In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes. Despite overall similarities between genomes, initiation of DNA replication and speed of duplication in different parts of the genome differs amongst EBV strains Biochemical studies performed in higher eukaryotes have shown that DNA replication initiates at specific sites, or within initiation zones, suggesting the involvement of particular DNA sequences called replicators  genome in human cell lines. During latent replication, the EBV genome is maintained as a circular episome (∼175 kb in size), and the host cell provides both the replication machinery and the licensing apparatus that limit the genome's duplication to once per cell cycle . By utilizing a different procedure to stretch DNA molecules we were able to collect a large number of images of the EBV genome representative of different stages of duplication. This allowed us to determine how DNA replication initiates, progresses, and terminates throughout the EBV genome and to precisely measure the duplication time of specific portions of the EBV genome.These improvements allowed us to obtain important new results as well as to extend previous observations. Here we show that initiation events are not limited to a specific portion of the EBV genome (namely the initiation zone detected by 2D gel electrophoresis), but, unexpectedly, take place throughout the EBV genome. Multiple initiation events were also detected in individual EBV episomes. Hence, if the initiation sites do correspond to replicators, the latter must necessarily be highly redundant (present at a frequency of one or more every 20 kb).Our new results also indicate that, in these two EBV strains, both the frequency and the order of activation of the initiation sites vary considerably throughout the viral genome. This variation involves initiation sites such as oriP, the sequence of which is highly conserved in the two EBV strains . Hence, In order to study DNA replication, we used a procedure that we call single molecule analysis of replicated DNA (SMARD). This procedure labels the replicating DNA in a way that allows us to determine the position, the direction, and the density of the replication forks in a steady-state population of replicating molecules . This in turn allows us to determine how DNA replication initiates, progresses, and terminates throughout the genomic region analyzed. SMARD presents several advantages over procedures that utilize different labeling schemes and allows us to overcome most of the limiting factors that have traditionally affected studies of replication performed on DNA fibers see .In our procedure, an asynchronous population of cells is sequentially labeled with 5′-iodo-2′-deoxyuridine (IdU) and 5′-chloro-2′-deoxyuridine (CldU) . The lenIn the experiments described in this study, agarose-embedded total DNA was prepared from cells labeled with halogenated nucleotides. The circular EBV episomes were converted to linear molecules by digestion with a restriction endonuclease (PacI or SwaI). After pulsed field gel electrophoresis, the EBV DNA was recovered by agarase treatment  and streIn order to define precisely how the Raji EBV genome replicates, we recovered the images of 245 PacI-linearized EBV episomes that incorporated halogenated nucleotides along their entire length . The results of this experiment are shown in However, the progression of DNA replication throughout the EBV genome is better described by the replication profile of the molecules analyzed C. This pThe RRF contains the initiation sites most frequently utilized to begin the duplication of the Raji EBV episomes. More than 80% of the molecules analyzed were stained in red throughout intervals 1–7 and 31–35 , and a small portion of the RRL. We recovered 209 fully substituted 105-kb fragments . These molecules were analyzed as described for the PacI-linearized EBV episomes . We founThe movement of the replication forks throughout the EBV genome is described by the profiles of replication fork abundance  can also be calculated . More details are provided later in the text. However, it is important to note that the highest Sd values were detected within the central portion of the Raji RRF . This result can be explained in two ways. First, replication forks may move faster throughout the RRF. Alternatively, the RRF could contain a significant level of multiple initiation events. For the reasons mentioned below we favor the second possibility.We found that different portions of the EBV genome replicate at different speeds, with values that range from a minimum of 0.3 kb/min ; the third is discussed in the last section of Results (differences in duplication speed across segments of the RRF of different sizes).The first line of evidence is provided by the immunostaining pattern of the EBV molecules. Although discontinuities in the immunostaining make it difficult to detect multiple initiation events when the distance between converging forks is 5 kb or less, the replication patterns of some of the molecules are compatible with the presence of multiple initiation events within RRF (molecule 8 in (a–i). We also reexamined the hybridization patterns of ten different fragments analyzed in a previous study  are used at a different frequency in different EBV strains . HoweverThe most striking differences in the replication of the two EBV strains were found in the order of activation of the initiation sites. In Raji episomes, primary initiation events are restricted to an 80-kb region approximately corresponding to the RRF  could potentially explain the different location of the RRF in Raji and Mutu I episomes. However, the following considerations indicate that this is not the case. Initiation events occurring at oriP take place in the vicinity of the dyad symmetry (DS) element and produce replication forks that stall in the leftward orientation at the family of repeats (FR) element . InitiatSimilar conclusions apply to the RRF of the Raji EBV genome (∼80 kb in size), in which primary initiation events were detected at various locations . This is very similar to the range of speeds found in the Raji episomes (0.3–4.7 kb/min). Therefore, DNA replication appears to progress with similar kinetics in the two EBV strains.In the previous sections we have shown that the order of activation of the initiation sites in Raji and Mutu I EBV episomes is not conserved. Here, we wanted to determine whether the quantitative aspects of DNA replication were also different. SMARD was used to calculate Sd values in the two viral strains. Segments A′–G′ divide the Mutu I EBV genome in seven parts of identical size . Instead, the RRF contained some of the lowest Sd values (segment B′). This is probably due to the presence of strong pausing sites within this portion of the Mutu I EBV genome. Nevertheless, segment B′ replicates faster than the corresponding portion of the Raji EBV genome . This allowed us to measure the average speed of the replication forks in various portions of the EBV genomes in which these conditions are satisfied.Previous observations have suggested that in mammalian cells the speed of the replication forks can vary . Here, wH and segment J  replicates even faster , nd G see  are compSd of a genomic segment and its location within the RRFs. For example, the high Sd value for segment G′ indicates the presence of active initiation sites outside the Mutu I RRF. Therefore the factors that influence the order of activation of the initiation sites are at least partially distinct from the factors that control their frequency of activation.In this study, we determined how DNA replication initiates and progresses in EBV episomes latently replicating in two human Burkitt's lymphoma cell lines (Raji and Mutu I). Previous experiments had suggested that some variability in the utilization of oriP might exist among different EBV strains . Here, hA′ , segment C′ (molecule 34), segment D′ (molecule 8), segment F′ (molecules 5 and 15), and segment G′ (molecules 4 and 31). Similarly, in Raji episomes, replication bubbles were detected within every restriction fragment analyzed by 2D gel electrophoresis throughout a region of about 80 kb  is reduced by one unit in the Mutu I strain (not shown), while the Raji EBV genome contains two short deletions see A Polack. In prinThis study also revealed modifications in the pausing of the replication forks in the oriP region. Accumulation of replication forks is clearly present in both EBV strains within this genomic region. However, only 25% of replicating Mutu I episomes contain replication forks at this location see D, comparWe also found that some features of the episomal duplication do not vary. In both Raji and Mutu I cells, replication forks move freely throughout the EBV genome , several aspects of their duplication are not conserved between Raji and Mutu I. In mammalian cells, prereplication complexes are believed to form at the end of mitosis, when general transcription is shut off . HoweverOne of the questions we tried to answer is whether initiation of DNA replication is regulated at the level of individual initiation sites. Clues to a possible regulatory mechanism can be found in the replication profiles of the EBV episomes. The RRFs are localized in specific portions of the EBV genome that differ in the two EBV strains. These regions are tens of kilobases in size (about 80 kb in Raji episomes and 20 kb in Mutu I episomes) and encompass multiple initiation sites. The early activation of an individual initiation site could be sufficient to generate a RRF. However, our results have demonstrated that within each RRF various initiation sites have a similar tendency to