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  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  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 , TFGFGF-2ZEO , TFGEMCVZEO  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 , 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 , 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) . NIH-3T3 cells and the cysteine-rich intestinal protein (ψCRIP) and 293T packaging cell lines, were purchased from the American Type Culture Collection . 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 . During the first 4 days after the first passage, cells were cultured with 0.1 mg/ml of G418  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 . 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)  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  were transduced with the retroviral vectors using polybrene (8 μg/ml) . 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)  or Zeocin (0.2 mg/ml) . 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 . RNA solutions were treated with 5 units of DNAse 1  to remove any contaminating genomic DNA. mRNA was transcribed into cDNA using the Ready-to-go™ kit  and random primers were purchased from Life Technologies. Amplification of cDNA was carried out using 1U/100 μl of Taq DNA polymerase  in a PTC-100™ Programmable Thermal Controller  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  were separated by electrophoresis on 0,8% agarose , Tris Borate EDTA  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  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 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  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 = 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 . The complete-lesioned animals were at chance. Analysis of percentage time in zone , 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 . With respect to the performance of the partial lesion group alone on this quadrant measure  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 . The same pattern of results is apparent in the zone data  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  . Conversely, sham-lesioned animals exposed to the novel location exhibited modest reversal learning, increasing their time in the opposite quadrant . The same pattern of results was obtained from the analysis of time in the training zone .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 , performing significantly better than the lesioned groups on PTn2 . 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 . 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 . 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 , 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  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 . 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 . 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 . 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 , 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


Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators , in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish,  Highly conserved non-coding sequences in vertebrate genomes are frequently located around genes involved in development and can direct tissue-specific gene expression in functional assays. The idea that animal development is controlled by cis-regulatory DNA elements (such as enhancers and silencers) is well established and has been elegantly described in invertebrates such as Drosophila and the sea urchin [cis-regulatory elements and their genomic locations. Unfortunately, initial in silico identification of such sequences is difficult, as current knowledge of their syntax or grammar is limited. By contrast, computational approaches for protein-coding exon prediction are well established, based on their characteristic sequence features, evolutionary conservation across distant species, and the availability of cDNAs and expressed sequence tags (ESTs), which greatly facilitate their annotation.Identification and characterisation of a urchin ,2,3,4. Tcis-regulatory elements. Comparing DNA sequences from different organisms provides a means of identifying common signatures that may have functional significance. Alignment algorithms optimise these comparisons so that slowly evolving regions can be anchored together and highlighted against a background of more rapidly evolving DNA that is free of any functional constraints.The completion of a number of vertebrate genome sequences ,6,7,8,9,One of the key decisions inherent in comparative genomics is the choice of organisms for which the comparison will be made. A number of successful pairwise and multiple-species sequence comparisons have already been carried out to identify novel enhancer elements in mammalian genomes ,18,19,20(Fugu rubripes) gene regions has readily identified highly conserved non-coding sequences that have been shown to have some function in vivo [Fugu last shared a common ancestor around 450 million years ago [Fugu genome has the added advantage of being highly