In vivo studies were conducted to quantify the effectiveness of low-level direct electric current for different amounts of electrical charge and the survival rate in fibrosarcoma Sa-37 and Ehrlich tumors, also the effect of direct electric in Ehrlich tumor was evaluate through the measurements of tumor volume and the peritumoral and tumoral findings.6 and 1 × 105 viable tumor cells, respectively. For each type of tumor four groups (one control group and three treated groups) consisting of 10 mice randomly divided were formed. When the tumors reached approximately 0.5 cm3, four platinum electrodes were inserted into their bases. The electric charge delivered to the tumors was varied in the range of 5.5 to 110 C/cm3 for a constant time of 45 minutes. An additional experiment was performed in BALB/c male mice bearing Ehrlich tumor to examine from a histolological point of view the effects of direct electric current. A control group and a treated group with 77 C/cm3 (27.0 C in 0.35 cm3) and 10 mA for 45 min were formed. In this experiment when the tumor volumes reached 0.35 cm3, two anodes and two cathodes were inserted into the base perpendicular to the tumor long axis.BALB/c male mice, 7–8 week old and 20–22 g weight were used. Ehrlich and fibrosarcoma Sa-37 cell lines, growing in BALB/c mice. Solid and subcutaneous Ehrlich and fibrosarcoma Sa-37 tumors, located dorsolaterally in animals, were initiated by the inoculation of 5 × 103, respectively.Significant tumor growth delay and survival rate were achieved after electrotherapy and both were dependent on direct electric current intensity, being more marked in fibrosarcoma Sa-37 tumor. Complete regressions for fibrosarcoma Sa-37 and Ehrlich tumors were observed for electrical charges of 80 and 92 C/cmHistopathological and peritumoral findings in Ehrlich tumor revealed in the treated group marked tumor necrosis, vascular congestion, peritumoral neutrophil infiltration, an acute inflammatory response, and a moderate peritumoral monocyte infiltration. The morphologic pattern of necrotic cell mass after direct electric current treatment is the coagulative necrosis. These findings were not observed in any of the untreated tumors.The data presented indicate that electrotherapy with low-level DEC is feasible and effective in the treatment of the Ehrlich and fibrosarcoma Sa-37 tumors. Our results demonstrate that the sensitivity of these tumors to direct electric current and survival rates of the mice depended on both the amount of electrical charge and the type of tumor. Also the complete regression of each type of tumor is obtained for a threshold amount of electrical charge. Several investigators have reported encouraging results from experimental low-level direct current therapy (DEC) in different types of tumor [The use of electric current in the treatment of malignant tumors has been known since the beginning of the 19of tumor . These sThe dose-response relationships obtained in these studies indicate that the DEC effectiveness depends on both the type of tumor and therapeutic scheme . Lack of guidance has become an obstacle to introduce the electrochemical treatment (EChT) in the clinic oncology. This is due to the lack of standardization of the EChT method regarding DEC doses and electrode array. Ren et al.  studied In spite of these results, the efficacy of DEC treatment has been controversial since an optimum electrode array and a threshold amount of electrical charge for each type of tumor have not been established. We believe that the procedure to determine the amount of electrical charge for each type of tumor is completely destroyed is more feasible to implement than that for the optimum electrode array, which involves several variables, such as polarity, number, and orientation of the electrodes. The knowledge of the optimum values of these parameters may lead to maximize the antitumor effectiveness of DEC and minimize their adverse effects in the organism. This allows the establishment of a therapeutic procedure for the tumor treatment in animals and in clinical oncology.The aim of this study is to test the hypothesis that the responses of the tumors treated with DEC is dependent on dose. Ehrlich and fibrosarcoma Sa-37 tumors were used. The survival rates of the mice bearing of these two types of tumor were determined. The antitumor effects of DEC were also evaluated through the peritumoral and tumoral findings in Ehrlich tumor.The experiment was run in accordance with Good Laboratory Practice rules and animals protection laws. The experiment was approved by the ethical committee of Oriente University, which follows the guideline from the Cuban Animal Ethical Committee. BALB/c male mice, 7–8 week old and 20–22 g weight were used. They were supplied from the National Center for Production of Laboratory Animals (CENPALAB), Havana City, Cuba, and were kept in standard laboratory conditions with water and food ad libitum. Animals were healthy  and were maintained in plastic cages inside a room at a constant temperature of 23 ± 2°C and relative humidity of 65 %, and a natural day-night cycle. During therapy the animals were firmly fixed on wooden boards, so all treatments were performed in the absence of anesthesia. All treated animals showed uneasy and quick breathing during fixation.Ehrlich and fibrosarcoma Sa-37 cell lines, growing in BALB/c mice, were received from the Center for Molecular Immunology, Havana City, Cuba. Both cell lines are being maintained in the Cell Culture Collection of the Department of Pathologic Anatomy, Hospital "Conrado Benítez", Santiago de Cuba, Cuba.6 viable tumor cells in 0.2 ml of 0.9 % NaCl, while fibrosarcoma Sa-37 solid and subcutaneous tumors located dorsolaterally in animals, were initiated by the inoculation of 1 × 105 viable tumor cells in 0.2 ml of 0.9 % NaCl. For both tumors, the viability of the cells was determined by Trypan blue dye exclusion test and it was over 95 %. Cell count was made using an hematocytometer.The Ehrlich and fibrosarcoma Sa-37 ascitic tumor cell suspensions, transplanted to the BALB/c mouse, were prepared from the ascitic forms of the tumors. Ehrlich solid and subcutaneous tumors, located dorsolaterally in animals, were initiated by the inoculation of 5 × 10. The mean tumor volume with the corresponding standard deviation of three determinations was calculated in each experimental group. Mice with non-palpable tumor at day 60 after the treatment were designated as cured.Tumor growth was followed by measuring three perpendicular tumor diameters (a, b and c, where a > b > c) with a vernier caliper. The tumor volume was estimated using the equation days) was determined for each individual tumor as the time needed to double the initial tumor volume. For each experimental group the mean DT and its standard deviation were calculated.Tumor doubling time , slight (+), moderate (++) and severe (+++).To supply electrochemical treatment, a high stability and low noise DEC source was built at the National Center for Applied Electromagnetism (CNEA). The electrode configuration consisted of a multi-electrode array formed by two anodes and two cathodes inserted into the base perpendicular to the tumor long axis keeping about 3 mm distance between them. Cathode and anode were connected in alternate sequence. This multi-electrodes array was proposed taking into account the results reported by Chou et al. . All ele3 were used. From this range of electrical charge three values were chosen to show the DEC effectiveness in both types of tumors. When the tumors reached approximately 0.5 cm3 in BALB/c mice, a single shot electrotherapy was supplied (zero day). For each type of tumor four groups consisting of 10 mice each randomly divided were formed. For Ehrlich tumor the groups formed were: control group (CG1), treated group with electrical charge of 36 C/cm3 (18.0 C in 0.5 cm3) and 6.7 mA for 45 min (TG1-1), treated group with 63 C/cm3 (31.5 C in 0.5 cm3) and 11.7 mA for 45 min (TG1-2), and treated group with 92 C/cm3 (46.0 C in 0.5 cm3) and 17 mA for 45 min (TG1-3). For fibrosarcoma Sa-37 tumor the groups formed were: control group (CG2), treated group with 36 C/cm3 (18 C in 0.5 cm3) and 6.7 mA for 45 min (TG2-1), treated group with 63 C/cm3 (31.5 C in 0.5 cm3) and 11.7 mA for 45 min (TG2-2), and treated group with 80 C/cm3 (40.0 C in 0.5 cm3) and 14.8 mA for 45 min (TG2-3).In order to find the thresholds of the electrical charge for which Ehrlich and fibrosarcoma Sa-37 tumors are completely destroyed, different amounts of electrical charge in the range of 5.5 to 110 C/cm3 (52.5 C in 0.5 cm3) and 19.4 mA for 45 min was supplied to 10 mice (5 mice bearing Ehrlich tumor and 5 mice bearing fibrosarcoma Sa-37 tumor). Also the dose of 110 C/cm3 (55 C in 0.5 cm3) and 20.3 mA for 45 min was supplied to 10 mice (5 mice bearing Ehrlich tumor and 5 mice bearing fibrosarcoma Sa-37 tumor). These doses were used to evaluate the therapeutic and adverse effects of the DEC above 100 C/cm3. For each type of tumor was formed a control group of 10 mice.The dose of 105 C/cm3 (27.0 C in 0.35 cm3) and 10 mA for 45 min (TG-A). This treated group was divided in three subgroups TG1-A, TG2-A and TG3-A to show the tumor and peritumoral findings at 1, 2 and 4 days after DEC treatment. Each experimental group was formed by 6 mice. When the Ehrlich tumor volumes reached 0.35 cm3, two anodes and two cathodes were inserted into the base perpendicular to the tumor long axis and a single shot electrotherapy was supplied (zero day).In order to examine from the histolological point of view the effects of direct electric current in Ehrlich tumor two experimental groups were formed: a control group (CG-A) and a treated group with 77 C/cmIn all experiments, before treatment the DEC was increased gradually step by step for two minutes until the desired intensity. During treatment it was constant and continually monitored. The voltage was also continually monitored. It varied, in accordance with the change of tissue resistance during the current application, between 5 and 25 V. The total electrical