be activated at the beginning of the duplication of each episome. Therefore, the order of activation of the initiation sites varies at the level of genomic regions rather than at the level of individual initiation sites and might reflect the presence of a particular chromatin organization.Recent findings have shown that histone acetylation can influence the timing of replication origin firing in yeast . In thisSd detected within the RRF compared to the RRL  encompass known common fragile sites in mammalian genomes , but none of the Raji EBV episomes showed forks paused in this orientation compare D. TherefVarious groups have suggested that different cellular proteins could participate in regulating the activity of oriP . HoweverIn this study we have shown that, while the basic features of DNA replication are conserved , the activity of the initiation sites (order and frequency of activation) varies significantly in different EBV strains and across different portions of the EBV genome. Importantly, using SMARD we are now beginning to detect similar modifications in the utilization of initiation sites across transcriptionally active chromosomal loci of the mouse genome (data not shown). Additional mechanisms could regulate DNA replication at transcriptionally silent loci, as suggested by the complete absence of initiation events throughout an approximately 450-kb portion of the mouse IgH locus in non–B cell lines . It is t5 and 8 × 105 cells/ml. The experiments presented in this manuscript were performed at approximately 5 × 105 Raji cells/ml, using two labeling periods (240 min each) with 25 μM IdU (first label) and 25 μM CldU (second label). IdU was added directly to the growing culture, followed by low-speed centrifugation of the cells at the end of the first labeling period and resuspension in warm medium containing CldU.Raji cells were grown in exponential phase (as described in 5 and 8 × 105 cells/ml) before the replicating DNA was labeled. The conditions used for growth and labeling were the same as those used for Raji cells, with the exception that the labeling periods were only 210 min each. This Mutu I cell line is characterized by the presence of a small fraction of cells in which EBV replicates lytically, producing molecules linearized at the terminal repeats. However, this did not affect our analysis of the latently replicating episomes because only the DNA molecules that were circular before the digestion with PacI were recovered from the agarose gels and analyzed by SMARD.Early passages of the Mutu I cells , type II , and type III . Although both Raji and Mutu I are human Burkitt's lymphoma cell lines, their transcription profiles are different. The Mutu I cell line used in this study was an early passage of a type I clone isolated in the Alan B. Rickinson laboratory. Raji cells, instead, have a type III-like transcription pattern and they also carry a deletion of the EBNA3C gene (Only ten EBV genes (out of about 100) can be expressed during latency  and rat anti-CldU  were used as primary antibodies , while Alexa Fluor 568-conjugated goat anti-mouse (Molecular Probes) and Alexa Fluor 488-conjugated goat anti-rat (Molecular Probes) were used as secondary antibodies. The immunostaining has almost no background. As described previously , the speStudies performed by fiber autoradiography have previously shown that the results obtained using DNA fibers (such as the average size of the replicons) are significantly affected by the length of the labeling period utilized to label the replicating DNA  of the restriction fragments analyzed by 2D gel electrophoresis were as follows: fragment a, DraI (79202)–EcoRI (82920); fragment b, EcoRI (82920)–DraI (88865); fragment c, DraI (88865)–EcoRI (91421); fragment d, EcoRI (93162)–EcoRI (95239); fragment e, EcoRI (95239)–DraI (103226); fragment f, EcoRV (100583)–EcoRV (116863); fragment g, HindIII (110942)–EcoRI (125316); fragment h, XbaI (161383)–EcoRI (1); and fragment I, EcoRV (126415)–EcoRI (137221). The probes used to detect the restriction fragments were as follows: pHindLHI for fragments a–c, pHindE for fragments d–e, pSalF for fragment f, the pHindC fragment XbaI (121146)–BglII (120341) for fragment g, the p107.5 fragment XhoI (169423)–XhoI (167487) for fragment h, and the pHindC fragment HpaI (131959)–XbaI (133151) for fragment i. The plasmids pHindLHI, pHindE, pSalF, pHindC, and p107.5 were kindly provided by John L. Yates. Two different preparations of replication intermediates were used to study the replication patterns of fragments a–c.The procedures for the enrichment of replication intermediates, 2D gel electrophoresis, and Southern analysis were essentially as described previously . Prepara


Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5′ ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3′ intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3′ ends of genes. Thus, recent intron dynamics do not support a model whereby 5′ intron positional bias is generated solely by 3′-biased intron loss. A comparative study of four fungal genomes reveals the patterns of intron gain and loss over several hundred million years of evolution Saccharomyces cerevisiae. It has been suggested that both the paucity and positional bias of introns in yeast may be due to intron loss through a mechanism of homologous recombination of spliced messages reverse-transcribed from the 3′ poly-adenylated tail , to roughly seven introns per gene on average for some basidiomycetes . Finally, fungi display a strong 5′ bias in intron positions, enabling us to investigate the processes underlying this phenomenon.Fungal genomes are in many ways ideal for exploring questions of intron evolution. The fundamental aspects of intron biology are shared between fungi and other eukaryotes, making fungi appropriate model organisms for intron study. They are gene dense with relatively simple gene structures compared with plants and animals, making gene prediction more accurate. Fungi also display a wide diversity of gene structures, ranging from far less than one intron per gene for SRY. Evidence for the occurrence of multiple independent intron losses has also been reported in studies such as those by Caenorhabditis elegans.In principle, a 5′ intron bias could arise through various combinations of intron gain and loss, and a complete understanding of intron positional bias requires an assessment of the contributions of both of these processes. A number of studies demonstrate the occurrence of intron gain and loss in individual genes or gene families. Fugu as an outgroup) and between mouse and rat (with human as an outgroup), and observed a sparseness of intron loss and complete absence of intron gain in these closely related organisms. On the other hand, More recently, a number of genome-wide studies of intron dynamics have been conducted. Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, and Neurospora crassa. Ortholog sets composed of one gene from each of the four genomes were identified as pairwise best bidirectional BLAST hits satisfying stringent overlap criteria. Orthologs in each set were subsequently aligned, and the locations of introns were marked. These intron positions  were subjected to rigorous alignment quality filtering to eliminate alignment and annotation errors  passed the quality filter. The complete set of aligned orthologs with passing and failing intron positions is provided in N. crassa (blue box), and the third is present only in A. nidulans, and, because of the ambiguity in inferring gain or loss in this case, is classified as “Other” (black-edged gray rectangle). Examining the region around the one raw gained intron in N. crassa at the nucleotide level  and M. grisea (M) but absent from F. graminearum (F) and N. crassa (N).where r is defined for a given organism as the fraction of introns in the parent that did not survive in any sibling. We define “sibling raw losses” for an organism as the number of introns that are present in the organism and at least one cousin but in no sibling. This quantity is then divided by the number of introns present in that organism and at least one cousin to give the sibling loss rate. For example, the sibling loss rate for F. graminearum is given byThe sibling loss rate, m, and subtract this quantity from the raw gains to obtain “inferred gains.” To calculate m we first count B0, the number of introns conserved in the organism and at least one sibling, but in no cousin. The quantities m and B0 are related through the variablen0, the number of introns present in an organism's parent but not in any cousin, by the equationsWe next correct the raw gains for each organism. Raw gains include some introns that were in fact lost in all but one lineage. We use the loss rates to calculate the expected number of these multiple losses, andThis follows from our assumption of independent gains and losses. Thus, we can calculate the expected number of multiple losses asn1, and the number not present in any cousin n0 (introduced above). To estimate the size of the set of parental introns present in at least one cousin, we first count the subset of these introns that are presently observable. An intron is in this set if it is present in at least one cousin and at least one sibling, or is present in at least one cousin and in the organism in question. We call this number of introns B1. By the assumption that gains and losses are independent, we haveWe use the loss rates to estimate the number of introns in each organism's parent. To do so, we estimate separately the number of parental introns present in at least one cousin Using this relation and the one in F. graminearum as an