Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues  from three non-astrocytoma patients were also included as the control.FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.Frequencies of methylation in GCTs were 7/25 (28 % for These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment. Ovarian cancer is one of the most common cancers in women of all age groups. Among ovarian neoplasms, granulosa cell tumors (GCTs) are relatively rare, accounting for approximately 3% of all ovarian cancers. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. During cancer development, there is a shift in methylation patterns and some promoter region CpG islands become methylated leading to silencing of the adjacent genes  and thisFHIT gene is a member of the histidine triad gene family [FHIT gene has been shown to be hypermethylated in oesophageal, lung, breast, prostate, bladder, cervical, and oral cancers [cyclin D2 was also demonstrated in human ovarian granulosa cell tumors and testicular germ cell tumor cell lines [cyclin D2 gene promoter region. Several studies have indicated that methylation of cyclin D2 and its mRNA and protein were absent in most breast cancer cell lines examined and in primary breast cancers although normal breast epithelial cells had abundant expression [BRCA2 in breast cancers cell lines and other normal human breast, bladder, colon, and liver tissues [The human e family , the fun cancers -9. Recen cancers . Methylall lines . In breapression -14. BRCA tissues . RUNX3 i tissues ,17.FHIT, FANCF and RUNX3 gene promoters were frequently methylated. Methylation status was correlated with histologic characteristics. We also found evidence that promoter methylation inactivates gene expression in GCTs and exposure to methylation and/or histone deacetylase (HDAC)-inhibiting agents reactivate the gene expression.The role of epigenetic gene inactivation in GCTs of ovarian origin is yet not fully understood. Previously published reports on GCTs and its precursor lesions showed varying degree of promoter methylation of many tumour suppressor genes -20. To iFHIT, FANCF, Cyclin-D2, BRCA2 and RUNX3 in 25 ovarian GCTs and ovarian cell line DNAs using the MSP assay . Hypermethylation of FANCF was significantly more frequent in stage IC . Cyclin-D2 methylation was found only in patients with IC type of cancer . No FANCF mRNA in TOV-21G, Cyclin-D2 in C13* and OV-90, and RUNX3 in C13* and KGN was detected in untreated cell lines  studied extensively in these tumours are in fact localised on these chromosomes.Cancer cells especially of ovarian origin keep on accumulating genetic changes that allow them to evade various chemotherapeutic drugs and hence become increasingly dangerous. We try to answer some of the questions by exploring the role of methylation mediated gene silencing in five tumour suppressor gene in the present study. We asked a question whether hypermethylation of comatous . Taken tFANCF gene disrupts the FA-BRCA pathway, resulting in cisplastin resistance. We found FANCF promoter hypermethylation in 24% of the tumours. This is in line with the previous findings in squamous cell carcinomas of lung and oral cavity and, in cervical and ovarian cancer [BRCA2 after treatment with DAC. DNA hypermethylation-mediated gene silencing is closely associated with histone modifications such as methyl-H3-K9. In this regard, DNA-demethylating agents 5'-aza-2'deoxycytidine (DAC) and trichostatin (TSA) reactivates expression of epigenetically silenced genes [FHIT, cyclin-D2 and RUNX3 in C13*, FANCF in TOV-21G and cyclin-D2 in OV-90 cell line after exposure to DAC in the absence of promoter methylation suggests that key histone modifications, either by direct or indirect involvement of promoter methylation, also play a role in down-regulating FANCF gene expression in this cancer type. It is therefore assumed that FANCF silencing might be considered a candidate-mechanism underlying the state of genomic instability may be considered to be a rate-limiting in the origin of GCTs of ovarian origin. FANCF gene silencing has been shown to revert in vitro as a result of demethylation in some ovarian cell lines as seen in the present study also [FANCF methylation. The present finding along with the results from other cancer types suggests the presence of genomic instability due to the methylation mediated FANCF gene silencing. Cyclin D2 gene promoter is hypermethylated in 12% of GCTs, a number that is consistent with previous findings in other cancers [Cyclin D2 methylation is found only in IC type tumors.FANC genes are essential in DNA repair pathways and recently, it has been shown that promoter hypermethylation of n cancer -25. To ted genes . Althouged genes ,28. In tudy also . These r cancers ,29,30. WRUNX3 gene promoter frequently occurred in ovarian GCTs. In human gastric cancers it has been shown that promoter hypermethylation and hemizygous deletion of the RUNX3 gene correlated with a significant reduction in expression, and the tumorigenicity of cell lines in nude mice was inversely related to their level of RUNX3 expression, indicating that RUNX3 is a tumor suppressor involved in the development of gastric cancers [RUNX3 CpG island hypermethylation in GCTs, but not in normal ovarian tissue or peripheral blood suggests that RUNX3 hypermethylation might be associated with the genesis of this cancer type and this frequent methylation might serve as a biological marker. Loss of expression in RUNX3 gene in patient 11 which also lacks any PCR product with either of the unmethylated and methylated primers indicates that it could be due to the large deletion in the gene. However, studies involving the markers D1S234 and D1S199 showed the loss of heterozygosity which could to some extent explain the loss of expression in this patient. We could not confirm this due to the lack of further sample. However, the sample quality was good as indicated by the presence of band in GAPDH1 which was used as an internal control.The present study showed that hypermethylation of the  cancers . The preThe histological examination of granulosa cells revealed that these are small, usually round to polygonal, but may be spindle-shaped with scanty amphophilic cytoplasm, containing only occasional small lipid droplets, and having indistinct cell borders. Granulosa cells regularly express inhibin and contain vimentin and smooth muscle actin intermediate filaments and, less commonly, cytokeratins. However, during their malignant transformation, theses cells exhibit one or more of so-called 'microfollicular', 'macrofollicular', 'trabecular' or 'insular' patterns. The microfollicular variant is characterized by multiple small rounded spaces formed by cystic degeneration in small aggregates of granulosa cells and often fragments of nuclear debris or pyknotic nuclei. These spaces, known as Call-Exner bodies, are found in only 30–50% of tumors. The granulosa cell nuclei are oriented somewhat radially around these structures. The nuclei are typically bland and often grooved in granulosa cell tumors and have few mitoses, but show variation in size and shape, and marked hyperchromatism.There exist two different pathways that can contribute to the development of cancers; genome-wide hypomethylation may lead to the loss of chromosomes (as seen in these tumours) leading to chromosomal instability, whereas promoter methylation in tumour suppressor genes, which are responsible for gene silencing, can lead to the development of cancers in somatic cells. Therefore, the balance in DNA methylation is very important, and alteration in these may be protective in one pathway but deleterious in the other.In view of the high mortality rates associated with ovarian cancer, a better understanding of the molecular mechanisms underlying tumor progression in the disease could reveal novel pathways of high clinical relevance. It can therefore be concluded that promoter hypermethylation of these genes, and loss of expression in ovarian cancers (GCTs) is relatively common and this may also be useful as a tumour marker for early diagnosis and subsequent disease monitoring. Hence, these epigenetic signatures could play a decisive role in designing treatment options for this category of ovarian cancer. These results may also have implications in better understanding the underlying epigenetic mechanisms in GCT development thus can provide prognostic indicators, and identify important gene targets for treatment of this type of cancer in females.The subjects were 25 patients affected with GCTs of ovarian origin without a positive family history. The study was approved by the Health Research Ethics Board of the Faculty of Natural Sciences. All of them were untreated at the time of study. Fifty normal individuals ranging in age from 20–60 years were studied simultaneously under similar experimental conditions. The tumor grading is as follows: 11 FIGO stage IA, 10 FIGO stage IB and 4 FIGO stage IC. All these diagnoses were reviewed by a gynaecologic pathologist and the tumor were assessed using standard criteria. An informed consent was taken from all the subjects prior the study. Fresh cancer tissue specimens were received from all the patient and these were cultured as per standard protocol. A part of the fresh tissue was used to isolate DNA and RNA for further analysis where as the cultured cells were used for the 5'-Aza-2'-deoxycytidine experiments. Microscopically, GCTs are composed of granulosa cells, theca cells, and fibroblasts in varying amounts and combinations. The term granulosa-theca cell tumor had been applied to all tumors in which both cell types were identified, regardless of the amounts present.2 [Five human ovarian cancer cell lines (TOV21G, C13*, OAW28, OAW42, OV90 and KGN) from the American Type Culture Collection and European Collection of Cell Culture were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and were grown at 37°C in 5% CO2 . DNA wasFHIT gene, methylated CpG site, forward 5'-ttggggcgcgggtttgggtttttacgc-3' and reverse 5'-cgtaaacgacgccgaccccacta-3', unmethylated CpG site, forward 5'-ttggggtgtgggtttgggtttttatg-3', and reverse 5'-cataaacaacaccaaccccacta-3'; 189–262 bp relative to transcription start site, FANCF gene, methylated CpG site, forward 5'-tttttgcgtttgttggagaatcgggttttc-3' and reverse 5'-atacaccgcaaaccgccgacgaacaaaacg-3', unmethylated CpG site, forward 5'-tttttgtgtttgttggagaattgggttttt-3' and reverse 5'-atacaccacaaaccaccaacaaacaaaaca-3'; the primers corresponds to the position +280 to +432, BRCA2 gene, methylated CpG site, forward 5'-gacggttgggatgtttgataagg-3' and reverse 5'-aatctatcccctcacgcttctcc-3', unmethylated CpG site, forward 5'-agggtggtttgggatttttaagg-3' and reverse 5'-tcacacttctcccaacaacaacc-3'; these primers are 135 and 211 bp upstream of transcription start site and cyclin-D2 gene, methylated, forward 5'-tacgtgttagggtcgatcg-3' (-1427 to -1409) and reverse 5'-cgaaatatctacgctaaacg-3' (-1152 to -1171) and unmethylated, forward 5'-gttatgttatgtttgttgtatg-3' (-1616 to -1594) and reverse 5'-taaaatccaccaacacaatca-3' (-1394 to -1414). Each PCR reaction generated 