 TMaxSAPowerBSO→ 1_6MaxSAPowerBSO). Low values of SA maximum power during BSO also occurred in combination with low-to-medium couple values (2_6 MaxSACoupleBSO→ 1_6MaxSAPowerBSO) and early in the phase (2_6 TMaxSACoupleBSO→ 1_6MaxSAPowerBSO). The latter rules showed that before seat-off kinetic variables of the main actuator are strongly related to each other and their timing. Given a value of one of them, a limited range of values is to be expected for the others. Moreover, 'average' SA velocity at seat-off was found to be present in combination with low maximum SA power at BSO (3_6 SAVelSo→ 1_6MaxSAPowerBSO) showing that relatively high speeds at seat-off could be reached even when the power exerted before seat-off was low. The presence of low value partitions in the rules may suggest that most healthy adults tend to use the least amount of energy necessary to complete the first phase of the task, showing an effective strategy of reduction of the energy expenditure [Very interestingly, the relationships of the item enditure . A valid4_6 MaxLAForceASO→ 1_6MaxSAPowerBSO). Moreover, consistent with the relationship to the SA velocity at seat-off, low maximum power of the SA during BSO occurred in combination with low-to-medium maximum velocity values during ASO (2_6 MaxSAVelASO→ 1_6MaxSAPowerBSO) showing that after seat-off a low-to-medium SA velocity can be reached and kept during the remaining part of the task, even when a low power is exerted before seat-off. Finally, average timing of occurrence of maximum SA power after seat-off implied a low maximum SA power before seat-off (3_5 TMaxSAPowerASO→ 1_6MaxSAPowerBSO) showing that, when the task is performed with an 'average' distribution of the time instants, the power exerted before seat-off is at its lowest values.The rules relating the low maximum SA power during BSO to variables occurring during ASO allowed for BSO-ASO crossed inferences. When medium-to-high maximum LA force during the elevation of the centre of mass toward the standing position was found, a low maximum SA power was generated by the SA before seat-off (The results' representation of Figure The study focused on finding the most frequent patterns of biomechanical variables and parameters obtained from dynamometric measurements of healthy subjects performing the sit-to-stand motor task. Data collected in a large database underwent a knowledge discovery process. The size of the database is strongly related to the simplicity of the data acquisition procedures. Simple and less expensive experimental set-ups allow the gathering of more data and in more locations than high-cost experimental set-ups and procedures. Data acquired from force platforms, processed with specific biomechanical models, represent a favourable condition to apply knowledge discovery processes effectively. In this study, data from volunteers in a large age range and of both genders were analysed in order to extract the most common patterns of healthy people performing the task. The results of the knowledge discovery process showed that sit-to-stand time events were strongly interdependent. Low maximum sagittal power values before seat-off were strongly related to numerous parameters both before and after seat-off, highlighting, among other characteristics, that most often a low power before seat-off is related to a regular occurrence of time instants and to low-to-medium sagittal speed from seat-off to the end of the task. The patterns found may be considered as typical rules of the sit-to-stand motor task and could constitute the basis for comparisons of patterns characteristic of different groups. The knowledge acquired in this study is the first step in the direction of developing a robust clinical tool to evaluate subject mobility.