example, the true number of losses would also include some introns conserved in the patterns A, M, N, and MN. We calculate the number of inferred losses as ntotal q.Finally, we correct raw losses. Our definition of raw losses undercounts the true number by omitting those introns not conserved in at least one cousin and at least one sibling. Taking This method can be extended to any phylogenetic tree and to any organism with at least one cousin.M. grisea  than the average for other genes analyzed, resulting in unusually large numbers of introns in these genes  synthetase genes. These genes encode a widely conserved protein that catalyzes the production of PRPP, a precursor in the nucleotide biosynthesis pathway. In contrast to the majority of orthologs that displayed fewer than two gained introns, the set of PRPP synthetase genes displayed a total of 22 raw gains , we determined the frequency of phase 0, 1, and 2 introns in the set of all intron positions  based on parsimony. However, this does not accurately account for the possibility of multiple gain or loss events. We have developed a probabilistic model that allows for multiple events, providing a corrected estimate of the total number of gains and losses within the dataset. Our probabilistic method allows for a more accurate assessment of rates of gain and loss. In our dataset, allowing for multiple events results in only modest corrections to the rates estimated using parsimony.F. graminearum and M. grisea the number of introns gained was on par with the number lost and similar in magnitude to the number of introns gained in N. crassa.Our analysis demonstrates a significant role for intron gain over the past few hundred million years in the fungi analyzed. Previous analyses of specific gene families have provided evidence of specific instances of gained introns . HoweverThe mechanisms underlying intron gain are not known. We analyzed the set of predicted intron gains for possible signatures that might shed light on this process. No statistically significant bias was detected in the positions of gained introns along the coding sequence see , and no N. crassa and M. grisea suggests the possibility of independent increases in gain rate in the two species. Alternatively, the apparent high intron gain rate exhibited by this gene may have arisen just prior to the last common ancestor of N. crassa and M. grisea. Although it is premature to speculate about possible mechanisms, one possibility is that a factor or factors responsible for intron insertion evolved to associate with the PRPP synthetase gene locus, transcript, or message at this point, leading to a higher rate of intron insertion in this gene.Our data further indicate that intron gain can vary substantially between different gene families in a lineage-specific fashion. The PRPP synthetase gene is a particularly striking example, exhibiting significant increases in gained introns in two of the four lineages investigated. Moreover, the paucity of intron positions shared between Finally, our results do not support the mechanism commonly proposed to account for the 5′ positional bias of introns in intron-poor organisms . Contrarhttp://www.broad.mit.edu/annotation/fungi/fgi). The following datasets were used: A. nidulans , N. crassa , F. graminearum , and M. grisea .All sequences and annotations were taken from the Broad Institute Fungal Genome Initiative website . We repeated our analysis, requiring that each best bidirectional hit also be the only BLAST hit in each genome (spanning 60% the length of the longest protein). This protocol yielded only 1,178 ortholog sets, but gave qualitatively similar results for intron gains and losses .The proteins in each ortholog set were aligned using ClustalW 1.82 , and intRegions of low alignment quality were eliminated with a filter that required at least 30% identity and 50% similarity in a window of ten residues on each side of the intron position. These parameters were determined following manual classification of a set of 181 randomly selected intron positions as “clearly homologous,” “ambiguous/possibly homologous,” or “non-homologous” see B. Using To further exclude likely annotation and alignment errors, intron positions were also filtered by eliminating positions adjacent to gaps in the amino acid alignment and by eliminating positions with nearby introns but low evidence of homology in the intervening sequence. It is possible that some of these positions may in fact reflect intron gain or loss events that occurred simultaneously with coding sequence insertion or deletion. However, removing these positions did not significantly impact our results, as the number of positions adjacent to gaps was only about one-tenth of the number of positions that passed the quality filter, and the introns removed did not have an apparent positional bias see A. In the second case λ is given by the total number of raw gains observed in that organism multiplied by the length of that gene in amino acids and divided by the total number of amino acids in all genes in that organism . We reported the less significant of the two p-values in the results.We modeled the number of gains in a particular organism as a Poisson distribution under two different null hypotheses. One null hypothesis was that the gains were spread uniformly across all genes. The other was that the number of gains in each gene was proportional to the length of the gene. In the first case the Poisson parameter λ is given by the total number of raw gains observed in that organism divided by the total number of ortholog sets p-value for the significance of phase 0 bias was determined by the χ2 test with two degrees of freedom using equal expected phase frequencies. To detect sequence bias at intron insertion sites, we examined gained introns separately in F. graminearum,M. grisea, and N. crassa. For each gained intron, we extracted four bases upstream and downstream of orthologous sites in the other two sequences, consistent with For each of the three in-group lineages, the frequency of phase 0, 1, and 2 introns was determined for five different datasets: for each class of conservation , for all introns passing our filter, and for all introns in the ortholog set. The Figure S1Positional biases in intron gain, loss, and current distribution in three fungal genomes determined using orthologs predicted by a “bidirectional only hit” method. (A), (B), and (C) are roughly analogous to (D), (E), and (A), respectively, in (78 KB DOC).Click here for additional data file.Table S1http://genes.mit.edu/NielsenEtAl/.Also available at (4.3 MB ZIP).Click here for additional data file.


To evaluate the clinical outcome of primary endonasal laser assisted dacryocystorhinostomy (ENL-DCR) using the potassium-titanyl-phosphate laser.We retrospectively reviewed all primary ENL-DCRs performed within a period of twelve months by the same combined Ophthalmology and Otorhinolaringology team in Freeman Hospital, Newcastle upon Tyne, UK. The main outcome measure for success was resolution or significant improvement of epiphora. Details of surgery, intraoperative and postoperative complications, as well as pathology associated with failure were also studied. Patients were followed up for at least 12 months.A total of 41 consecutive ENL-DCRs on 29 patients  were analysed. All patients had bicanalicular silicone intubation for at least 4 months. The success rate at 12 months postoperatively was 78.1%. Pathology associated with failure included: intranasal pathology (12.2%), mucocele (7.3%), and systemic sarcoidosis (2.4%). No significant intra-operative complications were recorded.The ENL-DCR with potassium-titanyl-phosphate laser can be considered as a safe and efficient primary procedure for the treatment of nasolacrimal duct obstruction. External dacryocystorhinostomy (EXT-DCR) was first described by Toti ,2 in 190emostasis.Reported primary ENL-DCR success rates vary from 68% to 99% ,6,11, deThe records of all 47 ENL-DCR procedures that were performed in Freeman Hospital, Newcastle upon Tyne, United Kingdom, within a period of twelve months were retrospectively studied. The patients' main symptom was moderate to severe epiphora. Obstruction of the nasolacrimal system distal to the lacrimal sac was diagnosed with nasolacrimal syringing. Radiographic imaging was not part of the routine preoperative evaluation. Otorhinolaryngological preoperative assessment included full endoscopic examination of nasal cavities, looking for evidence of mucosal disease including polyps particularly in the middle meati. Exclusion criteria for ENL-DCR were: noticeable lower lid laxity, previous lacrimal surgery, suspicion of malignancy and previous radiation therapy. Each patient with primary nasolacrimal duct obstruction was counselled as to the advantages and disadvantages of EXT- DCR versus ENL-DCR, together with estimated success rates of the two different types of DCR. The operations were performed by the same ophthalmologist (C.N.) and ENT surgeon (S.C.). The majority of patients had surgery under local anaesthesia on an outpatient basis, except for one who opted for general anaesthesia. For local anaesthesia, Amethocaine drops were instilled in the conjunctival sac, followed by injection of Xylocaine 2% with 1:200000 Adrenaline in the medial third of both eyelids and transcaruncularly to the lacrimal sac. Cophenylcaine spray and intranasal cocaine 4% paste was applied to achieve anaesthesia and haemostasis. Dilatation of the lower punctum was performed and a 20G vitreoretinal probe was inserted in the lower canaliculus and advanced into the nasolacrimal sac. The light was directly visualised endonasally with a 0° rigid nasal endoscope, and the laser energy was delivered, with full laser precautions, via a KTP laser probe guided by the light. The nasal mucosa and lacrimal bone were ablated and the ostium was enlarged anteriorly as necessary with a microronguer. Bicanalicular O'Donoghue silicone tubes were inserted and secured with a Watzke sleeve. A course of topical Chloramphenicol drops was given for 1 week. Patients were examined 1 week postoperatively and then at 6 months for removal of tubes, or earlier, if discomfort was experienced. Mean follow up period was 16 months (range 12–24 months).Forty- seven consecutive primary ENL-DCR operations with lacrimal intubation were performed from March 2001 to February 2002 on 35 patients with primary acquired nasolacrimal duct obstruction. Five cases with incomplete follow up and one which had to be converted to EXT-DCR due to very thick lacrimal bone were excluded from the study. Forty-one ENL-DCR procedures on 29 patients  were included in the study. Mean age was 75 years . 12 patients (41.4%) underwent simultaneous bilateral surgery and 17 (58.6%) had unilateral ENL-DCR. In total, 19 right sided (46.3%) and 22 left sided (53.7%) procedures were recorded  and previous dacryocystitis in 4 (9.8%), while in another 3 cases (7.3%) these two conditions co-existed Table . Mild meDuring the operation the mean laser energy used was 400.2 joules . Serious intra-operative complications did not occur, although in some patients' records per-operative mild discomfort was documented.The removal of silicone tubes was scheduled at 6 months post-operatively, though in ten patients extubation was performed at 1–4 months due to discomfort. In two patients, mild nasal haemorrhage was noted on removal of tubes.The operation was defined as being successful if the patient was asymptomatic or if there was significant improvement of symptoms, not requiring any additional procedure. Success rate at 12 months postoperatively was 78.1%. No improvement of symptoms was noted in 8 patients (19.5%), while 1 patient reported worsening of epiphora Table . PatholoENL-DCR is a well established surgical technique with some advantages compared to the conventional EXT-DCR. These include limitation of tissue injury to the discrete fistula site, avoidance of a skin incision, excellent haemostasis, the ability to perform a lacrimal bypass operation on an outpatient basis, quicker patient rehabilitation, decreased overall health care expense and patients' preference ,13. MainENL-DCR avoiding the use of laser is a well described procedure. Several surgical instruments have been employed to remove the bone overlying the lacrimal sac, including drills, osteotomes, curettes and rongeurs. Weidenbecher reported resolution or improvement of symptoms in 95% of the patients. In anot2, Ho:Yag, Nd:Yag), have been employed in an attempt to achieve better bone ablation and haemostasis. Massaro et al [Various types of laser , where iThe optimum duration of stent retention following DCR is controversial. In published series it varies from 4 weeks to 6 months, though there is some evidence that prolonged silicone intubation may increase the incidence of DCR failure by inciting a granulomatous reaction at the internal ostium, with subsequent stenosis . All ourReported pre-operative risk factors for ENL-DCR failure include pre-existing sinus disease, mucocele, nasal septum deviation, connective tissue diseases such as sarcoidosis, previous EXT-DCR, other nasal surgery, nasal fracture, and thickened lacrimal bone . In our It has been proposed that the thermal energy produced by the laser may lead to scarring and subsequent blockage of the ostium . It has ENL-DCR performed under local anaesthesia is reported to be generally well tolerated by the patients. In our In our experience, the use of KTP laser in ENL-DCR under local anaesthesia with the adjunctive use of an osteotome is a safe and efficient technique, with good results. The particular advantages of this laser are its superior haemostatic properties and its diversity, which reduces the cost of the operation, as it is already employed in other procedures by the ENT surgeons. Significant complications are not common with this technique. In cases of failure, revision ENL-DCR or EXT- DCR can be performed. ENL-DCR with KTP laser is routinely performed under local anaesthesia, thus avoiding the risks of general anaesthesia usually required for EXT-DCR  The operENL-DCR using KTP laser appears to be an efficient technique, with low complication rate and it is well tolerated by the patients. It still needs refinement in order to achieve the higher success rate of the EXT- DCR, which remains the gold standard method for the treatment of nasolacrimal duct obstruction. At present, we believe that patients should be involved in the decision on the type of operation, after comprehensive consultation on the advantages and disadvantages of each technique.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression. A computational analysis of human RNA has identified 1,445 transcripts are edited mainly within non-coding Alu repeats, with the potential effect of regulating alternative splicing On the molecular level, the complexity of higher organisms is based on the number of different gene products available for structural, enzymatic, and regulatory functions. Posttranscriptional and/or posttranslational mechanisms have an important role in generating RNA and protein diversity . One posCaenorhabditis elegans resulted in transgene silencing, suggesting that the RNA editing and RNA interference (RNAi) pathways intersect  discrepancies in these cDNAs are the result of RNA editing involving intramolecular pairs of inverted Alu repeat sequences, repetitive elements that represent approximately 10% of the human genome and are concentrated in and around genes .We also characterize functional consequences of the observed editing events and the factors that determine editing levels in Alu repeats and their modification patterns. The prevalence of Alu elements in primate genes, together with our experimental and computational analysis, suggests that the vast majority of primary human gene transcripts (greater than 85% of RNAs with average structure) are subject to A-to-I RNA editing. We show how editing might influence the alternative splicing of exonized Alu elements and discuss the implications of this extensive modification of mRNAs bearing repetitive elements for the regulation of gene expression.A hallmark of an A-to-I RNA editing event is an AtoG transition when comparing genomic and cDNA sequences of the affected gene since inosine base-pairs with cytosine and therefore is replaced by guanosine during reverse transcription and PCR amplification. However, AtoG discrepancies between genomic and cDNA sequences can also be due to single-nucleotide polymorphisms (SNPs) or errors in databases. Therefore the search for edited sequences on a genome-wide basis is not feasible solely based on this single feature. However, in some cases of editing, not a single, but a cluster of AtoG discrepancies between genomic and cDNA sequences is evident within a stretch of a few hundred nucleotides . TherefoIn an initial screen for candidate genes, we used the Human Unidentified Gene-Encoded (HUGE) database of ca. 3,000 human cDNAs derived from the Kazusa cDNA sequencing project . SeveralC. elegans  containing pairs of Alu repeats as seen schematically in Many primary gene transcripts allow several energetically favorable foldback structures to be predicted for a given Alu that involve different combinations of Alu pairs. Do all these alternative Alu-pair foldback structures exist in vivo and are therefore subject to RNA editing? To address this question we examined the editing pattern of the G-protein-coupled receptor 81 . GPR81 contains four Alu elements, one sense and three antisense oriented, in the 3.6-kb pre-mRNA and was selected based on Alu repeat configuration and transcript length. If the alternative foldback structures depicted in These results suggest that Alu elements in human mRNAs are subject to RNA editing by ADARs because of foldback structures formed between two oppositely oriented Alus present within the same primary transcript.Exonic Alu repeat elements are predominantly located in the 5′- and 3′-UTRs of mRNAs, and as a result, most cases of Alu editing occur in noncoding regions. However, some editing events predict amino acid changes . Among tThe LUSTR1 cDNA codes for a G-protein-coupled seven-transmembrane receptor , with three AtoG discrepancies located within an alternatively spliced AluJo-derived exon that leads to the in-frame insertion of 29 amino acids between transmembrane regions V and VI of the protein see A. The exAnalyzing the RNA editing pattern of LUSTR1 pre-mRNA revealed additional intronic editing sites, one of which represents the splice acceptor adenosine (AG to IG) in intron 15  is generated in 57% of primary transcripts by RNA editing. This is predicted to give rise to alternatively spliced mRNA products represented by GenBank entry AF385431 see B. This iIt is intriguing that we find cases where editing in alternatively spliced Alu exons, or within adjacent splice sites, interferes with or counteracts exon formation of an Alu repeat. It suggests that RNA editing might be more generally involved in the regulation of Alu exonization. Recently, it has been shown that more than 5% of the alternatively spliced exons in the human genome are Alu derived . ExonizaFurthermore, RNA editing in Alus might be involved also in the generation of novel introns as seems to be the case in GPR81. Statistically, however, the exonization of intronic Alus would be much more frequent than the intronization of exonic Alus because of the abundance of Alus in introns.The results presented above show that clusters of AtoG mismatches in cDNA/gDNA sequence comparisons represent an effective way to identify authentic editing cases with a low rate of false positives. Since all clusters of AtoG discrepancies mapped to repeat elements, we wondered how prevalent the editing of Alu or other repeat elements is in the human transcriptome. Therefore, we devised a database search procedure to identify pairs of inverted repetitive elements in human mRNAs exhibiting AtoG transitions.Initially, a limited search was carried out for closely spaced (less than 2 kb) inverted pairs of human Alu, MIR, and L1 repeat elements that overlap with exonic sequences and for which an mRNA sequence can be found in GenBank entries. This search, involving about one-third of all repeat elements in the human genome, identified 71 mRNAs with exonic repetitive-element pairs . From those mRNAs, 27 displayed clusters of AtoG changes, all in Alu elements. Fourteen of these genes were chosen for experimental analysis, and all 14 proved to be subject to A-to-I RNA editing . Since tWe analyzed the total of 103,723 human mRNA sequences  are approximately three times more frequent than transversions is in line with results from previous studies analyzing gDNA sequences , there i2 test comparing the observed number of AtoG discrepancies with the expected number, based on the number of non-AtoG mismatches present in the same sequence. Elements with a χ2 higher than the critical value for α = 0.00001  were selected as “edited” and will be called so throughout the rest of the manuscript. Using this approach we found that out of those 17,406 mRNAs with one or more exonic Alu elements, 1,445 (8.0%) mRNAs are edited within one or more of the Alu sequences  between cDNA and gDNA. The probability that a cluster of several AtoG discrepancies is due to sequencing errors or SNPs  is negligible. Thus the number of clustered AtoG changes can be used to distinguish genuinely edited elements from elements with aberrant or non-editing-related base changes. For each Alu element with AtoG discrepancies, we computed the χIn order to validate our screening approach, we performed an identical analysis for GtoA, CtoT, and TtoC mismatches. Compared to the 1,925 AtoG-edited Alu elements in mRNA, we found 12 GtoA, 11 CtoT, and 11 TtoC cases of “editing.” These cases may represent false positives and thus set the error level of our screen to less than 0.6%.These results suggest that out of the 103,723 human mRNAs at least 1.4% are A-to-I edited within an exonic Alu element. Apart from Alu repeats, many more low- and high-frequency repeats exist in the human genome  and migh19.3 or 94.7% (85.0% with the 12.4 Alu/gene estimate). While this value is an approximation, assuming that all genes have similar structures, and does not take into account editing in other repeat elements, it does reflect the magnitude of repetitive-element editing.Most Alu repeats are located in introns, and it is there where the bulk of RNA editing is expected to occur. The average number of Alu repeats per gene is 12.4 estimated for Chromosomes 21 and 22 . This vaTo gain insight into the factors that determine which Alus are subject to RNA editing, and under what circumstances, the identified set of 1,925 high-confidence cases of editing in Alu elements  or its orientation in relation to the mRNA . No significant correlation of Alu editing was detected with any of these features (data not shown).The availability of such a large collection of A-to-I edited sequences resulting from this analysis allowed us to examine the modification pattern of edited Alu elements for potential editing hot spots or base preferences. To this end we first aligned all 141 edited Alu sequences (greater than 260 bp) in RNAs originating from Chromosome 1 and mapped the edited sites on their consensus sequence . InteresAs a result of the high CpG mutation rate, frequently the Alu foldback structure of the unedited RNA is predicted to carry A–C mismatches at these positions. Editing at these sites restores the CpG repeat (CA→CI) on the RNA level and converts the A–C mismatch to an I–C base pair. Energy calculations for several predicted Alu pairs show, surprisingly, that the stability of the foldback structure is not diminished by editing but often increased because of the high frequency of I/C pair formation (data not shown). It is therefore unlikely that in the case of Alu foldback structures, RNA editing serves to resolve RNA secondary structures that interfere with the processes of splicing or translation of these RNAs, as suggested previously . Two typcis preferences was carried out by extracting 14,774 pentanucleotide sequences with the edited adenosine as the middle base and estimating the frequency of each base at positions −2, −1, 1, and 2 relative to the editing site. To correct for Alu sequence bias we performed the same analysis for a randomly chosen adenosine for each edited adenosine in our sample. We then subtracted those frequencies to obtain unbiased editing preferences  are very low compared to editing levels seen in Alu repeats  generated by editing in Alu sequences. This estimation for Alu editing is in the range of one inosine in 17,000 nt (brain), one in 33,000 nt , to one inosine in 150,000 nt  as was experimentally determined by Bass and colleagues in the polyA-fraction of rat RNAs . Since tWhile a significant amount of editing occurs in mRNAs that contain repetitive elements in their exons, our results predict that the bulk of A-to-I editing takes place in intronic sequences missing from cDNA databases. This is suggested by the experimental results regarding the LUSTR, GPR81, p53, SIRT2, NFκB, and paraplegin genes, for which intronic data was available see , and 5A.It has been shown that hyperedited, inosine-containing RNAs are retained in the nucleus by a protein complex containing the inosine binding protein p54 . In viewC. elegans where inactivation of the editing machinery leads to transgene silencing . Total RNA isolation and reverse transcription have been described previously . Gene-spFor analysis of the pool of human cDNA sequences we developed a program named Procedures for Repetitive Element Foldback Analysis (PREFA). We used the set of cDNA sequences from the UCSC database (July 2003) comprising 103,723 sequences . The set of repetitive elements  and related information of the human genome  were discarded as misaligned or erroneous. From the repetitive elements showing at least a single AtoG change we selected those where mismatch distribution cannot be accounted for by SNPs and sequence errors using the following procedure:The overall expected ratio of AtoG discrepancies relative to the total number of mismatches was calculated from the whole sample, assuming the expected AtoG mismatches to be approximately equal to the average of the rest of the transitions:The expected probability for an AtoG mismatch at a single position in a given element was calculated from the total number of mismatches found in the element in cases where other mismatches were present (2) or from the whole sample where only AtoG mismatches were found (3): Here nAtoG and nOther is the total number of AtoG and non-AtoG mismatches found for this element:p for an AtoG mismatch to occur at any given position, the expected values for the number of AtoG were calculated:Given the probability 2 test was calculated for each element and those with a χ2 value exceeding the critical value (for α = 0.000001) were selected as edited, and these values correspond to approximately more than five AtoG changes in the absence of any other change in the approximately 300 bp of an Alu).A χFor each element in the high-confidence set the closest inverted element was identified among the elements present in the same gene boundaries. The distance separating the pair was calculated from the location of the first base of each element, according to the genomic sequence numbering and irrespective of their orientation. The divergence of each element was derived from the corresponding entry in the UCSC annotation database (ChrN_rmsk) representing mismatches per hundred bases. Tissue of origin of the RNAs was also derived from the UCSC mRNA annotation. For RNAs described to originate from multiple tissues, the corresponding RNAs were included in the count for each of those tissues. RNAs originating from a specific subregion of a tissue, such as subareas of the brain, were counted within the subregion but not in the whole-tissue set of RNAs.Alignment of the Chromosome 1-derived Alu sequences was performed with the MegAlign program of the DNASTAR  package (Lasergene) using the CLUSTAL algorithm . FurtherTable S1http://genome.ucsc.edu/cgi-bin/hgGateway and following the link to mRNA/Genomic alignment. We found that six cDNAs map on two chromosomes ; details are given for the most plausible assignment. We have observed that in the 43 cases that we experimentally analyzed, usually additional editing sites were identified when directly sequencing gene-specific PCR products.The database lists the GenBank accession numbers, gene names, gene product description, chromosome location, and type of Alu element and location within the mRNA sequence, the identity of the most likely pairing Alu elements within the same gene, and the distance in base pairs (bp) between the pairing Alus. The positions of all predicted editing sites within the individual sequences can be viewed by pasting the accession number into the USCS genome browser  at http:(276 KB XLS).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genetic sequences discussed in this paper are LUSTR (AB046844), KIAA0500 (AB007969), BTKI (AB037838), KIAA1497 (AB040930), and GPR81 (BC0067484).The GenBank ((http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) ID numbers for ADAR1, p53, SIRT2, NFκB, and SPG7 are 103, 7157, 22933, 4790, and 6687, respectively.The Entrez Gene (