74 bp products both with methylated and unmethylated primers for FHIT, 153 bp for FANCF both with methylated and unmethylated primers, 337 and 250 bp product with BRCA2 primers specific for methylated and unmethylated primers and 276 and 222 bp product for cyclin-D2 primers specific for methylated and unmethylated PCR reactions respectively. For the PCR reaction of 25 μl, 50 ng sodium bisulfite treated DNA was added to reaction buffer containing 0.2 mM dNTP, 16.6 mM (NH4)2SO4, 67 mM Tris pH 8.8, 10 mM β-mercaptoethanol, 1.5 mM MgCl2, 10 pmol of forward and reverse primers specific to the methylated and methylated DNA sequences and 1.25 units of AmpliTaq Gold (PE Biosystems, Foster City, CA, USA). The PCR reactions were cycled in a GeneAmp 9600 thermal cycler (Applied Biosystems) under the following conditions: preheat at 94°C for 3 min. followed by 40 cycles  methylation status using the primers: forward, 5'-ataatagcggtcgttagggcgtcg-3', and reverse, 5'-gcttctactttcccgcttctcgcg-3' , for methylated DNA of RUNX3; and forward, 5'-ataatagtggttgttagggtgttg-3', and reverse, 5'-acttctactttcccacttctcaca-3' , for unmethylated DNA of RUNX3 with annealing temperature of 55°C for 20 seconds. For each PCR set, DNA isolated from normal peripheral lymphocytes of healthy individuals served as a negative methylation control. Human placental DNA was treated in vitro with SssI methyltransferase (NEB, Beverly, USA) to create completely methylated DNA at all CpG-rich regions and served as positive methylation control. Methylation-specific PCR products were analysed on 3% agarose gel electrophoresis with ethidium bromide staining. A positive control and a negative control were included in each amplification reaction.DNA methylation patterns in CpG islands of tumor suppressor genes FHIT, FNACF and BRCA2 were determined by chemical modification with sodium bisulphite as described previously . BrieflyFHIT, FNACF and cyclin-D2 mRNA levels in cancer patients were compared to their expression in normal cells using semi-quantitative reverse transcriptase polymerase chain reaction. In brief, cDNA was synthesized using AMV reverse transcriptase (Promega, Mannheim, Germany) and amplified in duplex reactions in a total volume of 25 μl containing 150 μM of each dNTP, 1.5 mM MgCl2, 10 pmol of each primer pair and 1.0 units of Taq polymerase. After initial denaturation at 95°C (94°C) for 5 min, 30 cycles of 30 sec (45 sec) at 95°C (94°C), 30 sec (50 sec) at 58°C (55°C) and 45 sec (50 sec) at 72°C were performed followed by a final 10 min elongation step at 72°C. The values in the brackets correspond to FANCF. The following primers were used to amplify FHIT: forward 5'-gctcttgtgaataggaaacc-3' and reverse 5'-tcactggttgaagaatacagg-3' which yields 532 bp product spanning within exon 5 to exon 10, FANCF: forward 5'-ttcggaagtctttgctgcct-3' and reverse 5'-agtaataacacacgattgcc-3' which yields 413 bp product spanning from +733 to +1144. RT-PCR was performed for Cyclin D2 using the primers 5'-catggagctgctgtgccacg-3' (forward) and 5'-ccgacctacctccagcatcc-3' (reverse) with PCR conditions being: 1 cycle of 94°C for 3 min and 35 cycles at 94°C for 20 s, 55°C for 30 s, 72°C for 45 s followed by 72°C for 5 min. RT-PCR was also carried out for RUNX3 gene using the primers: forward 5'-aggcattgcgcagctcagcggagta-3' and reverse 5'-tctgctccgtgctgccctcgcactg-3' (152 bp). GAPDH1 was used as internal control. Each reaction was performed in triplicate. PCR products were electrophoresed on 2% agarose gels and quantified using densitometer (Molecular Dynamics). Fold increases in expression in cancer cells were calculated with respect to the levels of the transcripts in normal cells.Cell lines (TOV21G, C13*, OAW28, OAW42 and OV90), normal ovarian cells and tumour cells of ovarian GCTs were treated with demethylating agent 5-Aza-2' deoxycytidine (Sigma) for five days at a concentration of 2.5 μM, HDAC-inhibiting agent trichostatin (TSA) at a final concentration of 5 μM for the last 24 hours or a combination of both. Total RNA isolated from treated, untreated cell lines and cell of the cancerous tissue was reverse transcribed using random primers and the Pro-STAR first strand RT-PCR kit (Stratagene, La Jolla, CA). A semi-quantitative analysis of gene expression was performed in replicate experiments using 30 cycles of RT-PCR as described above.The authors declare that they have no competing interests.VSD for executing the MSP and RT-PCR experiments; completing manuscript; MS, for carrying out cell culturing; and SAH conceived and coordinated the study. All authors read and approved the final manuscript.


We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and  For example, in sea urchins, an animal's testes can be dissected and sperm is activated by exposure to seawater. Eggs can be released by injecting KCl into the perivisceral cavity, and mixing eggs and sperm  embryos . These s embryos , dealing embryos .in vitro before being implanted into surrogate mothers [Artificial reproduction has also been well-studied in mammals, and cloning of eutherians from somatic cells is now common -8. Harve mothers ]. Import mothers ,11.Xenopus laevis and the North American leopard frog, Rana pipiens [e.g. [R. pipiens with male pituitary glands to induce ovulation and deposition of unfertilized eggs. The eggs were mechanically activated by pricking with a needle, a process which brings the pronucleus immediately under the surface of the animal pole. Taking advantage of this situation, the pronuclei were extruded, along with a small amount of cytoplasm, using a glass needle. In other species, such as the Xenopus or the axolotl, UV radiation can be used to destroy the female pronucleus instead [Frogs have been favorite model organisms in reproductive and developmental biology for many years, mainly because of the ease with which they can be kept in captivity; their external fertilization; easily visible development in large, transparent eggs; and large numbers and ease of manipulation of their eggs. Consequently, research on frogs has often been in the vanguard of advancement in artificial reproduction techniques, and much is known about a few model species such as the African clawed frog, ns e.g. -15]. Ind. IndXenons [e.g. . Briggs  instead ,18. DeveXenopus: testes were macerated in solution and the sperm membranes partially dissolved, allowing access to the condensed chromosomes. Linearized bacterial plasmids containing genes of interest were mixed in with the sperm solution and recombinant ligase was used to covalently insert the bacterial plasmids into the sperm genomic DNA, resulting in the insertion of many copies of the plasmid construct into each genome. These nuclei were then microinjected into mature eggs, generating, under appropriate conditions, hundreds of nonmosaic, transgenic embryos. Such techniques allow the investigation of gene function in these species [e.g. [In another group of experiments, Kroll and Amaya  developees [e.g. ].Clearly, there are enormous advantages to being able to manipulate a species' reproduction in the laboratory. However, despite the multiplicity of studies concentrating on anurans, to date all model species are aquatic breeders. Yet amphibians have the largest diversity of breeding strategies among terrestrial vertebrates, and it is to be expected that species with different reproductive strategies will require different methods for their manipulation in the laboratory. Therefore, many species remain experimentally intractable. Notably, terrestrial-breeding frogs, a very large and diverse group of organisms, are largely inaccessible to reproductive investigations.Eleutherodactylus is characterized by terrestrial breeding and direct development without an aquatic larval stage. With more than seven hundred described species, this is the largest vertebrate genus [Eleutherodactylus frogs, ranging from basic developmental biology [in vitro fertilization in these frogs. The development of such techniques would allow additional investigations into the genetic regulation of direct development in these species and would also assist with conservation of declining populations, an important goal considering the fact that many species of Eleutherodactylus are declining, and several are already extinct [The neotropical frog genus te genus ,22. Ther biology -26; to e biology -30]; to  biology ,31. Howe extinct ,33.Eleutherodactylus coqui are small tree frogs with internal fertilization and direct development [Eleutherodactylus species, E. coqui embryos develop directly into tiny froglets in terrestrial eggs, without a tadpole stage [E. coqui is arguably the best-studied terrestrial-breeding frog, we have focused on this particular species as a model for the development of reproductive techniques.elopment . This spelopment . As withle stage . Protocole stage ,38. A mele stage . It is kle stage . Corticale stage , but theEleutherodactylus coqui frogs were collected in Puerto Rico near El Verde Field Station in the Luquillo mountains and transported to Tulane University where they were housed and fed as previously described [Adult escribed . All aniNatural matings were performed by placing a gravid female and a calling male together as described . Mature,In vitro fertilization (IVF) experiments using sperm in solution were carried out by macerating the testes from a single frog in 500 μL of sperm dilution buffer (SDB: 10 mM NaCl, 0.2 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes pH 7.5), and adding this dropwise over the tops of the eggs or injecting it under the jelly coat using a tuberculin syringe (28.5 gauge, 13 mm length). Alternatively, small pieces of macerated testes were placed directly on top of each egg without the use of buffer solution. Incubation of sperm with egg jelly was accomplished by vigorously vortexing the jelly from one egg in 100 μl of SDB. Sperm was then incubated for 10 min in the jelly/buffer supernatant.Sperm preparations were checked for morphology, movement and membrane integrity by fluorescent microscopy with Live/Dead sperm stain (propidium iodide and SYBR 14) (Molecular Probes, Eugene, OR) using an Olympus BH-2 microscope . Additio2 with 0.1 mM A23187 calcium ionophore. Scoring of activation was done by noting the appearance of the first cleavage furrow .E. coqui. This ionophore non-specifically activates the unfertilized eggs of a variety of species through stimulation of a calcium-dependent signalling cascade [Table  cascade . Ten hou cascade ; figure  cascade ). ActivaTo test whether the pseudocleavage response was an effect of our stimuli and not a reaction tied to other uncontrolled variables, we examined eight eggs that had remained undisturbed and that had not started pseudocleavage ten hours after laying. At this time, we poked them with fine forceps, and 6 of 8 (75%) began pseudocleaving six hours later table . This deE. coqui during natural matings because this species undergoes internal fertilization, and the eggs will have already been fertilized prior to deposition [E. coqui eggs are arrested or what signal takes them out of their arrest, we followed procedures used in Xenopus [An interesting observation was that one third of all eggs deposited in response to hormone treatment activated of their own accord. This may be due to the mechanical stress to which the eggs are exposed during oviposition. Clearly, mechanical stimuli are able to activate the eggs, and stress incurred in during transit from the ovisac may be sufficient to cause activation. This would presumably not affect position . In orde Xenopus . HoweverE. coqui eggs might be sensitive to aging, as has been reported under certain conditions for the externally fertilizing X. laevis [E. coqui has internal fertilization, we suspected that eggs laid unfertilized might degenerate rapidly, complicating the artificial manipulation of this species' reproduction. Consequently, we investigated the ability of eggs to be activated as a function of time. When we artificially activated eggs with the calcium ionophore at different time points and examined them six hours post treatment, the percentage that pseudocleaved was high even ten hours after being laid  – by simply mincing the testes and adding them directly over the eggs . We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization.Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development . A seconis sperm . However36 = 3.2 × 10-5, or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated.To test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none 0/36) developed (see table 6 developE. coqui, we have observed that a large proportion of eggs that are laid unfertilized auto-activate. We showed that E. coqui eggs are easily activated by mechanical stimuli, leading to a need for careful manipulation of unfertilized eggs in all reproduction studies. Further, the cleavage pattern seen in mechanically activated eggs is similar to that of both auto-activated and chemically activated eggs, suggesting that mechanical stress, probably incurred in during oviposition, is responsible for the auto-activation mentioned above (table E. coqui, this phenomenon may relate to internal fertilization, and it would be interesting to investigate auto-activation of unfertilized eggs in closely related, externally fertilizing species such as E. antillensis [E. coqui eggs, however, remain intact and can be manipulated, showing signs of activation both at the morphological level, through the initiation of development, as well as the functional, through the slow block to polyspermy. By careful handling, we are now able to fertilize over half of the remaining, functionally viable eggs using a simple procedure. Finally, we have shown that E. coqui eggs do not degenerate rapidly and are capable of undergoing activation up to ten hours after deposition, thus creating a window of time for carrying out experimental procedures. Our results demonstrate efficient IVF in an internally fertilizing, terrestrial-breeding frog and help lay the foundation for future research and conservation possibilities in this unusually large genus of amphibians.In an effort to conserve declining populations of animals, the development of protocols for the artificial manipulation of reproduction is of great interest. In the case of the neotropical frog ve table . Facile illensis . Some E.ET carried out the experiments and prepared the manuscript. SFM carried out preliminary experiments and supervised the manuscript. The study was conceived jointly. Both authors approved the final manuscript.


Analysis of DNA microarray data usually begins with a normalization step where intensities of different arrays are adjusted to the same scale so that the intensity levels from different arrays can be compared with one other. Both simple total array intensity-based as well as more complex "local intensity level" dependent normalization methods have been developed, some of which are widely used. Much less developed methods for microarray data analysis include those that bypass the normalization step and therefore yield results that are not confounded by potential normalization errors.Instead of focusing on the raw intensity levels, we developed a new method for microarray data analysis that maps each gene's expression intensity level to a high dimensional space of SEDs (Signs of Expression Difference), the signs of the expression intensity difference between a given gene and every other gene on the array. Since SED are unchanged under any monotonic transformation of intensity levels, the SED based method is normalization free. When tested on a multi-class tumor classification problem, simple Naive Bayes and Nearest Neighbor methods using the SED approach gave results comparable with normalized intensity-based algorithms. Furthermore, a high percentage of classifiers based on a single gene's SED gave good classification results, suggesting that SED does capture essential information from the intensity levels.The results of testing this new method on multi-class tumor classification problems suggests that the SED-based, normalization-free method of microarray data analysis is feasible and promising. DNA microarray technology is now playing an increasingly important role in biomedical research. Microarray technology gives one the opportunity to measure gene expression levels of thousands to tens of thousands of genes simultaneously, in order to study the differential gene expression pattern between different developmental stages, diseases states and samples treated with drugs or other compounds. Before comparing data from different arrays to address these biological questions, however, a "much more mundane but indispensable " normalization step  is curreBecause of the slight difference in RNA quantities, imaging settings and other variables, even in very controlled experiments the intensity levels from different arrays are of different scales and need to be normalized before they can be compared with each other. Various normalization methods have been developed and some are widely used. The simplest method is total intensity based normalization ; this apFor most current applications, these normalization methods seem to be adequate. However, the residual left by a less than perfect normalization procedure is another source of non-biological variation that is usually non-desirable, especially when the differences in expression levels are expected to be small . In addietc. We decided to use multi-class tumor classification as a test case to illustrate the power of our approach. We compared our results for a multi-class tumor classification problem with more conventional approaches published by Ramaswamy et al. [et al. [DNA microarray technology has been used widely in biomedical studies. One interesting application is in the area of molecular classification; one popular use is in the comparison of tumor samples. Since clinical and histopathological classification is sometimes difficult and labor-intensive, the use of genome wide expression patterns to classify tumor samples has recently become a very active research area -16. Althy et al.  and Yean [et al. . These ai (1<= i <= N) for each gene i on the array, where N is the total number of genes on the array. Without first doing some type of normalization, the intensity level of gene i from array A, xiA, cannot be directly compared with the intensity level of gene i from array B, xiB. In this study, we sought an alternative quantity or quantities that can be directly compared between different arrays without compromising important biological information. One obvious candidate is ri, the rank of intensity level of gene i on the array. However, we felt that rank is not an adequate measure because information about relative expression level is not represented explicitly. Instead, we decided to use the following measures.Generally, measurements on single microarrays give a real-valued intensity level xLetij = 1 if(xi - xj > 0)s0 otherwise     (1)ij is the sign of the intensity difference of gene i and j on a single microarray and therefore will remain unchanged under any monotonic transformation of x. Therefore, instead of computing with the absolute expression level of a gene, its relative level to all the other genes on the microarray is used. For each gene i, instead of one real valued xi, the approach uses si = (si1, ..., sij, ..., siN), a binary vector of size N. For ease of reference, we will simply refer to this value as the SED (Signs of Expression Difference) of gene i; and the entire matrix (sij) the SED of the array. Given (xi), sij is simply and uniquely defined but (sij) does not uniquely determine xi so some information is lost by only using (sij) instead of (xi)., where 1<= i, j <= N. Basically, si = Σj = 1,...,N sij is the rank of gene i in terms of intensity levels, rank information is preserved in (sij). What is lost in the transformation from (xi) -> (sij) is just the intensity differences between the closest ranked genes, which in most cases are small, considering that microarray data are generally considered "very noisy". It was our major goal to demonstrate that (sij) has indeed captured important components of the information from (xi).Since rA + μB)/(σA+ σB)], we will use (sij) to compare gene expression differences between arrays. Since we expect measurement variations within an array will be less than those between arrays and we take the signs of relative expression differences to get SED, we expect the SED will be less "noisy". However, the value of any single sij may still vary between technical and biological replicates. One would expect more sij would change values randomly if the technical replicates was done on arrays that were fabricated in a different run than arrays from same run, for example. Biological variations are expected to be even more frequent. However, we hypothesize that we can perform statistical analysis on the SED, which contains tens of thousands of sij for a single gene, and minimize the impact of such noises.Instead of directly using the intensity levels x, and its derivatives such as the mean μ, the standard deviation σ and the signal to noise ratio (S2N) between two sample groups A and B, [(μij), a natural generalization of the SED concept. Here, spij is the probability of xi > xj. In other words, imagining one can get a large number, n, of either technical or biological replicates of the sample of interest, then spij = m/n as n -> 8, where m = ΣK = 1,...,n sijK and sK is for replicate K. We will call (spi) = (spi1, ..., spij, ..., spiN) the SED probabilities of gene i. Note that in calculating both SED and SED probabilities, only intensity comparisons within arrays are involved and therefore forego the normalization step.We will also consider  spijA would be larger than spijB. Since rank can be calculated from SED, any rank based method can be expanded to use SED. A gene i's SED can be viewed in two different perspectives. On one hand, it provides information about gene i's expression level relative to every other gene on the array, and therefore can be used to examine gene i's expression patterns between samples. On the other hand, it