compact, reducing intronic and intergenic distances almost 10-fold [Another highly successful approach to increasing the resolving power of comparative analyses is to use multi-species alignments combining both closely related and highly divergent organisms ,22,23,24 in vivo ,32,33,34ears ago , predati 10-fold ,36. WithFugu. A general strategy for testing whether non-coding regulatory sequences of this type are functionally relevant involves assaying their ability to up-regulate reporter gene expression in vivo. “Enhancer” assays using transgenic animals, in particular mouse, are both slow and laborious, but have provided some exciting data on the function of non-coding DNA around developmental genes  embryos [As a first step towards identifying such networks in humans, we have used comparative genomics to identify and begin to characterise non-coding sequences that are highly conserved between human and s . An alt embryos ,38,39, wWe have adopted a medium-throughput strategy to test DNA sequences for enhancer activity in zebrafish embryos, whereby results may be cross-referenced and compared through a generalised scheme. We present functional data for 25 highly conserved non-coding sequences that are located around four unrelated developmental regulators, SOX21, PAX6, HLXB9, and SHH in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns.Fugu genome assembly [Fugu genome. This is due to additional gene or genome duplications in the teleost lineage [To locate conserved non-coding sequences, we masked the majority of the coding and tRNA content of the assembly  and compassembly  with theassembly . From th lineage , where r lineage . To avoiFugu sequence. The zebrafish, chicken, mouse, and rat genomes are at different stages of completeness, and therefore missing sequence information may account for the missing CNEs (as well as the lower percent identity in zebrafish), although it may also reflect regulatory differences between the lineages.The set of CNEs comprise a total of 273 kb of sequence, with a maximum length of 736 bp (average = 199 bp) and identity ranging from 74% to 98% (average = 84.3%). This is considerably higher than the level of identity seen between coding regions in these two organisms. Unsurprisingly, virtually all of the CNEs are conserved in rodent and chicken genomes, as well as a majority in the zebrafish genome. Of the 1,373 CNEs, 1,365 are conserved in the mouse, 1,316 in rat, and 1,310 in chicken, aligning to the human sequence with average identities of 97% for mouse and rat and 96% for chicken; 1,093 are also found to be conserved in the zebrafish genome, aligning with an average identity of 87.6% to the −76  relate to transcriptional regulation and development . Over 93% of the clusters (154/165) have a trans-dev gene located within 500 kb of one or more of its CNEs . Finally, there are three clusters that associate with two or more zinc finger genes.Whilst most of the clusters can be associated with one Trans-dev genes associated with CNE clusters tend to be located in regions of low gene density. We counted the number of genes situated within 500 kb upstream and downstream of a trans-dev gene, and compared this with the average for all human genes. Whereas the average for all human genes is 17, it is just six for the trans-dev genes. This is similar to the “gene desert” phenomenon described around the DACH gene [ACH gene . Interestrans-dev genes that we identified have previously been shown to have highly conserved cis-regulatory elements associated with them, including the Hox clusters [Fugu genomes and are located in a large gene desert on human Chromosome 22. Given that gene annotation and genomic sequencing of parts of the human genome are not yet fully complete, the discovery of CNEs here may well point to the existence in this region of an important transcriptional or developmental regulation gene with which they are associated. Indeed we find the largest number of CNEs (48) clustered around a relatively uncharacterised gene with zinc finger domains, ZNF503 on human Chromosome 10, the rat orthologue of which was recently characterised as a probable transcriptional regulator in brain development [A number of the clusters ,33, PAX6clusters , PAX9 [3clusters , SOX9 [2clusters , OTX2 [3clusters , SHH [30clusters , and DACclusters . Five CNelopment .All CNEs were compared with each other to look for local similarities. Forty-three elements show significant similarity to at least one other CNE, and in each case are situated close to genes with clear paralogous relationships, e.g., the HOX and IRX clusters. The remainder of the sequences appear to be unique in the human genome.trans-dev genes associated with CNE clusters, stringent whole-genome alignment located 408 CNEs, whereas MLAGAN identified over twice as many conserved regions (871) of at least 100 bp in length. The whole-genome analysis was more stringent in that we used a minimum exact word match of 20 bp, whereas MLAGAN uses short inexact words to create anchors from which a more sensitive (Needleman–Wunsch) alignment is carried