WRKY proteins are newly identified transcription factors involved in many plant processes including plant responses to biotic and abiotic stresses. To date, genes encoding WRKY proteins have been identified only from plants. Comprehensive search for WRKY genes in non-plant organisms and phylogenetic analysis would provide invaluable information about the origin and expansion of the WRKY family.Giardia lamblia, a primitive eukaryote, Dictyostelium discoideum, a slime mold closely related to the lineage of animals and fungi, and the green alga Chlamydomonas reinhardtii, an early branching of plants. This ancestral WRKY gene seems to have duplicated many times during the evolution of plants, resulting in a large family in evolutionarily advanced flowering plants. In rice, the WRKY gene family consists of over 100 members. Analyses suggest that the C-terminal domain of the two-WRKY-domain encoding gene appears to be the ancestor of the single-WRKY-domain encoding genes, and that the WRKY domains may be phylogenetically classified into five groups. We propose a model to explain the WRKY family's origin in eukaryotes and expansion in plants.We searched all publicly available sequence data for WRKY genes. A single copy of the WRKY gene encoding two WRKY domains was identified from WRKY genes seem to have originated in early eukaryotes and greatly expanded in plants. The elucidation of the evolution and duplicative expansion of the WRKY genes should provide valuable information on their functions. Transcriptional control is a major mechanism whereby a cell or organism regulates its gene expression. Sequence-specific DNA-binding transcription regulators, one class of transcription factors , play anIpomoea batatas) [Arabidopsis thaliana [Avena fatua) [Dactylis glomerata) [Hordeum vulgare) [Nicotiana tabacum) [Matricaria chamomilla) [Oryza sativa) [Petroselinum crispum) [Retama raetam) [Saccharum hybrid cultivar) [Solanum dulcamara) [Solanum tuberosum) [Triticum aestivum) [Caenorhabditis elegans and Drosophila melanogaster) and yeast (Saccharomyces cerevisiae) [WRKY proteins are recently identified transcriptional regulators comprising a large gene family . The firbatatas) . Numerouthaliana ,6, wild a fatua) , orchardomerata) , barley vulgare) , tobaccotabacum) -13, chammomilla) , rice (O sativa) ,15, parscrispum) ,17, a de raetam) , sugarcaultivar) , bitterslcamara) , potato berosum) ,22, and estivum) . In addiestivum) . To datecis element, as a transcription activator [The WRKY family proteins contain one or two highly conserved WRKY domains characterized by the hallmark heptapeptide WRKYGQK and a zinc-finger structure distinct from other known zinc-finger motifs . To reguctivator .In plants, many WRKY proteins are involved in the defense against attack from pathogenic bacteria ,23,25-27Based on the number of WRKY domains and the pattern of the zinc-finger motif, Eulgem et al.  classifiGiardia lamblia, a primitive protozoan, and Dictyostelium discoideum, a slime mold. The data indicate an early origin of WRKY genes in eukaryota and tremendous gene amplifications in the plant lineage. We then cataloged the WRKY genes from the rice genome and compared them with Arabidopsis WRKY genes. We also identified WRKY genes from expressed sequence tags (ESTs) and EST-assembled sequence contigs from nineteen plant species. The result suggests that WRKY gene duplication events correlate with the increasing structural and functional complexities in land plants. We propose a model for the evolution of WRKY genes.Given the large family of WRKY genes with divergent regulatory functions in important plant processes, it would be desirable to understand the evolutionary origin and gene duplications leading to the multi-member WRKY family. The clarification of the phylogenetic relationships among WRKY genes in model plants will also assist understanding of the functions of these genes in important crops. We have comprehensively searched all currently available sequence data for the existence of WRKY genes outside the plant kingdom. Homologues of WRKY genes are found from two eukaryotic species: We searched for WRKY genes in two comprehensive datasets, GenBank's non-redundant (nr) and dbEST of all species. Together these datasets contain over 13 million sequence records from more than 110,000 organisms . HomologGiardia lamblia [Giardia is one of the most primitive organisms that represent the earliest branching among extant eukaryotes [Dictyostelium discoideum [D. discoideum belongs to the Mycetozoa, a lineage more closely related to animals and fungi than to green plants [Interestingly, two WRKY homologues were identified from non-plant eukaryotic species, and both have two WRKY domains [see Additional files  lamblia . The unikaryotes ,40. The scoideum . The genscoideum , indicatn plants ,43.Chlamydomonas reinhardtii is a unicellular green alga with a cell wall. It also has chloroplasts for photosynthesis. The evolutionary position of the species is located before the divergence of land plants [d plants ,45. The d plants . Since td plants  and 2. MRice, one of the most important crops for world agriculture, is recognized as a model monocot for the study of cereal crop genomes. A comprehensive catalog of rice WRKY genes would provide a basis for investigating the evolutionary patterns of the gene family and for transferring knowledge of the functions of these transcription factors from Arabidopsis to rice and from rice to other cereal crops.Giardia lamblia, and the conserved WRKY domain patterns can be found in A. thaliana [We identified the members of the WRKY family in rice (Japonica variety) from its published genome sequence . The WRKthaliana  as the rthaliana  . We then estimated the number of unique WRKY genes for 17 species using their Gene Indices, which are assembled EST sequence contigs with the minimal redundancy, provided by The Institute for Genomic Research (TIGR) . The anaable see . For theg plants .ESTs encoding WRKY proteins were identified in all the 19 species. Moreover, multiple WRKY genes are represented in the EST or contig sets for most plants including the moss and pine, with the most WRKY genes (109) from soybean see . AlthougThe abundance of WRKY ESTs in the total EST set is lower for pine, fern and moss than for flowering plants, as the percentage of WRKY ESTs in the total EST set for the three non-flowering plants is among the lowest see . The WRKThe identified WRKY genes were phylogenetically classified into five groups see . In six G. lamblia, the slime mold, the green alga, Arabidopsis and rice. The phylogenetic relationships were also inferred with the programs of the least squares and parsimony from PAUP 4.0 for the corresponding nucleotide sequences. We also did the same analysis for the rice dataset alone. The topology of trees obtained from these analyses is essentially the same, and the neighbor-joining tree is shown in Figure To examine the evolutionary relationships among the WRKY domains, we estimated the phylogeny by using the neighbor-joining program from PHYLIP 3.57 for the amino acid sequences of WRKY domains from In flowering plants, genes encoding WRKY domains appear to have been duplicated independently in monocots and dicots. For Group 3 domains, three subsets each of which consists of five or more members only from rice can be distinguished from the phylogram shown in Figure G. lamblia and the mycetozoan D. discoideum, but not in fungi and animals. G. lamblia is a primitive unicellular eukaryote diverged ~ 1,500 million years ago (mya) [WRKY genes seem to be an innovation in eukaryota after the divergence of eubacteria – archaea – eukaryota. In eukaryotes, the WRKY genes are present in the green plants as well as in the ancient eukaryote go (mya) . Originago (mya) ,3, the Wgo (mya) ,43, the G. lamblia, the slime mold and the green alga seems to be a Group 1 gene encoding two WRKY domains. The conservation of the C- and N-terminal domains suggests that they are derived from a single domain by domain duplication. Therefore we hypothesize that the earliest WRKY factor had one WRKY domain and the gene was innovated post the first appearance of eukaryotes ~ 2,500 mya [Giardia protist, ~ 1,500 mya. Second, our data and the previous results by Eulgem et al. [Based on the current data, we propose a model for the origin and evolution of the WRKY factor family Figure . First, ,500 mya  but priom et al.  suggest m et al. . Third, m et al.  and the m et al.  suggestsThe classification of the WRKY family in Arabidopsis by Eulgem et al.  is not cOur evolutionary analysis of WRKY transcription factors in this study may be important to the understanding of the overall mechanisms of biodiversity in the plant kingdom and the particular functions WRKY genes play in plant regulatory networks. First, the comparative analysis of WRKY factors in lower and higher plants indicates that the WRKY family expands as plants evolve from simpler, unicellular to more complex, multicellular forms. Since WRKY genes seem to play important regulatory roles in plants under abiotic and biotic stresses, and flowering plants which have the largest WRKY family are dominant over non-flowering plants in their distribution on the earth, WRKY genes might be essential for much of the enhanced adaptability of flowering plants to the environment. In comparison with pine, fern and moss, WRKY ESTs of flowering plants seem to be over-represented see , suggestOriginally believed to be plant-specific, WRKY transcription factor family has an early origin in eukaryotes and is also present in a slime mold which is more closely related to the lineage of fungi-animals than to plants. WRKY genes have been duplicated many times during evolution in plants, resulting in a large gene family for WRKY proteins in flowering plants. The elucidation of the evolutionary pathway of WRKY family and a new classification system we proposed based on phylogenetic analysis, conserved WRKY domain structures and intron positions should assist the functional characterization of WRKY genes.Oryza sativa spp. japonica)  and Arabidopsis  were downloaded from TIGR [Chlamydomonas reinhardtii, the genome sequence release 1.0 on 2/4/2003 was used [Giardia lamblia genome sequence released on 1/1/2003 [Dictyostelium discoideum were downloaded from TIGR [The annotated genome sequences of rice  were parsed and manually checked for the presence of the characteristic features of the WRKY domain.We searched 'nr' and dbEST datasets for WRKY genes in species outside the plant phyla. The dbEST dataset was also used to survey the expressed WRKY genes in plant species. We aligned the sequences in the datasets with WRKY-seed using BLAST programs . To deteG. lamblia, we searched their genome sequences with blastp and PSI-BLAST [-4 for blastp and PSI-BLAST and E < 0.1 for HMMER were manually compared to remove non-WRKY hits. We also used the same strategy to identify the set of WRKY genes from the Arabidopsis genome.To systemically catalog the WRKY genes for rice and SI-BLAST  using WRSI-BLAST . HMMER, SI-BLAST , is avaiSI-BLAST . The sea-4) were then subject to WRKY domain and gene predictions. The WRKY domain was predicted with the Pfam's DNA SEARCH [To identify WRKY genes from the green alga, we first BLASTed its genome sequence against the WRKY-seed. The significantly aligned sequences , > 97% for 300 ~ 399 nt, > 98% for 200 ~ 299 nt, > 99% for 100 ~ 199 nt, and = 100% for 50 ~ 99 nt. The alignment with < 50 nt was discarded.We also searched ESTs and EST-assembled contigs for the identified WRKY genes of rice, the green alga, The WRKY domain boundary was defined as by Eulgem et al. . The pepLW initiated the study. YZ and LW carried out the analyses, and YZ drafted the manuscript.Giardia lamblia, Dictyostelium discoideum and Chlamydomonas reinhadrtiiWRKY genes from Click here for fileMultiple alignments, domain classification and sequence conservation patterns of WRKY domains from rice (OsWRKY), Arabidopsis (AtWRKY), the green alga (ChrWRKY), the slime mold (DsWRKY) and Giardia lamblia (GlWRKY)Click here for fileIdentified members of the WRKY superfamily in the rice genomeClick here for fileSurvey of WRKY genes from ESTs or their assembled gene indices for 19 plants and the phylogenetic classification of the genesClick here for file


S. aureus, one of the main etiologic agents of brain abscess in humans, is a potent stimulus for proinflammatory mediator production. Recent evidence from our laboratory indicates that Toll-like receptor 2 plays a pivotal role in the recognition of S. aureus and its cell wall product peptidoglycan by glia, although other receptors also participate in the recognition event. This review will summarize the consequences of S. aureus on CNS glial activation and the resultant neuroinflammatory response in the experimental brain abscess model.Brain abscess represents a significant medical problem despite recent advances made in detection and therapy. Due to the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, the occurrence of brain abscess is likely to persist. Our laboratory has developed a mouse experimental brain abscess model allowing for the identification of key mediators in the CNS anti-bacterial immune response through the use of cytokine and chemokine knockout mice. Studies of primary microglia and astrocytes from neonatal mice have revealed that  The leading etiologic agents of brain abscess are the reported ,2. Brainreported . The mosAt the histological level, brain abscess is typified by a sequential series of pathological changes that have been elucidated using the experimental rodent models described in detail below -7. StagiS. aureus, one of the main etiologic agents of brain abscess in humans. The mouse brain abscess model accurately reflects the course of disease progression in humans, providing an excellent model system to study immunological pathways influencing abscess pathogenesis and the effects of therapeutic agents on disease outcome. We have successfully utilized this model to characterize inflammatory mediators induced in the brain immediately following S. aureus exposure [in vivo [S. aureus leads to the immediate and sustained expression of numerous proinflammatory cytokines and chemokines in the brain including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1α,β, macrophage inflammatory protein-2 (MIP-2/CXCL2), monocyte chemoattractant protein-1 (MCP-1/CCL2), MIP-1α/CCL3, MIP-1β/CCL4, and regulated upon activation T cell expressed and secreted (RANTES/CCL5) [Although case reports of brain abscess in humans are relatively numerous, studies describing the nature of the ensuing CNS and peripheral immune responses are rare. Therefore, our laboratory has developed a mouse experimental brain abscess model to elucidate the importance of host immune factors in disease pathogenesis ,7-9. Ourexposure  as well [in vivo . For exaS. aureus infection persists, effectively augmenting damage to surrounding normal brain parenchyma [S. aureus exposure [S. aureus infection was found to induce a chronic disruption of the blood-brain barrier, which correlated with the continued presence of peripheral immune cell infiltrates and glial activation [As mentioned earlier, lesion sites in both our experimental model and in human brain abscess are greatly exaggerated compared to the localized nature of bacterial growth, reminiscent of an over-active immune response. To account for the enlarged region of affected tissue involvement associated with brain abscesses compared to the relatively focal nature of the initial insult, we have proposed that proinflammatory mediator production following renchyma . Specifiexposure . Concomitivation . CollectBesides the potential detrimental roles cytokines may exert on surrounding normal brain parenchyma during the later stages of brain abscess, numerous proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 may have beneficial effects on the establishment of host anti-bacterial immune responses. These cytokines exert numerous functions within CNS tissues including modulation of blood-brain barrier integrity, induction of adhesion molecule expression on cerebral microvascular endothelial cells, and subsequent activation of resident glia and infiltrating peripheral immune cells -17. We rS. aureus-induced acute cerebritis was demonstrated by Lo et al. where transient neutrophil depletion resulted in enhanced pathology [S. aureus-specific lymphocytes [Neutrophils are potent bactericidal effector cells and represent the major peripheral cell infiltrate associated with developing brain abscesses ,9. Neutrathology . In addiathology ,8. The pathology  that participate in the initiation of the anti-bacterial adaptive immune response remain to be elucidated. Evidence to support the establishment of an adaptive immune response is provided by our recent findings that elopment . It is nS. aureus and its cell wall product peptidoglycan (PGN) serve as potent stimuli for proinflammatory mediator production in primary microglia [S. aureus infection, well before the significant accumulation of peripheral immune cell infiltrates [S. aureus induces IL-1β expression in neonatal rat microglia [Relevant to our experimental brain abscess model, recent studies from our laboratory have established that both intact icroglia ,10,25. Siltrates . Anothericroglia .S. aureus and PGN are capable of inducing microglial MHC class II [S. aureus to augment the expression of receptors that are important for antigen presentation suggests that the ability of microglia to present bacterial peptides to antigen-specific T cells may be greatly enhanced following an initial exposure to S. aureus. The effects of S. aureus and PGN on microglial CD40, CD80, CD86, and MHC class II expression may either be a direct consequence of bacterial stimulation or indirect via the autocrine action of cytokines produced by activated microglia.Microglia represent one of the main antigen presenting cells in the CNS ,27. To aclass II ,25, CD40class II . The abiS. aureus and PGN with significant proinflammatory mediator expression, and many of these same mediators are persistently elevated in brain abscess. Drawing on this relationship, we have proposed that chronic microglial activation may contribute, in part, to the excessive tissue damage characteristic of brain abscess. Therefore, attenuating chronic microglial activation subsequent to effective bacterial elimination in the brain may result in attenuation of the structural and functional damage associated with brain abscess. We have recently examined the efficacy of the cyclopentenone prostaglandin 15d-PGJ2 to modulate microglial responses to S. aureus [2 was found to be a selective and potent inhibitor of S. aureus-dependent microglial activation through its ability to significantly attenuate the expression of numerous proinflammatory cytokines and chemokines of the CC family including IL-1β, TNF-α, IL-12 p40, MCP-1, and MIP-1β. In addition, 15d-PGJ2 also selectively inhibited the S. aureus-dependent increase in microglial TLR2, CD14, MHC class II, and CD40 expression whereas it had no effect on the co-stimulatory molecules CD80 and CD86. The ability of 15d-PGJ2 to modulate the expression of these receptors may serve as a means to regulate microglial and T cell activation during gram-positive bacterial infections in the CNS. Preventing microglial activation by 15d-PGJ2 or related compounds may help to resolve inflammation earlier, resulting in reductions in brain abscess size and associated damage to surrounding normal brain parenchyma.Microglial activation is a hallmark of brain abscess ,5,9. The. aureus . 