charge was calculated in real time. After a single application of the intended dose, the treatment was stopped. In this case, the current was decreased step by step for two minutes until its intensity was 0 mA. During electrotherapy, mice were firmly restrained, without obvious discomfort; therefore no anesthesia was necessary.In the control groups, four electrodes were placed into the base perpendicular to the tumor long axis without applying any direct current (0 mA). The animals of this group were firmly fixed but without DEC and showed uneasy and quick breathing during their fixation.Survival rates of the mice bearing both types of the tumors were determined for each experimental group. The survival rate (in %) was defined as the ratio between the number of live animals and the total number of animals, multiplied by 100 %. Survival checks mortality were made daily.The histologic cuts from each tumor were made according to the largest diameter. They were fixed in a 10 % formal solution and processed by the paraffin method.Hematoxylin and eosin staining was used. Each cut was divided into four microscopic fields in order to calculate the necrosis percentage through panoramic lens. This percentage was calculated as the ratio between the necrosis area and the tumor total area, multiplied by 100 %.The nonparametric statistical criterion of one-tailed Wilcoxon-Mann-Whitney rank sum was used to compare volumes between the treated groups with DEC and their respective control groups. Survival curves for the three different mice treatment groups for each tumor type were estimated by using the Kaplan-Meier product limit estimator .McNemar's statistical criterion was used for comparing the main histopathological findings in peritumoral zones in animals from CG-A and TG-A. P values of less than 0.05 were considered significant. The mean value and its mean standard error were reported for each experimental group.As it is shown in Table 3 that the tumors partially regressed for 2 and 4 days, respectively. For these same doses the fibrosarcoma Sa-37 tumor reached their respective partial regressions for 4 and 5 days. Both eventually outgrew again.The results shown in this study revealed that the sensitivity of the Ehrlich and fibrosarcoma Sa-37 tumors was dose dependent. The sensitivity to DEC of both types of tumors increased with the increase of the amount of electrical charge Table  and 2. T3 ; however, for the other doses there were significant differences (P < 0.05).The overall survival curves of the mice bearing Ehrlich and fibrosarcoma Sa-37 tumors are shown in figures The cured mice were sacrificed at 100 days post-treatment. Before sacrifice, the animals were active and in good physical condition with adequate body weight. They had good posture and coats of hair. After sacrifice, the histopathological findings in each of these mice showed complete disappearance of the tumor and evidence of healing. In the treated mice a very little necrotic tissue remained within a fibrous scar. Serology and histological finding of the organs did reveal neither abnormalities nor metastases (results not shown).3, during the first 24 hours after DEC treatment. The histological findings showed both severe alterations in liver and kidney and an increase in the weight of these organs. Metastases were not observed in any of these mice.The death of a mouse 1-day after DEC treatment was observed in TG1-3. The histological findings revealed damages in the lungs due to hemorrhage and a small circular necrosis. Metastases were not observed in this mouse. It was also observed the death of a mouse 25 days post-treatment in TG1-3 and 50 days in TG2-3 due to the cannibalism shown by the mice, probably because of the blood present in the tumors after DEC treatment. All the mice died for amounts of electrical charge above 100 C/cmThe histopathological findings revealed that in the Ehrlich untreated tumors (CG-A) the necrotic area was mainly central and it constituted approximately from 20 % of the tumor total area Fig. . HoweverThere was a lack of well defined necrosis zones surrounding the electrodes. The morphologic pattern of the necrotic cell mass observed is the coagulative necrosis. The dead tissue becomes both swollen and firm in consistency. Preservation of the basic profile of the coagulated cancerous cell and nuclear karyolysis were also observed. The lysed erythrocytes was also observed. This type of necrosis was accompanied by accumulation of neutrophil polymorphonuclear leucocytes.Lymphocytes (L) and plasmatic cells, named CP, were observed in all the tumors in CG-A and TG-A but there were no significant differences Table , Fig. 8.In this experiment no mouse died from intercurrent disease during or after the treatment. Before sacrifice, the animals were active and in good physical condition with adequate body weight. They had good posture and coats of hair.The results of this study demonstrated that DEC has a marked antitumor effect because a single-shot electrotherapy delivered via four platinum electrodes inserted into the base of the fibrosarcoma Sa-37 and Ehrlich murine tumors significantly retarded their growths when compared with their respective control groups. The fact that tumor regression increases with the increase of the amount of electrical charge may be explained because the induced necrosis by DEC into the tumor depends directly on its intensity, a matter that is in agreement with the results of Robertson et al. [We believe that the necrosis is the predominant mechanism of cell death, by the cellular tumefaction (or cellular swelling), cell rupture, breakdown of organelles and acute inflammatory response observed during the first 4 days post-treatment in all treated tumors, result that agrees with that previously reported by Dodd et al.  and HolaThe necrosis may be due to the ischemia observed in all tumors treated with DEC, which could lead to an irreversible cell injury of the tumor cells and therefore to cellular death. This fact could be related with other experimental findings found after DEC treatment, such as: degradation of phospholipids, lost of high energy phosphate and increase of the intracellular calcium , membranThe prolonged acute inflammation observed during 4 days after DEC treatment may be explained by the persistent leukocyte infiltrate also observed in the peritumoral findings. This persistent leukocyte infiltrate  becomes a harmful agent because during the chemotaxis they amplify the effects of the initial inflammatory stimulus through the liberation of potent mediators  that lead to both endothelial and tissue damages. This leukocyte infiltrate may also activate the immune system . In all 3, respectively, may suggest that each tumor model has its threshold of electric charge from which it is completely destroyed. This threshold depends on the electric nature of the tumor and their physiological characteristics (stage, volume and histogenic characteristics). This fact explains the cure of the mice and why the tumors do not duplicate their initial volumes during the observation time  declare that they have no competing interestsHCC conceived the study, and participated in its design and coordination. Also, he carried out the inoculation of the tumor cells in the mice, the measure of the tumor volumes and the survival rate of mice as well as elaborated the manuscript. MCSQ participated in the design of the study and participated in the measure of the histological findings of the organs and tumor and peritumoral findings and as well as elaborated the manuscript. LEBC carried out the inoculation of the tumor cells in the mice, conceived and participated in the design of the study and performed the statistical analysis as well as elaborated the manuscript. RNPB and DSL participated in the design of the study and contributed to elaboration of this manuscript. MFS participated in the design of the study and carried out the serology. All authors read and approved the final manuscript. OGP and TRG participated in the design of the study and contributed to elaboration of this manuscript. All authors read and approved the final manuscript. JLMF participated in the design of the study and performed the statistical analysis.The pre-publication history for this paper can be accessed here:


Functional information is lacking for many of the hypothetical proteins encoded within sequenced genomes. Richard Roberts proposes that a community-based approach might offer an effecient way to fill the function gap During the last few years, we have seen enormous strides in our abilities to sequence genomes, and the information that has poured out of these sequences is quite astonishing. With more than 150 complete genome sequences now available and many laboratories rushing into microarray analysis, proteomic initiatives, and even systems biology, it seems an appropriate time to consider not just the opportunities those sequences present, but also their shortcomings. By far the most serious problem is the quality and degree of completeness of the annotation of those genomes. Most troublesome are the large numbers of open reading frames that have been identified by computer programs, but remain labeled as a “conserved hypothetical protein” when they occur in more than one genome or simply a “hypothetical protein” when they appear unique to the genome in question. Between them, these two categories of annotated open reading frames often represent more than half of the potential protein-coding regions of a genome.These annotations highlight just one portion of our ignorance about the information content of genomes and our lack of fundamental knowledge about the function of so many of the building blocks of cells. Unless we rectify this situation, it is likely to undermine many of the other “-omic” efforts currently underway. Here I advocate a rather straightforward approach to address this problem—focused initially on the bacterial genomes. In contrast to the numerous proposals for big science initiatives to understand the fundamental workings of biological organisms, I