also provides information about the expression levels of all the other genes on the array, using the gene i as a control, in essence. Therefore, SED can be used to study questions either at the gene level or at the array level. In this paper, we focus on solving a simpler problem at the array level where it is not necessary to decide whether the expression level of an individual gene is increased or decreased between array A and B and by how much. Rather, it is focused on whether the overall expression patterns are different at all between array A and B.For example, if gene i is more highly expressed in sample A than B we would expect that more sij) and (spij) extracts most of the information from (xi), we used these values in a test case of a multi class classification problem described by Ramaswamy et al. and Yeang et al [ijC) for each class C (1 <= C <= 14) using training samples, i.e. the 144 samples with T taken away (for details see methods). Then sample T is classified according to:To test whether ,     (2)score(T, C)ij = spijC if sijT = 1where pijC otherwise1 - spT is simply classified to the class C that has the maximum score.In the NN method, we compute instead, for each training sample t,i = 1,...N Σj = 1,...,N δ(sijT, sijt),     (3)matches(T, t) = Σwhere δ(x, y) = 1 if x = y.0 otherwiseThen T is classified to class C of the sample t that has the maximum matches.ij) as defining a multi-binomial probability model. In addition, one could consider each sij as a draw from a binomial distribution with probability pij = spij. Then, the score(T, C) is simply a logarithm of the probability p to get all the sij exactly the same as sijT under the probability model C where pij = spijC. (Since each class defines a different probability model, score(T,C) for difference class C, in theory, should not be directly compared. Instead, a P value should be calculated from the probability model for Pr(p<exp(Score(T, C))) and used to evaluate the closeness of sample T to each class C. For simplicity, we are not considering such issues here.)If one is to give a statistical interpretation of these scores, one can simply view  = Σj = 1,...,N log(pij), where pij is as same as mentioned previously. Similarly, matchesi(T, t) = Σj = 1,...,N δ(sijT, sijt) defines a classifier based on gene i's SED. Fig. In these cases, we define a classifier based on gene i and its relative expression to every other gene. Therefore, the scoreFrom the analyses described above, we noticed that there was a significant difference in accuracy between different tumor classes. For 3 classes (LY, LE, CNS) we obtained either 100% or close to 100% accuracy , in addition to being normalization free, still convey the essential information in the original attribute, the intensity x.Although we used the multi-class tumor classification problem as our test case, our major goal was to illustrate the feasibility of the normalization free SED approach, and not in sample classification Since the data representation is quite different between the intensity x  and SED , it is difficult to directly compare the two. No obvious yet non-trivial algorithms work with both representations; even if there were such an algorithm it is not clear that it would be the right one to use for comparison as it might well be the case that different data representation works best with different algorithms. Here, we have limited the presentation to some empirical results with SED representation, which are comparable with results using several different algorithms that are based directly on raw intensity ,20. Our Certainly, SED probability is more information rich than SED. We expect that an SED probability based analysis would perform better than the simple binary valued SED. In this paper, we mainly tested the SED. SED probability is only used for a group of samples, not for single samples. If one limits oneself to use only raw data, then for single arrays one can only get SED. However, if some assumptions about the patterns of gene expression levels can be made, one can certainly get an estimation of SED probability even for a single array. For example, as in some nonlinear normalization algorithms, if one assumes that the variation of expression levels are similar for genes with similar expression levels, then one can estimate SED probability from a probability model. Also, the magnitude of the intensity difference can also be used to help such an estimation. Alternatively, as more and more microarray data become available, one can use other similar samples to get an estimation of a prior SED probability, and then use a Bayesian approach to estimate the sample's SED probability.The obvious disadvantage of our SED based approach is that for each gene expression level, one is not dealing with a single real number but instead a vector of size N, where N is in the tens of thousands. This could significantly increase both computing time and memory requirement (however, see methods for details) On the other hand, it also has certain advantages: 1) It is free of normalization noise. Since it is generally believed that biological variation is larger than technical variation and normalization noise is just another source of technical variation, the benefit here is only of a limited scope. However, it may be important when the expression level difference one is interested in is small. 2) In addition to being normalization free, SED and SED probability also have the advantage in being distribution free, and therefore could perform better if the intensity levels were non-normal. 3) SED and SED probability are easier to interpret. SED values can easily be checked against raw intensity levels according to Eq. (1). While SED probability is one step further away from intensity levels, one could still have an intuitive sense of it and make comparisons between different experiments. It would be much harder to have a real grasp of the absolute gene expression level, except that it is "high" or "low" or somewhere "in between"; it is certainly harder to compare between experiments intuitively.We have only tested the SED approach on datasets that are from the same chip format. Data from different chip formats or complete different technology platforms, of course, would be harder to compare. But they are also challenge for normalization based method. It would certainly be interesting to compare SED and normalization based method under these more challenging conditions.i.e., to find similar microarray results in the database that are "similar" to one's own, independent of array annotations.If this normalization-free approach (SED) proves to retain the essential biological information in general, its application may be extended to meta-analysis where different datasets could be integrated and intervalidated. The method could also be used when the number of arrays is a limiting factor for experiments. For example, one could take advantage of the massive amount of public array data, obtain prior distribution of SED probabilities from datasets with similar conditions, and analyze new data within a Bayesian framework. If the performance of the nearest neighbor method in general is anywhere close to what we demonstrated in the multi tumor classifications here (as is clear from Eq. 3) and Fig.  and Fig.It might also be worth noting that the SED approach could easily be applied to other kinds of comparative data analysis for samples with very large numbers of "noisy" attributes. The SED approach may also perform better when between-sample-variation is large, especially if such variation contains some rather uninteresting technical measurement errors that would not affect within-sample-variations.We have proposed a new approach to analyze microarray data and tested the method on a set of publicly available datasets. The results were comparable to those obtained with some widely used normalization based algorithms. We hope that we have demonstrated that this normalization free method is feasible and promising. We think the SED based, normalization free approach could be used to complement the more popular normalization based approaches in microarray data analysis.. Naive Bayes and Nearest Neighbour Classifiers were implemented in the Java programming language. Ad hoc analysis was done with perl scripts. Graphics were generated using the R computing environment.Microarray data for multiple tumor samples were downloaded from ij. Assuming a prior probability of 0.5, spij was estimated by Bayesian posterior probability (m+1)/(n+2) where n is the total number of samples in the class and m is the total number of samples where xi > xj. For classes that were over-represented (sample size > 8), the threshold of spij was set to [0.125, 0.875], since the NB method is sensitive to the extreme values of sp, and samples can be over-predicated without thresholds.Because of the uneven and relatively small sample sizes for each tumor class (mostly 8 but up to 24), extra care was taken in computing spIn addition, several alternatives were tested to demonstrate that our results were reasonably robust and not sensitive to the particular choices we made:i.e. only the first 8 samples were used to calculate spij with no significant change of results observed;1) To examine the influence of the sample size, in a separate analysis the sample size of ME, LE, CNS class was artificially reduced to 8, ij depends on the sample size n for each tumor class, we have applied a "sample replacement" strategy in addition to the usual "take-one-sample-out" approach for cross-validation, i.e. when one sample is taken out as the test sample, another sample from the same class is duplicated to take its place to keep the sample size constant. Essentially the same results were obtained. Results reported are from the sample replacement runs.2) Since spij between all 14 classes was calculated as:In Feature Filtering, the variance of sp2 = (ΣC = 1,...,14,spijC * spijC)/14 - ((ΣC = 1,...,14 spijC)/14)2     (4)σwith the test sample taken out, and used as the criterion for feature exclusion.Feature filter was done as in the Naive Bayes Method.The analysis was done on a computer (Pentium M 1.5 GHz) operating under Microsoft XP. Both Naïve Bayes and Nearest Neighbor Classifiers are implemented in Java. Since SED can be easily calculated from the raw intensities only the later are kept in memory and SED are computed from the intensities on an as-needed bases. Memory needed to analysis the 144 samples is less than 64 MB.The most computationally intensive algorithm that we tried is the Nearest Neighbor method without any feature selections and it takes about 10 sec to calculate SED score for one pair of tumor samples with about 16000 genes.hw14@columbia.edu.A Java program named SED (including source code) to perform nearest neighbor analysis of microarray samples is freely available by contacting author at H.W. conceived of the SED study and performed implementations. H.H. refined the approach and provided additional statistical insight on SED. Both authors read and approved the final manuscript.