15d-PGJS. aureus and respond with robust production of numerous proinflammatory mediators. However, to date, the receptor repertoire responsible for bacterial recognition remains to be defined. In macrophages, numerous receptors have been implicated in bacterial phagocytosis and subsequent activation leading to proinflammatory mediator release including Toll-like receptors (TLR), scavenger receptors, and mannose receptors. The fact that microglia and macrophages share many functional and phenotypical characteristics supports the contention that these receptors may play an important role in microglial responses to bacteria.As detailed above, our laboratory has established that microglia are capable of recognizing S. aureus and PGN lead to significant increases in TLR2 mRNA and protein expression, which may enhance microglial sensitivity to bacteria during the course of experimental brain abscess development [S. aureus . These findings indicate that an alternative receptor(s) is involved in mediating responses to intact bacteria. Candidates include the mannose receptor and members of the scavenger receptor family.Toll-like receptors are a family of surface receptors expressed on cells of the innate immune system that allow for the recognition of conserved structural motifs on a wide array of pathogens (referred to as pathogen-associated molecular patterns) ,33. To delopment . Recent S. aureus [S. aureus and PGN differentially modulate the expression of several distinct scavenger receptors that may influence the nature and extent of phagocytosis . Scavenger receptors have been implicated in β-amyloid phagocytosis by microglia in the context of Alzheimer's disease, in part, by the finding that microglia associated with senile plaques express a high degree of scavenger receptor immunoreactivity [S. aureus phagocytosis by microglia remains to be established.Scavenger receptors encompass a broad range of molecules involved in receptor-mediated phagocytosis of select polyanionic acids such as lipoteichoic acid of . aureus . Althoug. aureus . In the . aureus . Prelimiactivity ,43. In aactivity -47. The S. aureus exposure, suggesting that the regulation of mannose receptor expression is conserved among diverse stimuli [S. aureus exposure remain to be defined. In addition to the receptors described above, there are additional candidates that may serve as receptors for S. aureus phagocytosis in microglia including complement receptor 3  and CD14, the latter of which we have shown to be expressed on microglia and significantly upregulated following activation with either S. aureus or PGN [Microglia have been shown to express functional mannose receptors that are responsible for the binding and phagocytosis of mannosylated and fucosylated ligands of bacteria ,49. Inte stimuli . Followi stimuli -52. In a stimuli . The funs or PGN ,25.Astrocytes play a pivotal role in the type and extent of CNS inflammatory responses. These cells likely play an important role in the initial recruitment and activation of peripheral immune cells into the CNS during neuroinflammation through the production of several cytokines and chemokines, such as IL-1, IL-6, IL-10, TNF-α, IFN-α/β, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), transforming growth factor-beta (TGF-β), RANTES, MCP-1, and IFN-γ-inducible protein-10 (IP-10/CXCL10) ,54.S. aureus and PGN and that they respond with vigorous proinflammatory cytokine and chemokine production [S. aureus-activated astrocytes are NO, TNF-α, IL-1β, MIP-2, MCP-1, MIP-1α, and MIP-1β. These proinflammatory chemokines may serve as signals for neutrophil (MIP-2), monocyte and lymphocyte  recruitment in vivo, whereas IL-1β and TNF-α likely alter blood-brain barrier permeability and induce the expression of critical adhesion molecules on CNS vascular endothelium required for immune cell extravasation into brain abscesses.Various studies have documented the ability of LPS to induce nitric oxide (NO), cytokine, and chemokine production in astrocytes ,56. In coduction . Among tS. aureus and PGN and in subsequent cytokine and chemokine expression by astrocytes [S. aureus. For example, TLR2 has been shown to form functional heterodimers with TLR1 and/or TLR6 [S. aureus and PGN is currently not known.Astrocytes have recently been shown to express TLR2 ,58, and trocytes . Interes/or TLR6 ,60, ther/or TLR6  and enha/or TLR6 -64. Rece/or TLR6 ,65. Alte/or TLR6 -68 may bS. aureus [S. aureus, indicating that bacterial uptake occurs via a phagocytic pathway and is not simply the result of productive infection by live organisms. Interestingly, TLR2 is not a major receptor for bacterial phagocytosis in astrocytes since both TLR2 KO and wild type astrocytes were equally capable of phagocytosing intact S. aureus organisms in vitro [S. aureus phagocytosis by astrocytes and the optimal conditions required for bacterial uptake are currently ongoing in our laboratory. Issues such as whether bacterial internalization is serum-dependent or requires other bacterial binding proteins must also be addressed.Although astrocytes have been shown to possess phagocytic activity in response to β-amyloid , apoptot. aureus . An actiin vitro . The recS. aureus experimental brain abscess model provides an excellent tool for deciphering the importance of various mediators in disease pathogenesis. Especially appealing is the ability to examine the role of specific factors using transgenic and knockout mice because, in our experience, all of the mouse strains examined with this model have qualitatively similar inflammatory profiles following bacterial challenge. In addition, the consequences of S. aureus infection do not appear to be influenced by gender, as the responses of female and male mice are similar- another advantage when performing studies with knockout or transgenic mice where animal numbers are often limiting.The incidence of brain abscess is expected to persist in the human population due to the ubiquitous nature of bacteria coupled with the recent emergence of antibiotic-resistant bacterial strains. Therefore, understanding the roles of both host anti-bacterial immune responses along with bacterial virulence factors may lead to the establishment of novel therapeutic treatments for brain abscess. The mouse S. aureus have been elucidated in terms of proinflammatory mediator expression and in general, have been found to be qualitatively similar to those observed following LPS exposure. Although studies with primary microglia and astrocytes from TLR2 KO mice reveal an important role for this receptor in mediating S. aureus-dependent activation, it is clear that additional receptors are also involved in glial responses to this bacterium. This functional redundancy is not surprising because these pathogens have the potential for devastating consequences in a tissue that has limited regenerative capacity such as the CNS.The responses of microglia and astrocytes to S. aureus bactericidal activity in vitro, suggesting that they may also participate in the initial containment of bacterial replication in the CNS. However, their bactericidal activity in vitro is not comparable to that of neutrophils or macrophages, suggesting that this activity may not be a major effector mechanism for microglia during acute infection. Third, activated microglia have the potential to influence the type and extent of anti-bacterial adaptive immune responses through their upregulation of MHC class II and co-stimulatory molecule expression. Finally, if glial activation persists in the context of ongoing inflammation, the continued release of proinflammatory mediators could damage surrounding normal brain parenchyma. Indeed, inappropriate glial activation has been implicated in several CNS diseases including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis as well as Alzheimer's disease. The continued use of transgenic and knockout mice for in vivo studies will facilitate our understanding of immune mechanisms contributing to brain abscess pathogenesis.The implications of glial cell activation in the context of brain abscess are likely several-fold. First, parenchymal microglia and astrocytes may be involved in the initial recruitment of professional bactericidal phagocytes into the CNS through their elaboration of chemokines and proinflammatory cytokines. Second, microglia exhibit BBB blood-brain barrierCCL CC chemokine ligandCD cluster of differentiationCSF cerebral spinal fluidCXCL CXC chemokine ligandCXCR CXC chemokine receptorGFAP glial fibrillary acidic proteinGM-CSF granulocyte-macrophage colony-stimulating factorIFN interferonIL interleukinIP-10 interferon-inducible protein-10KO knockoutLPS lipopolysaccharideM-CSF macrophage colony-stimulating factorMCP monocyte chemoattractant proteinMHC major histocompatability complexMIP macrophage inflammatory proteinNO nitric oxidePGN peptidoglycanRANTES regulated upon activation T cell expressed and secretedTGF transforming growth factorTNF tumor necrosis factorNone declared.