propose a small science, relatively low-tech approach that could have a dramatic pay off. A relatively small investment could yield a massive amount of information that would greatly enhance our current efforts to use genomic approaches to study life.The initial proposal is directed at deciphering the role of the “hypothetical proteins” encoded in the microbial genomes and would involve a community-wide approach to determine the function of these hypotheticals based on solid, old-fashioned biochemistry. The essence of the idea is to undertake an interdisciplinary effort that couples our current bioinformatics capabilities to predict protein function with a directed exploration by experimental laboratories to test those predictions. I would encourage a consortium of bioinformaticians to produce a list of all of the conserved hypothetical proteins that are found in multiple genomes, to carry out the best possible bioinformatics analysis, and then to offer those proteins to the biochemical community as potential targets for research into their function. To energize laboratories with appropriate expertise to participate in this community-wide effort, I suggest that a special program be set up by one or more of the funding agencies so that laboratories undertaking the investigation of any particular protein receive a small grant upfront as a supplement to an existing grant. Upon completion of the project and the identification of the function, they would receive a further supplement to that grant as a reward. In this way, one might hope to rally some of the best biochemical talent and apply it to this problem of determining function for a wide range of new proteins. The cost of such an operation could be quite minimal, and the bureaucracy and review process could be equally simple. Here is a case where a modest infusion of funds could greatly enhance our ability to annotate both existing and new genome sequences and ensure that our current investments in genomic sequences yield the richest biological harvest possible. There are two key steps in the proposed plan.The first step is to encourage some bioinformaticians with appropriate expertise in the functional annotation of genomes to form a consortium and undertake the assembly of a list of prime targets for which an experimental demonstration of function would be most valuable. Three general classes of such genes come to mind: (1) The conserved hypothetical genes. These belong to the set of genes that have orthologs in many other genomes, but for which no function has been experimentally determined in any case. A recent success among such genes is illustrated in These sets of targets would be combined and arranged into a prioritized list in which each was accompanied by the best assessment of potential function. The priorities would be based on which genes were most likely to prove broadly informative. For instance, a conserved hypothetical gene that occurred in most genomes would be of higher priority than one that had only two orthologs. The list would be on a public Web site where these targets and the predicted functions could be examined and modified by alternative or additional predictions from other groups to guide future experimentation. As function was derived, that information could be presented and the target removed from the main list.The second step would be to invite experimentalists to peruse the list and find those potential genes whose protein products might lie within their realm of expertise so that they could use their experimental knowledge and reagents to quickly test for function. Initially, I would advocate allowing laboratory teams to pick and choose among the list and sign up to study just one of these open reading frames. I would recommend allowing one laboratory per open reading frame in the initial stages. A laboratory wishing to sign up would generate a short document highlighting why its expertise might be suitable for a particular protein. A one-page proposal should suffice, with no experimental plan demanded. At this point, a small panel could choose among competing efforts and the laboratory chosen would be given a small grant and up to six months to carry out its analysis. If it was successful in delineating the function of their target protein, a paper would be written and submitted for peer review. If the paper was accepted for publication, then an additional sum would be allocated as a supplement to the laboratory's existing grant. If, after six months, a laboratory had not managed to delineate the function, it would submit a short report describing the approaches that have been tried, with the results of its analyses. This would be posted on the public Web site and that target would then become open for analysis by other laboratories, under the same conditions as before.Escherichia coli, I would not demand that the biochemical experiments be done on the E. coli gene. Any of the orthologs would do, so long as the similarity was sufficiently strong to give high expectations that function would be conserved. In fact, for a laboratory that happened to be already working on one of the homologs, this program might provide an added bonus and greatly speed its work. I would also encourage both biochemical and genetic approaches, since one can never be certain when one method might be better than another. The list would, of course, also include conserved genes not found in E. coli, but commonly distributed in other genomes. In particular, I would make a pitch for including all genes in Mycoplasma genitalium, which, as the free-living organism with the fewest genes, might be the most suitable as a model system for in-depth understanding of its biology.While the initial list of target genes should probably be based on a well-studied and experimentally tractable organism such as Saccharomyces cerevisiae genome (This proposal for experimental attack on hypothetical genes is really a very traditional approach that becomes large-scale simply because of the parallel nature of the implementation. It resembles the successful approach used by the Europeans to achieve the complete sequence of the e genome . The resE. coli, one gene, hemK, was found that had no immediately known protein product (hemK gene might encode this enzyme. Subsequent hemK homologs were annotated as putative protoporphyrinogen oxidases and soon the “putative” was dropped. Then one group noticed that the gene product contained protein sequence motifs typical of DNA adenine methyltransferases. From then on, the annotations in GenBank alternated between these two assignments.During studies of the genetics of heme biosynthesis in  product . Since ohemK gene is of ubiquitous occurrence from humans to Chlamydia, and yet until 2002 its true biochemical function was unknown. At that point, two groups (E. coli the HemK gene product was an N5 glutamine methyltransferase that transferred a methyl group from S-adenosylmethionine to the amide nitrogen of a specific glutamine residue in the protein chain release factors prfA and prfB. Particularly noteworthy was the observation that the hemK gene is positioned immediately adjacent to the prfA gene in many microbial genomes! Here is a case where bioinformatics suggested strongly that the hemK gene encoded a methyltransferase, but an experiment was needed to identify the substrate. Since the adjacent gene in the genome encoded the substrate, it might have been possible to make that prediction too. There are additional paralogs of hemK in several genomes, but their biochemical activity and substrates remain to be identified.This o groups  demonstr


Benign fibrous histiocytomas (BFH) usually presents as a small benign lesion that predominantly occurs in the skin. Only few cases of BFH arising from bone have been reported, its occurrence in pelvic bones is even rarer.os ilium which was larger than earlier reported BFH of the bone. Surgical resection was performed successfully including resection of the inner pelvic ring and reconstruction of the linea terminalis using a non-vascularized fibular autograft. At 18 months after tumor resection and reconstruction of the pelvic ring, with interposition of a free vascularized fibula graft patient has an excellent clinical oncological and functional outcome.A 34-year-old female presented with BFH at a rare anatomical location on both sides of the Non vascularized fibular autograft is a useful reconstructive procedure in select patients. Benign fibrous histiocytoma (BFH) is a tumor that occurs predominantly in the skin  and most commonly in younger individuals. The tumor typically presents as a painless nodule varying in size from a few millimeters to several centimeters.BFH of the bone has been a subject of increasing interest within the past few years. The term was initially introduced by Dominok in 1980 to describe a cystic lesion in the femur of a 66-year-old man -6.Histologically, BFH arising from soft tissues cannot be distinguished from those arising from bone . Pain isOn radiography the lesion is characterized by osteolytic lesions with a well defined sclerotic margin . MagnetiA local recurrence rate of 5 to 25 % is reported in literature. The recurrence rate is typically related to the size of the tumor . Consequilium with a large extra- and intrapelvic soft-tissue component . We are of the opinion that stabilization of the inner pelvic ring is necessary to prevent stress fracture of the remaining bone bridge.After mobilization of the patient an increasing diastasis of the symphysis was noted Figure . This miOncologically the marginal tumor resection has proved to be an adequate treatment. Despite high recurrence rates, especially in the case of large BFH , no locaThe author(s) declare that they have no competing interests.PN prepared the manuscript and participated in the operations.KL carried out the radiographies and MR imaging and contributed the section on imagingMW performed histological analysis and wrote pathology part of manuscriptLB and DP critically reviewed the manuscript for its scientific content and performed surgery.