Plasmodium genus that are under consideration as candidates for inclusion in transmission-blocking vaccines. Previous research using transgenic rodent malaria parasites lacking P25 and P28 has demonstrated that these proteins have multiple partially redundant functions during parasite infection of the mosquito vector, including an undefined role in ookinete traversal of the mosquito midgut epithelium, and it has been suggested that, unlike wild-type parasites, Dko P25/P28 parasites migrate across the midgut epithelium via an intercellular, rather than intracellular, route.P25 and P28 are related ookinete surface proteins highly conserved throughout the via an intracellular route similar to wild-type parasites and subsequently develops into oocysts.This paper presents an alternative interpretation for the previous observations of Dko P25/P28 parasites, based upon a recently published model of the route of ookinete invasion across the midgut epithelium. This model claims ookinete invasion is intracellular, with entry occurring through the lateral apical plasma membrane of midgut epithelial cells, and is associated with significant invagination of the midgut epithelium localised at the site of parasite penetration. Following this model, it is hypothesized that: (1) a sub-population of Dko P25/P28 ookinetes invaginate, but do not penetrate, the apical surface of the midgut epithelium and thus remain within the midgut lumen; and (2) another sub-population of Dko P25/P28 parasites successfully enters and migrates across the midgut epithelium These hypotheses are tested by showing how they can account for previously published observations and incorporate them into a coherent and consistent explanatory framework. Based upon these hypotheses, several quantitative predictions are made, which can be experimentally tested, about the relationship between the densities of invading Dko P25/P28 ookinetes in different regions of the midgut epithelium and the number of oocyst stage parasites to which these mutant ookinetes give rise.The recently published model of ookinete invasion implies that Dko P25/P28 parasites are greatly, although not completely, impaired in their ability to enter the midgut epithelium. Therefore, P25 and/or P28 have a novel, previously unrecognized, function in mediating ookinete entry into midgut epithelial cells, suggesting that one mode of action of transmission-blocking antibodies to these ookinete surface proteins is to inhibit this function. P25 and P28 are related major ookinete surface proteins under consideration as candidates for inclusion in transmission-blocking vaccines -4. ConsePlasmodium genus [P25 and P28 are structurally similar proteins, highly conserved throughout the um genus ,31,35-43um genus , putativum genus ,28,29, tum genus ,17-24. Pum genus -21,44,45Plasmodium berghei rodent malaria parasites lacking P25 and P28 demonstrated that these proteins have multiple and partially redundant functions during parasite infection of the mosquito vector [P. berghei parasites exhibit greatly reduced levels of oocyst infection compared to wild-type or Sko P25/P28 parasites, ookinetes lacking both P25 and P28 are still able to cross the midgut epithelium and establish oocyst infections [P. berghei ookinetes migrate intracellularly through the midgut epithelium causing significant damage to invaded midgut epithelial cells [via a solely intercellular route [Previous research using transgenic o vector ,27. Althal cells -48, whical cells -48, inclal cells ,45, protal cells ,45,48 anal cells ,49 and Sal cells . Furtheral cells ,45. Dko al cells ,45,48. Car route  suggestsin vivo support this model [P. berghei ookinetes [The route of ookinete migration across the midgut epithelium of the mosquito vector has long been controversial . The majis model . Accordiis model ,51,52. Sis model  and Figuis model ). Ookineokinetes ,55. Subsokinetes ,52,56, mP. berghei parasite infection of the mosquito vector are proposed. First, some Dko P25/P28 ookinetes invaginate, but are unable to penetrate, the apical surface of the midgut epithelium. Second, other Dko P25/P28 parasites are able to successfully enter and migrate across the midgut epithelium via an intracellular route, in a manner similar to wild-type parasites.Following the model of ookinete invasion of the midgut epithelium outlined above . If Dko P25/P28 ookinetes do migrate across the midgut epithelium via a solely intercellular route there is no known reason why these parasites should not also be melanized in the basal region of the midgut epithelium. Consequently, if solely intercellular migration does occur melanized parasites should be found that are not associated with any morphologically abnormal midgut epithelial cells.First, all melanized Dko P25/P28 parasites in the refractory via a solely intercellular route greatly exceeds the number of intracellular ookinetes [via a solely intercellular route, the number of oocysts should greatly exceed the number of ookinetes migrating via an intracellular route .Second, there should be a quantitative relationship between the density of Dko P25/P28 ookinetes associated with morphologically abnormal midgut epithelial cells and the number of oocysts that subsequently develop on the basal surface of the midgut epithelium. Specifically, the number of oocyst stage parasites should be equal to or less than the number of Dko P25/P28 ookinetes associated with morphologically abnormal midgut epithelial cells, as only ookinetes migrating intracellularly are predicted to become oocysts. The Dko P25/P28 ookinetes located between morphologically normal midgut epithelial cells are not expected to transform into oocysts, as these parasites do not enter, and hence cross, the midgut epithelium. The number of ookinetes apparently migrating okinetes . ConsequP. berghei parasites implies that there are two sub-populations of Dko P25/P28 ookinetes, neither of which migrate across the midgut epithelium via a solely intercellular route (Figure An. gambiae and do not give rise to oocyst parasite stages. These parasites remain surrounded by morphologically normal midgut epithelial cells, which do not exhibit the morphological abnormalities associated with parasites invading intracellularly [An. gambiae [The re-interpretation presented here of previously published work on Dko P25/P28 llularly . A seconllularly , GNBP [2llularly , NOS andllularly , undergo gambiae , and for gambiae . Accordi gambiae . HoweverP. berghei ookinetes is unknown. One explanation is that loss of P25 and/or P28 impedes, but does not entirely prevent, penetration of the apical plasma membrane of midgut epithelial cells. Consequently, the entry of Dko P25/P28 ookinetes into the midgut epithelium may be protracted, prolonging the period of exposure to the hostile environment of the midgut lumen, which results in the death of most parasites before completion of midgut epithelial cell penetration. This interpretation is consistent with the observation of lysed Dko P25/P28 parasites on the luminal side of the midgut epithelium [The reason for the existence of the two distinct sub-populations of Dko P25/P28 ithelium .In summary, the unified model of the route of ookinete invasion across the mosquito midgut epithelium suggests a novel, previously unrecognized, function for P25 and/or P28 in mediating ookinete entry into the midgut epithelium. Specifically, the interpretation presented implies that the loss of P25 and/or P28 greatly impairs, but does not entirely abolish, ookinete entry into midgut epithelial cells and probably has relatively little effect on the ability of ookinetes to traverse through the cytoplasm of midgut epithelial cells. A role for P28 in parasite entry into the midgut epithelium is suggested by the deposition of this molecule at the site of ookinete penetration into midgut epithelial cells ,45. ThisCTRP = circumsporozoite and thrombospondin-related anonymous protein-related protein; Dko P25/P28 = double knockout of P25 and P28; GNBP = gram-negative binding protein; MAOP = membrane-attack ookinete protein; NOS = nitric oxide synthase; Sko P25/P28 = single knockout of either P25 or P28; SRPN10 = serine protease inhibitor 10.LAB wrote the manuscript and prepared the figures. LRC revised the manuscript. Both authors read and approved the final version of the manuscript.


Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636.Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family.Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from  The PD-(D/E)XK domain is ubiquitously found in enzymes involved in metabolism of nucleic acids, mostly in nucleases with diverse biological functions. The first structurally characterized members of the PD-(D/E)XK superfamily were restriction enzymes (REases) (reviews: ,2). CrysD...Xn...(D/E)XK (where X is any amino acid). The Lys residue serves to position a water molecule for an in-line attack on the scissile phosphodiester bond, while the carboxylate residues coordinate a Mg2+ ion, which acts as a cofactor. Despite the wealth of structural and biochemical data, obtained mainly for REases (summarized in a collection of reviews: [All members of the PD-(D/E)XK superfamily share a common structural core, comprising a mixed β-sheet of 4 or 5 strands flanked on both sides by α-helices ,2,12. Threviews: ), there reviews: ,15. Morereviews: -19. In areviews: -22.2+ binding site common to its cousins that cleave phosphodiester bonds in DNA. Remarkably, on the opposite side of the common fold, EndA developed a different active site, whose geometric configuration is very similar to that of a His-Tyr-Lys triad in structurally unrelated RNase A [Saccharomyces cerevisiae exhibits perfect conservation of the restriction enzyme-like structure, but lacks any catalytic residues – it is postulated that it functions as a nucleic acid binding domain devoid of any catalytic activity [Crystallographic analyses have also revealed the PD-(D/E)XK fold in proteins that do not function as deoxyribonucleases at all and exhibit no conservation of the active site with the above-mentioned enzymes. The structure of the C-terminal catalytic domain of tRNA splicing endoribonuclease (RNase) EndA is identical to the minimal core of the PD-(D/E)XK fold , yet thi RNase A . Finallyactivity .The divergence exhibited by the members of the PD-(D/E)XK superfamily is remarkable. Even enzymes with very similar biological functions, such as REases that recognize and cleave the same substrate, can exhibit little or no significant sequence similarity. Thus, most of the afore-mentioned enzymes were considered unrelated until the corresponding crystal structures were solved. Only in a few cases the membership in the PD-(D/E)XK superfamily was successfully predicted using bioinformatics  before the actual structures were determined -29. The In the course of analyses of proteins with unknown structures, we came across a family of sequences grouped together in the Clusters of Orthologous Groups (COG) database  as COG46In order to test the hypothesis of the evolutionary connection between COG4636 and the PD-(D/E)XK superfamily we carried out the fold-recognition analysis, which allows to predict the three-dimensional fold of the target protein by matching its sequence with the available protein structures and assessing the sequence-structure compatibility using a combination of criteria, such as sequence similarity, match of secondary structure elements, compatibility of residue-residue contacts, etc. (review: ). SequenNostoc sp. PCC 7120, GI: 17231142) and used it as a platform to study the three-dimensional arrangement of conserved residues. A homology model of all3650 was constructed using the "FRankenstein's Monster" approach (see Methods and ref. [In order to identify the putative active site of newly predicted members of the PD-(D/E)XK superfamily, we modeled the structure of one of the COG4636+ members, whose sequence was close to the consensus calculated for the whole family , startiand ref. . The cooand ref.  and on-lThe model of all3650 reveals a typical PD-(D/E)XK nuclease-like spatial arrangement of one Lys ε-amino group (from the residue K127) and two carboxylate groups (from residues D83 and E111) Figure . The modG. violaceus, GI: 37520478) the Lys residue is present both at the "classical" and alternative position, thereby forming a "PD-EXK-K" variant of the active site. This arrangement resembles a putative evolutionary intermediate between the "classical" active site and the newly discovered rearranged variant. In another lineage of the COG4636+ family, an Asp residue appears in the position normally occupied by Lys in the C-terminal half-motif. Some of the members of this lineage (exemplified by glr2344 from G. violaceus, GI: 37521913) exhibit therefore the "PD-EXD-K" motif, but the majority  lack the Lys residue and exhibit only the "PD-EXD" variant. In another lineage (represented by gll1896 from G. violaceus, GI: 37521465) the Lys residue is replaced by Asn to form the "PD-EXX-N" variant of the predicted active site. The conserved His residue  is present in most members of the COG4636+ family, with the exception of a small lineage of closely related proteins (represented by gll1896 from G. violaceus, GI: 37520579) in which it is substituted by Gln, and a larger group of more diversified sequences, in which it is substituted by Thr or Ser. Most members of the latter group possess a Lys or Arg residue in the "catalytic" position and hence exhibit "PD-EXK-K" (see above) or "PD-EXR-K" variants of the active site. It will be very interesting to determine experimentally, which of those residues in different configurations are involved in catalysis, and which are only auxiliary. In particular, it would be interesting to find if both or either of the Lys residues present in the potential "intermediate" versions of the active site are required for catalysis.Inspection of the multiple sequence alignment reveals that only two carboxylates  are practically invariant in the COG4636+ family, while all the others undergo various substitutions . It is astonishing that members of COG4636+ represent over 2% of all protein-encoding genes of G. violaceus PCC 7421  originate from Cyanobacteria; of these, 84% were found in just 6 genomes , other 30 total  and depoD. hafniense DCB-2 (Firmicutes), 7 sequences from C. aurantiacus , and 6 from S. coelicolor (Actinobacteria) each form a separate species branch on the phylogenetic tree, while 14 sequences from T. thermophilus HB27 (Deinococcus-Thermus lineage) form three separate branches. Several monophyletic groups of closely related sequences are also observed in G. violaceus (e.g. a sub-family comprising 7 sequences with GI numbers: 37522824, 37520777, 37522646, 37521452, 37522233, 37520151, 37522558). There is also one branch comprising 6 closely related sequences in C. watsonii, GI numbers: 45527153, 45527776, 45524526, 45527777, 45527775, 45527774). Other statistically significant branches, however, comprise members from different species, suggesting that they were either formed prior to speciation or that their members were transmitted horizontally between different genomes of already existing species.In order to reconstruct the evolutionary history of the COG4636+ family, we calculated the phylogenetic tree, based on the same reliable section of the multiple sequence alignment that was used for protein structure prediction (see Methods). Unfortunately, in all trees obtained with different methods and parameters, the majority of deep branches received very low bootstrap support (data not shown), hence the relationships within the whole family must be regarded as unresolved. We were able, however, to identify a number of branches with bootstrap support >90%. Many of such branches comprise members from one species only. This situation is characteristic for sequences found in a few non-Cyanobacterial species; for instance 8 sequences from C. watsonii WH8501, all from the above-mentioned branch of 6 closely related sequences (the other two relatives are located elsewhere on the chromosome). We also found a few tandem duplications: 9 in C. watsonii WH8501 and 5 in G. violaceus PCC7421, 5 in Nostoc sp. PCC6803, 2 in N. punctiforme PCC73102, 2 in A. variabilis ATCC 29413, 2 in Synechocystis sp. PCC6803, 2 in T. thermophilus HB27, 1 in T. erythraeum IMS101 and 1 in M. magnetotatcticum MS-1. In general, however, tandem duplications are rare and the distribution of COG4636+ family members along the chromosomes of Cyanobacteria with completed genomes seems completely erratic  in the range of 3000 bp upstream and downstream, we weren't able to identify any recurrent type of proteins, either with respect to the molecular or cellular function or the predicted three-dimensional fold (data not shown). Also no preference for occurrence of COG4636+ family members within or near any apparent mobile genetic elements (putative prophages etc.) was observed. Thus, insertion of the genes encoding putative COG4636+ nucleases seems virtually random. The only notable exception is a neighborhood of another member of COG4636+, suggesting tandem duplication. We identified one instance of 4 consecutively arranged genes in the genome of Our results suggest that functionally uncharacterized proteins grouped together in COG4636 are a branch of the PD-(D/E)XK superfamily, which has not been identified to date due to a presence of an unusual variant of the active site, which lacks the conserved Lys residue at the typical position in the primary sequence. That the catalytic Lys can migrate in the framework of the active site of PD-(D/E)XK nucleases has been suggested earlier, based on the sequence analysis of another nuclease domain found in site-specific, non-long terminal repeat retrotransposable elements , but to C. watsonii WH 8501, which was isolated from tropical waters of the Western Atlantic and Pacific oceans. It is remarkable that members of COG4636+ are almost absent from the genomes of Synechococcus and Prochlorococcus species thriving in the Sargasso sea, as well as in the environmental samples isolated from that region. On the other hand, in G. violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. This phylogenetic distribution resembles that of mobile genetic elements such as introns or insertion sequences (reviews: [T. thermophilus – three independent successful colonizations).Phylogenomic analyses show that putative nucleases grouped in the COG4636+ family are exceptionally abundant in genomes of certain Cyanobacteria, but absent in others. They are typically abundant in the sequenced genomes of freshwater species, but scarce in the genomes of marine species, with the exception of reviews: ,42) and Anabaena PCC 7120 [We hypothesize that the mechanism by which these putative nucleases induce their proliferation in a genome is similar to that displayed by homing nucleases and restriction enzymes , namely PCC 7120 , and theAnabaena sp. PCC 7120 [Microcystis sp. [C. watsonii WH 8501). Summarizing, it is plausible that members of COG4636+ fulfill an important role in the genome dynamics of Cyanobacteria and other species they colonize. We hope that our predictive study will facilitate experimental determination of the molecular and cellular function of members of this intriguing protein family.One possibility is that COG4636+ members serve as a part of the restriction barrier, similarly to the unrelated NucA family of extracellular nucleases found in Cyanobacteria, e.g. PCC 7120  and Micrstis sp. . They costis sp. . From thstis sp. . ConversSearches of the non-redundant (nr) database were carried out at the NCBI using PSI-BLAST  with def. The "coded_by" field from each GenPept file was used to identify the corresponding DNA