Pseudonocardia). Specifically, we examine conflict between the ants and their Pseudonocardia symbionts maintained to derive antibiotics against parasitic microfungi (Escovopsis) infecting the ants' fungus garden. Symbiont assays pairing isolates of Pseudonocardia spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with Acromyrmex leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of Pseudonocardia symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of Pseudonocardia within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria ( Although mutually beneficial associations appear congenial, selection favoring individual partners using resources to facilitate their own selfish interests is predicted to destabilize cooperation The regulation of conflict may be of particular importance within mutualistic associations, where partners are unrelated so cooperation cannot be explained by kin selection Although studies exploring mutualisms in a pair-wise framework have provided important insights into mutually beneficial interactions, the costs and benefits of cooperation and cheating within mutualisms can be significantly altered by the interactions that take place within the more complex communities in which they occur Pseudonocardia association.This study evaluates the expression of conflict within a broader symbiotic community context, as well as across symbiont lineages, using the fungus-growing ant–microbe symbiosis, a model system in mutualistic cooperation. This system provides a relatively unique opportunity for studying the complexity of symbiont conflict expression, because: i) it is a complex community involving at least three obligately associated mutualists and one specialized parasite Acromyrmex, competitive conflict between genetically distinct fungus clones is apparently prevented through behavioral recognition and removal of non-native fungi by the ants Fungus-growing ants , a monophyletic group of more than 210 New World ant species, are characterized by their obligate dependence on fungi for food Escovopsis  Escovopsis infections the ants employ specific behaviors, including grooming their fungal mutualist and weeding out heavily infect garden fragments Pseudonocardia), which produce antibiotics capable of inhibiting the growth of the specialized parasite PseudonocardiaEscovopsis and Pseudonocardia symbionts are phylogenetically diverse and have an ancient and coevolved association with fungus-growing ants and their fungal cultivar Pseudonocardia symbionts Pseudonocardia occur over evolutionary time and disrupt strict cocladogenesis .The ants and their fungal cultivar are only two members of a more complex symbiotic community. The fungal mutualist is parasitized by specialized and coevolved microfungi in the genus Pseudonocardia within individual ant colonies Pseudonocardia decouples the reproductive interests of the ants and the bacteria, and can result in the presence of more than a single genotype of Pseudonocardia within individual nests Pseudonocardia produces antibiotics to help defend the ants' fungus garden from the horizontally transmitted Escovopsis parasite Pseudonocardia strains suggests that the actinomycetous symbionts are involved in Red-Queen-like antagonistic co-evolution with the parasite Escovopsis , inferring that individual ant colonies would benefit from maintaining more than a single genotype of Pseudonocardia or from being able to easily acquire novel strains when confronted with resistant parasite strains. Replacement by, or mixing with, a foreign strain of Pseudonocardia would incur a direct cost to the resident Pseudonocardia strain, which would either be discarded or have to share both the energy allocated by the ants for growth and the transmission to the next generation on the cuticle of prospective ant queens Pseudonocardia strain would therefore benefit from preventing foreign strains from invading its niche, and one likely mechanism to do so would be the production of compounds that inhibit other bacteria. Such antagonistic interactions are common in free-living bacteria Although the ant-actinomycete mutualism is expected to be stabilized by the alignment of interests of specific symbiont pairs through vertical transmission and the rearing of single strains of Pseudonocardia symbionts originating from different fungus-growing ant colonies. We explore this through Petri dish bioassays pairing symbionts spanning the phylogeny of Pseudonocardia associated with fungus-growing ants as well as free-living (non-ant associated) actinomycetes. In addition, we conduct a second set of bioassay challenges specifically focusing on a phylogenetically narrow group of strains isolated from sympatric and allopatric species of the leaf-cutting ant genus Acromyrmex. We evaluate the factors contributing to the observed patterns of antagonistic interactions, including the species or genus of ant host for the symbiotic Pseudonocardia strains. Additionally, we examine the relationship between antagonism and relative genetic difference between pairs of strains inferred from patterns of DNA sequence divergence analyses.In this study we examine the presence, frequency, and intensity of antagonistic interactions between pairs of Pseudonocardia symbionts associated with fungus-growing ant species spanning most of the phylogenetic diversity of the ant-microbe mutualism (cross-phylogeny bioassay experiment). Fourteen Pseudonocardia isolates were obtained from eight species of fungus-growing ants, including: Apterostigma dentigerum , Cyphomyrmex costatus, C. muelleri, C. longiscapus , Trachymyrmex zeteki, T. cornetzi , Acromyrmex echinatior and A. octospinosus (Acromyrmex being one of the most derived attine ant genera (a leaf-cutter) http://nrrl.ncaur.usda.gov; see Streptomyces griseus, Pseudonocardia saturnae, P. thermophilia, and P. halophobica), and a Nocardiodes sp., culture isolated from a colony of A. dentigerum, were also included in this experiment. The second bioassay experiment examined possible antagonism between bacterial symbionts associated with sympatric and allopatric species of the leaf-cutting ant genus Acromyrmex (within-Acromyrmex bioassay experiment). Specifically, we used 12 Pseudonocardia symbionts obtained from ant colonies from two geographically distant locations: Panama and Argentina. Eight strains originated from two sympatric Panamanian ant species (A. echinatior and A. octospinosus) collected from 2001–2005, and four strains were isolated from three ant species collected in Argentina in 2003 . The species, colony code, and geographic origin for each ant colony from which Pseudonocardia symbionts were isolated are provided in We conducted two sets of bioassay experiments. The first examined interactions between strains of Pseudonocardia symbionts used in this study were obtained using a modified method from Cafaro and Currie PseudonocardiaPseudonocardia strain was obtained; this meant performing isolations on between one and ten ants per colony. In the ant genera Apterostigma and Cyphomyrmex, actinomycetes were isolated by placing individual ants, either whole or ground-up, in 750 µl autoclaved water in a 1.5 ml eppendorf tube. The tube was mixed for ∼30 seconds using a vortex, and 100–250 µl of the suspension was subsequently spread onto chitin-agar plates containing antifungals . To obtain Pseudonocardia symbionts from ants in the genera Trachymyrmex and Acromyrmex, bacteria were either isolated following the protocol describe above, or by aseptically scraping bacterial cells from the propleural plates with a scalpel Pseudonocardia colonies were transferred to Petri plates containing yeast malt extract agar (YMEA) with antifungals (concentrations as above) The Acromyrmex experiment, and three replicates were performed for each pairing. In the cross-phylogeny bioassay experiment the three replicates for each pairing were applied on three succeeding days, because the application of the second strain was labor intensive.In both experiments, biossay challenges involved co-culturing two strains of actinomycetes on YMEA with antifungals (concentrations as above) in 8.5 cm diameter Petri dishes. The first strain (hence forth referred to as the “resident” strain) was point inoculated in the center of the Petri dish and left to grow at room temperature for three weeks . The secPseudonocardia strains used in the experiments, and to determine if genetic distance between strains influences antagonistic reactions, we did partial sequencing of ribosomal 16S rDNA and nuclear Elongation Factor-Tu (EF-Tu) regions using standard PCR and sequencing techniques http://www.biotech.wisc.edu). The resulting sequences were corrected for mismatches using Sequencher 4.6 for Windows , and aligned using Clustal X 1.83 Acromyrmex bioassay, with R[A-C] = 0.5817, R[A-G] = 0.7977, R[A-T] = 0.5239, R[C-G] = 1.0821, R[C-T] = 1.8745, and R[G-T] = 1.0000, I = 0.7055, and G = 0.6718. Finally, the frequency of base pair differences between pairs of actinomycete strains (relative genetic distance) was determined in PAUP* 4.0b 10 To infer phylogenetic relationships between Due to the non-normality of the data, and the high sensitivity of 0-values in log-linear models, the statistical approach used to analyzing the bioassays was a generalized linear model design with an underlying gamma distribution. The exact values of the zoi measurements were inserted in the analyses, except that 0-values were imputed with 0.0001. All analyses were performed using SAS/STAT software, Version 9.1.3 of the SAS System for Microsoft Windows , and significance of terms in the models was determined through Likelihood Ratio Statistics for Type III ANOVAs.Acromyrmex bioassay), resident and intruder actinomycete strains nested within their respective ant origins, and the interaction between ant hosts of the two strains. The minimum diameter of the resident actinomycete at the time the zoi of the intruder actinomycete was scored, and the relative genetic distance between pairs of actinomycetes were inserted as continuous covariates. Due to the nested design of the models, they could not include the interaction term between the resident and the intruder actinomycete strains. An additional model was therefore built for each bioassay, including only resident actinomycete strain, intruder actinomycete strain, the resident*intruder interaction term, and the size of the resident actinomycete inserted as a continuous covariate.Two models were built for each of the bioassay experiments. A nested model contained ant host of resident and intruder actinomycetes , Mantel tests of matrix associations with 10,000 permutations were performed on reactions displayed in both bioassays using Arlequin 2.0 Pseudonocardia isolated from different colonies of attine ants spanning most of the diversity of the tribe and some non-ant associated free-living isolates, revealed a high proportion (208 out of 342) of pairings of actinomycetes with detectable levels of inhibition of the intruder strain by the resident strain  exhibiting inhibition  as compared to pairings in the within-Acromyrmex bioassay experiment (12.1% of pairings displayed antagonism), where both the number and strength of antagonistic interactions were substantially lower. This is further supported by the level of antagonism in the cross-phylogeny experiment being significantly affected by the host ant genus origin of the bacterial symbionts. However, this effect may be at least partially due to the significant effect of genetic distance between pairs of strains .Although our results indicate that  strains , 3A, becPseudonocardia symbionts, we found some strains that were highly potent , as well as particularly resistant strains . In both experiments the primary factors contributing to the variation in antagonism were i) the strength of inhibition by the residing bacterium, ii) the degree of susceptibility of the intruder, and iii) the combination of inhibitory capabilities and susceptibility  suggest that actinomycete-Escovopsis interactions are governed by dynamics of antagonistic coevolution. This predicts that antibiotics targeted at Escovopsis are subject to specialization over evolutionary time and that inhibition of other microorganisms in the association, such as the mutualistic fungus and the actinomycete bacteria, should be diminished. Third, in a separate study, some of the same Pseudonocardia strains were used in symbionts-pairing assays with Escovopsis to examine the specificity of activity of antifungal against diverse strains of the parasite . When comparing individual strains in these two studies, there was no correspondence in the inhibition profiles against other strains of Pseudonocardia and Escovopsis . Since the chemical composition of secretions from Pseudonocardia remains unknown, it is not currently possible to determine whether bacteriocins or antifungals are responsible for the actinomycete-actinomycete antagonism. However, even if antagonism is a bi-product of antibiotics targeting Escovopsis, activity against other strains of Pseudonocardia would still shape the dynamics of the association by precluding the rearing of bacterial strains exhibiting antagonistic interactions within individual ant colony.The observed antagonistic reactions resemble interactions in free-living actinomycetes Pseudonocardia, known to take place on both an ecological and an evolutionary time scale Acromyrmex experiment, supports relatively more potent reactions between actinomycetes originating from different host ant genera. This indicates that the potential for conflict is likely less pronounced if mixes occur between more closely related ants, while stronger reactions between actinomycetes reared by different ant genera could restrict switching of bacterial lineages across the attine tribe, contributing to the maintenance of host-mutualist cocladogenesis . While single-strain rearing within individual colonies nevertheless is apparent, the extent to which horizontal switches of the bacterial mutualists take place appears to be symptomatic of the role they play in the association. Even though ant-actinomycete phylogenetic comparisons reveal some degree of host-mutualist cocladogenesis, actinomycete switches appear to take place more frequently than ant-fungus exchanges, including across the broad groupings governing ant-fungus associations  that likely adjust the dynamics of cooperation and conflict within associations previously examined in a bipartite framework. Future studies exploring ant-bacteria and bacteria-cultivar conflict will inevitably provide additional insights into the expression and regulation of conflict within complex mutualistic associations.Conflict is an inherent major force shaping mutualistic interactions


An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review. The antisense RNA (asRNA) mechanism comprises all forms of sequence-specific mRNA recognition leading to reduced or altered expression of a certain transcript . NaturalFor the asRNA as well as other antisense compounds to be able to anneal to an mRNA or functional RNA, such as ribosomal RNA, the chosen RNA target region must be accessible. Determining target accessibility is the first step in designing antisense molecules, and both experimental and computational approaches have been applied. A brief and inexhaustive presentation will be given here.When antisense molecules anneal to a complementary mRNA, translation can be disrupted as a result of steric hindrance of either ribosome access or ribosomal read-through Fig. . This inAlthough the basic principle of the antisense inhibition mechanism is the same for all three kingdoms, the conditions affecting the efficiency of the particular antisense molecule are different in each system. Antisense gene silencing in bacterial systems will be the focus of this review. Application of antisense inhibition methods in eukaryotic systems, including siRNA and the RNA interference mechanism, is reviewed elsewhere -7. An RNIt is not possible to design antisense molecules without investigating the RNA target. The target is an unchangeable RNA molecule with a defined structure, which must be examined in order to detect appropriate local target sites suited for invasion of an antisense strand . ConsequThe only method that will give you the precise answer, as to which target region is best suited for antisense inhibition, is empirical screening. However, this may be a tedious task, and ways of limiting the search area have been suggested.in vitro screening assays using antisense oligodeoxyribonucleotides (ODNs). The ODN-mRNA duplexes are subjected to RNase H cleavage followed by gel electrophoresis ) and S. enterica serovar Typhimurium appeared more susceptible to peptide-PMO inhibition than wildtype E. coli, possibly due to different extents of peptide hydrolysis in the different strains. EPEC growth in tissue culture could also be inhibited by the peptide-PMOs, but their efficiency in reducing bacterial viability was orders of magnitude greater in tissue culture than in liquid culture. This may be due to differences in bacterial growth in the two types of culture.Using the 11-mer anti-to cells . In thisacpP PMO in a mouse model of E. coli peritonitis was the first demonstration of antisense DNA analogues inhibiting bacterial growth in an animal model [3RXB-peptide was shown to be about 50–100 times more potent than the single PMO in the mouse model [E. coli peritonitis, the anti-acpP peptide-PMO was found to reduce the number of colony-forming units (CFU) in the blood and promote survival at a potency at least 15 times greater than ampicillin. Though the results suggested high doses of the conjugate to be toxic, preliminary toxicology data on treatment with about 20 times higher doses of anti-acpP peptide-PMO showed no apparent toxicity [The inhibitory effect, though modest, of the anti-al model . The conse model . In the toxicity . Since ttoxicity ), furtheThe therapeutic use of antisense PMOs seem highly possible, and further pharmokinetic and toxicological studies will help improve this compound for antibacterial treatment.The apparent and alluring simplicity of antisense sequence inhibition of bacterial growth has turned out to be simply apparent, and the antisense approach now appears far more complicated and challenging than first thought. Parameters such as degradation resistance, binding efficiency, solubility, intracellular concentration and cellular delivery must be considered carefully, and further optimization on these parameters is still required despite the emergence of the first antisense-based drug almost a decade ago. Pharmaceutical formulations of antisense compounds with the same potency as antibiotics have not yet been developed, though they become increasingly more needed as bacterial resistance spread. The substantially modified PNAs and PMOs show promising results and may in time become the new medical weapons against the elusive pathogenic bacteria. In any case, we can not afford not to continue this research.O-methylribonucleotide2'-OMe 2'-asRNA antisense RNAcat chloramphenicol acetyltransferaseCFU colony-forming unitsCPP cell-penetrating peptidedsRNA double-stranded RNAEGS external guide sequenceE. coliEPEC enteropathogenic FDA The Food and Drug AdministrationHH hammerheadi.p. intraperitoneal50 concentration required for 50% inhibitionIC70/90 70%/90% lethal doseLDLNA locked nucleic acidsMALDI-TOF matrix-assisted laser desorption/ionization time of flightMIC minimal inhibitory concentrationMoMuLV moloney murine leukemia virusS. aureusMRSA methicillin-resistant ODN oligodeoxynucleotidePBP2a penicillin-binding protein 2aPBS primer binding sitePMO phosphorodiamidate morpholino oligomerPNA peptide nucleic acidPS-FANA phosphorothioate-linked 2'-deoxy-2'-fluoro-D-arabinonucleic acidPS-ODN phosphorothioate oligodeoxynucleotideptRNA precursor tRNARBS ribosome binding regionRNAi RNA interferencerRNA ribosomal RNAssDNA single-stranded DNAThe author(s) declare that they have no competing interests.LCVR formulated the content, performed the literature search, drafted the manuscript and created the illustrations. KKM contributed to revising the manuscript and created the illustration of chemical structures. HUSP contributed to revising the manuscript. All authors read and approved the final manuscript.


Two cases of gestational choriocarcinoma have been examined for the expression of HLA and trophoblast antigens using the indirect immunoperoxidase technique on frozen tissue sections. Approximately 40-70% of tumour cells express MHC Class I antigen as detected by a panel of antibodies to monomorphic, or framework, MHC Class I antigenic determinants. Evidence in one case suggests that most of these cells may not express the paternal, polymorphic HLA antigenic determinants but that a small subpopulation do carry the fully antigenically active molecule. These latter may give rise to patient anti-paternal HLA antibodies. Class II (HLA DR or DC) antigens are expressed by none, or very few, tumour cells.


The relationship between severity of preoperative radiographic findings and surgical outcomes following decompression for lumbar degenerative spinal canal stenosis is unclear. Our aim in this paper was to gain insight into this relationship. We determined pre-operative radiographic severity on MRI scans using strict methodological controls and correlated such severity with post-operative outcomes using prospectively collected data.Twenty-seven consecutive patients undergoing decompression for isolated degenerative spinal canal stenosis at L4-L5 were included. We measured cross-sectional area on MRI using the technique of Hamanishi. We categorized the severity of stenosis using Laurencin and Lipson's 'Stenosis Ratio'. We determined pre-operative status (prospectively) and post-operative outcomes using Weiner and Fraser's 'Neurogenic Claudication Outcome Score'. We determined patient satisfaction using standardized questionnaires. Each of these is a validated measure. Formal statistical evaluation was undertaken.No patients (0 of 14) with a greater than 50% reduction in cross-sectional area on pre-operative MRI had unsatisfactory outcomes. In contrast, outcomes for patients with less than or equal to 50% reduction in cross-sectional area had unsatifactory outcomes in 6 of 13 cases, with all but one negative outcome having a cross-sectional area reduction between 32% and 47%.The findings suggest that there appears to be a relationship between severity of stenosis and outcomes of decompressive surgery such that patients with a greater than 50% reduction in cross sectional area are more likely to have a successful outcome. The prognosis for a satisfactory outcome following lumbar decompressive surgery for degenerative spinal canal stenosis depends upon several factors such as comorbid diabetes, peripheral vascular disease, and cardiopulmonary insufficiency which are known to have a negative impact . AnotherThe question is an important one which deserves re-evaluation. Faced daily with patients who have severe stenosis, it is unclear whether such severity makes a difference in how they will respond to surgical intervention. On one hand, animal model research has demonstrated that prolonged neurologic compression results in irreversible damage including intraneural fibrosis at the root level and plastic changes in nociceptive transmission at the cord level -13. ThisAccordingly, the aim of this study was to better delineate the relationship between pre-operative radiographic severity and post-operative outcomes by paying strict attention to methodological controls to limit confounding factors outside of degree of compression.Twenty-seven consecutive patients undergoing surgery between January 1998 and January 2000 who satisfied the following criteria were included:1. They had isolated spinal canal stenosis at the L4-L5 level and underwent a single level decompression. The study was limited to single level cases to avoid quantification problems associated with multi-level stenosis with varying degrees of severity.2. The stenosis was degenerative; defined as isolated to one segment (L-4-L5) and compression most significantly due to disc bulge and hypertrophic/buckled ligamentum flavum (the classic 'napkin ring' configuration). This afforded the use an internal control to determine the 'stenosis ratio'[is ratio'.3. They had neurogenic claudication with no radicular component. The clinical syndromes and surgical outcomes appear to differ between stenosis patients with neurogenic claudication versus those with acute monoradiculopathies. The syndrome of neurogenic claudication is characterized in Table 4. They had MRI's with a minimum 1.5 Telsa and axial images obtained at right angles to the anatomic segment measured to facilitate accurate measurement of cross-sectional areas,5. They had pre-operatively filled out the Neurogenic Claudication Outcome Score(NCOS) and were available for a minimum twenty month post-op follow-up. The NCOS[ The NCOS has been6. They did not have comordities including diabetes, peripheral vascular disease, cardiopulmonary insufficiency, severe hip disease, or a degenerative spondylolisthesis.The NCOS questionnaire is shown in Figure We used the technique described by Hamanishi Figure  to detert-test to measure significance of stenosis ratio versus change in NCOS. We used the chi-square test to measure significance of stenosis ratio versus patient satisfaction. To test the correlation between the two independent readers of MRI's, we used the Pearson correlation coefficient.We used the Student All patients underwent a lumbar decompression at L4-L5 using a previously described technique  performeTwenty-seven patients were studied. Their demographic data, area measurements, stenosis ratios, and NCOS scores are shown in Tables no patients with greater than 50% reduction in cross-sectional area had unsatisfactory outcomes, whereas those with less than or equal to 50% reduction in cross-sectional area had unsatisfactory outcomes in 6 of 13 cases---suggesting a potential threshold effect. That is, for cases with less than or equal to 50% reduction in cross-sectional area, greater variability in outcomes (greater likelihood of unsatisfactory outcome) can be anticipated. Cases with unsatisfactory outcomes, however, were clustered between a 32% and 47% reduction in cross-sectional area and, accordinlgy, the relationship between severity of stenosis and outcome does not appear to be linear. There was no statistical difference between cases with satisfactory outcomes and those with unsatisfactory outcomes in regards to duration of symptoms but power may be insufficient.As can be seen from the data, For the fourteen patients with greater than 50% reduction in cross-sectional area, NCOS improved an average of 75 points (range 52 to 94 points) and 100% were satisfied with the outcome. Of the thirteen with less than or equal to 50% reduction in cross-sectional area, the NCOS improved an average of 49 points (range 16 to 85 points) and only 50% were satisfied with the outcome. These findings were statistically significant at p < 0.05. It is of note that the starting point of the two groups was quite similar; there does not appear to be a ceiling effect.Several animal models have demonstrated that rapid application of severe, prolonged compression of nerve roots may result in intraneural fibrosis which, despite decompressive intervention, may be irreversible -13,18. Tintrinsic to the roots may diminish their ability to physiologically adjust to compression. Based upon the findings in the current study, one might hypothesize that those patients who present with more severe spinal canal stenosis have roots which physiologically are better able to withstand progressive neurologic compression (hence they present later in the process) and, acordingly, these roots are better able to recover physiologically following decompression. By the same token, patients presenting with neurogenic claudication with milder amounts of stenosis may have roots which are physiologically more susceptible and such roots may be less likely to recover following decompression---the development of neurogenic claudication in these patients may relate more to poor physiologic reserves than the actual severity of compression.Degenerative spinal canal stenosis with neurogenic claudication, however, is physiologically distinct from these more acute types of neurologic compression. The slowly progressive compression appears to afford the roots time to physiologically adjust to the changing situation such that many patients with severe narrowing of the spinal canal remain asymptomatic. There is, however, a subgroup of patients with milder degrees of stenosis who clearly present with neurogenic claudication suggesting that factors This also appears to be the case in patients with comorbidities (who were excluded from the current study) such as significant diabetes , vascular disease, and cardiopulmonary insufficiency. Physiologic changes in baseline nerve root nutrition may inhibit recovery following decompression.In summary, we have found that patients with a greater than 50% reduction in cross-sectional area using the described technique of measurement appear to have a better (more predictably positive) outcome following decompressive surgery. Accordinlgy, those patients presenting with true neurogenic claudication but milder degrees of stenosis deserve greater attention. Checking EMG/NCT's to rule out neuropathy, checking non-invasive arterial studies to rule out vascular disease, re-reviewing hip x-rays and lateral flexion lumbar films to rule out degenerative joint disease or spondylolisthesis is appropriate. One might also consider attaining upright/weight-bearing MRI studies (as these become more readily available) which may provide better insight into the dynamic aspects of stenosis and may have prognostic importance. If these are negative, a realistic picture regarding potential outcomes of surgical intervention should be presented---acknowledging that the anticipated outcomes in this subgroup may be worse than those in whom more severe stenosis is present.None of the authors has any financial or non-financial competing interest in this study.NP and MW collected and statistically analyzed the data. BW conceived, designed, and wrote the paper. Each author read and approved the final manuscript.


Stanley Riddell and Frederick Appelbaum review progress in preventing graft-versus-host disease following allogeneic hematopoietic cell transplantation for malignancies or other life-threatening blood diseases. This year approximately 20,000 individuals will receive an allogeneic hematopoietic cell transplant (HCT) as treatment for a malignant, or life-threatening non-malignant, hematopoietic disease. The process of HCT generally begins with administration of a preparative regimen to eradicate the underlying disease and immunosuppress the patient in order to prevent rejection of the subsequently transfused hematopoietic stem cells. Following HCT, donor T cells transplanted with or developing from the hematopoietic stem cells react with cells of the human leukocyte antigen (HLA)-matched but genetically non-identical host, providing a beneficial graft-versus-tumor (GVT) response but also resulting in possibly life-threatening graft-versus-host disease (GVHD). The manifestations of GVHD vary over its course. Acute GVHD usually appears within several weeks of HCT and is characterized by a diffuse maculopapular rash, mucosal inflammation causing crampy abdominal pain and diarrhea, and elevated liver function tests . GVHD thIf no immunosuppression is given after allogeneic HCT, life-threatening or fatal GVHD inevitably develops. The first successful application of allogeneic HCT to treat human leukemia in the early 1970s was made possible by the use of methotrexate, administered early after transplantation as prophylaxis against GVHD ,3, and sAn elusive goal of research has been to find ways to prevent GVHD without dramatically increasing other transplant complications. Most clinical studies to date have focused on the use of alternative immunosuppressants or removal of T cells from the donor stem cell source. More intensive post-transplant immunosuppressive regimens and T cell depletion are both capable of dramatically reducing the incidence and severity of GVHD, but do so at the cost of an increased incidence of fatal post-transplant infections and tumor recurrence. Increased graft rejection may also occur if donor T cells are removed from the donor stem cell graft, because the reaction of these cells against residual host immune cells contributes to engraftment. More intensive preparative regimens can overcome this problem but are associated with increased toxicity. More potent post-transplant immunosuppressive regimens and T cell depletion suppress or remove immunocompetent cells responsible for protection against many pathogens as well as cell populations that contribute to the beneficial GVT response. Accordingly, in retrospective analyses and prospective randomized trials, neither more intensive post-transplant immunosuppression nor T cell depletion has been demonstrated to improve survival following allogeneic HCT . While iOne area of progress, as noted above, has been in HLA-typing technology and donor selection. Historically, HLA typing was conducted using serologic methods, but with the advent of the polymerase chain reaction assay in the 1980s, it became possible to perform molecular typing of donor and recipient. When patients previously transplanted from serologically matched donors were retrospectively analyzed using molecular typing, approximately 30% were found to be mismatched with the donor for one or more alleles, and such mismatching was shown to lead to more GVHD and poorer survival . Thus, mIL-10 promoter region as well as in the donor IL-10 receptor beta gene have each been found to be associated with a lower risk of GVHD and non-relapse mortality [The pathogenesis of GVHD involves the expansion and differentiation of donor T cells reacting in peripheral lymphoid tissues against host antigen-presenting cells that display disparate minor histocompatibility antigens. These antigen-presenting cells are activated as a consequence of tissue injury and the resulting release of proinflammatory cytokines . Polymorortality ,9. PolymFerrara et al., 2006 [l., 2006  This revWagner et al., 2005 [l., 2005  A multicAnderson et al., 2003 [l., 2003  Stem celDickinson et al., 2002 [l., 2002  This papBonnet et al., 1999 [+ minor histocompatibility antigen–specific T cells recognize and eliminate human acute myeloid leukemia stem cells.l., 1999  The firsIn an effort to provide a more global assessment of whether a donor is likely to induce GVHD, Baron et al. have examined gene expression profiles of CD4 and CD8 T cells from donors and report that it is possible to segregate donors into those likely to cause GVHD and those who are relatively safe . If thisN), antigen-experienced memory T cells, (TM), and regulatory T cells (TREG). Recent studies have begun to dissect the contribution of these individual T cell subsets to GVHD and have identified opportunities for more refined manipulation of the T cell content of stem cell grafts that may reduce GVHD without the severe T cell deficiency associated with complete depletion (N from allogeneic stem cell grafts abrogated GVHD in both CD4- and CD8-dependent multiple minor histocompatibility antigen–mismatched mouse models, and the remaining TM provided reconstitution of immunity to pathogens [N and TM can also be distinguished based on phenotype— TN are CD45RA+ and CD62L+, while TM are CD45RO+ and either CD62L+ or CD62L-, and emerging data suggest that alloreactivity for minor histocompatibility antigens is predominantly contained in the TN repertoire subset [M comprises less than 1% of the overall T cell receptor diversity and consists of large numbers of T cells specific for cytomegalovirus, Epstein Barr virus, and other pathogens that cause opportunistic infections in HCT recipients [CD4+CD25+Foxp3+ TREG cells suppress T cell responses in vitro and in vivo suggests another attractive approach to donor graft manipulation for preventing GVHD. The importance of TREG in GVHD is supported by murine studies showing that their depletion from stem cell grafts exacerbates GVHD and that the infusion of additional TREG at the time of HCT reduces lethal GVHD, apparently by limiting the initial activation of alloreactive T cells in lymph nodes [REG confer a lower risk of GVHD [REG that might be used to supplement stem cell grafts and abrogate GVHD [Stem cell grafts contain distinct functional and phenotypic subsets of T cells, including antigen-inexperienced naïve T cells (Tepletion . For exaathogens ,13. Humae subset . The humcipients . Thus, uph nodes ,17. Clin of GVHD , and effate GVHD .Although these new approaches to allogeneic HCT are likely to reduce the severity of GVHD, an important concern for patients undergoing HCT for a malignant disease is whether reducing GVHD might increase the risk of tumor recurrence. Like GVHD, GVT is the result of donor T cells reacting with disparate minor histocompatibility antigens, and elimination of GVHD would seem almost certain to diminish the GVT effect. Elucidation of the molecular structure, HLA restriction, and tissue expression of human minor histocompatibility antigens, and the identification of non-polymorphic leukemia-associated antigens that can be recognized by T lymphocytes offers the exciting prospect that targeted T cell therapy after HCT might selectively augment GVT activity . An incrN subsets, retaining TM subsets, and possibly supplementing TREG subsets are just being initiated. In the long term, perhaps the most exciting potential lies in the segregation of GVHD from the GVT reaction based on increased recognition of minor histocompatibility antigens exclusively expressed on normal versus malignant hematopoietic tissues. Whether these new advances will lead to the hoped-for clinical victories will become apparent in the next few years.The human graft-versus-host reaction continues to both fascinate and frustrate clinical investigation with its lack of predictability, possibly lethal toxicity, but potentially lifesaving anti-tumor effects. The ability to select optimal donors based on improved HLA-typing technologies and better understanding of non-HLA contributions to immune reactivity seems at hand, and should substantially reduce the risk of developing severe GVHD. Clinical studies of donor graft manipulation removing T


Methylation at certain human CpG rich sequences increases with age. The mechanisms underlying such age-related changes are unclear, but methylation may accumulate slowly in a clock-like manner from birth and record lifetime numbers of stem cell divisions. Alternatively, methylation may fluctuate in response to environmental stimuli. The relative stability of methylation patterns may be inferred through serial observations of the same colon.A 22 year-old male with attenuated familial adenomatous polyposis received neo-adjuvant chemotherapy and radiation prior to surgery for rectal adenocarcinoma. Colon crypt methylation patterns before and after neo-adjuvant therapy (62 days apart) were essentially identical with respect to percent methylation and diversity. Consistent with previous studies, methylation patterns recorded no evidence for enhanced colon crypt stem cell survival with a germline mutation (codon 215) proximal to the mutation cluster region of APC.The inability of neo-adjuvant therapy to significantly alter crypt methylation patterns suggests stem cells are relatively protected from transient environmental changes. Age-related methylation appears to primarily reflect epigenetic errors in stem cells that slowly accumulate in a clock-like manner from birth. Therefore, life-long human stem cell histories are potentially written within and may be read from somatic cell epigenomes. Recent studies reveal methylation of certain CpG rich sequences increases with age in normal human colon ,2. TheseRandom epigenetic errors potentially records cell histories because methylation exhibits somatic inheritance . DaughteAlthough crypt methylation patterns from multiple different aged colons are consistent with a clock-like accumulation of epigenetic errors, environmental factors may also alter methylation. Age-related methylation levels differ between individuals of the same age ,2,4, whiA 22 year-old male presented with anemia. Colonoscopy revealed eight hyperplastic to adenomatous polyps ranging in size from 0.6 to 3.0 cm scattered through the colon, and a four cm, locally invasive, ulcerative rectal adenocarcinoma five cm from the anus. The clinical impression was attenuated familial adenomatous polyposis (AFAP), with a germline heterozygous truncating APC mutation at codon 215 . The first sample for methylation analysis (Day 1) was a biopsy of normal appearing sigmoid colon.The patient received neo-adjuvant chemotherapy consisting of three, two week cycles of oxaliplatin and 5-fluorouracil, with the last cycle ending two weeks before colectomy. The patient exhibited Grade 3 toxicity with mucositis, stomatitis, diarrhea, and parasthesia. He also received radiation therapy to the pelvis , ending 19 days before colectomy. The specimens obtained for analysis were outside of the primary radiation field.Colectomy revealed approximately 50 scattered small hyperplastic to adenomatous polyps (0.1 to 0.3 cm), and a 0.7 cm ulcerative lesion at the site of the rectal adenocarcinoma containing a few residual neoplastic glands with superficial invasion of the muscularis propria. Lymph nodes were negative for metastatic carcinoma. The second specimen for analysis (Day 62) was taken from a normal appearing region of the sigmoid colon.2 patch of mucosa) specimens using an EDTA-containing solution as previously described [Individual crypts were isolated from the first (a colonoscopic biopsy) and last . The BGN (biglycan) tag is on the X-chromosome  and has nine CpG sites located just distal to the promoter and 512 ([9) possible patterns. These tags are not expressed in the colon and therefore any methylation is unlikely to confer selection [Crypt patterns (5' to 3' order of methylation) before and after neo-adjuvant therapy were complex and seemingly random, consistent with stochastic epigenetic errors Figure . The CSXelection . Crypt mMethylation tag values differed between crypts sampled before and after neo-adjuvant therapy, but these differences were not significant and did not follow a trend Figure . AverageIn addition to numbers of divisions since birth, methylation patterns Figure  may alsoThe ability to extract historical information from normal appearing tissue is limited. However, recent advances in molecular phylogeny illustrate that genomes automatically record ancestry and time . AlthougSequences comparisons depend on relatively constant error rates (a molecular clock hypothesis ) becauseA general problem with the use of sequences to reconstruct ancestry is the stochastic nature of most errors. It is difficult to prove stochastic sequence errors actually accumulate in a clock-like manner, and error rates may potentially fluctuate in response to environmental changes. Highly variable error rates would obscure ancestral information because many errors may accumulate in relatively short periods.Although it is difficult to test a molecular clock hypothesis for species evolution, it is possible to test a somatic epigenetic molecular clock through serial examinations of the same individual. This Case Report experimentally tests the hypothesis that age-related methylation predominantly reflect stem cell errors that start to slowly accumulate from birth. Consistent with this hypothesis, methylation patterns were not significantly different after 62 days despite intervening neo-adjuvant therapy. Neo-adjuvant chemotherapy targets rapidly dividing cells and normal colon is relatively unaffected, although diarrhea (Grade 3 toxicity) may and did occur in this patient. However, the therapy did not appear to alter stem cell methylation patterns, suggesting stem cells are protected from this type of therapy. Therefore, age-related methylation appeared to be relatively stable and primarily a function of the time or number of stem cell divisions since birth. Further serial examinations with more patients and longer intervals may provide additional evidence supporting or refuting this hypothesis.In summary, age-related methylation appears to slowly accumulate in a clock-like manner in stem cells from birth. The stochastic nature of somatic epigenetic errors requires an algorithm for interpretation, which essentially translates the sophisticated methods used to extract ancestry from species genomes to reconstruct somatic cell histories. The unseen fates of human crypt stem cells may be potentially read from anyone because methylation automatically records their autobiographies.The author(s) declare that they have no competing interests.Dr. Kim performed most of the experiments and helped write the manuscript. Dr. Beart provided the specimens and clinical information. Dr. Shibata analysed the data and wrote the manuscriptThe pre-publication history for this paper can be accessed here:


The 2006 Cold Spring Harbor Laboratory meeting on the Mechanisms and Models of Cancer was held August 16–20. The meeting featured several hundred presentations of many short talks (mostly selected from the abstracts) and posters, with the airing of a number of exciting new discoveries. We will focus this meeting review on models of cancer (primarily mouse models), highlighting recent advances in new mouse models that better recapitulate sporadic tumorigenesis, demonstrations of tumor addiction to tumor suppressor inactivation, new insight into senescence as a tumor barrier, improved understanding of the evolutionary paths of cancer development, and environmental/immunological influences on cancer. Traditional mouse models of cancers initiated by oncogene activation or tumor suppressor inactivation have yielded valuable insight into tumorigenesis, but have suffered from concerns that the mutation in either all cells of the mouse or in all cells of a particular tissue fails to properly recapitulate natural cancer initiation. In the last few years, new models have been developed that rely on Cre recombinase mediated deletion of floxed sequences to activate an oncogene or inactivate a tumor suppressor in only a subset of cells of a tissue at a defined point in time. Published studies have used Cre mediated activation of mutant K-Ras , with or without similar mutation in p53, to generate models of lung adenocarcinoma in mice -3. TheseD. Dankort (A. McMahon lab) showed that activation of oncogenic BRAFV600E via adenoviral delivery of the Cre recombinase in the mouse lung leads to frequent neoplastic lesions, albeit benign with proliferation indexes that decrease with time, suggesting that additional oncogenic hits are required for progression to full malignancy. A. Puzio (C. Abate-Shen lab) developed a mouse model for bladder carcinoma, histologically similar to the human disease, using adenoviral delivery of Cre to induce simultaneous p53 and PTEN deletion in the bladder in vivo. As will be discussed in the next section, conditional models for gene inactivation can also be used to determine the importance of maintaining tumor suppressor inactivation in an established tumor.Analogous to the Ras model, R. Wechsler-Reya showed that conditional deletion of Patched (resulting in activation of the Hedgehog pathway) in lineage-restricted granule neuron precursors (GNPs), while not sufficient for transformation of all cells, does lead to medulloblastomas in all mice. Thus, these cancers can arise in more lineage-restricted neuronal progenitors, although further studies will be needed to determine whether they actually do in humans. Unlike the Lox-Cre models discussed above, Patched should be deleted in most GNPs, and since GNPs without Patched are continuously replenished, there is ample opportunity for additional mutations that result in transformation.While stem cells are thought to represent the targets for tumorigenesis for at least some cancers, more committed progenitors may also be targets, particularly if early mutations confer self-renewal. Genetic analysis of cancers reveals a number of defined mutational events that appear specific for particular cancers, indicating that cancers follow certain evolutionary paths. However, the fact that a particular mutation is selected at a certain step in cancer development does not necessarily imply that the same mutation is required for tumor maintenance. The question of whether a particular mutation is important for maintaining a full-blown malignancy is important not only for understanding the biology of cancer, but also for devising rational treatment strategies. For several oncogenes the requirement for maintenance of an initiating mutation for cancer viability has been experimentally demonstrated, leading to the term "oncogene addiction" , and thuC. Martins (G. Evan lab)explored the importance of loss of p53 function for leukemia maintenance using tamoxifen (TAM)-inducible p53 (p53ERTAM) [A. Ventura (T. Jacks lab) employed a knock-in mouse experimental system, where endogenous p53 expression was prevented by Lox-flanked transcriptional termination sequences, in order to address the consequence of re-expressing p53 in spontaneous or irradiation induced malignancies that arise on a p53 null background. In this system, endogenous p53 expression can be restored by action of Cre recombinase conditionally activated by tamoxifen expression. Restoration of p53 expression led to dramatic inhibition of tumor progression and regression of the tumors. Interestingly, the effect of p53 restoration appeared to be tissue specific: p53 re-expression in lymphomas induced rapid apoptosis, while in carcinomas it induced permanent senescent-like arrest. In both cases, restoration of p53 function delivered a fatal blow to the tumor.S. Lowe's lab reached similar conclusions using conditional expression of an shRNA against p53. W. Xue used a liver progenitor cell transplant model developed by L. Zender in the Lowe lab [in vitro for a very long time, in vivo the tumors regressed very rapidly. Expression profiling revealed that the senescent tumor cells dramatically upregulated expression of pro-inflammatory leukocyte-attracting cytokines and immune receptors. Activation of p53 resulted in infiltration of the tumor mass with immune cells , and application of chemical inhibitors of macrophages and neutrophils substantially delayed tumor regression, revealing the importance of the innate immune system in removing senescent cells. Analogous to results from T. Jacks lab, restoring p53 expression by silencing the shRNA silencer in B-cell lymphomas induces apoptotic death of tumor cells (R. Dickins in Lowe lab). In summary, results from the Jacks, Lowe and Evan labs using different conditional models all validate p53 as an attractive therapeutic target.Two reports from Lowe lab  to exploG. Evan presented evidence that p53 stabilization due to the absence of Mdm2, even without numerous modifications of the p53 protein induced by stress, can rapidly induce senescence or apoptosis in an adult animal. Protein levels of p53 are held in control by its own target MDM2, which both squelches protein activity and down-regulates the protein levels via proteosomal degradation. Null mutation of MDM2 leads to early embryonic lethality unless rescued by p53 mutation [In a related story, mutation ,8. Evan L. Beverly (A. Capobianco lab) presented evidence that Notch (which is mutated in 50% of human T-ALL) inactivates p53 via repression of ARF-dependent MDM2 regulation. Inactivation of Notch in spontaneous tumors arising in mice that conditionally express Notch in the T-cell compartment leads to rapid tumor regression via p53 dependent apoptosis. Importantly, p53 can be reactivated using either irradiation or Nutlin even in the presence of sustained Notch signaling, resulting in apoptosis and lymphoma regression. Thus, it is proposed that inhibition of p53 activity by Notch signaling might be an important step in the evolution and maintenance of the resulting T cell malignancies. These findings provide a basis for the high responsiveness of tumors with aberrant Notch signaling to agents that re-activate p53, such as Nutlin or irradiation.It appears that in some cases "oncogene addiction" might be tightly linked with "suppressor inactivation addiction". K. Podsypanina (Varmus lab) demonstrated that in mammary tumors resulting from coexpression of activated K-Ras with either Myc or Wnt-1, deinduction of K-Ras (using tetracycline regulation) leads to rapid tumor regression. Thus, oncogene addiction extends to contexts where multiple defined initiating events are involved in tumorigenesis, which is perhaps not surprising given that oncogene addiction demonstrated in other models is on the background of additional, albeit undefined, oncogenic hits.While others have shown that persistent expression of Myc or Ras oncogenes is required for tumor maintenance , K. PodsJ. Jonkers reported on the development of conditional mouse models of breast cancer. By K14-Cre (expressed in epithelium) mediated deletion of p53, together with either E-cadherin or BRCA1, they demonstrated enhanced breast and skin carcinogenesis relative to p53 deletion alone. However, the mechanisms may be distinct, with E-cadherin loss abrogating detachment induced apoptosis (anoikis) and BRCA1 loss resulting in increased genomic instability. As the resulting adenocarcinomas resemble their human counterparts, these models should be useful for testing therapeutics and drug resistance mechanisms. Indeed, in their BRCA1/p53 mammary tumor model, the development of drug resistance appeared to derive from enhanced multidrug resistance pump activity.Finally, From its discovery, cellular senescence has been thought to be an important barrier for cancer development. Recent reports -14 have While permanent senescent growth arrest has unambiguous effects at the cellular level, it also leads to dramatic changes in gene expression. The classical marker for senescence has been the expression of senescence associated β-galactosidase activity, and more recently senescence has been associated with DNA damage foci . One impJudith Campisi's lab has previously shown that the secretory phenotype displayed by senescent fibroblasts can substantially promote tumorigenesis by stimulating the proliferation and tumorigenicity of pre-malignant cells [nt cells ,18. Her W. Xue and S. Lowe using a hepatocarcinoma model revealed that secretion of inflammatory cytokines mobilized the innate immune system to eliminate senescent tumor cells, thus perhaps preventing the emergence of mutants escaping the senescence. D. Peeper's lab reported an important role of cytokines secreted by senescent cells in maintaining the senescent phenotype. This group recently published that human naevi  display senescence-like growth arrest [V600E expressed in p16 mutant fibroblasts). Gene-expression arrays identified potent induction of interleukins . Importantly, they showed that expression of IL-6 and its receptor were required for maintenance of the senescent phenotype. C/EBPβ was identified as a common denominator responsible for induction of cytokines and maintenance of senescence. The use of bar-coded shRNA library screen for senescence-escape mutants allowed identification of additional genes implicated in maintenance of senescence, and thus potentially oncogenic events that can allow malignant transformation.Other reports however, implied anti-tumor functions of the secretion phenotype. As described above, studies by h arrest . In his M. Bissell overviewed critical roles that the extracellular environment has on the development of cancers, highlighting evidence that alteration of physiological communication of cancer cells with the environment is not only a required step in cancer evolution, but also that the disruption of this communication impacts other hallmarks of cancer, such as proliferation and genetic instability [The last few decades have seen dramatic progress in advancing a gene-centered understanding of molecular mechanisms of cancer development, including elucidation of pathways that regulate cellular proliferation, apoptosis and growth arrest. While better understanding of cell-intrinsic mechanisms and pathways is undoubtedly a step toward better clinical strategies, excessive "gene-centrism" initially gave insufficient attention to the fact that the evolution of cancer is shaped not only by cell-intrinsic events, but also to a large extent by the environment. tability . ImportaJ. DeGregori's lab has previously demonstrated that a background of impaired cellular proliferation engendered by drugs or genetic alterations substantially increases the selective advantage provided by the Bcr-Abl oncogenic translocation, resulting in dramatic increases in leukemogenesis [A. Marusyk from the DeGregori lab presented evidence supporting the validity of this hypothesis. Using a bone marrow transplantation model for leukemia initiation, Bcr-Abl was demonstrated to provide a much stronger selective advantage in the background of aged hematopoiesis compared to the background of young hematopoiesis, leading to a substantial increase in leukemias. Importantly, this age-dependence appeared largely non-cell autonomous, since introduction of young competitors efficiently reversed the competitive advantage conferred by Bcr-Abl expression. These findings reveal a novel factor contributing to age-related increase in cancer incidence, highlighting the importance of non-cell autonomous forces driving cancer evolution.Cancer progression can undoubtedly be regarded from the perspective of principles of Darwinian evolution. In order for an oncogenic mutation to be selected it should increase the fitness of the cell. But the fitness of a cell is determined by not only cell-intrinsic properties but also by the environment where the cell exists and by the relative fitness of other cells in the same population, since selection works by competition. ogenesis . Aging iB. Amati's lab suggest that tumors could select for the restoration of partial loss of function events that favored tumor initiation. C. Gorrini presented evidence that the acetyl-transferase Tip60 is important for Myc-induced double-stranded DNA damage response, as heterozygosity for Tip60 substantially attenuates activation of p53 and ATM. Tip60 heterozygosity accelerates the onset of Eμ-Myc driven lymphomagensis, suggesting the importance of DNA damage response in the suppression of tumorigenesis. Although Tip60 heterozygosity prevents activating phosphorylation of p53, lymphomas originating in a Tip60+/- background still retain selective pressure to lose p53. Interestingly, while Tip60 heterozygosity dramatically shortens the onset of lymphomas, the lymphoma cells duplicate the remaining Tip60 allele, restoring Tip60 expression to wildtype levels. These studies suggest an interesting scenario, whereby haploinsufficiency is important in the initial stages of tumor development, but at some point in cancer evolution there is selective pressure to restore gene function.While experiments described above showed that mutations (such as in p53) involved in the genesis of cancer can also be required for tumor maintenance, an interesting presentation from G. Evan has previously shown that activation of Myc on the background of suppressed apoptosis not only drives cell proliferation and expansion of β-cells, but also concomitantly induces features of advanced neoplasia such as increased invasiveness and vascularization [Evolution of cancer involves acquisition of multiple new traits, the so called "hallmarks of cancer" . Each harization . Here Evrization . This reJ. Allison presented a report of the successful mobilization of the immune system to target cancer cells by capitalizing on a better understanding of the complex regulatory pathways that coordinate T-cell immune responses. The immune system is extremely efficient in eliminating cells that express foreign or abnormal proteins. Extensive research efforts have been applied to harness the immune system to recognize and eliminate cancer cells, and although T-cells are capable of recognizing tumor-specific antigens, attempts to mobilize T-cells to kill tumor cells have thus far met with very limited success. Part of the problem appears to lie in the existence of intricate control mechanisms that negatively regulate T-cell proliferation in order to prevent autoimmune disease. One such control mechanism involves upregulation of CTLA-4 receptor in response to antigen receptor signaling [ignaling . CTLA-4 might possibly be happening but what really is happening (to paraphrase M. Bissel). In addition, the cancer field is showing steady progress towards a better understanding of the evolutionary forces that drive tumorigenesis, the intricate and inherent tumor suppressive mechanisms that prevent cancer, and the alterations that allow tumor cells to escape these controls. Building upon this knowledge will allow a more intelligent identification of targets for anti-cancer therapies and strategies for cancer prevention.The results presented above highlight substantial progress in developing and refining experimental models of cancers. The studies reviewed here emphasize the importance of the research shift towards animal modeling in order to understand not what The author(s) declare that they have no competing interests.AM and JD both researched and wrote this meeting review.


These incidentally discovered renal tumours are generally slower growing, are detected at an earlier stage, and are localized to the kidney rcc) are asymptomatic at presentation.With widespread utilization of noninvasive cross-sectional abdominal imaging, small solid renal masses are being found with increasing frequency rccs, but a shift has occurred toward treating small, incidentally found renal neoplasms in a nephron-sparing manner. Nephron-sparing techniques have been shown to offer oncologic and functional outcomes that are equivalent to those with radical nephrectomy for patients with renal tumours 4 cm or smaller in size The radical nephrectomy has been the “gold standard” for the treatment of clinically localized in situ ablative technologies Since the mid 1990s, the movement toward minimally invasive alternatives has meant the replacement of open surgery  with laparoscopic techniques and now with ca), radiofrequency ablation (rfa), microwave rfa and ca are promising, but long-term studies are ongoing to validate their oncologic efficacy and durability.Several ablative technologies have been investigated, among them, cryoablation  and tumours in the hilum or the collecting system.Radiofrequency ablation is a heat-mediated method of tissue destruction. The technology was initially developed for treating primary and metastatic liver lesions Radiofrequency ablation works by transmitting a high-frequency electrical current through an electrode placed directly into the renal tumour. Alternating current delivered through the probe causes ions in the surrounding tissues to vibrate, creating frictional heat that results in heat-induced tissue damage. The mechanism of tissue destruction has been extensively reviewed At a molecular level, the heat generated by the high-frequency electrical current causes tissue destruction in three phases. Immediately post-ablation, molecular friction produces some combination of destruction of cellular structure, protein denaturation, membrane lipid melting, and cellular vaporization rfa depends on factors including probe temperature, generator power, temperature distribution, and targeting of the tumour The success of tumour ablation with rfa generators have been replaced by new generators with upwards of 200 W that can consistently achieve temperatures above 100°C. However, temperatures higher than 105°C cause immediate vaporization and boiling of tissue, which creates gas bubbles, tissue carbonization, and eschar formation at the electrode. These effects increase impedance and reduce the extent of tissue ablation For the cellular changes to occur as described earlier, temperatures above 50°C must be achieved. Earlier underpowered rfa may be applied percutaneously or laparoscopically ct, and magnetic resonance imaging (mri) have all been used to target lesions. Now, with the advent of fluoroscopic ct and open interventional mri, real-time ablation monitoring can be achieved.Many studies have aimed to achieve electrode temperatures between 50°C and 100°C. Innovations to reduce the impedance created at high temperatures include infusion of hypertonic saline into the target tissue during ablation. Electrodes are also designed in variously-sized configurations from single and multiple tines to expandable hooks. The radiofrequency may be applied using a temperature-based or impedance-based system - rfa. To date, Matsumoto et al. have reported the largest series: 109 tumours treated with percutaneous rfaet al. reported 100 tumours treated with percutaneous rfa ct at a mean follow-up period of 28 months.Similarly, Gervais et al. reports the ablation of 56 tumours with a mean tumour size of 2.2 cm. No residual tumour was detected on ct for 47 lesions at a mean follow-up time of 27 months The most recent study by Varkarakis rfa procedure is not without complications. In a multi-institutional review of complications of cryoablation and radiofrequency ablation of small renal tumours, Johnson et al. reported 11 complications in 133 cases (8.2%) rfa The in situ. The tumour is rapidly frozen, creating a cryolesion, which then undergoes necrosis over time and eventually heals by secondary intention. At a molecular level, the damage induced by the cryo-energy is two-fold Cryoablation (or cryotherapy) involves freezing the target tissue with a cryoprobe Argon or nitrogen are the cryogens most commonly used for cooling to a temperature of –40°C, and their effect usually extends 1 cm beyond the lesion margin rfa in that the extremes of temperature alone are not enough to completely destroy cells; the effects of delayed microvasculature failure are also required. The contraindications for cryotherapy are similar to those for rfa.Cryoablation differs from rfa, cryoablation requires real-time monitoring of the ice ball to ensure that the tumour is completely frozen and to minimize injury to the surrounding healthy tissue. To date, most cryoablation has been performed using laparoscopic techniques under ultrasound monitoring. An open or interventional mri has been used to permit real-time monitoring of the ice ball in a percutaneous approach ct Cryoablation can be performed by open et al. published the first series of patients undergoing cryoablation in 1998 Gill et al. ct-guided biopsy.Gill et al. reported a series of 37 patients undergoing laparoscopic cryoablation. The mean follow-up time was 20.5 months, and 25 patients who underwent the postoperative ct-guided biopsies had negative results.Cestari et al. Most recently, Lawatsch rfa of small renal tumours, Johnson et al. reported complications in 139 cases (13.6%)rfa, pain and paresthesia at the site of probe insertion were the most commonly reported complications In a multi-institutional review of complications of cryoablation and With the number of incidentally detected small renal tumours increasing and minimally invasive techniques for treating those tumours becoming more common, investigators have turned toward energy-ablative technologies. In particular, small asymptomatic renal masses in older patients or in those who are poor candidates for surgery require treatment in a minimally invasive fashion with minimal morbidity.rfa being the least cumbersome approach. Percutaneous cryoablation requires real-time monitoring of the ice ball, and because of the need for open mri or fluoro-ct few centers have performed this technique to date. The early results appear promising; however, long-term follow-up data are needed to prove the efficacy and durability of both ablative technologies.Radiofrequency ablation and cryoablation both appear to be safe and effective methods of treating small renal tumours. Both can be deployed in a minimally invasive fashion, with percutaneous


It has been linked with a number of different stress responses in cyanobacteria.The dspA (ycf26) in Synechocystis 6803. We found little phenotypic effect during nitrogen starvation. However, when the mutation was combined with deletion of the pta gene coding for phosphotransacetylase, a more significant phenotype was observed. Under nitrogen starvation, the pta/dspA double mutant degrades its phycobilisomes less than the wild type and still has about half of its chlorophyll-protein complexes.We constructed an insertional mutant of Synechocystis 6803.Our data indicates that acetyl-phosphate-dependent phosphorylation of response regulator(s) overlaps with DspA-dependent signalling of the degradation of chlorophyll-protein complexes (and to a lesser extent phycobilisomes) in  The assembly, composition and maintenance of photosynthetic pigment-protein complexes are regulated by many different factors including light quality, light intensity, temperature, water availability and nutrient status . Microor2PO3) and carbamyl phosphate [pta gene encodes phosphotransacetylase which catalyses the production of acetyl phosphate, the activated acetate intermediate that occurs during conversion of acetyl-CoA to acetate [pta gene abolished PhoP*-mediated transcription. Most response regulators can be phosphorylated by acetyl phosphate in vitro [Km for each individual response regulator for acetyl phosphate. It may be that acetyl phosphate maintains a low level of phosphorylation for most response regulators. The pta gene has been identified in Synechocystis 6803 (slr2132) and Crocosphaera watsonii by homology to other bacterial pta sequences (up to 47% sequence identity by BLASTP), however pta has not been identified in the genome sequence of the other cyanobacteria.Response regulators are generally phosphorylated by their cognate sensor kinases, but phosphorylation can also be mediated through low molecular mass phosphate donors, such as acetyl phosphate, phosphoramidate  codes for a putative sensory histidine kinase that is conserved in plastids and cyanobacteria. The deduced amino acid sequence of DspA contains 663 amino acids in Synechocystis sp. PCC 6803 (Synechocystis 6803). DspA has a HAMP [ycf26 is also the only histidine kinase gene present in the plastid genomes of the red algae Porphyra purpurea, Gracilaria tenuistipitata and Cyanidium caldarium [The gene s a HAMP  and a PAs a HAMP . We havealdarium . This suSynechocystis 6803 is an ORF designated sll0698 in the CyanoBase genome database  which has also been called Hik33 [Synechocystis 6803 [Synechococcus sp. PCC7942 (Synechococcus 7942), the probable orthologue for Ycf26 was shown to regulate phycobilisome degradation during nutrient starvation and in high light. The gene was therefore designated nblS (non-bleaching mutant sensor) [Synechocystis 6803 when grown in high-light [Synechococcus 7942 for NblS, however, no orthologue for NblR has been identified in Synechocystis 6803  and it is likely that NblS also interacts with other response regulators [The probable orthologue for Ycf26 in the cyanobacterium ed Hik33  and DspAed Hik33 . It was ed Hik33 . It was tis 6803 ,17,18. Dtis 6803 ,20 and i sensor) . DspA isgh-light . One posgulators . NblS hagulators . Thus itSynechocystis 6803. We have constructed an insertional deletion mutant in this ORF, which we will refer to as dspA. We show that the inactivation of dspA has little phenotypic effect under nitrogen starvation, unless the mutation is combined with deletion of the pta gene. In the double mutant there is a dramatic effect on the degradation of chlorophyll-protein complexes under nitrogen starvation, which is not seen in either of the single mutants, however phycobilisomes are degraded, though not to the same level as in wild type. We conclude that acetyl phosphate masks DspA signalling under our laboratory conditions. The results indicate that the degradation of photosynthetic complexes during nutrient stress is controlled by multiple inputs, including acetyl phosphate level.A role for the sll0698 gene product in controlling the degradation of the photosynthetic apparatus during nutrient stress has not yet been demonstrated in Porphyra purpurea, Gracilaria tenuistipitata and Cyanidium caldarium were analysed by Pfam and SMART. This identified a HAMP, PAS (low confidence in Cyanidium), HisKA and HATPase_c domain  are probably orthologues (see supplementary data online). This is supported by Tu et al., [dspA null mutant in Synechocystis 6803 with the nblS gene from Synechococcus 7942.The deduced amino acid sequences for Ycf26 from 15 available cyanobacterial annotated genome sequences and the red algal species PD339187 ) and it PD339187 ,25,26), PD339187  that cou et al.,  who wereMutants were routinely characterised by whole cell absorption spectra, which can be used to give an estimate of the amount of chlorophyll and phycocyanin per cell . Mutants-2 s-1) the absorption spectra were similar, indicating little difference in the chlorophyll and phycocyanin content of the cells (data not shown). When cells were grown under higher light (80 μmol m-2 s-1), only slight differences in absorption spectra and Chl/PC ratios were seen .When cells were grown under low light , but containing no NaNO3.An rticular . We ther-2 s-1), the wild-type and the dspA and pta single mutants were almost completely bleached . Thus the pta/dspA double mutant retains its photosynthetic apparatus during nitrogen starvation, in contrast to the wild-type and both single mutants, where nearly all pigment is lost.After three days nitrogen starvation in moderate light , and a double mutant deficient in both genes. We have used the mutants to examine the possible roles of DspA and acetyl phosphate in controlling cell responses to nitrogen starvation. We find strong evidence that DspA and acetyl phosphate act cooperatively as signalling inputs into pathways controlling photosystem stoichiometry under nutrient-replete conditions, and photosystem degradation during nitrogen starvation. Under nutrient-replete conditions, the dspA and pta single mutants both show a decreased PSII/PSI ratio  other than those phosphorylated by DspA.In perature ,31,32, tand OmpR in vivo, rylation . In the pyruvate . There hSynechococcus 7942 as a factor important for initiating the degradation of the photosynthetic complexes (in particular the phycobilisomes) during nitrogen starvation [Synechocystis 6803 there is strong evidence for DspA as a global regulator controlling the expression of many genes involved in photosynthesis, carbon metabolism and stress responses [dspA mutations in Synechocystis 6803 have not previously been shown to induce a non-bleaching phenotype like that seen in Synechococcus 7942. Here we have shown that such a phenotype can be seen in the dspA mutant of Synechocystis 6803, provided that the pta gene is also deleted. The phenotype is still not identical to that seen in Synechococcus 7942, however, since in Synechocystis 6803 it is not only the degradation of the phycobilisomes which is affected but also the degradation of chlorophyll-protein complexes (Table NblS (DspA homologue) was originally identified in arvation . In Syneesponses -19,21,22dspA single mutant  and the dspA open reading frame (sll0698) were amplified by PCR from Synechocystis 6803 DNA, using primers shown above. The PCR products were cloned into pBluescriptII KS +. The slr2132 sequence was cut at a PstI site at position 872 in the coding sequence, and an erythromycin resistance cassette (pBSEmEco1) was ligated in. The sll0698 sequence was cut at an EcoRI site at position 1384 in the coding sequence, and a kanamycin resistance cassette (pUC4K) was ligated in [Synechocystis 6803 was transformed with the slr2132::EmR construct as described by Porter [R locus, and hence that the pta gene had been completely inactivated generating a fully-segregated pta mutant. A similar procedure was followed with the sll0698::KmR construct, which was used to transform both the wild-type and the pta mutant, to generate a sll0698 single insertional mutant (dspA) and a sll0698:slr2132 double insertional mutant (pta/dspA). These mutants were shown to have segregated after six rounds of streaking to single colonies on kanamycin (plus erythromycin for pta double mutants) plates as has been observed by Hsiao et al. [The gated in ,49. Slr2y Porter . After so et al. , though 3. This was then divided into two 5 ml portions, which were then used to inoculate parallel 20 ml cultures.50 ml cultures were grown to mid-log phase in BG11 medium. Each culture was then harvested by centrifugation, resuspended in 30 ml of wash medium, harvested a second time and resuspended in 10 ml of BG11 medium supplemented with glucose but lacking NaNOa concentration of 5 μM [a was excited at 435 nm: light at this wavelength is not significantly absorbed by the phycobilisomes [a concentration was estimated from the absorption of methanol extracts at 665 nm [et al. [Fluorescence emission spectra were measured at 77 K in a Perkin-Elmer LS50 luminescence spectrometer equipped with a liquid-nitrogen sample holder. Cells were harvested by centrifugation and resuspended in growth medium to a chlorophyll  of 5 μM . Re-abso of 5 μM . The samt 665 nm . Spectra [et al. . Cell nuhistidine kinases, adenyl cyclases, methyl binding proteins and phosphatases; NblS – non-bleaching mutant sensor, PAS – PER-ARNT-SIM; RR – Response regulator.aa – amino acids; Dsp – drug sensory protein, HAMP – SSM performed the spectroscopy, analysed the segregation of the mutants and drafted the manuscript. CWM supervised SSM at UCL and drafted the manuscript. MKA conceived the study, constructed the interposon mutants and drafted the manuscript.ClustalW alignment of NblS  putative amino acid sequences.Click here for file


Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, micro-exons and partial gene structure predictions that span across several genomic clones.We present a mRNA/DNA homology based gene structure prediction tool, GIGOgene. We use a new affine gap penalty splice-enhanced global alignment algorithm running in linear memory for a high quality annotation of splice sites. Our tool includes a novel algorithm to assemble partial gene structure predictions using interval graphs. GIGOgene exhibited a sensitivity of 99.08% and a specificity of 99.98% on the Genie learning set, and demonstrated a higher quality of gene structural prediction when compared to Sim4, est2genome, Spidey, Galahad and BLAT, including when genes contained micro-exons and non-canonical splice sites. GIGOgene showed an acceptable loss of prediction quality when confronted with a noisy Genie learning set simulating ESTs.GIGOgene shows a higher quality of gene structure prediction for mRNA/DNA spliced alignment when compared to other available tools. A vast amount of genomic data, including most of the human genome , is now Most of the human genes have piecewise structure with a number of exons separated by introns. Introns are excised from original gene transcripts (pre-mRNA) to form mature mRNA. By aligning mRNA with originating genomic clones, we can reconstruct gene structure.Several fast and efficient tools, such as BLAST  and BLASspliced alignment, are often classified according to the homology type they employ  . The result of this step is a set of unambiguous HSP sequences.Step 5 Build an interval graph of overlapping unambiguous HSP sequences. The interval graph captures intersection relations of nodes (unambiguous HSP sequences) as we put edges between nodes when nodes belong to different genomic clones, while their mRNA composite segments intersect. Edges between HSP sequences from the same genomic clone are not allowed.Step 6 Occasionally, short exons missed by the BLASTN algorithm or dust low-complexity filtering result in interrupted unambiguous HSP sequences. Their fragments reside in different interval graph nodes marked with the same genomic clone and transcript. We merge these nodes to form longer, original, uninterrupted unambiguous HSP sequences. An intuitive interpretation of this step is in Figure Step 7 Compact the interval graph, as discussed [see Subsection Joining unambiguous HSP sequences]. This results in the biggest composite genomic clone containing the maximum number of possible exons.Step 8 Use splice-enhanced affine gap penalty global alignment to identify possible intron/exon boundaries in the composite genomic clone, as discussed [see Subsection Splice-enhanced affine gap penalty global alignment].Step 9 Extract intron and exon segments from the composite genomic clone and print a report.It is well-known that genes, or parts of genes, are duplicated during the course of evolution. This can result in ambiguities during the assembly of a complete gene structure from HSP sequences, as illustrated in Figure The problem arises when a segment in an mRNA transcript matches multiple segments in a genomic clone. To address this our algorithm for finding an unambiguous HSP sequence (a chain of putative exons) adheres to the following biological principles:sequential rule).1. Transcripts are always linear. Thus, we require the set of HSPs to be sequential  or paralog(s), as schematically shown in Figure 5. Pre-mRNA splicing results in mature mRNA, with exons arranged side by side. In the case of an HSP sequence containing potential exons, we require the entire mRNA transcript to be covered with segments continuously, without breaks.Disambiguation of an HSP set is shown in Figure For the purposes of the disambiguating algorithm we build a bipartite graph structure, where segments are nodes and HSPs are edges connecting the nodes. A dynamic programming disambiguation procedure with an affine gap penalty Figure  is then weight where the weight of an HSP is a tradeoff between its identity and size:In our experiments, we noticed cases in which HSPs from the real gene match have a smaller identity compared to HSPs originating from paralogs and pseudogenes. Thus, the disambiguation procedure finds the unambiguous HSP sequence covering the longest mRNA segment with the minimum number of HSPs. For the HSP sequences of equal length with the same number of HSPs, we compare the maximum total weight = size·mx 100-    (1)x is the BLASTN-assigned percent identity for an HSP, size is the HSP length, and m <1 is the decay rate to ensure substantial weight loss for identity lower than the threshold value. The value m = 0.85 produced good results in our experiments. Weight function (1) characterizes the importance of any given HSP in a global solution.Here Iy is visited when a sequence of genomic segments is interrupted. This subtracts D, and E for every additional genomic segment missed, from the total weight. If a continuously overlapping transcript-side sequence of segments is broken, the total weight is nullified by visiting state Ix. Weight is gained at state M with a normal transcript-side overlapping sequence of HSPs.The disambiguation procedure can be represented as a series of transitions between states Figure . State IF of dimensionality 2 × (# of RNA segments) × (# of DNA segments) and a matrix C of the same size to record the intermediate HSP sequences in the dynamic programming procedure. For the convenience of indexing we introduce aliases M ← F0 and Iy ← F1. We ignore matrix Ix as being unnecessary. The boolean function CONNECTED determines the overlap between segments i and j in the transcript.For this algorithm we must allocate a score matrix C using the recursive algorithm shown in Figure To generate the final set of unambiguous HSP sequences for a given BLASTN result, the HSP sequences are restored from matrix Algorithm for an unambiguous HSP sequences allocation]. In order for the nodes to be connected by an edge, they must contain overlapping HSP sequences coming from different genomic clones. To join the nodes, a Floyd-Warshall all-pairs-longest-path algorithm , the small exons are recovered by processing the expanded anchors with our spliced alignment procedure.As an example of a successful anchor expansion, consider an HSP sequence interrupted by undetected short exon(s) Figure . After cSimilarly, low-complexity regions may interrupt HSP sequencing in BLASTN results due to dust filtering. In this case, interrupted unambiguous HSP sequences are combined and the gap will be closed by sequence matching, resulting in a monolithic exon Figure .. We used transcripts corresponding to mRNA or CDS features in the Genie learning set annotation.GIGOgene was tested, along with Spidey, est2genome, Sim4, Galahad and BLAT on 462 mRNA transcripts of the human Genie multi-exon annotated learning set Sensitivity (ESn) and specificity (ESp) were calculated according to the formulasTE is the number of accurately predicted exon boundaries, AE is the number of annotated exon boundaries in the Genie learning set, and PE is the number of predicted exon boundaries. Only internal exonic boundaries were considered. Results are summarized in Table Here This test is designed as evidence of general prediction quality of different gene structure annotation tools. GIGOgene has the highest sensitivity and specificity in this case, which highlights advantages of the approach we use.We followed the Sim4 prediction compensating procedure described in  to identThis study shows that the GIGOgene program has the highest structural prediction sensitivity and specificity in this case. BLAT recovered 96.69% true exonic boundaries in the micro-exonic set, while other programs had fraction of true splice sites recovered no more than 90%, i.e. they most likely miss micro-exon(s) from their prediction.According to  approximIn this study est2genome made a mistake in annotating virtually every non-canonical splice site while reinforcing canonical splice rule. Although BLAT was very sensitive in this experiment, it makes mistakes occasionally.Experiments with Genie learning set]. Noise was equiprobably distributed between random nucleotide insertions, deletions and substitutions. Results of a simulated EST experiment are presented in Table In order to research the EST-related performance of different programs we introduced 4% noise in the Genie experiment discussed [see Subsection Experiments with micro-exon detection], where they sacrifice micro-exons to reinforce canonical splice rule.With simulated EST study our program performed worse than Sim4 and est2genome, about as well as Galahad, and substantially better than BLAT and Spidey, the programs that were specifically designed for mRNA/DNA spliced alignment. The reason for substantial quality loss with GIGOgene is in splice site annotation strategy. If we get a number of nucleotide inserts between exon boundaries in mRNA, they can be easily interpreted as micro-exon(s) with non-canonical splice sites, rather than reinforcing the GT-AG rule in a genomic clone as Sim4 and EST2genome do. That is why these two applications have rather poor performance in micro-exonic testing [see Subsection Experiments with micro-exon detection].In Table Run time comparison on the set of micro-exons indicates that our program runs faster than est2genome but slower than other tools we have looked at. By using splice-enhanced affine gap penalty global alignment we traded execution time for quality, compare to simpler heuristics used to predict splice sites in other tools.For this experiment we chose human chromosome 22 whole draft sequence NC_000022.8 from NCBI Genbank. A total of 506 transcripts were mapped to the chromosome by parsing human RefSeq flatfiles, but only 430 transcripts have corresponding genes annotated in NCBI Genbank.We report running time for all 506 transcripts mapped to chromosome 22. For the GIGOgene program it took 12 hours 16 minutes 42 seconds to parse BLASTN results, while BLASTN took 9 days 18 hours 9 minutes 3 seconds to align transcripts to the chromosome (without dust filtering). Such a long running time could be explained by extensive low-complexity domains duplicated across the chromosome. BLASTN with low-complexity filtering took only 13 hours, 33 minutes and 18 seconds, but the following GIGOgene gene structural prediction was inferior to the results reported in Table We report exon-level comparative performance of BLAT and GIGOgene in Table Results of BLAT and GIGOgene comparison on Chromosome 22 whole draft sequence annotation agree well with the previously observed tendency: with GIGOgene, gene structural prediction takes longer, compared to BLAT, and has higher prediction quality.Using a homology-based approach, we have designed a program for eukaryotic gene structural annotation. In case of mRNA/DNA spliced alignment we have been able to improve on exon-level sensitivity and specificity by addressing several possibilities of error. Program domain is limited to mRNA/DNA spliced alignment with a reasonable fraction of sequencing errors. Experiments on running time position our tool as a relatively slow utility for annotating specific cases of gene structural prediction.Background]. Our splice-enhanced affine gap penalty global alignment in some ways similar to the spliced alignment of protein/DNA blocks described in the Procrustes paper [Several published spliced alignment algorithms were mentioned [see Section es paper . The keyes paper  and Genoes paper , are lim.The stand-alone program version, web implementation interface, test results and manual for GIGOgene are available at Project name: Good In Good Out gene structural prediction tool (GIGOgene)Project home page: .Operating system: Platform independentProgramming language: JavaOther requirements: Java 1.4.1 or higherLicense: GNU Lesser General Public LicenceAC and DQ conceptualized the project and set up computational facility. DQ implemented BLASTN SAX parser and made many valuable suggestions through the progress of our study. AC implemented and evaluated GIGOgene Java code. MP extensively edited the manuscript and made many important changes. HA helped to conceptualize the tool, provided general support and gave final approval of the version to be published. All authors read and approved the final manuscript.


Ichthyosis uteri is an exceedingly rare condition in which the entire surface of the endometrium is replaced by stratified squamous epithelium. Originally described as an endometrial response to iatrogenically-introduced caustic substances, similar changes have since been described in association with a variety of inflammatory conditions of the endometrium. We describe herein a heretofore undescribed example of a moderately differentiated squamous cell carcinoma of the uterine cervix associated with extensive ichthyosis uteri-like changes of the entire adjacent endometrium. Additionally, the squamous epithelium of the latter also showed multifocal changes diagnostic of a low-grade squamous intraepithelial lesion. The potential genesis of this composite of findings is discussed, as is the neoplastic potential of ichthyosis uteri. It is concluded that a squamous cell carcinoma of the cervix extended proximally into the endometrium, and that there was a colonization of a pre-existing ichthyosis uteri by associated human papillomavirus. The possibility of significant cervical pathology should be considered when plaques of squamous epithelium with low grade dysplastic changes are identified in an endometrial biopsy or curettage. A 38-year-old nulligravid female presented with complaints of a vague pelvic heaviness and a vaginal discharge. Her past medical history is significant for acquired immune deficiency syndrome (diagnosed 9 months previously) and Hepatitis A, B and C infections. Physical examination revealed a massively enlarged, barrel-shaped cervix with areas of ulceration on the ectocervix and a fungating mass protruding from the endocervical region. Following a biopsy, the patient underwent a type III radical hysterectomy, bilateral salpingo-oophorectomy and pelvic/paraaortic lymph node sampling. The procedure was well-tolerated and without any complications. She has had no evidence of tumor recurrence at last follow-up, 9 months after her surgery.Upon external inspection of the uterus, both the cervix and the inferior portion of the lower uterine segment were significantly enlarged. Sectioning revealed a 7 cm × 4 cm exophytic, friable, tan to tan-brown mass obliterating the endocervix and extending into the lower uterine segment Figure . SectionThe tumor was a moderately differentiated, large cell, keratinizing squamous cell carcinoma as previously described . NotablyThe term "ichthyosis uteri" was initially coined in 1885 by Zeller to refer to extensive squamous metaplasia of the surface endometrium following iatrogenically-introduced caustic substances such as formalin or iodine . Since tThe second, albeit unlikely, potential genesis of this tumor (i.e a squamous cell carcinoma arose form ichthyosis uteri in the lower uterine segment) caused us to re-evaluate the literature on the neoplastic potential of ichthyosis uteri. In the experience of some authors , ichthyoIn summary, a case of squamous cell carcinoma of the cervix with probable proximal extension into, and colonization of, a pre-existing ichthyosis uteri by associated HPV is described. The possibility of significant cervical pathology should be considered when plaques of squamous epithelium with low grade dysplastic changes are identified in an endometrial biopsy or curettage.The author(s) declare that they have no competing interests.Not Applicable