An increasing number of studies have profiled tumor specimens using distinct microarray platforms and analysis techniques. With the accumulating amount of microarray data, one of the most intriguing yet challenging tasks is to develop robust statistical models to integrate the findings.n = 305 samples) on a common probability scale, we developed a 90-gene meta-signature, which strongly associated with survival in breast cancer patients. Given the set of independent studies using different microarray platforms which included spotted cDNAs, Affymetrix GeneChip, and inkjet oligonucleotides, the individually identified classifiers yielded gene sets predictive of survival in each study cohort. The study-specific gene signatures, however, had minimal overlap with each other, and performed poorly in pairwise cross-validation. The meta-signature, on the other hand, accommodated such heterogeneity and achieved comparable or better prognostic performance when compared with the individual signatures. Further by comparing to a global standardization method, the mixture model based data transformation demonstrated superior properties for data integration and provided solid basis for building classifiers at the second stage. Functional annotation revealed that genes involved in cell cycle and signal transduction activities were over-represented in the meta-signature.By applying a two-stage Bayesian mixture modeling strategy, we were able to assimilate and analyze four independent microarray studies to derive an inter-study validated "meta-signature" associated with breast cancer prognosis. Combining multiple studies (The mixture modeling approach unifies disparate gene expression data on a common probability scale allowing for robust, inter-study validated prognostic signatures to be obtained. With the emerging utility of microarrays for cancer prognosis, it will be important to establish paradigms to meta-analyze disparate gene expression data for prognostic signatures of potential clinical use. DNA microarray analysis has been shown to be a powerful tool in various aspects of cancer research . With thτ2. The effect size was defined to be the difference between the tumor and normal sample means divided by pooled standard deviation. From a Bayesian perspective, Wang et al. [More systematic approaches have been proposed for integration of findings from multiple studies using different array technologies. Rhodes et al.  have prog et al.  used datg et al. . The larpoe (i.e., probability of expression) was introduced as a new scale and used in the context of molecular classification [poe in a framework to meta-analyze microarray data. Several desirable features of using poe as a new expression scale include the following: 1. poe provides a scaleless measure and thereby facilitates data integration across microarray platforms; 2. poe is a model-based transformation with direct biological implications in the context of gene expression data, as it is estimated based on a method that adopts an underlying mixture distribution that accommodates over-, under-, and unchanged expression categories; 3. poe unmasks differential expression patterns in microarray data by offsetting the influence of extreme expression values [poe allows merging of samples on the unified scale rather than using gene-specific summaries.A recent study proposed a Bayesian mixture model based transformation of DNA mi-croarray data with potential features applicable to meta-analysis of microarray studies . The basfication . The plan values ; 4. DataIn recent publications of breast cancer microarray studies, several groups have explored the hypothesis that the capacity to metastasize is intrinsic to the tumor and therefore can be revealed by gene expression pattern. Four independent studies have correlated gene expression profiles generated from distinct DNA microarray platforms to breast cancer prognosis -13. Amonpoe. We applied our method to the aforementioned breast cancer DNA microarray data sets. With the strength of the poe transformation and data integration, our goal was to develop an inter-study validated meta-signature that predicts relapse-free survival in breast cancer patients with improved statistical power and reliability.In this proof-of-concept study, we propose a two-stage meta-analysis of microarrays based on poe. The Bayesian hierarchical model employed for estimation borrows strength across genes by assuming further distributions for the gene-specific parameters (see Methods). For data integration purposes, we focused on a common set of 2,555 genes that were profiled in each of the four studies. Although the cost for compiling common genes is a loss of potential predictive features, it is not unreasonable to assume, given the analogous hypothesis explored in each study, that the common set represents the most relevant genes of interest for breast cancer prognosis. The resulting values of poe represent signed probability of differential expression for gene j in sample i, and thus provide a unified measure across studies. Further, the transformation improves contrast in each data set by removing the influence of extreme expression values. In stage 2, the expression profile of tumor samples from multiple studies were combined on the poe scale to generate a meta-cohort. The benefit of data integration using poe is twofold. First, it improves power of statistical analysis by increasing the sample size. Such integration of independent data sets renders sensitivity to those small yet consistent expression changes for certain genes. Second, it reduces the chance of false positive features due to artifacts from a single study, and allows reliable findings across studies. In this paper, we integrated four breast cancer microarray data sets of distinct platforms , not all studies have the time to recurrence information available and instead provide data on recurrence within a certain time interval (e.g., recurrence within five years versus no recurrence within five years). To deal with this issue, we utilized a dichotomization where a bad outcome is recurrence during followup and a good outcome is remaining recurrence-free for at least three years. The additional constraint for the good outcome group is to reduce potential bias introduced by short censoring due to insufficient length of follow-up. This is particularly relevant in cross-study analysis, given the heterogeneity in patient recruitment criteria and study designs. Accordingly, of the combined meta-cohort (n = 305) of breast cancer patients, 48.9% were in the poor outcome group, whereas 51.1% in the good outcome group. The sample sizes for each study are shown in Table P ≤ 0.001) in all cross-validation steps, representing a cohort of essential genes strongly associated with breast cancer recurrence. By random chance, there would be on average 2.5 genes to be found significant at P ≤ 0.001 in a set of 2,555. By finding 23 genes with a P ≤ 0.001, it is clear that there are much more predictive features than would be expected by random chance.Each gene was then associated with the recurrence status by a logistic regression within a leave-one-out cross validation scheme, and rank-ordered by the significance level of the coefficient. As a result, 23 genes held up as significant predictor of recurrence  as a linear combination of the P < 0.0001). In spite of the heterogeneity of the combined patient population, the meta-signature predicted the odds of recurrence for a patient showing a high risk signature as four times of the odds of recurrence for a patient showing a low risk signature. Several studies have implicated that the lymph node status is one of the principal clinical factors to classify patients in relation to the risk of relapse of breast cancer [By minimizing the misclassification error, we obtained a 90 gene meta-signature that reliably predicts outcome in the meta-cohort. This meta-signature classified 122 patients into a high risk group, where 84 (69%) of them had a recurrence. On the other hand, the signature classified 183 patients into a low risk group, where 118 (64%) of them did not recur by the end of the followup. By cross-tabulating the risk groups predicted by the meta-signature and the actual recurrence status, we obtained an estimated odds ratio of 4.0 (95% CI: 2.5–6.5, t cancer -16. AlthP < 0.0001). Similarly for nodal positive patients, a cohort thought to be at high risk of recurrence, the meta-signature identified 79 (51.6%) of the LN+ patients to have, in fact, lower recurrence risk over time . A multivariate logistic regression model suggested that the meta-signature is an independent predictor of the recurrent status with respect to nodal status in the meta-cohort (OR = 3.7(2.3–6.1), P < 0.0001).Kaplan-Meier analysis provides further evidence that the meta-signature was a significant prognostic index of breast cancer recurrence in the meta-cohort Figure . The estTo comprehend the potential gains of such two-stage meta analysis over individual analysis in each single study cohort, we constructed study-wise gene expression signatures using the same method. By minimizing the misclassification errors, we obtained a signature consisting 10, 60, 100, and 130 genes for Sorlie, van't Veer, Sotiriou, and Huang study cohort respectively . The resMeta-analysis accounts for such heterogeneity of the individual signatures in two ways. First its overlap with the study-specific signatures ranged from 3–40%. The excluded genes are likely to be cohort-specific findings that can not be replicated. Second, the meta-signature recruited 41 genes not previously picked by any of the single cohort signature, likely representing predictive features with small but consistent effects previously masked in single studies. When examining the performances of the gene signatures, the meta-signature showed a comparable or better performance compared with the individually optimized signatures both in the odds ratio estimates  terms and then grouped by functional classes. Based on the hypergeometric distribution, we calculated the significance of over-representation of a particular process in the signature. Figure poe scale. In particular, poe provides a unified platform-free scale, and simultaneously enhances the intrinsic contrast in the expression data. Furthermore, combining sample pools on the poe scale mitigates the influence of potential artifacts from a single study. The benefit of such data integration is reflected on two counts. One, integrated sample cohorts improve the reliability of the findings by guarding against false positive results from a single study. Two, it increases the statistical power to detect small consistent effects that can be otherwise masked by inadequacy of the sample size of an individual data set. By implementing this modeling approach, we were able to combine information from four microarray studies to build an inter-study validated meta-signature for predicting survival in breast cancer patients.Several important issues to consider when integrating microarray studies include use of different gene expression measurement scales, varying analytical power and reliability of the results for individual studies. To account for these issues, we proposed a two-stage mixture modeling strategy, the strength of which was built on the mixture model based transformation and the subsequent data integration on the As described earlier, a common set of 2555 genes was used in this meta-analysis, as it is important to provide the same context for data-driven estimation of the posterior probabilities. Although we assume the common set comprises the most biologically relevant genes, the loss of potential predictive genes, however, may offset the statistical power of the analysis. For example, one of our recent studies has established the polycomb protein EZH2 to be an independent predictor of breast cancer survival