Retrograde amnesia can occur after brain damage because this disrupts sites of storage, interrupts memory consolidation, or interferes with memory retrieval. While the retrieval failure account has been considered in several animal studies, recent work has focused mainly on memory consolidation, and the neural mechanisms responsible for reactivating memory from stored traces remain poorly understood. We now describe a new retrieval phenomenon in which rats' memory for a spatial location in a watermaze was first weakened by partial lesions of the hippocampus to a level at which it could not be detected. The animals were then reminded by the provision of incomplete and potentially misleading information—an escape platform in a novel location. Paradoxically, both incorrect and correct place information reactivated dormant memory traces equally, such that the previously trained spatial memory was now expressed. It was also established that the reminding procedure could not itself generate new learning in either the original environment, or in a new training situation. The key finding is the development of a protocol that definitively distinguishes reminding from new place learning and thereby reveals that a failure of memory during watermaze testing can arise, at least in part, from a disruption of memory retrieval. Teasing apart memory defects in animal models is not an easy task. A new protocol reveals that failure of memory can arise, at least in part, from a disruption of memory retrieval We are not in a position to decide between these two hypotheses.”For more than a century, the phenomenon of retrograde amnesia (RA)—the loss of memory for events that occur prior to a variety of precipitating brain insults—has provided the foundation for theories of memory consolidation and the locus of trace storage . HoweverStudies of RA have favoured a memory-consolidation interpretation in instances in which systematic variation of the time interval between experience or training and the subsequent brain insult has revealed a temporal gradation of RA . ComputaIn studies of spatial memory using the watermaze, amnesia for the location of the escape platform in posttraining probe trials (PTs) has generally been interpreted as a failure of learning, consolidation, or storage . To inveA summary of the experimental design is provided in F[7.78, 412] = 30.4, p < 0.001). Only animals that reached the acquisition criterion received lesions (69 out of 73 rats trained). The prospective lesion groups, trained as normal animals, did not differ .During cued pretraining, the rats quickly learned to search for, and climb onto, the visually cued escape platform. In the main spatial training phase, the animals rapidly learned to locate and raise the platform in order to escape from the pool , as indiOf the 69 animals that received lesions, one died after surgery and nine were excluded based on strict histological criteria, leaving a total of 59 animals  involving two between-subject factors: lesion group and platform location during the reminder treatment , and two within-subject factors: PT (PT1 and PT2) and quadrant (training versus opposite). In both figures, the initial memory expressed during PT1 is shown in the left lane. This reveals that the partially lesioned rats were at chance, whereas the sham-lesioned rats could remember the location of the platform (t = 6.15, df = 21, p < 0.005, paired-sample t-test, training versus opposite quadrant). The complete-lesioned animals were at chance. Analysis of percentage time in zone (t = 4.18, df = 21, p < 0.005, one-sample t-test, comparison with chance = 50%), but not in the two lesion groups.The key new findings are shown in  in zone  likewisePT1 ended with the animals finding the platform in the original training location, or in a novel location in the ‘opposite' quadrant of the pool (middle lane in F[2, 53] = 19.28, p < 0.001). With respect to the performance of the partial lesion group alone on this quadrant measure (see F[1, 16] = 7.98, p < 0.02) and no difference between novel and original reminding locations (F < 1). The partial lesion group also showed a highly significant preference for the training quadrant versus the opposite quadrant on PT2 (F[1, 16] = 16.83, p < 0.001). The same pattern of results is apparent in the zone data (see F[1, 16] = 7.64, p < 0.02) that also did not differ between ‘novel' and ‘original' groups (F < 1). Because a bias for the training location appeared even in the animals that were exposed to a novel platform position, memory on PT2 cannot be attributed to relearning of the platform location.The key new finding is that the partial lesion group displayed a bias for the training quadrant that was equivalent whether the animals had found the platform in the original training location or in the novel opposite location, at the end of PT1. The overall ANOVA of the PT2 quadrant data revealed a triple interaction of lesion group × quadrant (training versus opposite) × platform location during reminding  (data see  where, oF[1, 11] = 12.41, p < 0.005). Conversely, sham-lesioned animals exposed to the novel location exhibited modest reversal learning, increasing their time in the opposite quadrant (F[1, 9] = 9.35, p < 0.02). The same pattern of results was obtained from the analysis of time in the training zone (F[1, 20] = 5.46, p < 0.05).In contrast, sham-lesioned animals behaved quite differently in PT2 as a function of whether the platform was presented in the original or the novel location during the reminder treatment. Performance showed a further bias towards the training location between PT1 and PT2 following the event of climbing onto the escape platform in its original location, but