out. It is important to note that similar alignments on genes that are not implicated in developmental regulation do not identify conserved non-coding sequence  alignment tool kit . This toe e.g., ,51)..trans-deFugu sequence, one CNE (SOX21_19) has 90% identity over 558 bp whilst another (SOX21_1) contains a 112-bp region of 100% identity  A. In mamidentity B, demonsCiona intestinalis, Drosophila melanogaster, and Caenorhabitis elegans, to see whether we could identify any of these highly conserved vertebrate sequences within the invertebrate lineage. Although many of the genes identified in our analysis have clear homologues within these genomes, we found no significant matches to any CNEs. More sensitive alignment using MLAGAN also failed to identify any conserved non-coding sequence similarity between vertebrates and non-vertebrates , whilst in each case the coding sequences were identified. This is surprising, given that the degree of identity between CNEs in vertebrates is higher than that of the coding regions for these genes. Thus, it is unlikely that the same set of sequences that appear to regulate important vertebrate trans-dev genes are found in invertebrates.Finally we searched invertebrate sequence databases, including the whole-genome sequences of We have assayed the ability of conserved non-coding sequences identified both from the whole-genome MegaBLAST analysis (CNEs) and from regional MLAGAN alignments (rCNEs) to up-regulate green fluorescent protein (GFP) reporter expression in zebrafish embryos see . We chosA total of 25 conserved non-coding regions were selected , and S1,Controls in which no element was injected , in which non-conserved, non-coding genomic DNA from the PAX6 or SOX21 regions was co-injected with the GFP reporter, or in which conserved, coding DNA from PAX6, SOX21, or SHH exons was co-injected with the GFP reporter produce essentially no up-regulation of GFP expression . When coIn order to build up a comprehensive picture of the GFP expression pattern induced by each of the elements, the expression profiles from multiple embryos positive for a given element were overlaid onto a schematic diagram, so providing a composite overview for each element . This alOf the eight SOX21-associated elements tested in our functional assay, seven enhance GFP expression . Three oThree elements strongly enhance GFP expression in the sense organs: SOX21_4 and SOX21_19 direct GFP expression to the developing eye . PAX6 is associated with the loss-of-function disorder aniridia. Some aniridia cases show chromosomal rearrangements downstream of an intact PAX6 gene, indicating that cis-acting elements can influence PAX6 gene expression in the eye at a significant distance from the coding region [cis-acting elements in upstream, intronic, and downstream positions. For example, 5′ elements drive expression in the lens, pancreas, and parts of the neural tube [Significantly, PAX6 is a paired-box-containing transcription factor, expressed in and playing essential roles in the developing eye; it is also expressed in the forebrain, hindbrain, and spinal cord  and thWe assayed six elements associated with a genomic region containing the HLXB9 and KIAA0010 genes . Each ofhb9, is expressed in the notochord, hypochord, tail mesoderm, and tailbud [HLXB9 is a Mnx-class homeobox gene associated with autosomal dominant caudal defects . The zeb tailbud , paralleTwo of the four SHH-associated elements tested in this study  direct Gshh and its co-orthologue twhh are both expressed predominantly in midline structures, i.e., floorplate and notochord. Later expression domains include the branchial arches, pectoral fin buds, and the retina [shh/twhh expression overlap in the floorplate; however, most of the other domains of GFP expression . In zebe retina ,63. GFP Understanding the intricate and finely tuned process of gene regulation in vertebrate development remains a major challenge facing post-genomic research. In order to begin to understand how genomic information can coordinate regulatory processes, we have adopted an approach integrating comparative genomics and a medium-throughput functional assay. Nearly 1,400 non-coding DNA sequence elements were identified that exhibit extreme conservation throughout the vertebrate lineage. Despite a degree of overlap, less than half of the non-coding ultra-conserved regions (109 out of 256) identified from the mouse and human genomes  are presIn agreement with current GRN models, and given the function of many of the genes we have identified in our analysis, it is logical to speculate that CNEs consist of modules of binding sites for transcription factors. However, the model of CNEs as transcription factor binding sites, even for large numbers of transcription factors, does not fully explain their high sequence identity across vertebrates, given that transcription factor binding sites are generally rather short and exhibit a level of redundancy. Consequently, we have not ruled out the possibility that the CNEs may have a completely different mode of action or act in numerous different