outcome. This geFunctional annotation of the meta-signature revealed genes such as Cyclin E and BCL2, which were previously shown to be correlated with survival outcome in breast cancer ,24. A stIn conclusion, a distinction of the analysis presented here relative to those by other authors ,6 is thaThe breast cancer microarray data sets were obtained at the author's websites from four recently published studies -13. EachX with elements xij, where i = 1, ..., mk, j = 1, ..., n (k = 1, .., 4 and n = 2,555). The expression measurement xij can be the ratio of the two fluorescent dye hybridization intensities for the spotted cDNA arrays[E be a latent class variable, and eij indicates over-, under- or normal expression for each entry of the R matrices. We have:Each of the four raw data sets was treated as an expression matrix NA arrays,12 and tNA arrays, or averNA arrays. Let E beij are latent and not directly observed from the data. We were interested in estimating the probabilities of eij being 1 or -1 given the observed raw expression xij, which were denoted as  and . Estimates of these latent quantities were obtained under a Bayesian mixture model setting. In particular, we assume the raw expression xij falls into one of the three expression categories. For each gene j, the expression then arises from a mixture of three distributions:The values of xij|eij = 1) ~ fj1,(·), (xij|eij = 0) ~ fj0,(·), and (xij|eij = -1) ~ fj-1,(·). and  = Peij = -1) are the multinomial probabilities for eij. With the specifications of models, we can calculate the latent quantities by Bayes' rule:respectively. Here, U refers to a uniform distribution and N refers to a normal distribution. By noting that the supports for the two uniform distributions are disjoint, the probabilities of differential expression are mutually exclusive with the forms:poe = p+ - p-. As a result, poe ranges from -1 to 1, and can be interpreted as the signed conditional probability of differential expression.A one dimension measure can thus be constructed as To borrow strength across genes, the estimation of the gene-specific parameters was formulated under a Bayesian hierarchical model setting. Given the large amount of parameters, prior distributions were specified to model the variation of the gene-specific parameter estimates, in particular, were obtained for each of the five data sets , we defined outcome groups as recurred during followup and remained relapse-free for at least 3 years. In particular, Let t* can be specified with clinical knowledge. We chose t* = 3 years in this study. We then consider constructing classifiers using y; note that y = 1 corresponds to the poor outcome group and y = 0 to the good outcome group. The sample sizes for each study are shown in Table where j, we fit the following univariate logistic regression model using data from all studies:Logistic regression was used to build a classifier for prognosis. For each gene x* is the rescaled value that allows data integration across multiple studies. The esti-mated values of βj, , are then used to form a risk score using a variation of the compound covariate predictor method [x1, ..., xD, the risk index is given as .where r method ,29; for , where , and  is the effect estimate for gene j in the combined meta-cohort without the ith sample. The risk index for sample i is a weighted linear combination of the expression profiles of the top D genes, where the ranking of the genes is based on their corresponding significance in the univariate logistic model fit. Classification of sample i to the risk groups is then based on the ith leave-one-out risk index, i.e.,  = I{RIi >c} with c being the empirical quantiles (40th - 70th) of the RI's. The number of genes D in a classifier is treated as a parameter and optimized to minimize the prediction error rates.If we want to assess the performance of the classifier, we must deal with the issue of training and testing the model using the same data. An "honest" estimate of the prediction error rate is obtained using leave-one-out cross-validation. Define a risk index The list of the top cumulative genes in the meta-signature was obtained by ranking all 2,555 genes by the number of times in the leave-one-out cross-validation steps that each one had a P-value from the univariate logistic regression less than 0.001.poe pro-files of the meta-signature. Yellow represents high probability of over-expression and blue represents high probability of under-expression. For heat maps of raw data matrices, we preprocessed the data by mean centering and then dividing by the standard deviation for each row. The means and the standard deviations used in the normalization were the relapse-free (RF) sample means and variances for each study data. The values for the recurrence (R) samples after standardizing then represented the number of standard deviations over or under the mean RF sample expression.We used the treeview software  to generRS, DG, and AC designed the study. RS carried out the statistical analysis and prepared the manuscript. DG and AC supervised the analysis and participated in the manuscript preparation. All authors read and approved the final manuscript.Plots of misclassification rates. The PDF file lists plots of misclassification error rates for classifiers identified in each individual study cohort and the meta-cohort.Click here for filePlots of Kaplan-Meier curves. The PDF file lists Kaplan-Meier plots for study-wise cross-validation of the individually identified signatures. A gene signature was trained in one study cohort and used to validate in each of the other study cohorts as testing sets.Click here for fileMeta-signature gene list. The excel file contains a list of Unigene ID, gene symbol, and full name of the 90 genes in the meta-signature.Click here for fileet al. study data (2,555 times 58 in dimension).POE imputation of the Sorlie data. The excel file contains a table of imputed signed probability matrix transformed from the Sorlie Click here for fileet al. study data (2,555 times 78 in dimension).POE imputation of the van't Veer data. The excel file contains a table of imputed signed probability matrix transformed from the van't Veer Click here for fileet al. study data (2,555 times 98 in dimension).POE imputation of the Sotiriou data. The excel file contains a table of imputed signed probability matrix transformed from the Sotiriou Click here for fileet al. study data (2,555 times 71 in dimension).POE imputation of the Huang data. The excel file contains a table of imputed signed probability matrix transformed from the Huang Click here for file


Aromatic L-amino acid decarboxylase (AADC) enzymes catalyze the synthesis of biogenic amines, including the neurotransmitters serotonin and dopamine, throughout the animal kingdom. These neurotransmitters typically perform important functions in both the nervous system and other tissues, as illustrated by the debilitating conditions that arise from their deficiency. Studying the regulation and evolution of AADC genes is therefore desirable to further our understanding of how nervous systems function and evolve.C. elegans, the bas-1 gene is required for both serotonin and dopamine synthesis, and maps genetically near two AADC-homologous sequences. We show by transformation rescue and sequencing of mutant alleles that bas-1 encodes an AADC enzyme. Expression of a reporter construct in transgenics suggests that the bas-1 gene is expressed, as expected, in identified serotonergic and dopaminergic neurons. The bas-1 gene is one of six AADC-like sequences in the C. elegans genome, including a duplicate that is immediately downstream of the bas-1 gene. Some of the six AADC genes are quite similar to known serotonin- and dopamine-synthetic AADC's from other organisms whereas others are divergent, suggesting previously unidentified functions. In comparing the AADC genes of C. elegans with those of the congeneric C. briggsae, we find only four orthologous AADC genes in C. briggsae. Two C. elegans AADC genes – those most similar to bas-1 – are missing from C. briggsae. Phylogenetic analysis indicates that one or both of these bas-1-like genes were present in the common ancestor of C. elegans and C. briggsae, and were retained in the C. elegans line, but lost in the C. briggsae line. Further analysis of the two bas-1-like genes in C. elegans suggests that they are unlikely to encode functional enzymes, and may be expressed pseudogenes.In the nematode bas-1 gene of C. elegans encodes a serotonin- and dopamine-synthetic AADC enzyme. Two C. elegans AADC-homologous genes that are closely related to bas-1 are missing from the congeneric C. briggsae; one or more these genes was present in the common ancestor of C. elegans and C. briggsae. Despite their persistence in C. elegans, evidence suggests the bas-1-like genes do not encode functional AADC proteins. The presence of the genes in C. elegans raises questions about how many 'predicted genes' in sequenced genomes are functional, and how duplicate genes are retained or lost during evolution. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.The  Drosophila, a single gene encodes the serotonin- and dopamine-synthetic AADC [Aromatic L-amino acid decarboxylase (E.C. 4.1.1.28, AADC) catalyzes the second enzymatic step in synthesis of the neurotransmitters dopamine and serotonin, which are found in neurons of all animals , trypto [mod-5; ), dopami [dat-1; ) and ves [cat-1; ). Postsy [cat-1; -32 and i [cat-1; -36.C. elegans is being facilitated by genomic sequencing of related nematodes. A whole genome shotgun sequence of Caenorhabditis briggsae was recently completed; the sequence is estimated to be 98% complete [C. briggsae and C. elegans is estimated between 80 – 110 million years ago [C. elegans that are more closely related than C. briggsae, which will enhance our ability to analyze the genes of C. elegans. We have used genomic sequences of both C. elegans and C. briggsae to help identify and characterize another component of the serotonin and dopamine signaling systems – the bas-1 gene – and to examine the evolution of this and related genes.Further analysis of gene function, regulation and evolution in complete . The divears ago ,38, althears ago . This isbas-1 [biogenic amine synthesis abnormal] mutant is serotonin- and dopamine-deficient, and displays several behavioral abnormalities [bas-1 mutants are unable to convert exogenous 5-hydroxytryptophan (5HTP) into serotonin (5-hydroxytryptamine, 5HT), as assessed by serotonin antiserum staining. Because of this phenotype, we have previously proposed that the bas-1 gene likely encoded the AADC enzyme of C. elegans [The malities ,16,17. Ubas-1 gene maps to chromosome III, between dpy-17 and unc-32. When this region was sequenced by the C. elegans Genome Sequencing Consortium, two AADC-homologous predicted genes, designated C05D2.4 and C05D2.3, were found to be located close together on a single cosmid, C05D2  plasmid DNA as a co-injection marker. We isolated transgenic Roller progeny (expressing the rol-6 (dom) marker phenotype) of the injected worm and propagated strains that transmitted the marker, then tested these worm strains using serotonin antibody staining. We found that 3 of 3 independent Roller transgenic lines were rescued for serotonin immunoreactivity, confirming that the bas-1 gene was located within this 46 kb of genomic DNA , as did smaller subclones of C05D2XN, including an 11.3 kb subclone (pCL3001, n = 11/11) and an 8.8 kb subclone (pCL7001, n = 1/1). These results confirm that at least one of AADC-homologous genes likely corresponds to the bas-1 gene.The 5D2 Fig. . This suDNA Fig. . We thenbas-1 mutants, we prepared two constructs from C05D2XN, one mutated in C05D2.4, the other in C05D2.3 , whereas the construct mutated in C05D2.4 failed to rescue (n = 0/5 rescued). A construct containing a GFP gene inserted into the C05D2.3 coding sequence (and disrupting the gene) also rescued bas-1 mutants (n = 2/2). In Roller transgenic lines lacking rescue, we confirmed the presence of the injected construct by PCR. Therefore, an intact C05D2.4 gene is necessary to rescue bas-1 mutants, whereas the C05D2.3 gene is not. In all rescued transgenic lines, we saw the complete set of known serotonergic neurons, although not necessarily all cells in every animal – mosaicism from somatic loss of extrachromosomal DNA is expected in these transgenics. This result suggests that no critical cell-specific regulatory sequences were missing from even the smallest construct we injected.To determine which of the two predicted AADC sequences was needed to rescue 2.3 Fig. . In eachbas-1 gene, we identified the mutations in four bas-1 mutant alleles; we also examined the phenotypes of deletion mutants in C05D2.4 and C05D2.3 generated by the C. elegans Gene Knockout Consortium (GKC). We found that the bas-1 alleles pa4, n2948, and n3008 contained point mutations in C05D2.4 coding sequence resulting in premature stop codons  had a 4268 bp deletion from the second exon of C05D2.4 to the final intron of C05D2.3; therefore, ad446 is a knockout of both predicted genes. We examined the phenotypes of GKC-generated deletion mutants in each predicted gene. The C05D2.4 knockout (tm351) removes the entire predicted second exon. We found that both tm351 homozygotes and tm351/ad446 worms were deficient in serotonin immunoreactivity. On the other hand, a knockout of C05D2.3 (ok703) is wildtype for serotonin staining. Therefore, tm351 is a fifth mutant allele of the bas-1 gene, and C05D2.4 corresponds to the gene bas-1.To confirm further that C05D2.4 is the ons Fig. . We founbas-1 gene, we isolated cDNAs using RT-PCR; we also obtained cDNA clones from the C. elegans EST/Transcriptome project (courtesy of Yuki Kohara) and the ORFeome project [bas-1 cDNAs are trans-spliced to SL1 just 15 nucleotides upstream of the predicted translation start site. The consensus sequence from our clones and others we examined predicts a 514 amino acid, 58 kDa protein product  and human (480 aa). The predicted protein possesses a conserved lysine PLP binding site at residue 343, and has other amino acids identical to those shown to be essential for rat AADC function [To continue our characterization of the  project . We founuct Fig. . This isfunction ,42. A nuC. briggsae ortholog of bas-1 in genomic sequence . We found a single clone that used an alternative splice acceptor 60 bp upstream of the usual splice site for exon 3; this alternate splice introduces a premature stop codon in the coding sequence. This transcript may be a rare, aberrant splice form without functional significance.We found two splice variants different from the Genefinder-predicted cDNA described above, which was the predominant form. About 20% of clones we sequenced had a 27 bp microexon inserted between the predicted exons 2 and 3 Fig. . The 27 nce Fig. , althougbas-1 translation start site and an in-frame fusion with the 2nd exon, injected with rol-6(dom) plasmid into wild-type worms (kindly provided by Ian Hope). Two independent transgenic Roller lines with extrachromosomal arrays had the same pattern of expression. The reporter was reliably expressed in several easily identified cells including the paired serotonergic neurons NSM and HSN and the dopaminergic PDE postdeirid sensory neurons  may be expressed from a single promoter initially as a single primary transcript [bas-1. We were unable to isolate either SL1 or SL2-spliced transcripts from C05D2.3 by RT-PCR, although we did isolate a partial cDNA using internal primers. DNA microarray experiments suggest the gene is not expressed above background levels, unlike C05D2.4/bas-1  gene, unc-25 [C. elegans genes are clearly closely related to other AADCs, whereas others are more divergent  used for tyramine and octopamine synthesis, which both appear to be used as neurotransmitters in C. elegans [tdc-1 is a predicted Drosophila AADC-homologous protein of unknown function (G30446). Interestingly, K01C8.3/tdc-1 shows a stronger match to known DDCs than any of the other C. elegans AADCs, including C05D2.4 (Table bas-1 (outside of nematodes) is to insect and mammalian DDCs, but again the match is only slightly better than to HisDCs. The predicted genes C05D2.3, F12A10.3 and ZK829.2 also have about the same level of identity and similarity to known AADCs and HisDCs. The ZK829.2 predicted protein, however, is much larger (830 AA) than a typical AADC, having extended N- and C-terminal domains not found in other PLP-dependent DCs. Most of ZK829.2 predicted coding sequence is confirmed by cDNA sequences, suggesting that the predicted protein 'extensions' likely are real.The protein predicted from K01C8.3 is now believed to encode a tyrosine decarboxylase  include other group II PLP-dependent enzymes such as sphingosine-1-phosphate lyase and cysteine sulfinic acid decarboxylase. C09G9.4, however, has very little or no significant similarity to these other enzymes. The C. elegans GAD/unc-25 predicted protein has a strong match to identified Drosophila and mammalian GADs . Most of the divergence between K01C8.3 and its ortholog is in N- and C-terminal extensions that are not found in other AADC's. The C. elegans C09G9.4 and C. briggsae ortholog are even less similar to one another than are the bas-1 orthologs.We performed BLAST searches of a og Table . Using aC. briggsae appears to lack orthologs for the C. elegans predicted genes C05D2.3 and F12A10.3. This suggests that gene duplications giving rise to these two genes, which are most closely related to bas-1/C05D2.4, occurred either in the C. elegans lineage after its split with the C. briggsae lineage, or that C. briggsae lost both C05D2.3 and F12A10.3 orthologs (or their common ancestor) following the split. Using phylogenetic analysis of aligned amino acid and nucleotide sequences, we found that C05D2.3 and F12A10.3 share a common ancestor and that the gene duplication giving rise to bas-1 and C05D2.3/F12A10.3 likely occurred prior to the C. elegans/C. briggsae divergence. This is also suggested by the pattern of introns in the genes. [We have confirmed the splicing pattern of C. briggsae bas-1 by isolating a cDNA (S. DePaul & C. Loer, unpublished data).] The C. elegans and C. briggsae bas-1 genes have identical genomic structure which differs from that of C05D2.3 and F12A10.3, which are more similar to one another , we found that current splicing predictions for the gene were incorrect. We sequenced six F12A10.3 clones and found two slightly different splicing patterns, both different from Genefinder and Intronerator predictions. The two types of clones differed only in whether a final intron was removed or not. We found 4 clones with 9 exons, and 2 clones with 8 exons. The failure of the gene prediction programs in this case is likely to be due to their preference for creating functional transcripts. All F12A10.3 cDNAs instead appear to be non-functional: they have frameshifts relative to AADC-homologous reading frames. The first frameshift occurs in the second exon and quickly leads to a premature stop codon. At best F12A10.3 transcripts would result in a 158 amino acid protein that could not function as an AADC. F12A10.3 therefore appears to be an expressed pseudogene. DNA microarray experiments and representation in cDNA sequencing projects suggest that F12A10.3, like C05D2.3, is likely expressed at a low level  comparing the bas-1 orthologs and bas-1-like genes. We also calculated these values for the other AADC ortholog pairs from C. elegans and C. briggsae (Table A/KS < 1 indicates purifying (negative) selection, KA/KS = 1 indicates no selection (as in true pseudogenes), and KA/KS < 1 indicates Darwinian (positive) selection. KA/KS for 8179 C. elegans and C. briggsae ortholog pairs had a mean value of 0.06, indicating most genes are under purifying selection [bas-1 genes are under purifying selection (KA/KS = 0.039), but the bas-1-like genes appear to be under reduced selective pressure; the average KA/KS for comparisons with bas-1-like genes was 0.148, more than three times the value of the bas-1 ortholog comparison. The proportion of observed to potential non-synonymous substitutions (pN) among the bas-1-like gene comparisons was similarly much higher than for the bas-1 orthologs.In order to assess whether the election . We founbas-1 orthologs  was near mutational saturation (pS > 0.75); KA/KS cannot be calculated when pS > 0.75. Thus, a value could not be calculated either for full-length tdc-1 alignments, or using a tdc-1 core sequence. We were able to calculate a value by aligning the N- and C-terminals sequence of the tdc-1 orthologs , corresponds to the genetically-defined eficient , and neibas-1 gene is expressed in at least two alternatively spliced forms, one of which appears to be less common and contains a small additional 27 nucleotide exon. The short segment of protein encoded by the additional exon, and the surrounding region are not found in other AADC proteins, suggesting a novel function for this region of the AADC protein. In other organisms, the serotonin- and dopamine-synthetic AADC genes have alternative splicing that result in tissue-specific protein isoforms. Currently we have no indication that bas-1 is expressed in any cells other than serotonergic and dopaminergic neurons, and no information about the functional significance of this alternative splicing.The in vitro experiments [C. elegans AADCs by phosphorylation remains speculation.AADC has received somewhat less attention with respect to the regulation of serotonin and dopamine synthesis than the specific, rate-limiting synthetic enzymes tryptophan hydroxylase and tyrosine hydroxylase . This iseriments , althougeriments . Our exaC. elegans with those of other organisms in order to guess about their possible functions. This is particularly relevant because all bas-1 mutants retain weak, residual serotonin immunoreactivity ([tdc-1 [Drosophila (G30446), an uncharacterized AADC homolog, is likely to encode the fly's octopamine-synthetic tyrosine decarboxylase. It has long been known that a separate gene encoded this enzymatic activity in flies, since the activity is still detectable in Ddc deletion mutants [tdc-1 protein could be reflected in the much slower rate of amino acid substitution seen in its C. elegans & briggsae orthologs than in the bas-1 orthologs which encode more 'promiscuous' enzymes.We compared the protein sequences of other predicted AADCs in ctivity . As with tdc-1, C. elegans ZK829.2 and its C. briggsae ortholog have also evolved more slowly than the bas-1 orthologs. A recent analysis of eukaryotic AADC sequences that includes the C. elegans ZK829.2 and its C. briggsae ortholog as the only nematode representatives clearly demonstrates that AADC genes can evolve at very different rates, and that a constant "molecular clock" cannot be assumed in phylogenetic analyses [Whether  elegans , this obanalyses .Finally, since the C09G9.4 predicted protein is so highly divergent from the typical AADC, and lacks a critical lysine residue that binds the PLP cofactor, it is unlikely to be an AADC enzyme. It has a similar level of divergence from genuine AADCs as do other group II PLP-dependent enzymes such as cysteine sulfinic acid decarboxylase, to which it has little or no similarity. Whatever the function of a C09G9.4-encoded protein, it appears to represent a new PLP-DC-related protein; sequencing of more genomes may yet reveal additional members.bas-1 in C. elegans, the genes C05D2.3 and F12A10.3, are missing from C. briggsae. Furthermore, phylogenetic analysis indicates the two extra genes did not arise in the C. elegans line, but were present (or their commmon ancestor was present) in the species that gave rise to both the C. elegans and C. briggsae lines. Finally, careful