sequence, which were also downloaded into the database. The sequence in the range of 3000 bp upstream or downstream from the region encoding a COG4636+ member were scanned for the presence of annotated Open Reading Frames (ORFs). Initially, the functional categorization of these ORFs was carried out based on the automatic assignment into the PFAM and COG families. In the absence of any recurrent function, the annotations of all ORFs were carefully re-analyzed visually and in uncertain cases, additional searches against the CDD database were carried out [The Eutils module from the Biopython package was used as an interface to access remotely the NCBI databases . The Genried out . The dis[Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway at . Seconda, PROFsec, PROF [6, SABLE [, JNET [6, JUFO [6, and SAM. Solvent and JPRE. The fol, SAM-T02, 3DPSSM , BIOINBG, FUGUE [, mGENTHR, and SPA. Fold-re.Fold-recognition alignments to the structures of selected templates were used as a starting point for homology modeling using the "FRankenstein's Monster" approach , comprisThermus thermophilus Hb8 To be Published). Our analysis of the Tt1808 structure and its comparison with the model of all3650 confirms our predictions. Tt1808 does indeed exhibit the PD-(D/E)xK fold: the DALI [After submission of this manuscript, a crystal structure of one of the COG4636+ members was released in the Protein Data Bank ; bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; ORF, product of an open reading frame,MF carried out all sequence analyses and structure predictions using fold-recognition methods and ROSETTA. JMB built the homology model, analyzed spatial vs. sequential conservation of the putative active site, and wrote the manuscript. Both authors have read and accepted the final version of the manuscript.The additional data file all3650.pdb contains the coordinates of the original all3650 model (obtained before the Tt1808 structure was published) in the PDB format.Click here for file


Plasmodium falciparum was based on the amplification of MSP1, MSP2 and GLURP genes. This method enabled confirmation of the same P. falciparum genotype in all three patients as well as in a fourth one (index case). The presence of nosocomial malaria was confirmed and it was concluded that the most likely source of transmission was through multi-dose preparations of heparin applied to heparin locks.Three cases of nosocomial malaria are reported from patients of the Internal Medicine Ward of a tertiary University teaching hospital in Medellin, Colombia. Epidemiological research, based on entomological captures, medical records review and interviews of nursery staff about patient care practices potentially involving contact with blood, were carried out. Molecular characterization of  Plasmodium falciparum-infected patient.The city of Medellin is located in the north-western region of Colombia and, although malaria is endemic in the country, absence of anophelines within the city makes vector transmission of the infection impossible. However, this city, located within the Andes Mountains (6°13'N, 75°36'W) at 1,588 m above sea level (a.s.l.), is surrounded by highly endemic regions and imported cases are often referred to the University hospital for treatment. Here, three cases of nosocomial malaria, which occurred in hospitalized patients in the Internal Medicine Ward of Hospital A, are described. These patients were being treated for diverse conditions and they shared the hospital facilities during different periods. One of these patients was in the Emergency Ward at the same time as a P. falciparum genomic DNA was amplified using specific primers for polymorphic streches of Merozoite Surface Antigen-1 (MSP-1), Merozoite Surface Antigen-2 (MSP-2) and Glutamate Rich Protein (GLURP), and the profile observed among the different isolates was compared.In order to confirm an association between all the cases, P. falciparum asexual forms (>100,000 per μl). A post-mortem confirmed as cause of death complicated malaria. Dengue and yellow fever IgM antibodies were absent. The patient remained in the emergency ward for 18 hours prior to his death.A 23-year-old male woodcutter, resident in Vigia del Fuerte (6.35N, 76.53W), was referred to the hospital on 11 April 2001, diagnosed with yellow fever. Vigia del Fuerte is a highly endemic malaria region located in the Atrato River basin with a mean Annual Parasite Index (API) of 72.6 during the past five years. The patient presented 15 days with fever, headache, chills, jaundice, arthralgia, melena, dark urine and bilious vomiting. He was given a heparin lock for administration of I.V. treatment. The patient deteriorated throughout the night and died before thick smear results were available. Peripheral blood thin and thick smears confirmed the presence of th April 2001, and remained there for 24 hours before being transferred to the Internal Medicine Ward with diagnosis of systemic lupus erythematosus (SLE). In addition to symptoms and signs of SLE, he presented fever on 23 April, and received treatment with fenitoin, dipirone, ampicillin, ranitidine, ceftriazone, methilprednisolone, oxacillin, amikacin and enoxaparine (via an heparin lock). As part of the treatment for his condition, he was administered chloroquine 150 mg daily from 24th April 2001. He was discharged on 19th May 2001. Then, on 6th June 2001, he was hospitalized with a diagnosis of a febrile syndrome. P. falciparum malaria was confirmed on 20th June 2001 after observation of the parasite in a blood cell count. A thick smear revealed the presence of 14,440 asexual forms per μl in peripheral blood. The patient was administered quinine and sulphadoxine-pyrimethamine with good treatment response.A 19 year-old male, unemployed, resident in Angelopolis (Antioquia, 6.06°N 75.42°W), a non-malarious area, located 1,900 m a.s.l., attended the emergency service of the hospital on 10th May with diagnosis of status asthmaticus. He was administered aminophyllin, hydrocortisone, ranitidine, enoxaparine (via a heparin lock) and was discharged on 15th May 2001. He returned to the hospital on 5th June 2001 complaining of fever, chills and adynamia and was admitted with diagnosis of toxic hepatitis due to the presence of dark urine, jaundice and hepatomegaly. On 6th June 2001, diagnosis of complicated malaria was confirmed by observation of the parasite in a blood cell count. Later, the presence of 87,500 trophozoites of P. falciparum per μl was confirmed by a thick smear. He received treatment with quinine, sulphadoxine-pyrimethamine and was discharged on June 23rd 2001A 46 year-old male, unemployed, resident of Santa Barbara (Antioquia, 5.57°N 75.91°W), a non-malarious area located 1,800 m a.s.l., was hospitalized in the Internal Medicine Ward on 8th May 2001 with superior vena cava syndrome (100% obstruction) and was applied a heparin lock for administration of I.V treatment. The patient was diagnosed with a mediastinal mass, compatible with lymphoma, and received treatment with dexametasone (I.V.) and radiotherapy. On June 9th 2001, he evidenced fever and on 9th June 2001, he was diagnosed as complicated malaria by a cell blood count. A thick smear confirmed the presence of 166,440 asexual forms of P. falciparum per μl. He received treatment with quinine, sulphadoxine-pyrimethamine with good treatment response. However, on 1st July 2001, he died as a consequence of multiple non-malaria related complications.A 41 year-old male, flower-grower, resident of El Carmen del Viboral (Antioquia, 6.09°N 75.34°W), a non-malarious area located 2,150 m a.s.l.., was admitted directly to the Internal Medicine Ward of the hospital on 7Anopheles was carried out by expert personnel from the Secretariat of Health, both within the ward and in the surrounding gardens, around the time of malaria diagnosis of patients 2 and 4. These confirmed the absence of the vector in the hospital area.Search for P. falciparum genomic DNA was extracted from whole blood collected onto filter paper or from paraffin embebbed brain tissue (for the index case). Paraffin was removed from post-morten material using xylene, followed by 2 washes with 100% ethanol. DNA was isolated by proteinase K digestion, followed by four rounds of phenol-chloroform extraction. Purified DNA was stored at -20°C until amplification.The Region 2 of MSP-1 and the central region of MSP-2 were amplified by a nested Polymerase Chain Reaction (PCR); the region RII of GLURP was amplified by a semi-nested PCR [P. falciparum infection by matching strains in all 4 patients  gel (2,5% for MSP-1 and 2% for MSP-2 and GLURP), stained with ethidium bromide and visualized under ultraviolet light. Size analysis of the amplified fragments revealed identical pattern distribution for all the examined markers assayed, confirming the presence of P. falciparum malaria infection in three patients without history of malaria or travel to malaria-endemic regions is described. The first of these patients (Patient 2) shared the Emergency Ward with a fatal case of falciparum malaria. The incubation period for this nosocomial infection was 12 days, however, since he was administered high doses of chloroquine, fever receded for about two weeks. Resistance of P. falciparum to chloroquine is highly prevalent in the Antioquia region [The presence of  Patient  shared tPatient 2 shared the Internal Medicine facilities with patients 3 and 4 at least for one week during May. Patient 3 evidenced an incubation period of 22 days, while in Patient 4 this was 26 days.Since patients 3 and 4 had no contact with the index case, further analysis of the incubation periods allowed us to conclude that transmission occurred from index case to patient 2 and the latter was the source of infection for patients 3 and 4. This means that at least 2 separate episodes of contamination were involved in transmission to the different patients (see figure Transmission of malaria via heparin locks has been reported previously by other authors, the source of contamination was a multidose heparin container . AlthougP. falciparum to confirm nosocomial transmission of malaria [P. falciparum observed in the Antioquia region [P. falciparum among unrelated parasites may be possible, but unlikely, since examination of these 3 different genes has been routinely used to discriminate unrelated parasites [Other researchers have reported on the use of molecular typing of  malaria . Genotypa region , the prearasites . MoreoveThese observations are of major relevance to sanitary and health authorities, since they confirm the importance of biosafety during minor procedures, such as application of heparin to peripheral locks. In addition, it highlights the need to rule-out malaria infections in all febrile patients sharing hospital facilities with malaria patients as well as in those not responding as expected to antimicrobial therapy.GL was involved in the review of clinical records, staff interviews and preparation of the manuscript. OJ coordinated the epidemiological and entomological researches. FL coordinated the microscopy diagnosis and following of the patients. AM participated on the epidemiological research. RE carried out the molecular analysis of the samples. BS, the Grupo Malaria leader, participated in the treatment and following of the patients. MA attended the patients, coordinated the clinical and molecular aspects of the research and prepared the manuscript.