ways.Fugu) together with some degree of proportional compaction in the Fugu genome. This suggests that the CNEs might play a role in structuring the genomic architecture around trans-dev genes, which in turn may lead to an additional level of transcriptional control. Further evidence that genomic architecture may be important comes from the fact the trans-dev genes are generally located in regions of low gene density.The relative positions and order of CNEs within a cluster is completely conserved in all vertebrate genomes we have analysed .Having established a “map” of the major locations of CNEs in the genome, we were able to take a more sensitive alignment approach in a number of these regions in order to identify additional CNEs (rCNEs). The distinction between CNEs and rCNEs is purely a bioinformatics one, based on our search parameters, and we have no reason to believe that there is any functional distinction between the two sets of elements. We selected a number of elements (both CNEs and rCNEs) as candidates for functional analysis. Data from our functional assay of 25 elements from four different developmental genes demonstrate that a significant proportion can act as enhancers, inducing expression of a GFP reporter gene in a tissue-specific manner. The observed expression patterns differ among elements, but are reproducible for individual elements. Enhanced GFP expression domains frequently coincide with endogenous expression domains of the Fugu genomes. This is exacerbated by the fact that some CNEs may also be found within or around neighbouring genes. This phenomenon has been described for both the PAX6 [Fugu) than to SHH . Furthermore, a number of elements are located directly 5′ of the HLXB9 gene, whilst others are found located further upstream, in introns of the next gene, KIAA0010. Although we strongly suspect that all these elements are associated functionally with the HLXB9 gene , we cannot rule out the possibility that they may associate with the SHH gene , which lies a few genes downstream. There are a number of cases where a CNE cluster is located close to more than one trans-dev gene, illustrating the value of correlating endogenous expression pattern with CNE enhancer activity. However, it should be noted that in order to build GRN maps for the elements, it is desirable but not essential to know which element associates functionally with which gene.Whilst it is straightforward to assign CNEs unequivocally to the SOX21 and PAX6 genes based on their location in the genome, the situation is more complex for elements in the vicinity of the SHH and HLXB9 genes, which are situated in close proximity to each other in the human, rodent, and the PAX6  and PAX9the PAX6  genes, athe PAX6 , where atrans-dev gene density.Our confidence in the correctness of our gene assignment for the elements tested in this study is borne out by the results of our functional analysis. For the elements that we have associated with PAX6 and SOX21, there is a good correlation between tissues that express the gene endogenously and tissues induced by the associated co-injected elements to express GFP, i.e., the major sites of endogenous gene expression are highly represented in our mosaically expressing embryos  to around 44% (SHH_6); this is probably due to many factors, e.g., variations in the embryonic stage at the time of injection and stochastic variations from embryo to embryo with regard to which cells the injected DNA is segregated into during cleavage. However, by combining expression data from a number of expressing embryos (an average of 30 embryos per positive element), we can gain insight into the overall pattern of reporter gene expression prescribed by each element.In addition to seeing GFP expression in “expected” domains (with respect to the associated gene), GFP expression was also often detected in tissues in which the associated gene is not normally expressed , subtle differences between CNE sequences may be tested functionally in this system. Zebrafish embryos are both readily produced and easily visualised, allowing convenient live screening throughout development. Their transparency makes the embryos ideally suited to GFP analysis and the problems associated with mosaicism in this screen are relatively easily overcome by injecting large numbers of embryos. Technical advances, such as the use of meganuclease injection, may facilitate this further.Fugu to the EMBL nucleotide database and are developing a publicly available relational database in order to store, curate, and analyse data from this study as well as data generated from ongoing identification and characterisation of rCNEs surrounding trans-dev genes.The combination of a comparative genomics approach together with functional screening of conserved elements produces a large and complex dataset. Efficient access, integration, and interrogation of this bioinformatics and functional data is crucial, and of increasing interest to the scientific community, to begin to characterise GRNs in vertebrates. To this end, we have submitted all CNE DNA sequences from We have identified an important set of highly conserved non-coding vertebrate sequences that associate with developmental regulators and have provided evidence that at least some of them demonstrate regulatory function. They are likely to be implicated in genetic disease, as has already been shown for the SHH gene . Their dFugu draft genome assembly . These predicted sequences were then masked in the Fugu sequence by supplying them as a “repeat library” to Repeatmasker35. The masked sequence was similarity searched against human genomic sequence from the Ensembl [Fugu sequences with alignments of 100 bp or over were selected to form the initial CNE sequence dataset.GENSCAN  . The GOstat program was used to find statistically over-represented GOs in this group of genes [The closest gene (using the transcription start site as defined in Ensembl) to the start of each CNE was determined from a list of all human genes supported by external evidence (“known” genes) downloaded using EnsMart, available from the Ensembl Web site  , D. melanogaster (v21.3.1), and Anopheles gambiae (v21.2.2) was carried out using MLAGAN as above.To look for the presence of CNEs in other available vertebrate genomes, CNEs were similarity searched against Ensembl mouse (v19.32.2), rat (v21.3.2), chicken (v22.1.1), and zebrafish (v21.3.2) genome sequences using BLASTn with default parameters. All invertebrate sequences in the EMBL database were searched in the same way using BLASTn with non-stringent parameters . More sensitive alignment of flanking orthologous sequence around the SOX21 gene (up to the coding sequence of the genes on either side) from Ensembl Zebrafish were raised and bred and embryos staged following standard protocols ,78; stagWe assayed for enhancer activity in embryos co-injected with candidate enhancer elements or control DNA and a minimal promoter–reporter construct in a method adapted from Muller and colleagues  as descrFugu genomic DNA , injected embryos were anaesthetised in Tricaine  and analmscooper@uwashington.edu;data relating to tissue category was also recorded on a spreadsheet.GFP-expressing cells were classified according to the following tissue categories: forebrain, midbrain, hindbrain, spinal cord, eye, ear, notochord, muscle, blood (circulating)/blood islands, heart/pericardial region (Please note: Some cells classified in this category may be circulating blood cells), epidermis/EVL, or fins. Cells that did not fall into one of these major expression categories  were categorised as “other”. The location and tissue category of each GFP-expressing cell for each embryo was recorded schematically using Adobe Photoshop software , by manually drawing colour-coded schematised cells in appropriate positions onto an overlay of a camera lucida drawing of a 31-hpf embryo  using standard protocols  and the Figure S1Fugu DNA.Each sequence represents the PCR product used in the functional assay. Sequence in bold type represents the position of the conserved element or elements within the PCR product. All PCR products were generated from (61 KB DOC).Click here for additional data file.Table S1(40 KB XLS).Click here for additional data file.Table S2(67 KB DOC).Click here for additional data file.All 1,373 CNEs (CR846105 to CR847477) and 16 rCNEs (CR847478 to CR847493) have been submitted to the EMBL database.


We set out to explore regulatory domains of the cIRK1 promoter that enhance or inhibit expression of the gene in different cell types.Inward rectifier potassium channels (IRK) contribute to the normal function of skeletal and cardiac muscle cells. The chick inward rectifier KcIRK1. cIRK1 contains two exons with splice sites in the 5'-untranslated region, a structure similar to mouse and human orthologs. cIRK1 has multiple transcription initiation sites, a feature also seen in mouse. However, while the chicken and mouse promoter regions share many regulatory motifs, cIRK1 possesses a GC-richer promoter and a putative TATA box, which appears to positively regulate gene expression. We report here the identification of several candidate cell/tissue specific cIRK1 regulatory domains by comparing promoter activities in expressing (Qm7) and non-expressing (DF1) cells using in vitro transcription assays.We cloned and characterized the 5'-flanking region of cIRK1 expression are similar to those seen in mKir2.1, the cIRK1 promoter differs by the presence of a putative TATA box. In addition, several domains that regulate the gene's expression differentially in muscle (Qm7) and fibroblast cells (DF1) were identified. These results provide fundamental data to analyze cIRK1 transcriptional mechanisms. The control elements identified here may provide clues to the tissue-specific expression of this K+ channel.While multiple transcription initiation sites and the combinatorial function of several domains in activating  Reaction mixtures were fractionated on 7% polyacrylamide (PAA)/7 M Urea gels. The 3'-most transcription initiation site was numbered +1.Total RNA was extracted from tissues and cells using TRIZOL reagent (Invitrogen) and treated with DNaseI. Three micrograms of total RNA  were reverse transcribed with cIRK1 cDNA [5'-GCA GAG TTA GCT TAA CAA GTA ACC G-3'] at 42°C for 1 hr. The PCR was performed in a reaction mix containing 1x PCR buffer, 200 nM each of 5'-forward primer [5'-CGA CTG GAG CAC GAG GAC ACT GA-3'] and R1, 100 μM dNTPs, 5 μCi 32P-α-dCTP, 5% DMSO, and 1.25 U of AmpliTaq (Perkin-Elmer). Reaction conditions were; 94°C for 3 min, 5 cycles at 94°C for 1 min, 57°C for 5 min, 72°C for 2 min, and then 30 cycles at 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, followed by 72°C for 5 min. Nested PCR was performed using the same conditions, with first round PCR products as template, and using the 5'-nested forward primer [5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3'] and R2 primer designed at 64-41 of cIRK1 cDNA [5'-GGG TGT TAA GAT CCG CGG GGA CAC-3']. Reaction products were eluted from the 7% non-denaturing PAA gel, re-amplified, then ligated into the pCR4-TOPO-TA cloning vector (Invitrogen) and sequenced.RNA ligase-mediated rapid amplification of 5' cDNA ends (5'RACE) was carried out using the GeneRacer kit (Invitrogen). In brief, 3 μg of total RNA were dephosphorylated, decapped and ligated with a 44-base RNA-oligo, according to manufacturer protocols. Next, the RNA was reverse transcribed using R1 primer designed at 146-122 of 32P-labeled cIRK1 cDNA fragment  overnight at 42°C, washed, and exposed to x-ray film. A quail glyceraldehyde-3-phosphate dehydrogenase (g3pdh) gene fragment  was used as an internal control. Message sizes were estimated by comparisons with RNA molecular weight markers (Invitrogen) run in the adjacent lane.Northern blotting was performed using poly A(+) RNA isolated using the FastTrak 2.0 kit (Invitrogen). Five micrograms of poly A(+) RNA were applied to each lane, fractionated on a 0.8% agarose gel, and transferred to a HybondN membrane. The membrane was hybridized with a random-primed cIRK1 5'-flanking region, AF375660), 3B-1229 (5228–6456 bp), 3B-531 (5926–6456 bp), and 3B-226 (6231–6456 bp) were generated by PCR, cloned into pBS-KS, and then subcloned into the multiple cloning site (between KpnI and XhoI) of pGL-3B (Promega). Constructs 3B-1089 (5368–6456 bp) and 3B-840 (5617–6456 bp) were obtained from a PCR fragment (1229 bp) with the 5'-region digested by SpeI and PvuII or XbaI, respectively. 3B-399 (6058–6456 bp) was derived from 3B-531 by removing the 5'-region by SacI and SmaI digestion, and 3B-206 was generated by exonuclease III digestion of 3B-226 followed by recircularization. 3B-109 (6348–6456 bp) was generated by SpeI and NheI digestion of 3B-206. Construction of 3B-206 with a mutated putative TATA box (3B-206dT) was accomplished by replacing the putative TATA box (TATTAA) with a StuI site (AGGCCT) by amplifying the original construct using primer sets designed to be complementary to sequences just outside the elements of interest, yet including StuI sites at their 5'-ends.Primers were designed to amplify each promoter region of interest. Constructs 3B-2256  with 1.5 g/L sodium bicarbonate, 10% fetal calf serum, and 100 U/ml penicillin/streptomycin. The quail myoblast cell line Qm7 [2 at 37°C. The ability of Qm7 cells to differentiate into myotube-like morphology was confirmed periodically as previously described [2HPO4, 6 mM dextrose, 25 mM HEPES at pH = 7.15, and 120 mM of CaCl2), and were incubated with 2 × 104 cells in 24-well dishes for 8 hrs in 500 μl of DMEM. The transfection mixes were replaced with fresh growth media and incubation was continued for 48 hrs. In vitro promoter activities were determined using the Dual-Luciferase Reporter Assay System (Promega). Briefly, transfected cells in each well were lysed in 100 μl of 1x Passive Lysis Buffer for 15 min at room temperature. Lysates were centrifuged briefly, and 20 μl of the supernatant was mixed with 100 μl of Luciferase Assay Reagent II, followed by 100 μl of SG reagent. Luciferase activities reflecting cIRK1 promoter activities and control thymidine kinase promoter activities were measured for 10 seconds after premeasurement periods of 2 seconds. All experiments were performed in duplicate and repeated at least 3 times (n= 3–6). The data were analyzed using a t-test assuming equal variance between two samples. Results are shown as fold increases ± standard error when compared with the basal luciferase activities from cells transfected with mock vector pGL-3B.The chick fibroblast cell line DF1 was purchased from ATCC and cultured at 5% COline Qm7  was cultescribed . One miccIRK1. He also conducted 5'RACE, Northern blot, cell culture, construction of deletion mutant, in vitro transcription analysis, and drafted the manuscript. LCK mapped much of the genomic structure and determined some of the sequence. EL and NK participated in maintenance of the cell lines and in vitro transcription analysis. JCO participated in design and coordination of the study and finalized the manuscript.HM obtained the sequence of cIRK1 genomic DNA and did all the database searches to identify motifs and control regions in the 5'-flanking region of