examination of the cDNAs and predicted protein sequences of C05D2.3 and F12A10.3 reveals that neither is likely to be functional as an AADC: the former lacks critical amino acids and the latter can encode only a truncated protein. Both are expressed, based on the presence of cDNAs, but probably at a very low level, which is not above background in microarray experiments. It is possible that the duplicate genes are functionally 'lost' in C. elegans as well.We found that the closest relatives of C05D2.4/C. elegans. Taking a random sampling of predicted genes and generating transgenics with reporter fusion constructs (in order to determine a pattern of expression), Mounsey and colleagues [C. elegans may be pseudogenes. In fact, careful inspection of recently duplicated genes showed that many were actually pseudogenes, like we found to be the case for F12A10.3. Overall, close inspection of predicted genes revealed at least 4% were pseudogenes.The features of C05D2.3 and F12A10.3 raise a number of interesting questions about the fate of duplicate genes, and the true nature of many 'predicted genes' in lleagues  found thC. elegans if they lack a function? C. briggsae and C. elegans may have diverged 80 – 110 million years ago [bas-1-like gene or genes were likely present in the common ancestor of C. elegans and C. briggsae, then there seems to have been ample time for loss in the C. elegans line. Under a simple model of gene loss following duplication, only a few million generations would be the mean time to fix a null allele of the gene duplicate [Caenorhabditis, a million generations could be completed in 10,000 years or less. This seems to suggest that the downstream duplicate of bas-1 (ancestor of C05D2.3) may have continued to function for a considerable time after the duplication, perhaps by gene conversion which might have continued until sufficient divergence from bas-1 [bas-1, but has not accumulated stop codons and frameshifts expected for a pseudogene. Walsh [So, why are C05D2.3 and F12A10.3 still present in ears ago ,38. Sincuplicate . In Caenom bas-1 . Loss ofe. Walsh  has propbas-1::GFP construct is expressed well without such sequences in cis.C05D2.3 and F12A10. 3 seem to have been retained longer than expected. Lynch and Force  proposedbas-1-like genes C05D2.3 and F12A10.3 are under relaxed selection relative to bas-1 and other AADCs. It should be noted that precise quantitative comparisons cannot be made with the results presented in the C. briggsae whole genome analysis [A/KS; however our calculations indicate that bas-1 and the other AADC's, like most genes in the Caenorhabditis genomes, are under strong purifying selection. Even if both C05D2.3 and F12A10.3 are now pseudogenes, some significant period of time during which they functioned and were under purifying selection could act to obscure this fact in an analysis of KA/KS. Even if C05D2.3 has acquired a new, adaptive function, such a new function might result from changes in only a few sites in the protein, and so again this could be obscured by a majority of sites under purifying selection. With the sequencing of three related Caenorhabditis species it will be interesting to learn of the fates of bas-1 and the bas-1-like genes in other lines.Our analysis of synonymous vs. non-synonymous substitutions indicates that the analysis , since wbas-1 gene encodes a serotonin- and dopamine-synthetic AADC enzyme in C. elegans. The C. elegans genome possesses five other AADC-homologous genes, two of which are closely related to bas-1. These bas-1-like genes are missing, however, from the congeneric C. briggsae, and evidence suggests that, despite their persistence in C. elegans, the genes do not encode functional AADC proteins. Since one or more of the bas-1-like genes was likely present in the common ancestor of C. elegans and C. briggsae- which may have diverged over 80 million years ago – it is unclear why the bas-1-like genes have been retained in the C. elegans line. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.The Caenorhabditis elegans was performed as described by Brenner [C. elegans genetics conforms to the conventions set forth by Horvitz et al. [him-5(e1490)V; MT7988: bas-1(ad446)III; MT7990: bas-1(n2948)III; MT8002: bas-1(n3008)III; LC7: bas-1(pa4)III; LC33: bas-1(tm351)III. The him-5(e1490) strain generates approximately 30% males by increased X chromosome non-disjunction [Routine culturing of  Brenner . Nomenclz et al. . Strainsjunction , but is bas-1 mutant strains  and then PCR-amplified small segments of C05D2.4 and C05D2.3 protein-coding sequence. Five pairs of primers were used to survey the C05D2.4 gene  and 4 pairs for C05D2.3 (C+D, K+L, M+N, P+Q). Bands of the predicted size were excised from 2% agarose gels and purified with GeneClean (Bio101/Qbiogene), then sequenced. All mutations were confirmed by sequencing both strands for two independent PCR reactions. All PCR and sequencing primers were designed using the program Primer3  from four imer3 . Primerrol-6(su1006) [bas-1 mutant worms. Progeny of injectees that express the rol-6 dominant plasmid have the easy-to-identify Roller phenotype which results in worms with a helically-twisted body along the anterior-posterior axis. Roller transgenic worms typically carry co-injected DNAs. Transgenic Roller progeny were isolated and propagated; rescue of bas-1 mutants was scored by staining with serotonin antiserum as described previously[All DNAs for microinjection were purified with Qiagen tip20 columns. Experimental DNAs were co-injected with the dominant marker plasmid pRF4 containing the mutant gene (su1006)  into N2 reviously,67.bas-1 mutants. Clones were analyzed by restriction digests. We also made C05D2XN derivatives in which either C05D2.4 and C05D2.3 was mutated to introduce a premature stop in coding sequence. Clones were sequenced to determine the nature of the introduced mutation. Clone pCL6991 had a 4 bp deletion in the second exon of C05D2.4; pCL7991 had a 2 bp insertion in the first exon of C05D2.3; each causing a frameshift mutation. In the clone pCL7003, derived from the rescuing plasmid pCL3001, almost the entire C05D2.3 coding sequence is deleted. The bas-1 GFP reporter construct (within transgenics) was kindly provided by Ian Hope, and consists of a PCR-generated fragment of C05D2 with 4595 bp upstream of the predicted translation start site and 403 bp protein coding region to make an in-frame protein fusion in the 2nd exon. The construct was created by a multi-site recombination reaction with the C05D2 fragment, PCR-generated GFP, and vector using the Invitrogen Gateway cloning system  as continuation of a project to determine expression patterns for C. elegans genes through reporter gene technology [The rescuing plasmid C05D2XN contains a 15.8 kbp genomic DNA insert (XhoI to NheI) derived from the cosmid C05D2, and contains both C05D2.4 and C05D2.3 predicted genes. A number of deletions of C05D2XN were made to test for rescue of chnology .bas-1 mRNA.) Worms were isolated from six 100 mm NGM plates, washed several times with M9 buffer, and pooled to form a ~100 μl pellet of worms. The pellet was mixed with 175 μl RNA lysis buffer , frozen and ground to a fine powder with a pestle and mortar cooled with liquid nitrogen. The powder was recovered in a fresh microfuge tube, mixed with 350 μl SV RNA dilution buffer and centrifuged to remove debris. The cleared lysate was transferred to a new tube and precipitated with 200 μl 95% ethanol and applied to the SV spin column assembly. The remaining RNA purification was performed exactly as described for the SV System "RNA Purification by Centrifugation." Purified RNA was eluted from the spin column with 100 μl nuclease-free water. RNA was converted to cDNA in a 80 μl reaction containing 800 Units of M-MLV Reverse Transcriptase (Life Technologies/BRL), 16 μl 5X RT buffer, 25 mM dNTPs, 80 Units RNAsin (Promega) 2.0 μg random hexamer primers (Life Tech), and 50 μl purified RNA (from above). Bas-1(C05D2.4) and C05D2.3 cDNAs were amplified by PCR from him-5 cDNA produced as described above, typically using Failsafe PCR mixes (Epicentre Technologies). Conditions for PCR were 94°C (1 min.), 50°C (1 min.), 72°C (3 min.) for 40 cycles, then 72°C (10 min.). Primers used to amplify C05D2.4 cDNAs were SL1-B + C05D2-L and SL1-B + C05D2-B; a partial C05D2.3 cDNA was amplified with primers C05D2-M and -N. Spliced leader primer sequences were as follows: SL1-B: AAAGGATCCTTTAATTACCCAAGTTTGAG; SL2-B: AAAGGATCCTTTTAACCCAGTTACTCAAG. Appropriately-sized bands were isolated with GeneClean or Ultrafree-DA (Millipore), then cloned directly into the pCRII-TOPO vector using the TOPO-TA Cloning kit (Invitrogen). From seven of our RT-PCR derived clones we sequenced, we found two different splice variants different form the Genefinder prediction (see results).We isolated RNA for RT-PCR from mixed stage CB1490 worms. . DNA we received from the ORFeome project is purified from a pool of transformants derived from ligation and transformation of their original RT-PCR, thereby allowing the isolation of internal splice variants from the mix. We transformed with this DNA and isolated several clones. We sequenced two ORFeome clones completely; six more clones were partially sequenced. Interestingly, each of the 8 clones appeared to have at least one mutation (compared to known genomic sequence). Since we found one splice variant containing the 27 bp microexon from among the eight clones that we sequenced, we analyzed 29 additional ORFeome project-derived clones by PCR from single isolated bacterial colonies. Amplification of the region between primers C05D2-T and -L allowed us to distinguish between clones with or without the 27 bp microexon based on product size (464 bp versus 437 bp). Seven of 29 clones were the larger size, so likely contained the 27 bp microexon. None of the ORFeome clones analyzed by sequence or PCR appeared to use the alternative exon 3 splice acceptor noted above. Finally, the two 'YK' cDNA clones from the C. elegans EST project that we examined are from a 'full-length' capped library, and each has an SL1 leader and a poly-A tail. We found that each bas-1 'YK' clone, however, contained a different internal deletion . Each of the deletions was adjacent to a short repeated sequence.We also obtained cDNA clones from the ORFeome project  and the C. elegans genomic and predicted cDNA sequences were retrieved from ACeDB, WormBase, and/or GenBank. Blast searches and Blast2 comparisons were performed using the NCBI Blast server. C. briggsae genomic sequence was searched using TBLASTN of the 7/12/02 shotgun assembly . Assembly and consensus sequence determination of our own cDNA sequences was done using the program SeqMan (DNAStar, Madison, WI). Some information about ORFeome clones was retrieved from WorfDB . Phosphv.1.1 (, ). Multipociates, ). Some aociates, . EstimatGojobori .AADC – aromatic amino acid decarboxylaseDDC – dopa decarboxylaseHisDC – histidine decarboxylaseGAD – glutamic acid decarboxylaseTrpDC – tryptophan decarboxylasePLP – pyridoxal 5'-phosphateNSM – neurosecretory motoneuronHSN – hermaphrodite-specific neuronPDE – postdeirid sensory neuronADE – anterior deirid sensory neuronRN – ray sensory neuronCEPD, CEPV – dorsal or ventral cephalic sensory neuronADF – amphid sensory neuron, dual cilia, designation FAIM – ring interneuron, designation MRIH – ring interneuron (unpaired), designation HCP – posterior daughter of designation C cell, male-specific ventral cord motoneuronEH prepared deletion and expression constructs, cloned and sequenced bas-1 cDNAs and mutant alleles, participated in anti-serotonin staining and genetics, and drafted the manuscript. CL conceived and directed the study, generated transgenics by microinjection, performed sequence and phylogenetic analyses, and completed the manuscript. Both authors read and approved the final manuscript.


A prominent and influential hypothesis of vision suggests the existence of two separate visual systems within the brain, one creating our perception of the world and another guiding our actions within it. The induced Roelofs effect has been described as providing strong evidence for this perception/action dissociation: When a small visual target is surrounded by a large frame positioned so that the frame's center is offset from the observer's midline, the perceived location of the target is shifted in the direction opposite the frame's offset. In spite of this perceptual mislocalization, however, the observer can accurately guide movements to the target location. Thus, perception is prone to the illusion while actions seem immune. Here we demonstrate that the Roelofs illusion is caused by a frame-induced transient distortion of the observer's apparent midline. We further demonstrate that actions guided to targets within this same distorted egocentric reference frame are fully expected to be accurate, since the errors of target localization will exactly cancel the errors of motor guidance. These findings provide a mechanistic explanation for the various perceptual and motor effects of the induced Roelofs illusion without requiring the existence of separate neural systems for perception and action. Given this, the behavioral dissociation that accompanies the Roelofs effect cannot be considered evidence of a dissociation of perception and action. This indicates a general need to re-evaluate the broad class of evidence purported to support this hypothesized dissociation. Paul Dassonville and Jagdeep Bala challenge a prominent hypothesis that proposes the existence of two separate visual systems within the brain, one creating perception and the other guiding action Several anatomical, neurophysiological, clinical, and behavioral investigations of human subjects and nonhuman primates have provided evidence for two separate and dissociable cortical systems for visual processing. One of these systems—the ventral stream—resides in a swath of cortex that extends in an anteroventral direction from primary visual cortex to the temporal lobe. The second system resides in a dorsal stream that roughly extends from primary visual cortex to the parietal lobe. These ventral and dorsal systems were originally thought to be dedicated to the visual processing required to determine an object's identity and location, respectively . HoweverMuch of the evidence for separate perception and action systems has come from behavioral studies of normal subjects. The general logic of these behavioral paradigms e.g.,  is as foAs a specific example of this type of evidence, Although these findings are compatible with the hypothesis of two separate visual systems that are differentially affected by Roelofs illusion, it is important to consider the possibility of an alternative explanation. While it is true that both perceptual and sensorimotor measures within the task of We first sought to replicate the findings of A second group of subjects was instructed to make open-loop saccadic eye movements to the target location. Saccades performed immediately after the frame and target were extinguished showed no significant effect of frame position  provided informed consent to participate and were compensated with either course credit or a small monetary payment, as per a protocol approved by the University of Oregon Committee for the Protection of Human Subjects/Institutional Review Board.Subjects were placed in a completely darkened room and presented with a visual display that was back-projected (Cine7 projector, Barco, Kuurne, Belgium) onto a screen measuring 128 × 96 cm, positioned 122 cm from the eyes. Visual targets were small  red spots, located −4°, −2°, 0°, 2°, or 4° from the subject's midline, at eye level. During experimental trials, targets were presented within a large red unfilled frame  that was either centered with respect to the subject's midline or shifted 5° left or right of the midline. All visual images were presented on a black background, with the high contrast of the Barco Cine7 projector preventing subjects from seeing the edges of the screen.Head and binocular eye positions were monitored at 250 Hz with an eye tracker  that allowed head-free measurement of gaze (precision = 0.01°); however, target placement was such that head movements contributed to only a small fraction of the total gaze displacement on any trial. To start each session (and as necessary throughout each session), eye-tracker calibration was performed using a 3 × 3 grid of targets spaced 13.5° apart in the horizontal dimension and 10.5° apart in the vertical dimension. For each subject, the average fixation error across the nine calibration targets was required to be 1° or less before beginning the subsequent practice and experimental trials; thus, absolute tracking errors within this calibration field were at most 1° in magnitude and were typically only 0.5°. In addition, subjects began each experimental trial by directing the eyes to a fixation point and pressing the space bar of a keyboard to indicate readiness. Upon this signal, the eye-tracker computer performed an adjustment of the calibration to correct for any drift that had occurred since the onset of the previous trial; that is, the calibration was adjusted so that the signal of eye position matched the known location of the fixation point. In experiments requiring eye movement responses, the gaze signals from the two eyes were averaged to yield a single representation of gaze direction as the dependent variable.Each experiment was preceded by a set of practice trials  in which subjects performed the appropriate task (see below) in the absence of the large Roelofs-inducing rectangular frame. Feedback was provided at the end of each practice trial to assist subjects in improving their performance. For all experiments, feedback included the illumination of a small circle at the target location. For the experiment in which subjects provided a button press to report the perceived target identity, a computer-generated voice also provided auditory feedback to indicate the correct target identity. For all experiments in which subjects indicated the target position with a saccadic eye movement, feedback following each practice trial also included the illumination of a small square to indicate the final gaze position; subjects were instructed to use the feedback in an attempt to minimize the distance between the target and final gaze positions. No feedback concerning response accuracy was ever provided during the experimental trials.To test the perceptual effects of Roelofs illusion see A, subjecSimilar trials were used to test the sensorimotor effects of Roelofs illusion see A, with tTo test the effects of Roelofs illusion on remembered visual space see A, subjecTo test the effects of Roelofs illusion on the apparent midline see A, subjecTo determine whether the delayed sensorimotor Roelofs effect was modulated by the presence of the frame during the imposed delay period see A, all trTo test the effects of Roelofs illusion on saccades directed toward targets defined allocentrically see A, stimulIn each experiment, the signed magnitudes of localization errors  were analyzed with a full-factorial analysis of variance, in a 5 (target location) × 3 (frame position) × 2 (response delay) design see , or a 4 Figure S1(A) Time line of task events for immediate (black) and delayed (gray) perceptual judgments of target location. Note that in this and all other experiments, feedback was presented only during practice trials, and the frame was presented only during experimental trials.(B) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of five target locations.(C) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of ten subjects.(579 KB TIF).Click here for additional data file.Figure S2(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses.(B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of five target locations.(C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of ten subjects.(1.1 MB TIF).Click here for additional data file.Figure S3(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward remembered reference-array locations.(B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference-array locations, for each of five target locations. The nonlinear effect of target location has been addressed elsewhere .(C) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference array locations, for each of ten subjects.(2.5 MB TIF).Click here for additional data file.Figure S4(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward the apparent midline.(B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward the apparent midline, for each of ten subjects.(856 KB TIF).Click here for additional data file.Figure S5(A) Time line of task events for delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, black) or was present throughout the delay (extended frame, gray).(B) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, dashed line), or was present throughout the delay (extended frame, dotted line), for each of five target locations.(C) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, dashed line) or was present throughout the delay (extended frame, dotted line), for each of ten subjects.(4.7 MB TIF).Click here for additional data file.Figure S6(A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses to targets defined allocentrically.(B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of five target locations.(C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of ten subjects.(1.8 MB TIF).Click here for additional data file.