exposure to the novel location resulted in a reduction in time spent in the training zone—a partial reversal. Supported by significant interactions in the overall ANOVA, analysis of time spent in the training quadrant revealed that, as expected, sham-lesioned animals reexposed to the original location increased their time there between PT1 and PT2 . However, escape from the water at the end of this PT might be sufficient to support new one-trial learning. Such learning was absent in the partial hippocampal lesion group (F < 1). The sham lesion group, in contrast, did learn (F[1, 21] = 4.51, p < 0.05), performing significantly better than the lesioned groups on PTn2 (post hoc Ryan–Einot–Gabriel–Welsch range test, p < 0.005). The complete lesion group again showed no evidence of learning in a new environment (F < 1).As an independent test of whether the reminder treatment of escape onto a platform could support new learning, all animals were taken to a second (‘downstairs') watermaze and given two PTs . This waThe key finding of this study is that rats with partial lesions of the hippocampus can be reminded of a preoperatively learned escape location in a watermaze by both correct and potentially misleading information. Whereas sham-lesioned rats showed new one-trial learning towards or away from the originally trained quadrant as a function of the type of reminder treatment to which they were exposed, partially lesioned animals were unable to learn. Instead, the first PT served only as a reminder of the original platform location irrespective of where in the pool the platform was raised at the end of this trial. Rats with complete hippocampal lesions showed neither new learning nor reminding.There is an extensive classic literature on the nature and effectiveness of reminder treatments . ExposurTwo features are distinctive about our study. First, unlike in many previous studies, the reactivated memory involves the recall and expression of highly specific information—a discriminable position in space, and not just a faster escape latency, or greater freezing. Second, despite exposure to a novel platform location leading to reversal learning in the sham lesion group, the partial lesion group displayed only reminding of the original platform location. This distinction is important because, with the current revival of interest in memory retrieval, our protocol circumvents the ambiguities involved in the use of relearning as an index of retention. One example of a study that used a reacquisition rather than a true reminding protocol  revealedNonetheless, the watermaze task is deceptively complex, and successful performance depends on the operation of several distinct memory systems . AccordiOur use of partial hippocampal lesioning introduces several other issues. First, it is a technique that is arguably more relevant to human amnesia, in which damage to a structure is typically incomplete. Second, it is also relevant to the many studies in which a pharmacological intervention is applied at a single site within a brain region—microinfusion into the dorsal hippocampus, for instance, is likely to have minimal effects on ventral hippocampal tissue see . Third, In raising many more questions than they answer, the present findings open a potential avenue of research into the neural dynamics of memory reactivation and retrieval. Specific interventions such as local AMPA receptor blockade cf.  might beDissociating the storage and retrieval functions of the hippocampus in memory is central to our understanding of the role of hippocampo–cortical connections. Many theories of hippocampal function are based on the idea that the hippocampus acts as a mediating link between different cortical regions during the interval before systems consolidation is complete . ParadoxWe used a total of 73 male Lister Hooded rats obtained from a commercial supplier (Charles River Laboratories, United Kingdom). They were pair-housed in plastic cages with sawdust bedding and ad libitum access to food and water. Their care and maintenance and all experimental procedures were carried out in accordance with United Kingdom Home Office Regulations.Behavioural testing was conducted using two separate circular pools, 2.0 m in diameter and 60 cm high, each located in well-lit rooms with numerous distal visual cues. One pool was used for training and retention (‘upstairs') and the other for new context learning (‘downstairs'). The pools were filled with water at 25 °C ± 1 °C made opaque by the addition of 200 ml of latex liquid (Cementone-Beaver, Buckingham, United Kingdom). We used the ‘Atlantis platform' , a polysTesting was carried out according to the schedule illustrated in This phase consisted of a single day of nonspatial cued training in the ‘upstairs' watermaze . The visible cue was suspended approximately 25 cm above the platform, which was moved every two trials to one of four possible locations, according to a pseudorandom schedule; the dwell radius was set at 20 cm, and the dwell time was 1 s.Training on a spatial reference memory task began 3 d later in the same watermaze. Rats received ten trials/day, in two sessions of five consecutive trials each , for 4 d. The dwell time was set to 0.5 s throughout training, but the dwell radius was gradually reduced over days . This schedule was intended to promote accurate and focused searching, but without generating the highly perseverative strategy that typically results from the use of long dwell times . Rats weSurgery took place 1–2 d after the end of training. Rats were given either partial or complete bilateral neurotoxic lesions of the hippocampal formation (DG and CA fields), or sham surgery. Complete lesions were intended to remove 85% or more of the total hippocampal volume. Partial lesions targeted the temporal two-thirds of the hippocampus, sparing the septal  third of the structure. The rats were assigned to groups of equivalent mean performance on the basis of their escape latencies during the final day of training. Lesions were made with ibotenic acid  following the protocol of This phase began 14 d after the end of training. It consisted of two PTs (PT1and PT2) spaced 1 h apart, with a reminder treatment occurring at the end of PT1.Each PT (PT1 and PT2) began with a standard 60-s swim with the platform unavailable. In each PT, the rats were placed into the pool in either the adjacent right or the adjacent left quadrants with respect to the training quadrant. Start positions were counterbalanced across PTs and across rats. At the end of the 60 s the platform was raised and the animals were allowed to find and climb onto it. The rats were allowed a further 60 s to locate the platform once risen (but still hidden just below the water surface); if unsuccessful within this period, they were guided to the platform. They then remained on the platform for 30 s.The raising of the platform at the end of PT1 constituted the reminder treatment; thus PT1 is sometimes referred to as the ‘reminder trial.' A key variable was that the platform was raised in either the original training location  or in a novel location in the centre of the opposite quadrant of the pool . Note that reminding using the original location always occurred in the training quadrant, and reminding using the novel location always occurred in the opposite quadrant. However, whereas the terms ‘training' and ‘opposite' are used to refer to physical areas of the pool, ‘novel' and ‘original' refer also to separate groups that received each type of reminder.For analysis of the different behavioural phases, several measures of performance were assessed, including escape latency, swim speed, and time spent within defined regions of the pool. Memory retention during PTs is inferred from the time spent in each quadrant of the pool as a percentage of the 60-s duration of the PT. A more sensitive measure can be obtained by analysing percentage time spent within a specified radius (zone) centred on the platform location . When tin1 and PTn2) spaced 1 h apart.New learning was assessed the next day in a separate ‘downstairs' watermaze that constituted a novel context. The protocol was identical to that used during ‘upstairs' retention testing, i.e., two rewarded PTs . Their brains were removed and stored in 10% formalin for 24 h before being blocked and embedded in egg yolk. The embedding procedure is described in The relative volume of spared tissue was calculated by measuring the area of hippocampus spared in each section of a particular brain according to the following protocol: Each coronal section containing hippocampus was placed under a photomacroscope (Wild, Heerbrugg, Switzerland), and the image taken by a mounted video camera was imported into NIH Image 1.63 . The area of spared hippocampal tissue in each section was then outlined and automatically calculated. Surrounding fibres such as the fimbria were excluded on the grounds that they would not be considered in a section were all the hippocampal cells dead. The sections were spaced 150 μm apart, yielding up to 32 sections in a sham lesion animal, and fewer in animals with acceptable partial lesions. For each rat, the total hippocampal ‘volume' was calculated by adding the area of hippocampal tissue spared in each successive section. The proportion of hippocampus spared for each lesioned animal was expressed as a percentage of the mean hippocampal ‘volume' for sham-lesioned animals. Values for the left and right hippocampi were initially calculated separately and then averaged see D.Strict criteria for acceptance of a lesion were used. The lesion had to be confined to the hippocampus in all cases, and leave intact tissue volumes of 25%–50% in the septal hippocampus with minimal sparing (less than 10%) elsewhere in the structure in the case of partial lesions, or less than 15% total hippocampal sparing in the case of complete lesions. Animals with minimal subicular damage, typically located at medial levels of the structure, were accepted.


Heptaplatin is a new platinum derivative with anticancer activity against various cancer cell lines, including cisplatin-resistant cancer cell lines .Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines has been investigated in connection with metallothionein (MT). Cytotoxicity was determined by an MTT assay. MT mRNA, was determined by RT-PCR assay. Transfection study was carried out to examine the function of MT.50 values of SNU-638 to cisplatin, carboplatin and heptaplatin were 11.2-fold, 5.1-fold and 2.0-fold greater than those of SNU-601, respectively. Heptaplatin was more effective against cisplatin-resistant and MT-transfected gastric cancer sublines than cisplatin or carboplatin was. In addition, heptaplatin attenuated cadmium, but not zinc, induction of MT.Of various gastric cancer cell lines, SNU-638 and SNU-601 showed the highest and lowest levels of MT mRNA, respectively, showing 80-fold difference. The ICThese results indicate that molecular mechanisms of heptaplatin effective against cisplatin-resistant gastric cancer sublines is at least in part due to the less involvement of MT in heptaplatin resistance as well as its attenuation of MT induction. Gastric cancer is the most frequently diagnosed and the second leading cause of cancer-related death in Korea. For many years, a few single agents such as 5-fluorouracil, doxorubicin, mitomycin C, and nitrosourea, have been considered to have significant antitumor activity in gastric cancer patients . HoweverDespite the efficacy of cisplatin against gastric cancer, there were two major problems with this agent. Firstly, there are significant side effects, such as severe nausea and vomiting, nephrotoxicity, and neurotoxicity . Secondlin vitro [Metallothioneins (MT) are a family of stress-induced proteins with a wide variety of physiological functions, including protection against metal toxicity and oxidants. They may also assist in regulating cellular proliferation, apoptosis, and malignant progression. MT has a high affinity for heavy metals since it contains many cystein residues, which account for approximately 30% of the total amino acids in this protein molecule . MT is ain vitro ,14 and Min vitro . The celin vitro .In this study, the effect of MT on the cisplatin analog-induced cytotoxicity was investigated in the gastric cancer cell lines. Since heptaplatin is less associated with MT, it is believed to be effective against cisplatin-resistant cells related to the high level of MT.MT cDNA, whose expression level was higher than the wild-type AML-2, were isolated from the paraquat-resistant AML-2 cells ,18. The Using the RT-PCR assay, the MT mRNA was determined in the 8 human gastric cancer cell lines, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668. The gastric cancer cell lines showed differential expression level of MT mRNA, in which the SNU-601 cells expressed the lowest MT mRNA level whereas SNU-638 expressed the highest level  and with the empty vector (pIRESneo2/Mock).Although the SNU-601/MT showed a 66% increase in MT expression when compared to the SNU-601/Mock cells, the SNU-601/MT cells scavenged significantly ROS generated by hydrogen peroxide or paraquat as compared with those in SNU-601/Mock using a fluorescence probe  to 2,000 ng/ml on an intermittent dosage schedule.The human gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668) were obtained from the Cancer Research Center in Seoul National University (South Korea). The cells were cultured in RPMI 1640 (GibcoBRL Grand Island, NY, U.S.A.) supplemented with 10% FBS . The cells were maintained as a monolayer culture and subcultured at confluence. The cisplatin-resistant gastric cancer cell subline was selected by chronic exposure to gradually increasing cisplatin concentrations ranging from 200 ng/ml  assay previously described by Pieters et al. [50) for a particular agent was defined as the drug concentration that causes in a 50% reduction in the number of cells compared to the untreated control for 3 days. The IC50 values were determined directly from the semilogarithmic dose-response curves. Cisplatin and carboplatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and heptaplatin (Sunpla®) was generously donated by SK Pharmaceutical (Seoul, Korea). Cisplatin analogs were dissolved in phosphate buffered saline and used immediately because of their instability in aqueous solution.The s et al. . The 50%32P] dCTP was added to each reaction mixture. Subsequently, the PCR products were combined and electrophoresed on 7.5% nondenaturing polyacrylamide gels. The bands were scanned using a densitometer (Pdi, Huntington Station, NY, USA). The quantity of each mRNA transcript was normalized with that of β-actin mRNA. Autoradiographic films of the RT-PCR assay were subjected to densitometric analysis using a densitometer.The total RNA was extracted from the cells using the acid guanidium thiocyanate-phenol-chloroform method . The MT 6 cells were prepared by lysing the harvested cells in an extraction buffer  supplemented with 2 mM phenylmethylsulfonyl fluoride  and 10 μg/ml leupeptin . The DNA was sheared by sonication. The sample buffer for MT analysis was used without 2-mercaptoethanol [et al. [The total cell lysates from 2 × 10oethanol . Western [et al. . The proGGATCCATGGACCCCAACTGCTCG-3' to introduce a BamH I site as underlined, and reverse primer: 5'ATAAGAATGCGGCCGCTCAGGCGCAGCAGCTGCA-3' to introduce a Not I site as underlined. The resulting MT PCR product was cloned to a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). After confirmation of the sequencing, the MT was cut from the TOPO plasmid vector (pCR 2.1-TOPO), purified, subcloning to the multicloning site of the expression vector pIRESneo2 and subsequently sequenced to confirm the correct open reading frame. Transfection was performed using the LipofectAmine Plus reagents (GIBCO BRL, Grand Island, NY, USA). Approximately 1 × 106 of the SNU-601 cells were plated into a 60 mm tissue culture dish and cultured overnight. Prior to transfection, the growth medium was replaced with the serum free RPMI 1640 media and cultured. The LipofectAmine reagent containing 2 μg of pIRES/Mock and pIRES/MT was combined with the Plus reagent and applied to the cells. After culturing for five hours, the media was replaced with RPMI 1640 containing 10% FBS. After selection by culture in the medium containing 0.2 mg/ml of G-418 (GIBCO BRL, Grand Island, NY, USA), the emerging colonies were isolated with cloning rings. The stable transfectants, SNU-601/Mock and SNU-601/MT, were obtained. The selection medium was changed every 2–3 days.Mammalian transfectants were produced as reported previously . The mam5/ml SNU-601/Mock or the SNU-601/MT cells was incubated with 1 μM DCFH-DA at 37°C for 4 hr. After incubation, the DCF fluorescence intensity was determined using a fluorometer at 485 nm for excitation and 530 nm for emission.Dichlorofluorescin (DCFH) was used to measure the ROS concentration. After 2', 7'-dichlorofluorescin diacetate (DCFH-DA) crosses the membrane, it is de-esterified to DCFH which is oxidized to fluorescent DCF by the ROS . Phospha