An obituary commemorates the life and works of Jacov Tal. At the time of his passing, Jacov was the Head of Virology at Ben-Gurion University Medical School, Israel. In brief, four of us, who befriended Jacov in different capacities, write our remembrances. It is appropriate to recall the words of a colleague who on another occasion upon the passing of a giant in American science remarked, "Well, ghosts can't make men do anything!" Thus, the true reflection of a person is not what (s)he through his/her prestige, wealth, and position makes others do in life, but how (s)he is remembered by others in death. Jacov started as a young retrovirologist in J. M. Bishop and H. Varmus' laboratory; and it is fitting that he is remembered by friends in Retrovirology.A friend's passing elicits a set of emotions. Collecting and sharing remembrances are steps of closure in bidding farewell. Here, we honor and remember Jacov Tal Fig.  who pass"I remember Jacov as my actual mentor in my first year as a graduate student in the laboratory of Uri Littauer that Jacov had joined a year earlier. Jacov took similar care of other new comers interested in nucleic acids and molecular biology including Hiroshi Inouye, Inder Verma and Jacque Beckman, even to the point of neglecting his own work. Ibelieve that Jacov's selfless involvement in public affairs and later decision to become a virologist date back to a childhood experience; namely, only a ruling elite and diplomatic corpse in a country that Jacov's parents were stationed in, knew about the polio epidemic, considered then an ideological insult.Jacov Tal studied at the Hebrew University in Jerusalem where he obtained the Bachelor degree in Biochemistry and Microbiology in 1964 and the Master degree in Biochemistry in 1966. He continued his studies at the Weizmann Institute of Science where he obtained his PhD degree in Biochemistry in 1971. Jacov Tal did his postdoctoral training first with Hershel Raskas in Washington University where he investigated adenovirus gene expression and later with Harold Varmus and Michael Bishop at USCF where he studied the relation between the retroviral genome and the genome of its cellular host. After these formative years as a molecular virologist Jacov Tal joined the newly founded Ben Gurion University in the Israeli desert city Beer Sheva where he initiated and led the Medical School's Virology Department." (Gabriel Kaufmann)"Professor Jacov Tal will be best remembered by the scientific community for his extensive studies of the parvoviruses adeno-associated virus (AAV) and minute virus of mice (MVM). One of his significant achievements was the determination of parameters of site-specific integration of AAV, leading to development of potential vectors for gene therapy. Other important contributions were his insights into MVM's ability to kill cancerous cells, while leaving normal cells unaffected. The students of Professor Tal will also remember him for his dedication to training them to be rigorous and discerning scientists, and his concern for their well being. His colleagues will miss his sharp wit and analytic acumen." (Maureen Friedman)"Jacov Tal and I collaborated closely to his last days. Together, we found that during embryonic development MVM somehow senses a differentiation signal, and we suggested a relation between this observation and the MVM's anti tumor cell activity. I recall Jacov's 'freshman' enthusiasm in private, and his public posture as one who speaks up his mind without hesitation; standing up against any perceived injustice." (Claytus Davis)"I met Jacov towards the latter years of his career. In 1993, Jacov came to the US to do a sabbatical in Peter Chiang's laboratory. By and by, he drifted into my laboratory and actually spent the entire year working with me. Jacov by then, already a senior scientist for many years, not unexpectedly struggled heroically (and largely unsuccessfully) at the bench; and certainly it was not his bench-skills that impressed me. What did impress me was Jacov's common sense and his very human and generous attitudes. I recall an incident during Jacov's first week when he had not yet gotten to know all the members of my lab. At that time, there was a tall, curly-haired, darkly-handsome and academically gifted young man, working as a post-doc with me, who had graduated from Yale, obtained his MD degree from Duke, and received house staff and infectious diseases training from the University of Virginia. This person also has four siblings who are MDs. Jacov upon meeting this young man, whispered to me excitedly, 'Now here is a nice Jewish boy who is going to make the mother of a Jewish daughter very happy!' Surprise, surprise...that person turned out not to be Jewish, but a Catholic Lebanese-American of Arabic descent. Afterwards, a sheepish Jacov explained to me that it is very difficult to nearly impossible to tell between an Arab and a Jew in Israel; and as far as he was concerned, it made no difference whether Arab-American or Jewish-American. I was struck by his frankness and openness. In his typically thoughtful and 'dovish' ways, over the next many years, Jacov would email me from Israel his periodic 'roadmaps' for peace in the Middle East, accompanied by his incisive commentaries. I will deeply miss my friend's common sense advice and humor." (Kuan-Teh Jeang)


The objective is to present the design of randomised clinical trial (RCT) on the effectiveness of physical therapy added to general practitioners management compared to general practitioners management only in patients with an acute lumbosacral radicular syndrome .Patients in general practice diagnosed with an acute (less than 6 weeks) lumbosacral radicular syndrome and an age above 18 years are eligible for participation. The general practitioners treatment follows their clinical guideline. The physical therapy treatment will consist of patient education and exercise therapy. The primary outcome measure is patients reported global perceived effect. Secondary outcome measures are severity of complaints, functional status, health status, fear of movement, medical consumption, sickness absence, costs and treatment preference. The follow-up is 52 weeks.Treatment by general practitioners and physical therapists in this study will be transparent and not a complete "black box". The results of this trial will contribute to the decision of the general practitioner regarding referral to physical therapy in patients with an acute lumbosacral radicular syndrome. Publishing the design of the trial has several advantages. It may prevent publication bias . A studyThe lumbosacral radicular syndrome (LRS) is a complex of symptoms related to the lumbosacral nerve roots. The LRS is a disorder with radiating pain in the leg below the knee in one or more lumbar or sacral dermatomes, and can be accompanied by phenomena associated with nerve root tension or neurological deficits (i.e. sensory deficits in the leg, decreased muscle strength in the leg, decreased reflexes, urinary problems) -7. A proMost patients seeking medical care in the Netherlands will first visit a general practitioner (GP), who is regarded as the 'gatekeeper' of the health care system. The majority of health problems presented to GPs are treated by the GPs themselves and they are responsible for most referrals to (para) medical specialists. In 1996 the Dutch College of General Practitioners published their clinical guideline for LRS . There iPrimarily, treatment consists of adequate pain medication, giving information about the natural course of LRS, which in general is favourable, and stimulating to continue the normal daily activities of the patient. GPs in the Netherlands largely comply with the recommendations stated in the clinical guideline regarding management in patients with LRS . HoweverThe LRS trial aims to assess the effectiveness of PTs management added to GPs management compared to GPs management only in patients with acute LRS. We will use a multicentre, randomised clinical trial as study design. Figure Participating GPs in and around Rotterdam, the Netherlands, will invite patients with suspected acute LRS to participate in the trial. GPs will invite patients from May 2003 till November 2004 if they have radiating (pain) complaints in the leg below the knee; duration of the (pain)complaints is less than 6 weeks, the age is above 18 years and they present one of the following symptoms: more pain on coughing, sneezing or straining, decreased muscle strength in the leg, sensory deficits in the leg, decreased reflex activity in the leg or a positive straight leg raising test. Patients will receive a letter of information about the LRS trial from their GP. Patients' name and telephone number will be faxed to the research institute. Subsequently, a researcher (PL) will screen eligible patients by telephone and make an appointment to check inclusion and exclusion criteria, to complete the informed consent procedure and to perform the baseline measurement. Figure Randomisation will take place after baseline measurement by the research assistant. We use a concealed randomisation procedure using a computer generated randomisation list developed by an independent person. Patients' specific and unique trial number will be typed in a special developed database  and the random allocation will appear on screen. In order to prevent unequal treatment group sizes, block randomisation will be used with blocks of 10 patients . This meFor obvious reasons GPs and PTs are not blinded for treatment allocation. But they are not involved with treatment effect measurements. The patients cannot be blinded because of the ethical reasons as stated by the Medical Ethical Committee. The researcher is involved in the statistical analysis, but the analysis and interpretation of the findings will be audited and verified by an independent and not involved statistician. In this trial the primary outcome measurement and most of the secondary outcome measurements will be scored by the patients. Studies from Ostelo et al  and SchoAll patients will be treated by the GP according to their clinical guideline  are not allowed in the PT treatment. This PT treatment protocol was accomplished in a consensus meeting with participating PTs. The PT will report what kind of information/ advice and what type of exercise the patient receives in each session. Both GP and PT intervention will be restricted to a maximum of 9 treatments/ consultations in the first 6 weeks after randomisation.In the Netherlands, PTs are mainly taught the biomechanic model . This moIt is possible that patients may suffer from a fear of movement because of pain . Good adThis trial attempts to enrol 182 patients with LRS, 91 patients in both treatment groups. This sample size is regarded sufficient to detect a difference of 20% (with a α of 0.05 and a power of 80%) in the primary outcome (GPE) between the two treatment groups. A difference of 20% is considered to be clinically relevant .Figure The primary outcome measure is the Global Perceived Effect (GPE), measured on a 7 points scale ranging from 1 = completely recovered to 7 = vastly worsened. It is regarded a clinical relevant outcome measure and is regarded valid and responsive to measure the patients' perceived benefit -20.Pain severity of the leg and the back will be scored on a 11 points Visual Analogue Scale (VAS) ranging from 0 = no pain to 10 = unbearable pain. Reliability, validity and responsiveness of the VAS have been shown -23.The functional status will be measured with the Roland Morris Disability Questionnaire (RDQ) for sciatica . The scoHealth status will be measured by the 36-item short form (SF-36)  and the Fear of movement will be measured by the Tampa scale for kinesiophobia (TSK) ,39. The Costs will be calculated and include LRS related sickness absence from work, medical consumption , out-of-pocket expenses and paid help. Patients' treatment preference will be evaluated at baseline and at 4 follow-up measurements.Baseline comparability will be investigated by descriptive statistics to examine whether randomisation was successful. If necessary, adjustments for baseline variables will be performed in the analysis. Group differences and 95% confidence intervals will be calculated for all outcome measures. The statistical analysis will be performed according tot the intention-to-treat principle, analysing the patients in the treatment group to which they were randomly allocated. Between group differences will be calculated using the Student t-test for continuous variables or Chi-Square for dichotomous variables. In addition a per-protocol analysis will be performed, analysing only those patients with no serious protocol deviations. Comparing the results of the intention-to-treat and the per-protocol analysis will indicate if and to what extent protocol deviations might have biased the results. Multivariate regression analysis will be conducted to examine the influence of baseline variables on outcome.This article introduces a design of a RCT to evaluate the additional effectiveness of PTs management added to GPs management in patients with LRS. The study is designed in a way that GP and PT treatment is transparent (according a guideline and a consensus meeting) and not a complete "black box". The results of this trial will contribute to the decision of the GP regarding referral of patients with LRS to PT. The inclusion of patients will run until the end of the year 2004. The follow-up measurements will be completed in the end of the year 2005.The pre-publication history for this paper can be accessed here: