However, some but not all studies in mice and humans (though none in rats) have found that CLA promotes insulin resistance. The molecular mechanisms responsible for these effects are unclear, and there are conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-γ (PPARγ) activation and expression. We have conducted three experiments with CLA in obese mice over three weeks, and one over eleven weeks. We have also investigated the effects of CLA isomers in PPARγ and PPARα reporter gene assays.Studies in rodents and some studies in humans have shown that conjugated linoleic acid (CLA), especially its trans-10, cis-12 isomer in the diet of female genetically obese (oblep/oblep) mice for up to eleven weeks reduced body weight gain and white fat pad weight. After two weeks, in contrast to beneficial effects obtained with the PPARγ agonist rosiglitazone, CLA or CLA enriched with its trans-10, cis-12 isomer raised fasting blood glucose and plasma insulin concentrations, and exacerbated glucose tolerance. After 10 weeks, however, CLA had beneficial effects on glucose and insulin concentrations. At this time, CLA had no effect on the plasma TNFα concentration, but it markedly reduced the plasma adiponectin concentration. CLA and CLA enriched with either isomer raised the plasma triglyceride concentration during the first three weeks, but not subsequently. CLA enriched with its trans-10, cis-12 isomer, but not with its cis-9, trans-11 isomer, stimulated PPARγ-mediated reporter gene activity; both isomers stimulated PPARα-mediated reporter gene activity.Inclusion of CLA or CLA enriched with its oblep/ob lepmice. Activation of both PPARγ and PPARα may contribute to the improvement in insulin sensitivity. In the short term, however, another mechanism, activated primarily by trans-10, cis-12-CLA, which probably leads to reduced adipocyte number and consequently reduced plasma adiponectin concentration, may decrease insulin sensitivity.CLA initially decreased but subsequently increased insulin sensitivity in  The major components of CLA, are the cis-9, trans-11 (c9, t11) and the trans-10, cis-12 (t10, c12) isomers, both of which have biological activities. The t10, c12-isomer is the one primarily responsible for the effects of CLA on weight gain and insulin sensitivity. The CLA used in the present study contains these isomers in roughly equal proportions.The term conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid (CLA may be of benefit in cancer, atherosclerosis and possibly some disorders of the immune system. In addition, it reduces weight gain and fat accretion in rats and mice. Some studies have found that CLA causes fat loss in humans -4, and toblep/oblep) mice, despite causing weight loss [falep/fa lepand Zucker diabetic fatty falep/fa leprats [In previous studies, CLA or t10, c12-CLA has exacerbated glucose tolerance and raised plasma insulin levels in normal and genetically obese α, since TNFα is associated with apoptosis of adipocytes but causes insulin resistance. TNFα mRNA levels were markedly increased in isolated adipocytes from normal mice that had been fed on CLA for as little as four days . Surprisdblep/db lepmice with CLA for eleven weeks improved glucose tolerance and reduced plasma insulin concentration during the glucose tolerance test, indicating improved insulin sensitivity. Treatment for five weeks also improved glucose tolerance but plasma insulin was raised [dblep/db lepmice because β-cell failure as well as insulin resistance affects glucose homeostasis, and glycosuria can cause weight loss. Therefore, to investigate the effect of CLA on glucose homeostasis further, we have conducted four experiments in oblep/ob lepmice. We have included rosiglitazone as a comparator in one experiment and compared the effects of t10, c12- and c9, t11-enriched CLA with CLA in another experiment. The first three experiments were over 22 days, whereas the fourth was extended to eleven weeks and included measurements of plasma TNFα and adiponectin. An intriguing finding is that whereas CLA initially exacerbated glucose tolerance and raised the plasma insulin level, after ten weeks it began to improve glucose tolerance and lower the plasma insulin levels.Although most studies in mice have found that CLA exacerbates glucose tolerance and raises the plasma insulin concentration, in one study treatment of s raised . It is sWe also describe activation of PPARγ by t10, c12 but not by c9, t11-CLA, whereas PPARα was activated by both isomers.oblep/oblep) mice reduced their body weight, or weight gain compared to mice fed on a diet containing a similar amount of sunflower oil  concentration in experiment 3 Table , and CLAAfter ten weeks CLA had no effect on the plasma TNFα concentration. The plasma adiponectin concentration, by contrast, was markedly decreased Table .CLA enriched with t10, c12-CLA (50 and 100 μM), but not with c9, t11-CLA, stimulated PPARγ-mediated reporter gene activity Figure . In contdblep/db lepmice [A number of studies have found that CLA reduces weight gain and fat accretion in both rats and mice. Those that have failed to show such effects are generally those that have used low levels of CLA or CLA that contained low concentrations of t10, c12-CLA . Studiespdb mice , show expdb mice -10. In hpdb mice , but doupdb mice -13, and pdb mice ,12,14. Tpdb mice .oblep/oblep) mice as our model of insulin resistance. There is only one previous report of the effect of CLA in oblep/ob lepmice [oblep/ob lepmice with CLA. These benefits were achieved faster with the higher (25 g/kg diet, including 40% t10, c12-CLA) than the lower dose (10 g/kg diet) of CLA.Our study raises the possibility that an initial exacerbation of glucose tolerance, apparently due to insulin resistance, might, after prolonged treatment with a high dose of CLA, be followed by improved insulin sensitivity and glucose tolerance. We used genetically obese  and maintained at 23 ± 1°C with lights on from 07:00 to 19:00 h. They were housed in plastic cages with bedding and fed 'rat and mouse standard diet' . Six days before the start of the studies they were allocated to treatment groups (6 mice per group), such that each group had a similar mean bodyweight. The CLA and other treatments were mixed with powdered diet and given ad libitum. The mice were killed by cervical dislocation in experiments 1 to 3; in experiment 4 they were anaesthetised with sodium pentobarbitone  and exsanguinated through an aortic catheter. All procedures were conducted in accordance with our Home Office, UK project licence under the Animals (Scientific Procedures) Act and as agreed by the University of Buckingham Ethical Review Board.Female C57Bl/6 CLA (in acid form), including isomer-enriched CLA, and sunflower oil were provided by Loders Croklaan, Wormerveer, The Netherlands. The CLA used in experiments 1, 2 and 3 was Clarinol™ A-60, containing 30% c9, t11-CLA and 31% t10, c12-CLA and 25% oleic acid. The CLA used in experiment 4 was Clarinol™ A-80, containing 40% c9, t11-CLA and 40% t10, c12-CLA. Rosiglitazone was synthesised by Dextra Laboratories, Reading, Berks, UK.ad libitum, and after 22 days when they had been fasted for 16 hours, immediately before termination of the experiment.At the start of treatment the mice weighed 34.5 ± 4.3 g (mean ± S.D.). The treatment groups were high oleic (83.5%) sunflower oil ; rosiglitazone (10 mg/kg diet) plus high oleic sunflower oil (15 g/kg diet); CLA (Clarinol™ A-60: 15 g/kg diet). An oral glucose tolerance test was conducted after 14 days as described below. 30 min prior to giving glucose, when the mice had been fasted for 4.5 hours, blood (100 μl) was taken for the measurement of plasma insulin and triglycerides. Plasma insulin and triglycerides were also measured after 21 days when the mice were feeding At the start of the treatment the mice weighed 44.0 ± 3.2 g (mean ± S.D.). A group fed on powdered chow alone was included. The doses of CLA (Clarinol™ A-60) and sunflower oil in the other two groups were increased to 25 g/kg diet. The oral glucose tolerance test and other measurements were carried out as described for experiment 1, except that the glucose tolerance test was one day earlier (i.e. after 13 days).At the start of treatment the mice weighed 34.1 ± 2.1 g (mean ± S.D.). The treatment groups were high oleic sunflower oil (25 g/kg diet); CLA (Clarinol ™ A-60: 25 g/kg diet); CLA with the CLA component of Clarinol enriched to 90% with t10, c12-CLA (25 g/kg diet); similarly, CLA enriched to 90% with c9, t11-CLA (25 g/kg diet). The oral glucose tolerance test and other measurements were carried out as described for experiment 2. In addition, the plasma non-esterified fatty acid concentration was measured in the blood taken prior to the glucose tolerance test, and the liver, pancreas, parametrial white adipose tissue depot and interscapular brown adipose tissue depot were weighed at termination.At the start of treatment the mice weighed 31.6 ± 3.8 g (mean ± S.D.). The treatment groups were high oleate (as glyceride) sunflower oil (25 g/kg diet); CLA (Clarinol A80: 10 g/kg diet) plus high oleic sunflower oil (15g/kg diet); CLA (Clarinol A80: 25 g/kg diet). Oral glucose tolerance tests preceded by blood sampling for the measurement of triglycerides, insulin and non-esterified fatty acids were taken after 14, 35 and 70 days of treatment. After 77 days of treatment the animals were anaesthetised in the fed state and blood was taken from the thoracic aorta for the measurement of plasma TNFα and adiponectin. Tissues were weighed as in experiment 3. Body length was measured from the tip of the nose to the anus.In each experiment oral glucose tolerance was measured after 13 or 14 days. In experiment 4 it was also measured after five and ten weeks. The mice were fasted for five hours before being dosed with glucose . Blood samples (20 μl) were taken from the tip of the tail after applying a local anaesthetic (lignocaine) and immediately before and 30, 60, 90, 120 and 180 min after dosing the glucose. They were mixed with haemolysis reagent and blood glucose was measured in duplicate using the Sigma Enzymatic (Trinder) colorimetric method and a SpectraMax 250 . Areas under the glucose tolerance curve (0–180 min) and other manipulations and analysis of the data were carried out using Prism software, version 3.0 (GraphPad Software Inc., San Diego, CA, USA).Blood was collected into EDTA-coated microcuvettes (Sarstedt microcuvette, Aktiengsellschaft & Co., Nämbrecht, Germany) for the measurement of plasma insulin, non-esterified fatty acids and triglycerides. Plasma was stored at -80°C. 5 μl plasma samples were assayed using kits for triglyceride (Sigma enzymatic colorimetric method), non-esterified fatty acids  and insulin .For the measurement of plasma TNFα  and adiponectin (Biosource UK, Nivelles, Belgium), blood was taken into tubes containing 200 units of heparin and plasma was stored at -80°C.MluI and BglII and ligated into pNF-κ B-luc that had been cleaved with the same restriction endonucleases. Ligated DNA was transformed into competent JM109, E. coli cells (Promega). pcDNA3/PPARα was prepared by removing the human PPARα cDNA insert from pUC18/hPPARα as a NruI/BamHI fragment and ligating it into EcoRV/BamHI cleaved pcDNA3.1(-) (Invitrogen). Ligated DNA was transformed into competent JM109, E. coli cells (Promega).pRSV/hRXRα and pcDNA3/hPPARγ1 were both obtained from Professor VKK Chatterjee . PPPRE3TK-luc was prepared by replacing the NF-kB enhancer element of pNF-κB-luc (Clontech) with a cassette of 3 PPAR Response Elements (PPREs). A double-stranded PPRE cassette was prepared using the 'Klenow fill-in' technique. An 113 bp oligonucleotide (PPRE3) 5'- GCATTCACGCGTCAAATATAGGCCATAGGTCATTCTCGAGCAAATATAGGCCATAGGTCATTCTCGAGCAAATATAGGCCATAGGTCAGATTCGATCAATATAGGCCATAGGTCACTCGAGGCAACAGATCTTACGCATG -3' containing a triplet of PPREs and appropriate restriction endonuclease sites was used as a template for synthesis of a second DNA strand. This was primed by PPRE3R, 5'-CATGCGTAAGATCTGTTGCC-3', which is complementary to the 3' region of PPRE3. 20 μl annealed PPRE3 and PPRE3R, 1.5 μl 2 mM dNTPs, 1 × Klenow Buffer and 5 units DNA Polymerase I (Klenow fragment), were incubated for 1 hour at 37°C and then 10 minutes at 75°C, purified by ethanol precipitation and resuspended in sterile water. The double-stranded PPRE cassette was digested with 2. Transient transfections were performed using LipofectAMINE as directed by the manufacturers (GibcoBRL). For the PPARγ assay Cos-7 cells were plated in 24-well plates at a density of 0.375 × 105 cells/well. Cells were transfected in serum-free medium (DMEM containing 2 mM L-glutamine) with pPPRE3TK-luc, pRLTK, pRSV/hRXRα and pcDNA3/hPPARγ 1 at concentrations of 0.4, 0.03, 0.02 and 0.02 μg/well, respectively. Five hours after transfection cells were fed with 250 μl/well of serum-free medium containing various concentrations (0–100 μM) of CLA (either the c9, t11 isomer or t10, c12 isomer prepared in 0.1% DMSO). Cell lysates were prepared after 46 hours using 100 μl 1 × passive lysis buffer (Promega) per well. Firefly and renilla luciferase activities were measured using a Dual Luciferase Assay kit (Promega), as described by the manufacturers. Measurements were performed on an MLX microtitre plate luminometer (Dynex). For the PPARα assay Cos-7 cells were plated in 24-well plates at a density of 0.5 × 105 cells/well. Cells were transfected in serum-free medium with pPPRE3TK-luc, pRLTK, pRSV/hRXRα and pcDNA3/hPPARα at concentrations of 0.4, 0.04, 0.03 and 0.03 μg/well, respectively. Five hours after transfection DMEM supplemented with 2 mM L-glutamine and 20% SBCS  was added to the cells. Following 18 hours incubation at 37°C/5% CO2 the medium was removed and replaced with medium (DMEM supplemented with 2 mM L-glutamine and 10% SBCS) containing various concentrations (0-100 μM) of CLA enriched to 85% with the c9, t11 isomer or 81% with the t10, c12 isomer prepared in 0.1% DMSO. After 24 hours of treatment cell lysates were prepared and luciferase activity measured as described above.Cos-7 cells (ECACC No. 87021302) were routinely cultured in DMEM containing 10% FCS, 2 mM L-Glutamine, 100 iu/ml penicillin and 100 μg/ml streptomycin at 37°C/5% COData were analysed by one-way analysis of variance followed by LSD test with the sunflower oil treatment as the control. Means are of 6 values with SEM.in vivo experiments and analysed materials from these. AM conducted the in vitro experiments. MS and MC conducted the initial analysis and interpretation of the data. JA reanalysed some of the data and wrote the manuscript with input from the other authors, and in particular from AE and IM with respect to interpretation and perspectives.MC, LB, IM and MS devised the experiments. EW, MS and CS, supported by JO and SW conducted the


Prochlorococcus sp. are marine bacteria with very small genomes. The mechanisms by which these reduced genomes have evolved appears, however, to be distinct from those that have led to small genome size in intracellular bacteria. Prochlorococcus species, the smallest and most abundant photosynthetic organism in the ocean, have recently been published. Comparative genome analyses reveal that genome shrinkage has occurred within this genus, associated with a sharp reduction in G+C content. As all examples of genome reduction characterized so far have been restricted to endosymbionts or pathogens, with a host-dependent lifestyle, the observed genome reduction in Prochlorococcus is the first documented example of such a process in a free-living organism.Three complete genomes of P. marinus SS120 that is even more pronounced in P. marinus MED4. This acceleration has affected every functional category of protein-coding genes. In contrast, the 16S rRNA gene seems to have evolved clock-like in this genus. We observed that MED4 and SS120 have lost several DNA-repair genes, the absence of which could be related to the mutational bias and the acceleration of amino-acid substitution.Our results clearly indicate that genome reduction has been accompanied by an increased rate of protein evolution in Prochlorococcus have to be selectively neutral, as the large size of populations imposes low genetic drift and strong purifying selection. We assume that the major driving force behind genome reduction within the Prochlorococcus radiation has been a selective process favoring the adaptation of this organism to its environment. A scenario is proposed for genome evolution in this genus.We have examined the evolutionary mechanisms involved in this process, which are different from those known from host-dependent organisms. Indeed, most substitutions that have occurred in  Mycoplasma genitalium [Buchnera [Nanoarchaeum equitans [Buchnera genome is devoted to biosynthesis of amino acids which are essential to its host [The size of bacterial genomes is primarily the result of two counteracting processes: the acquisition of new genes by gene duplication or by horizontal gene transfer; and the deletion of non-essential genes. Genomic flux created by these gains and losses of genetic information can substantially alter gene content. This process drives divergence of bacterial species and eventually adaptation to new ecological niches . In somenitalium  or phytonitalium  and symbBuchnera -8 or theequitans . In the equitans  and 35 kequitans , respectits host .Prochlorococcus [5 cells/ml) in all nutrient-poor areas of the world's oceans between 40°N and 40°S and is probably the most abundant photosynthetic organism on Earth [b to a ratio; the second inhabits the upper layer of the ocean and has a low divinyl-chlorophyll b to a ratio [Prochlorococcus marinus MED4 [P. marinus SS120 [Prochlorococcus species MIT9313 [So far, all characterized examples of genome reduction have been associated with a change from a free-living to a host-dependent lifestyle . It is trococcus -15. The on Earth ,17. It hon Earth . The fir a ratio . The gennus MED4 , and of us SS120  and Proc MIT9313 , have reProchlorococcus sp. MIT9313 branches at the base of the Prochlorococcus radiation, close to the Synechococcus group [Prochlorococcus HL clade, encompassing the MED4 strain, appears to be the most recently evolved Prochlorococcus group, consistent with the fact that this clade is much less diversified than are the LL clades.Phylogenetic trees based on 16S rRNA sequences  or 16S-2us group . In contSynechococcus sp. WH8102 genome [Despite the close relatedness of these strains, their genomes vary widely in terms of size, G+C content and the number of protein-coding genes Table . While t2 genome , MED4 ha2 genome . HoweverSynechococcus and Prochlorococcus but also between MIT9313 and the two other Prochlorococcus strains  and more amino acids encoded by A+T-rich codons (for example, isoleucine or lysine).The downsizing of MED4 and SS120 genomes during evolution is associated with a genome-wide adenine (A) and thymine (T) enrichment Table . Second,-12) for BLAST comparisons. In contrast, our analysis is based on identification of reciprocal best hits without the use of any particular threshold (apart from the default BLAST threshold) and consequently allows the detection of orthologous relationships whatever the gene lengths or the level of similarity. Still, our ortholog identification process is rather strict and the set of orthologs identified in this study probably corresponds to a lower estimate of the actual number of orthologs shared by the four genomes. This set of genes represents a substantial percentage of the total pool of all protein-coding genes in P. marinus MED4 (73.2%) and SS120 (69.2%) and about half of the gene set in Prochlorococcus sp. MIT9313 (56.2%) and Synechococcus sp. WH8102 (51.1%). These percentages are consistent with the differences in the respective number of genes within these genomes  and probably constitute an estimate of the core of genes conserved in all marine picocyanobacteria. This is sensibly more than the pool of around 1,000 orthologs identified by W.R. Hess . The difr genome .Prochlorococcus genomes, we have investigated whether it also affects the rate of protein sequence evolution in these genomes. We used the 1,306 orthologs common to the four genomes to estimate the amino-acid substitution rate in each genome. Branch lengths calculated for a given tree topology  indicate that protein sequences evolved at significantly different rates (P < 0.001) between MED4, SS120 and MIT9313. Therefore the hypothesis of a constant evolutionary rate between these strains can be rejected for protein-coding genes. The calculation of branch-length ratios reveals that the amino-acid substitution rate is 2.04-fold higher inMED4 than in SS120 (dA/dB) and 3.81-fold higher in MED4 than in MIT9313 ((dA+dX)/dC). This rate is also 2.31-fold higher for SS120 than for MIT9313 ((dB+dX)/dC). Computation of branch lengths for each functional category shows that the increased rate of amino-acid replacement in protein sequences concerns every category  to the rate of synonymous substitutions (dS) is commonly used to measure the relative rate of purifying selection acting at the protein level. We determined dS and dN for each gene pair of every group of orthologs and their values were averaged for each genome. Surprisingly, we observed saturation at synonymous sites for all genome pairs (dS > 2) and the calculation of the dN/dS ratio was thus impossible. Still, the average dN was higher between MED4 and SS120 (0.36) than between SS120 and MIT9313 (0.32). The lowest dN was observed between MIT9313 and WH8102 (0.24), a finding which is consistent with the relative acceleration of amino-acid substitutions in MED4 and in SS120.The ratio of the rate of nonsynonymous substitutions ), as well as several other marine Synechococcus spp. , it is reasonable to assume that the common ancestor of all Prochlorococcus species also had a genome size around 2.4 Mbp. Under this hypothesis, the genome reduction which has occurred in MED4 would correspond to around 31%. By comparison, the extent of genome reduction in the insect endosymbiont Buchnera, as compared to a reconstructed ancestral genome, is around 77% [P. marinus SS120 - and a fortiori the MED4 genome - is considered to be near minimal for a free-living oxyphototrophic organism [The process of genome reduction which has occurred within the ound 77% . The genorganism  and dataProchlorococcus lineages and host-dependent organisms have undergone genome reduction associated with accelerated substitution rates, these phenomena must have arisen from very different causes as the resulting gene repertoires of the two types of organisms differ tremendously. Indeed, the genome evolution of endosymbionts and obligatory pathogens is driven by two main processes which have mutually reinforcing effects on genome size and evolutionary rates. Being confined inside their host, these bacteria have tiny population sizes and are regularly bottlenecked at each host generation or at each new host infection. Consequently, they experience a strong genetic drift [If both ic drift  involvinic drift ,29 as weic drift . This geic drift ,31-33. Fic drift -6,8,9,32Prochlorococcus, the very large size of field populations [P. marinus SS120 only two such genes are missing . One noteworthy exception is the presence in Prochlorococcus, but not Synechococcus, of flavodoxin and ferritin, two proteins that possibly give Prochlorococcus a better resistance to iron stress. Apart from that, Synechococcus appears more like a generalist, in particular with regard to nitrogen or phosphorus uptake and assimilation [a priori be more suited to sustain competition. Hence, we assume that the key to the success of Prochlorococcus resides less in the development of a specific complex or pathway to cope better with unfavorable conditions than in the simplification of its genome and cell organization, which can allow this organism to make substantial economies in energy and material for cell maintenance.If it is neither the relaxation of purifying selection nor an increase in genetic drift that has been the main factor causing hococcus ,34, stromilation , and shoper se is a potential source of substantial economies for the cell, as it reduces the amount of nitrogen and phosphorus, two particularly limiting elements in the upper part of the ocean, which are necessary, for instance, in DNA synthesis. Another advantage is that it allows a concomitant reduction in cell volume. It has been previously suggested  that, fProchlorococcus genomes. Using a rate of 16S rRNA divergence of 1% per 50 million years [Prochlorococcus and Synechococcus. The ancestral Prochlorococcus cells must have developed in the LL niche, a niche probably left free by other picocyanobacteria. Given the considerable difference in genome size between the LL strains MIT9313 and SS120, it appears that genome reduction itself must have started in one  lineage(s) within the LL niche some time after Prochlorococcus differentiation from its common ancestor with marine Synechococcus species. Why the selection has affected only one (or some?) and not all Prochlorococcus lineages remains unclear. Examination of the gene repertoire of P. marinus SS120 [ada gene, which may be responsible for the shift in base composition, but also possibly several others, not necessarily involved in GC to AT mutation repair  and were secondarily lost in the LL-adapted lineages.Later during evolution (around 80 million years ago, according to the degree of 16S rRNA sequence divergence between MED4 and SS120) one LL population which probably already had a significantly reduced cell and genome size must have progressively adapted to the HL niche and eventually recolonized the upper layer. How this change in ecological niche was possible is still hard to define. Comparison of the gene set that differs between the LL-adapted SS120 and the HL-adapted MED4 shows that very few genes might be sufficient to shift from one to the other niche, including a multiplication of li genes  and the Prochlorococcus has similar features to that in host-dependent prokaryotes: genome reduction, bias toward a low G+C content, acceleration in the evolution rate of protein-coding genes, and loss of DNA-repair genes. In contrast to the latter organisms, however, in Prochlorococcus this evolution does not appear to be the result of genetic drift or relaxed selection being exerted on some gene categories. Indeed, purifying selection is very efficient in Prochlorococcus, as rRNA genes have evolved at a similar rate in all genomes. Despite the decrease in G+C content and an accelerated rate of evolution of protein-coding genes, purifying selection must also act on these genes and avoid potentially deleterious mutations. We hypothesize that a reduction in genome size  can constitute a selective advantage for life in the open ocean, both at depths where photon energy is low and in surface waters where nutrients are scarce.Genome evolution in the free-living genus Prochlorococcus has led to populations highly specialized to narrow ecological niches, at the expense of versatility and competitiveness in changing conditions. Indeed, not only is the distribution of the Prochlorococcus genus limited to low latitudes  but theProchlorococcus marinus MED4, P. marinus SS120, Prochlorococcus sp. MIT9313 and Synechococcus sp. WH8102 (accession numbers: NC_005071, NC_005072, NC_005042 and NC_005070 respectively) were downloaded from the Genome division of the NCBI Entrez system. A few additional genes which were modeled in at least one genome and were present in the other genomes but not modeled  were included in our dataset .The complete genome sequences and annotations of Genome sequences translated in their six reading frames were aligned with the Promer program of the MUMmer 3.0 system .Codon usage was computed for every open reading frame (ORF) of each genome with the EMBOSS program cusp. Amino-acid usage was derived from the results produced by cusp.We used a sequence-similarity based approach which is similar to the procedure used for the cluster of orthologous groups (COGs ). For eaProchlorococcus genomes (hypothesis of the molecular clock). The same analysis was applied to orthologs of each functional category.Protein sequences from each of the groups of four orthologous genes were aligned using ClustalW  with defdS) and non-synonymous (dN) substitution rates were obtained from the Yn00 program of the PAML 3.13 package [Nucleotide sequences of each group of orthologs were aligned with Protal2dna according to alignments of their corresponding amino-acid sequences . Pairwis package .The following additional data are available with the online version of this article. Additional data file The orthologous genes classified by functional categoryClick here for additional data fileA a fasta file of orthologous genes which were modeled in at least one genome and present but not modeled in the other genomesClick here for additional data file


Haemophilus influenzae. The list has grown considerably since then: add over 160 bacterial species (and counting), most major model organisms, and an ever-growing list of mammals—including, of course, humans. With 99% of our genome now fully sequenced, the Human Genome Project's next major goal is to identify all the functional elements contained in our 2.85 billion nucleotides. Such an effort is hardly trivial: producing the sequence of a mammalian-size genome can run from $10 to $50 million, the estimated price tag of the Cow Genome Project.It's hard to believe it was just ten years ago that scientists reported the first complete genome sequence of an organism, the bacterial pathogen In an ideal world, any organism would be fair game for sequencing, but in the real world, sequencing resources are scarce. Comparing genome sequences turns out to be a great way to identify regions that have important functions, but comparative genomics studies would be far more efficient if scientists could figure out in advance which genomes would reveal the most information about a particular question. Taking up that challenge, computational biologist Sean Eddy reports a statistical model that predicts how many genomes, and at what evolutionary distance, are needed for effective comparative genomic analyses. In addition to confirming some working principles of comparative genomics, the model also reveals a surprisingly simple guideline for future studies.Comparative genomics works by aligning sequences of different organisms to identify patterns that operate over both large and small distances. Aligning mouse chromosomes with human chromosomes, for example, shows that 99% of our protein-coding genes align with homologous sequences in mice. Underlying such analyses is the principle that DNA sequences that are highly conserved are likely to be functionally important. A common assumption is that adding more comparative genomes to the alignment helps distinguish functionally significant from irrelevant conserved sequences.How do you go about creating an abstract model that captures what Eddy calls the “essential flavor of comparative genomic analysis”? His model puts aside the specific characteristics of individual organisms, genomic features, and analysis programs in favor of identifying higher-level patterns and scaling relationships, specifically between the number of genomes, evolutionary distance, and feature size (features include genetic elements like exons and transcription factors).The model shows that the number of genomes required to identify conserved regions—that is, regions evolving under selection—scales inversely with the size of the feature being sought. Thus, to look for conserved sequences half as long, you need twice as many genomes, assuming a constant evolutionary distance and statistical power. For example, to identify a conserved human feature the size of a coding exon (about 50 nucleotides), it is sufficient to compare just the human and mouse genomes. But to identify conserved single nucleotides, you would need 55 comparative genomes at “mouse-like” evolutionary distances (roughly 75 million years).Things get a little trickier when varying evolutionary distance. We can see a substitution only at a given point in time: we can't tell how many times a site has changed, for example, or whether it changed at some point and then changed back. But at short evolutionary distances—where it's safer to assume no sites have changed more than once—the evolutionary distance is roughly the same as the fraction of sites identified as changed, and evolutionary distance and the number of genomes needed scale inversely. Therefore, the closer the evolutionary distance, the more genomes needed: one would need seven times as many comparative genomes using human/baboon distances, for example, compared to human/mouse distances. So when it comes to using primate sequences to study the human genome, our most distant relatives (such as lemurs) offer far more comparative analysis power than our next of kin (chimps and bonobos).While this model confirms the intuitive assumption that identifying smaller features requires more genomes, it reveals an inverse scaling relationship far more direct, and precise, than previously imagined. With the next phase of the Human Genome Project under way, Eddy's model offers valuable guidelines for identifying which genomes and how many might best meet this ambitious goal.


Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse  Through genetic manipulation, these authors have reduced signaling by bone morphogenetic factors in joint regions, and created a valuable model for the study of arthritis Thin layers of articular cartilage line the bones of synovial joints and provide a smooth, wear-resistant structure that reduces friction and absorbs impact forces . Loss orJoint formation begins during embryogenesis, when stripes of high cell density called interzones form across developing skeletal precursors . ProgramWnt14 is expressed in stripes at the sites where joints will form, and it is capable of inducing expression of other joint markers when misexpressed at new locations in the limb  containing the Gdf5 locus was modified by homologous recombination in bacteria to insert a cassette encoding Cre-internal ribosome entry site (IRES)-human placental alkaline phosphatase (hPLAP) into the translation start site of Gdf5  line used for all subsequent breeding experiments was seen to precede LACZ expression during successive development of joints in the digits  mice to analyze the pattern of Cre-mediated lacZ recombination throughout development. Joints in developing limbs begin forming in a proximal-distal pattern such that the shoulder joint forms prior to the elbow joint. In addition, three major stages of early joint development have been defined by histology as (1) interzone formation, (2) three-layer interzone formation, and (3) cavitation (Gdf5 in interzone regions during embryonic development (Gdf5 gene are not as well characterized, but Gdf5 expression has previously been detected in adult articular cartilage using both RT-PCR and immunocytochemistry (GAC(A) mice were crossed with vitation . Consistvitation B and 1C,vitation . Sectionvitation D and 1E elopment . Adult ehemistry .lacZ expression. Starting at E13.5, LACZ activity is detected in an anterior and posterior domain of the limb bud . Previous studies have shown that the recombined floxPBmpr1a allele mimics a null allele of the Bmpr1a locus when transmitted through the germline . BMP signaling is known to be required for growth of the external ear of mice . Finally, mutant animals showed obvious skeletal changes in whole-mount skeletal preparations. At some sites in the ankles, joints seemed to be missing entirely, with fusion of bones that would normally be separate. For example, the second distal tarsal was fused to the central tarsal bone in every conditional knockout animal examined (18 of 18), a phenotype not observed in controls (zero of 18) (Bmpr1a function is required for the formation of specific joints in the ankle region and for either generation or maintenance of articular cartilage in most other joints of the limb.The viable  of mice , and thio of 18) B and 3C.o of 18) B–3E  is down-regulated in the interzone region .In most joints of ions see C, no chaions see D, no difCol2a1 and Aggrecan (Agg), the genes encoding the major structural proteins of cartilage matrix , a gene expressed specifically in the periarticular and perichondral regions of developing joints . Less severe reductions were also seen in articular cells of tarsals and metatarsals in the hindfeet (unpublished data). By 2 wk of age, Col2a1 expression was reduced in most cells of the articular region  and Collagen 10 (Col10a1) (Collagen 1 (Col1a1) expression, increase proliferation, and result in cells with flattened, fibroblast-like morphology . While recombined LACZ marker expression was detected in most articular cartilage cells, it was also observed in scattered subarticular chondrocytes, growth plate chondrocytes, and osteoblasts . Together, these data suggest that BMPR1A activity is required in postnatal joint articular cartilage to maintain expression of many genes encoding structural components of cartilage matrix.By 1 wk after birth, obvious differences began to be detected in the articular regions of mutant animals. The expression of r region L and 5Q,r region K and 5P,Col10a1) M and 5R Col10a1) . Inhibitrphology . Howevereoblasts O and 5T Sox9 is required for normal cartilage differentiation, for expression of cartilage extracellular matrix (ECM) genes including Agg, and is a direct transcriptional regulator of the key cartilage matrix gene Col2a1 , toward the regions where surface articular cartilage was severely eroded or missing .Agg and Col10 expression were all reduced in mutant articular regions of the forefeet and hindfeet by 7 wk of age, and the beginning signs of cartilage loss were observed (unpublished data). By 9 mo of age, many regions of articular cartilage were completely missing or extremely fibrillated, leaving regions of exposed bone on the surface . The overall shape of mutant knee skeletal elements appeared similar to controls, although the fibrocartilaginous meniscus that resides between the femur and tibia appeared much less dense in mutants at E16.5. Some cartilage formed in the meniscus region, but the size of these elements was greatly reduced and contained abundant cells with fibrous, noncartilaginous appearance (unpublished data). This reduction of the meniscus can also be seen in sections from 7-wk- and 9-mo-old animals . By postnatal day 7, Safranin O staining and Bmpr1a is required cell-autonomously in articular cartilage .At 7 wk of age the normally domed tibial epiphysis was flattened and depressed in the knees of mutant animals, markedly reducing the distance between the growth plate and articular surface F and 7I.floxPGdf5-Cre/Bmpr1a mutant animals showed a highly significantly reduced ability to grasp and remain suspended on a slender rod . Mutant mice also showed a clear decrease in the maximum range of mobility of two different joints in the digits, as assayed by passive manipulation . The structural, histological, marker gene expression, and functional changes in mutant mice demonstrate that BMPR1A is required for normal postnatal maintenance of articular cartilage.The histological signs of joint arthritis were accompanied by functional impairments in both grasping ability and range of motion in mutant animals. Gdf5-Cre recombination system bypasses the early embryonic lethality of Bmpr1a mutations, and provides important new information about the role of this receptor in limb and skeletal development.Previous studies suggest that BMP signaling is involved in a large number of developmental events. Many of these events occur early in embryogenesis, and complete inactivation of BMP receptors causes death by E9.5 . The GdfBmpr1a with Gdf5-driven Cre include webbing between digits, lack of joint formation at specific locations in the ankle, and failure to maintain articular cartilage after birth, resulting in severe arthritis. Previous studies have shown that manipulation of BMP signaling alters interdigital apoptosis during development of the limb, but no experiment has identified a specific member of the BMP signaling pathway that is required for this process  and the R26R LACZ marker (expressed following Gdf5-Cre recombination) suggests that recombination-stimulated changes in gene expression may be delayed for a 0.5–1 d in the digit region  type II receptor also disrupts normal articular cartilage maintenance  followed by IRES-hPLAP (from plasmid 1726, gift of Oliver Bogler) directly behind the ATG start site of Gdf5. In the process, 583 bp of the first exon of Gdf5 was removed and no functional GDF5 protein is predicted to be produced. The 5′ homology arm was subcloned from a PCR product tailed with XhoI and Bsp120I restriction sites that contains 781 bp of 5′ genomic Gdf5 sequence ending at the ATG translation start site . Cre was subcloned from a 1.1-kb Bsp120I/EcoRI fragment of pML78. IRES hPLAP was subcloned from a 2.1-kb PCR product tailed with EcoRI and SpeI sites that contains the hPLAP translation stop site . The 3′ homology arm was subcloned from a 0.8-kb PCR product amplified from a 0.9-kb XhoI Gdf5 genomic subclone containing part of the first exon and downstream intron. The forward primer contains the 3′ end of the first exon and is tailed with a SpeI site; the reverse primer is from the T7 promoter of the vector containing the 0.9-kb subclone and flanks the intronic XhoI site . The targeting construct was built and verified in pBSSK , then digested with XhoI and subcloned into pSV1, the vector used for homologous recombination (A mouse 129x1/SvJ BAC library (Invitrogen) was screened to identify a 140-kb BAC from the  E. coli  to placebination . SoutherloxP site present in the BAC vector, pBeloBAC11, was removed to prevent the addition of undesired Cre target sites into the genome. To do this, BAC DNA was prepared by CsCl separation, digested with NotI to free the insert from the vector, and size-fractionated over a sucrose gradient. Aliquots of fractions were run on a pulse-field gel and Southern blotted using vector-specific DNA as a probe. Fractions containing unsheared insert and almost no detectable vector DNA were dialyzed in microinjection buffer (10 mM Tris [pH 7.4] with 0.15 mM EDTA [pH 8.0]) using Centriprep-30 concentrators (Millipore, Billerica, Massachusetts, United States). This purified insert DNA was adjusted to 1 ng/μl and injected into the pronucleus of fertilized eggs from FVB/N mice by the Stanford Transgenic Facility. Transgenic founder mice were identified by PCR using Cre-specific primers 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′, which amplify a 725-bp product, and were assessed for absence of BAC vector using vector-specific primers 5′-CGGAGTCTGATGCGGTTGCGATG-3′ and 5′-AGTGCTGTTCCCTGGTGCTTCCTC-3′, which amplify a 465-bp product. Three lines of Gdf5-Cre mice were established and maintained on the FVB background. Matings with R26R Cre-inducible LACZ reporter mice . Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount  and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the Tissue from animals ranging from stages E14.5 to P14 was prepared for analysis by fixing in 4% paraformaldehyde (PFA) in PBS for 45 min to 4 h depending on the stage; washing three times in PBS, once in PBS + 15% sucrose for 1 h, and once in PBS + 30% sucrose for 2 h to overnight depending on the stage; and then freezing in OCT. Tissue from animals aged 7 wk to 9 mo was processed similarly to earlier stages except that it was decalcified in 0.5 M EDTA (pH 7.4) for 4 d prior to incubating in sucrose. All solutions were prechilled and used at 4 °C with agitation, and skin from tissues of P0 or older mice was lacerated or removed prior to processing.Tissue was then cryosectioned at 12 μm and processed. Staining of sections with Safranin O, Fast Green, and Harris' hematoxylin was carried out using standard histological procedures. Detection of LACZ activity with X-Gal was performed as described  and was Bmpr1a .RNA in situ hybridization was performed as described , with thBmpr1a , Col2a1   in PBS (pH 5) at 37 °C for 30 min to 2 h depending on the stage. Slides were then washed in PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30 min, washed, blocked with PBS + 0.05% Tween20 + 5% goat or fetal bovine serum, washed again, and incubated with primary antibodies in PBS + 0.05% Tween 20 + 1% goat or fetal bovine serum overnight at 4 °C. Biotin-labeled secondary antibodies (Vector Labs) were tagged with HRP using the Vectastain Elite ABC kit (Vector Labs) followed by detection with DAB (Vector Labs). Primary antibodies and dilutions used were: goat anti-mouse MMP13, 1:100 ; rabbit anti-human SOX9, 1:500 ; rabbit http://www.ncbi.nih.gov/Genbank/) accession numbers for the genes discussed in this paper are Gdf5 (AC084323) and Bmpr1a (NM_009758).GenBank (


The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two  HCMV is a member of the betaherpesvirus family ,48. OtheSor OriL) and a DNA cleavage/packaging signal [cis-acting functions can be relatively small ranging from ca. 90–150 base pairs (bp) for the ori and ca. 250–300 bp for the a sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp [a sequence varies in size from ca. 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis-elements which determine the sites for cleavage of replicated viral DNA are present [Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level ,43,52,67g signal ,57,60-62 1500 bp ,37. HCMV 1500 bp . The HCM present ,58,64,65et al. 2003 [In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses . Howeveral. 2003 , describHCMV infects cells of the hematopoietic lineage ,39,55,68varRIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) , under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data).HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV Townea sequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2  between the EagI and SmaI site of the β2.7 gene taken from Toledo (G.M. Duke, unpublished data). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb DraI fragment of TownevarRIT (corresponding to nucleotides 91,166 – 95,909 relative to AD169) , and cloned into the HCMV amplicon Tn9-8 at the PstI site in both orientations. The gpt gene in Tn9-8-gpt was derived by cloning a PCR fragment from Escherichia coli DH5α using the primer pairs 5'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3'and 5'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3'.Plasmid pON205 (Spaete and Mocarski, 1985), contains the Towne strain ) Figure , spannin et al., . The pla4 precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 106 passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1.Plasmid DNA was transfected by CaPOViral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), , and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985).5 CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18–72 hours. At the end of the incubation the cells were harvested for CD34 staining.The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was >95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove's modified Dulbecco's Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection.a sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection.In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and EcoRI E fragment of Towne that was not present in the Toledo strain [EcoRI E region spans in part the complex oriLyt region [BamHI sites in Figure During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the o strain . The Ecot region ,2,24,37.t region . This secis-acting functions required for the propagation of the defective virus genomes in the presence of helper virus  [EcoRI site of pON205. The resulting amplicon was designated Tn9-8    cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with  blotted . Southera Figure , lane 2.r Figure . This regpt), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8-gpt amplicons did not enhance recovery.Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8-gpt), digested with Pst I and Hind III, respectively, in order to analyze monomeric repeat units. The Hind III-digested DNA was circularized by ligation and used to transform E. coli bacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input . Digestion with NaeI produced a fragment of the predicted size of a unit length a sequence (762 bp), and this product hybridized with an a sequence specific probe (PstI-SgrAI fragment from Tn9-8),  promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection  promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated  population infected with either TN9-8GF5 amplicon containing viral stocks (0.8%, 0.1%) or with the CMV-EGFP virus, RC2.7EGFP (3.6%)  Figure , &7b or ) Figure  &7c. The) Figure , and 7e.We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells.Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells CD34+) and other dividing and non-dividing cell types which may support HCMV infection + and oth,39,55,68in vivo and in vitro [ex vivo culture conditions, which preserve the developmental properties of the stem cells [in vitro with HCMV [in vitro culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitro showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population [in vitro culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vitro . Furtherin vitro . The HCMin vitro . The appem cells ,22. Umbiem cells . In our ith HCMV , reportepulation . In our tion Fig , &7c. Itcis-acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks [Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines ,46,50,66e stocks ,63. ThesThe authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest.a' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the '


Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.Although A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatusThe vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in  Aspergillus fumigatus is a saprophytic filamentous fungus that has become the leading mould pathogen in leukemia treatment centers and transplantation units in developed countries, second only to Candida spp. as a cause of systemic mycosis [Aspergillus case-fatality rate demonstrated that more than 50% of patients die with, or as a result of, aspergillosis, despite having received the reference standard of therapy [ mycosis . Despite therapy . The conA. fumigatus genome is expected to greatly facilitate efforts to determine the contribution of specific gene products to the virulence of this opportunistic pathogen. Unfortunately, the genetic tractability of A. fumigatus has lagged behind some other fungal systems, particularly in the area of conditional expression systems. Inducible promoter systems have proven to be instrumental for the elucidation of gene function in a number of species, most notably with essential genes. Experimental manipulation of gene expression in A. fumigatus is presently accomplished through the use of DNA cassettes that are introduced into the organism as transgenes [alcA promoter from A. nidulans has been successfully used in A. fumigatus [alcA promoter can have significant effects on the metabolism of the organism and thus remain a concern for many applications, particularly for in vivo studies.The completion of the annotated sequence of the ansgenes -5, inseransgenes ,6 or expansgenes . An induumigatus . HoweverE. coli tetracycline-resistance operon, a regulatory unit that detects minute concentrations of tetracycline and mounts an appropriate resistance response. Expression of the operon is controlled by a repressor protein, TetR that binds to operator sequences (tetO) in the promoter/enhancer region of the operon and prevents transcription. In the presence of tetracycline TetR is unable to bind tetO, which releases the repression and allows the operon to be expressed. This system has been adapted for experimental gene regulation in eukaryotes by fusing TetR to the VP16 transcriptional activating domain of herpes simplex virus VP16, thereby creating a synthetic tetracycline-regulatable transcriptional activator protein (tTA) that can be used to regulate a gene that is under the control of a tetracycline-responsive promoter  linked to a 175 bp minimal gpdA promoter from A. nidulans  and one showing moderate hygromycin sensitivity (tTA-1). Conidia from each of these transformants were spotted into the center of a plate of minimal medium containing both doxycycline and hygromycin and the radial growth of the colony was monitored with time. The pH of the medium in this experiment was adjusted to 8 in order to maximize the hygromycin toxicity. As shown in Fig. A. fumigatus .Inducible promoter systems are particularly useful for creating strains that can be inducibly depleted of an essential gene product . To modeulans p48, Fig. 2.tus Fig.  can be uhph RNA than tTA-2  and/or basal expression from one or more integrated copies of the tetO7-hph reporter gene (which would not be affected by doxycycline). Since there was a clear dose-response effect of doxycycline on hph expression and hygromycin resistant growth in this strain  that requires interaction of the transactivator with tetracyclines before 'tet-on' . Unfortunditions , we chosnditions . One paric cells .tetO7-hph reporter . Doxycycline was incorporated into the medium at 100 μg/ml to ensure that the tetO7-hph transgene would be expressed at sufficient levels to protect against hygromycin toxicity. Approximately 15% of 27 hygromycin resistant colonies showed reduced growth when shifted to hygromycin medium without doxycycline, one of which was selected for further analysis. As shown in Fig. tetO promoter in A. fumigatus. A further increase in hygromycin resistance was achieved at 15 μg/ml of doxycycline, but concentrations above 15 μg/ml had no additional effect. Northern blots analysis confirmed that the levels of hph RNA in the rtTA transformant were increased by the addition of doxycycline to the medium .A recent report has shown that iron blocks the accumulation and activity of tetracyclines in bacteria . Since iH20 Fig. . This inS. cerevisiae [Candida albicans and C. glabrata are the only pathogenic fungi in which the system has been successfully applied thus far, however neither of these studies used the tetR-VP16 fusions upon which the tTA and rtTA systems are based [The tetracycline-inducible method of gene regulation has become one of the most popular tools to manipulate gene expression in eukaryotes . The effrevisiae , the sysre based -17.A. fumigatus. Since the hph gene is essential in the presence of toxic levels of hygromycin, the ability to control hygromycin resistance by modulating the levels of hph transcription validates the system as a tool for analysis of essential genes in A. fumigatus. In the tTA system we found that individual transformants varied in the amount of doxycycline that was necessary to regulate expression of the tetO7-hph reporter gene. Since doxycycline prevents the tTA protein from binding to the tetO sequence, this is most likely due to variability in the amount of tTA protein that is expressed in each transformant. A limitation of the tTA approach described here is that the majority of the hygromycin-resistant transformants from the tTA/tetO7-hph co-transfection were not susceptible to regulation by doxycycline. This may be due in part to leaky expression of the tetO7-hph reporter, caused by enhancers in the proximity of the integration site [gpdA promoter used in this study were too high to be removed by non toxic concentrations of doxycycline. Since lower levels of tTA expression are more readily suppressed by doxycycline, it is conceivable that a weaker promoter used to drive tTA would increase the frequency with which doxycycline-regulatable transformants can be isolated. Lower levels of tTA expression could also be accomplished by using a shorter segment of the gpdA promoter used in this study.In this study we show that both the tet-off (tTA) and tet-on (rtTA) systems can be used to regulate the expression of a hygromycin resistance reporter gene in ion site ,25. A setetO7-hph reporter gene was also observed in a strain expressing the reverse transactivator, rtTA. Concentrations of doxycycline from 2 μg/ml to 15 μg/ml gave a graded response of hygromycin resistance, indicating that A. fumigatus is responsive to concentrations of doxycycline that are similarly effective in S. cerevisiae [C. albicans [A. fumigatus. Only 15% of the hygromycin-resistant colonies from an rtTA/tetO7-hph co-transfection showed doxycycline-dependent hygromycin resistance however, suggesting that some of the hygromycin resistance was due to leaky expression of the tetO7-hph gene. Leakage of tetO7-regulated genes has been described in other systems, and is attributed to enhancers located in the proximity of the integration site that increase expression of the tetO-linked gene [tetO7-controlled genes regardless of whether they are integrated randomly in the genome or targeted to specific loci.The ability to quantitatively control expression from the revisiae  and C. aalbicans , raisingked gene ,25. ThisA. fumigatus. A limitation of the system was that only 10–15% of the transformants could be regulated by doxycycline, either when tTA or rtTA were used, emphasizing the need to screen for regulatable transformants. A recent approach to limit the problem of leakiness of a tetO-driven gene is the use of trans-silencer proteins comprised of fusions between tetR and a transcriptional silencing domain [A. fumigatus-derived trans-silencer protein into the co-transfection approach described in this study would improve the efficiency of the system.This report establishes the utility of the tetracycline-regulated system as an approach to regulate gene expression in g domain ,27. It iAll vectors are based on the pBluescript plasmid (Stratagene) and were linearized prior to transfection. PCR amplification of components were performed using standard amplification protocols using PfuTurbo DNA polymerase (Stratagene).tet operator sequence (tetO7) was PCR amplified from pUHD10-3 [aagcttgcgtatcacgaggccctttc and the reverse primer 5'-aagcttctcgacccgggtaccgag (added HindIII cloning sites are underlined) and cloned into the HindIII site of pBluescript. A 1.6 kb fragment containing a minimal gpdA promoter from A. nidulans (-175 relative to the ATG of the hph open reading frame), the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans, was then PCR amplified from pAN7-1 [gagctccccatcttcagtatattcatc (added SstI cloning site underlined) and reverse primer 5'-tctagatcgcgtggagccaagagcgg (added XbaI cloning site underlined) and cloned downstream of tet07 into the SstI and XbaI sites of the plasmid, creating p482. To minimize read-through from flanking sequences into tet07, a 280 bp segment of the terminator region of A. fumigatus cgrA [tet07 PCR to create p500. The cgrA terminator was PCR amplified from genomic DNA of A. fumigatus isolate H237 using the forward primer 5'aagcttacagcagaagaatctctc (added HindIII cloning site underlined) and reverse primer 5'ctcgagatgattcatgacgtatattc (added XhoI cloning site underlined), cloned into pCR2.1-Topo (Invitrogen), excised with HindIII, and inserted upstream of tetO7 in p482 to create p500.A segment containing seven copies of the pUHD10-3  with them pAN7-1  with fortus cgrA  was inseA. nidulans gpdA promoter was amplified from pAN7-1 [hph open reading frame) using the forward primer 5'-aagcttcggagaatatggagctt (added HindIII cloning site underlined) and the reverse primer 5'-gaattcggtgatgtctgctcaag (added EcoRI cloning site underlined) and cloned into pBluescript at the same sites. The tTA gene was then PCR amplified from pUHD15-1 [gaattctggcaatgtctagattagataaaag (added EcoRI cloning site underlined) and reverse primer 5'-atcatgtctggatcctcgcg  and cloned into the EcoRI and BamHI sites downstream of the gpdA (-679) promoter. A 280 bp segment of the terminator region of A. fumigatus cgrA [actagtacagcagaagaatctctc (added SpeI site underlined) and reverse primer 5'-gcggccgcatgattcatgacgtatattc (added NotI site underlined) and inserted into the SpeI and NotI sites downstream of tTA. To introduce phleomycin selection into this construct, a phleomycin resistance cassette containing the A. nidulans gpdA promoter, the Streptoalloteichus hindustanus ble gene encoding resistance to phleomycin, and the S. cerevisiae CYC1 terminator was amplified from pBCphleo  using the forward primer 5'-cctcaggcggagaatatggagcttcatcg and the reverse primer 5'-cctcaggaattaaagccttcgagcgtccc. The PCR product was cloned into pCR-Blunt II-TOPO (Invitrogen), excised with KpnI and XhoI and inserted into the PgpdA-tTA construct to create p444. The phleomycin cassette was excised from p444 with HindIII and re-ligated to create p473. To introduce hygromycin selection into p444, the phleomycin cassette was excised with KpnI and HindIII and replaced with a hygromycin resistance cassette (containing the A. nidulans gpdA promoter, the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans) that was amplified from pAN7-1 [ggtacccggagaatatggagcttc (added KpnI cloning site underlined) and reverse primer 5'-aagcttgcttgagagttcaaggaag (added HindIII cloning site underlined) to make p434.A segment of the m pAN7-1  (positiopUHD15-1  with thetus cgrA  was thenEcoRI and BamHI and replaced with an EcoRI-BamHI fragment containing the rtTA2s-M2 variant of rTA from pUHrT62-1 (generous gift from C. Berens, Erlangen, FRG) to create p474. To introduce phleomycin resistance into p474, the phleomycin resistance cassette was excised from p444 with KpnI and HindIII and cloned into the same sites in p474 to create p480. To introduce hygromycin resistance into p474, the hygromycin resistance cassette described in p434 was excised from an unrelated plasmid as a HindIII fragment and cloned into the HindIII site of p474 to make p502.The tTA gene was excised from p473 with A. fumigatus strains used in this study are listed in Table-Aspergillus minimal medium plates [4·4H20. For low-iron minimal medium, the FePO4·4H20 concentration was reduced to 0.45 μM.The m plates . This miA. fumigatus protoplasts as previously described [tetO7-hph reporter construct was co-transfected with 5 μg of the linearized tTA plasmid (p444), or 50 μg of the linearized rtTA plasmid (p474).Plasmids were introduced into escribed . FollowiAspergillus minimal medium agar containing hygromycin and doxycycline at the concentrations specified in the Figure legends. The plates were then incubated at 37°C, and colony diameter was measured with time. Radial growth rates were calculated from the exponential part of the resulting growth curves.For experiments addressing the effects of doxycycline on hygromycin sensitivity, ten thousand conidia were spotted onto the surface of hph gene expression, RNA was isolated from overnight cultures in minimal medium supplemented with the indicated concentrations of doxycycline by crushing in liquid nitrogen and extracting RNA from the crushed mycelium with phenol/chloroform. Twenty micrograms of total RNA were fractionated by formaldehyde gel electrophoresis as previously described [32P-labeled hph DNA probe under stringent conditions in 50% (v/v) formamide/5XSSC (1X SSC is 0.15 M NaCl/0.015 M Na3·citrate, pH 7.6)/2X Denhardt's solution/10% (w/v) dextran sulfate/1% (w/v) sodium dodecyl sulfate (SDS). The hph probe was an 800 bp EcoRI-BamHI fragment from pAN7-1 [hph open reading frame. Hybridization intensity was quantified with a Phosphorimager (Molecular Dynamics) and normalized for differences in gel loading by quantitating the relative levels of SYBR-green II-stained rRNA (Molecular Probes, Inc., Eugene, OR, USA).For analysis of escribed , transfem pAN7-1  containitTA tetracycline transactivatorrtTA reverse tetracycline transactivatorTetR tetracycline repressortetO TetR binding sequencehph hygromycin resistance geneble phleomycin resistance geneA. fumigatus, screening of transformants and drafting the manuscript. RB participated in plasmid construction. JCR contributed to the planning of the study. DSA conceived of the project and directed its design and execution. All authors have read and approved the final manuscript.KV participated in vector construction, gene transfer into


Basal Cell Carcinoma (BCC) is the most common carcinoma in humans. It accounts for 20% of carcinomas in men and 10–15% of carcinomas in women. Despite its high incidence, metastatic events are exceedingly rare. The reported frequency of metastatic dissemination is estimated at 0.0028–0.5 percent. Once metastasis is detected, there is a high mortality rate of 50% within 8 months.In this study, we present a case of simultaneous lung and parotid metastases of giant BCC primary located on the right medial canthus of a 62 year old female.Examination of the tumor located on the medial canthus obtained showed "adenoid BCC". Computed tomography (CT) was performed to evaluate parotid region for evaluation of parotid gland and chest. Parotid and lung metastasis were detected in CT. Routine labarotory tests and radiological investigations were done. There was no abnormal finding. We also investigated this patient with a bone scan , abdominal and cranial CT scans .Although metastasis of BCC is a very rare condition, this study reports a case of simultaneous parotid gland and lung metastasis originating from a giant BCC primary that was located on the right inner canthus of a 62 year old female. Basal cell carcinoma (BCC) is the most common carcinoma in humans and accounts for 20% of carcinomas in men and 10–15% of carcinomas in women. Approximately 75–86% of primary BCCs are found on the head or neck. The most common location on the head is the nose, specifically the nasal tip and alae. It constitutes 90% of periorbital malignancies [BCC arising on the medial canthus tends to be deep and invasive and may result in perineural extension and loss of optic nerve function. Pieh et al reported that the highest recurrence rates of BCC following attempted excision, (approximately 60%), was seen with lesions arising from the medial canthus since these lesions tend to be more invasive and difficult to manage . ReclusiA 62-year-old woman was referred to the Plastic and Reconstructive Surgery Department for treatment of a bleeding exophytic tumor located on the right inner canthus. She had had the lesion for approximately 11 years. Initially, the patient was treated with excision and primary closure ten years ago. At this time the tumor had a diameter of 5 cm. The tumor was diagnosed as adenoid BCC microscopically and surgical margins were tumor-positive. The patient was operated on two years later when the diameter of the recurrent tumor was 15 mm. Histological examination of this second specimen revealed an "adenoid BCC" with clear surgical margins.Although the tumor recurred again after the second excision, the patient neglected medical advice and did not undertake any treatment Figure . More reWe also investigated this patient with a bone scan , abdominal and cranial CT scans  and a thoracic CT. Multiple metastatic lesions were seen in the chest CT Figure .Examinations of the cardiovascular, gastrointestinal, neurological, urogenital and hematological systems and other parts of the skin were performed by physical and routine laboratory and radiological techniques. There were no abnormal findings. Biopsy was performed from the tumor located on the inner canthus and revealed "adenoid basal cell carcinoma" Figure . Also, FThe patient did not accept the offer of surgical treatment for the tumor on the inner canthus. She was referred to the Oncology Department and treated with radiotherapy and chemotherapy. The patient received approximately 6000 cGy of external beam radiation over 3 weeks totally. Also, chemotherapy was initiated with cisplatin and 5-fluoruracil. She was followed up with physical examination and CT scans for six months and there were no metastases to other organs. She is still being followed.Spates et al. noted that metastatic BCC was first reported in 1894 . As outl1) the primary tumor must occur in skin containing hair follicles and not the mucous membranes;2) metastasis cannot be by simple extension, but occurring at a site distant from the primary tumor;3) the primary tumor and the metastasis must have similar histologic appearances of basal cell carcinoma; and4) squamous cell features must not be present in the lesions ,8. The cWhile the usual BCC that gives rise to metastases is a large, ulcerated, locally invasive BCC of the head and neck that recurs despite repeated surgical procedures or radiotherapy, these features are not absolute prerequisites for metastasis . ImmunosTumors greater than 3 cm in diameter have a 2 % incidence of metastasis and/or death. This increases to 25% in those lesions more than 5 cm in diameter and to 50% in lesions more than 10 cm in diameter . The proThe author(s) declare that they have no competing interests.EC, conceived the study and coordinated the write-up and submission. AA participated in the writing of the manuscript. All authors read and approved the final manuscript.


Cigarette smoking prevalence among gay men is twice that of population levels. A pilot community-level intervention was developed and evaluated aiming to meet UK Government cessation and cancer prevention targets.Four 7-week withdrawal-oriented treatment groups combined nicotine replacement therapy with peer support. Self-report and carbon monoxide register data were collected at baseline and 7 weeks. N = 98 gay men were recruited through community newspapers and organisations in London UK.At 7 weeks, n = 44 (76%) were confirmed as quit using standard UK Government National Health Service monitoring forms. In multivariate analysis the single significant baseline variable associated with cessation was previous number of attempts at quitting (OR 1.48, p = 0.04).This tailored community-level intervention successfully recruited a high-prevalence group, and the outcome data compares very favourably to national monitoring data (which reports an average of 53% success). Implications for national targeted services are considered. Analysis of tobacco marketing has demonstrated lesbian and gay youth as an emerging target community , therebyGay men have been disproportionately affected by HIV/AIDS disease in developed countries. HIV risk-taking behaviour is associated with cigarette smoking among HIV-negative gay men , and amoThe National Health Service (NHS) Cancer Plan, a UK Government health strategy, recommends that Primary Care Trusts (PCTs) take a commissioning lead in forming local alliances involving community groups, harnessing community efforts, and dissemination of effective interventions. . In ordeThis innovative pilot study aimed to design, recruit to, and deliver a series of pilot smoking cessation group interventions and to evaluate outcomes using standard UK Government assessment criteria.Setting up and running specialist Smoking Cessation Clinics", part of the Smoking Cessation Training and Research Programme (SCTRP) at St Bartholomew's and Royal London School of Medicine and St George's Hospital Medical School. The programme of withdrawal-oriented treatment combines groupwork, nicotine replacement therapy  and ongoing peer support throughout. An initial information session is followed by 6 closed group sessions, setting a quit data for week 3. This pilot consisted of 4 delivered groups, and each group consisted of 7 closed weekly meetings each of 2 hours.The intervention was developed and delivered by a community-based volunteer-led charity in London UK, with a remit to promote the health of gay men. Potential acceptability and effectiveness were maximised by providing an NHS-approved programme adapted for an appropriate service wholly facilitated and attended by gay men. Seven volunteers experienced in delivering group interventions within the organisation were trained in the 3 day course "The service principle was for a non-judgemental environment where gay men could address socialising and gay social spaces, recreational drug use, sexuality and HIV and the impact of these on their motivations, and ability, to quit smoking. Several specific modifications were made to the taught model. Our intervention modified the SCTRP program's use of "Quit buddies" which promoted partnered support, instead creating "Quit cells" of 3 or 4 participants. This design modification was made in the light of other group interventions delivered by this community organisation in which reliance of a participant on more than one person for support has found to be more reliable. The information on Zyban was expanded to address contraindications with HIV antiretroviral combination therapies. Exercises from assertiveness training courses were imported to assist participants in clearly communicating the intention to remain a non-smoker. In general, group discussion and processes were focussed on culturally-specific contexts to gay men. A detailed intervention programme was written in order to promote consistency across the cycles of intervention delivery.Week 1: information on the course content as well as expectations of quit date are given along with information regarding potential side effects and how to deal with them. Week 2: what to expect when you quit and how to deal with reactions, information on the effects of carbon monoxide, preparation for quit date, personal action plan and the how to use a smoking diary. Week 3: information on how to use nicotine replacements, role play of assertive refusal of cigarettes, selection and formation of quit support cells, and personal statements of cessation. Week 4: group review of challenges of the first week of not smoking with reference to smoking diary and personal action plan, exploration of potential "alternative" support such as meditation and exercise, discussion of the challenges of drug use with respect to smoking cessation. Week 5: group review of previous week's experience, information on health benefits achieved to date and weight gain issues. Week 6: review of previous week's experience, identification of future sources of support. Week 7: review of previous week's experience, information of health benefits to date, elaboration on support sources, small celebration of the group's achievement.Twenty-four recruitment advertisements were placed in free London-wide and national gay press, and accompanying editorial and articles were secured to support the recruitment process.Prior to the initial session, participants were sent the required UK Department of Health self-completion Smoking Cessation Service NHS Client Assessment Form. Carbon monoxide readings were taken at each session from week 2, using the "Smokealyser" calibrated carbon monoxide register, and readings were used in addition to self-report data to confirm smoking cessation at week 7. All intervention attendees were asked to give written permission for data collection purposes and were given guarantees of confidentiality.All data were entered into SPSS for windows V11. In line with NHS monitoring data requirements, the percentage of successful quitters was calculated as those who gave carbon monoxide readings and confirmed they had quit at week 7 as a percentage of those who set a quit date for week 3. Variables were entered individually into univariate binary logistic regressions, with cessation outcome as the dependent variable and participant baseline characteristics, attitudes and behaviour, and nicotine replacement methods as independent variables. Variables with p values below 0.25 were then entered stepwise into a multivariate logistic regression, with 95% confidence intervals (95% CI) reported.Ninety-eight men registered to attend the intervention, and of these 76 attended at least the first session. Sixty-nine of men returned the assessment sheet, and the outcome analysis is of those 69 men.The mean age of participants was 37.1 years (range 23–63, SD = 7.2 years), and n = 63 (90%) reported their ethnicity as White. Forty-four men (64%) had been educated to degree level or higher, and n = 52 (75%) were in full time employment with a further 9 (13%) men medically retired, n = 5 (7%) unemployed, n = 2 (3%) in full time education and n = 1 (1%) retired. Seventeen men (25%) were entitled to free prescriptions (i.e. the welfare state pays for their prescribed medications). Sixty-five men (94%) reported that they drink alcohol, consuming a mean of 22.8 units per week (median = 20, SD = 19, range 1–120).The daily number of cigarettes smoked was as follows: 1–5 (n = 3, 4%); 5–10 (n = 5, 7%); 11–20 (n = 27, 39%); 21–30 (n = 21, 30%); 31–40 (n = 8, 12%); 41+ (n = 5, 7%). The first cigarette after waking was smoked during the following number of minutes after waking: 5 minutes (n = 19, 28%); 6–30 minutes (n = 31, 45%); 31–60 minutes (n = 7, 10%); 61+ minutes (n = 11, 16%). Smoking motivations are summarised in Table Participants reported a mean 2.6 of consultations with their primary care General Practitioner (GP) in the previous year (median = 2, SD 3.5). Secondary/hospital consultations in the previous year were reported by n = 35 (52%) men, with a mean of 2.26 consultations for these men (median = 1, SD = 3.9). Thirty-four men (51%) had been recommended by their GP to give up cigarette smoking, and n = 26 (38%) men were currently on prescribed medication. Fourteen men (20%) were diagnosed HIV-positive, n = 25 (51%) HIV-negative, n = 16 (23%) untested and n = 4 (6%) refused to answer. The participants rated their health as follows: excellent n = 10 (14.5%); good n = 36 (52%); moderate n = 20 (29%); poor n = 2 (3%); very poor n = 1 (1%).Sixty-one men (90%) had made a previous attempt to quit, and of those who had made an attempt the mean was 2.85 attempts (median 3, SD = 1.4). Previously employed nicotine replacement methods were gum n = 30 (49%), patches n = 30 (49%), nasal spray n = 3 (5%), inhalor n = 12 (20%), microtabs n = 3 (5%), nicotine lozenges n = 4 (7%), and Bupropion (Zyban) n = 12 (20%).Participants described the importance of this current attempt to quit as extremely important (n = 33, 48%); very important (n = 27, 39%); quite important (n = 9, 13%); not at all important (n = 0). Participants rated their chances of quitting for good on this attempt as extremely high (n = 10, 15%); very high (n = 27, 39%); quite high (n = 24, 35%); not very high (n = 7, 10%); very low (n = 1, 1%).Attendance at sessions was consistently high, of 532 person-sessions 13 sessions were missed. Non-attendance did not apparently cluster around a particular session.At week 3, of the 69 men who gave data, n = 58 men (84%) set a quit date. At week 7 (4 weeks after the quit date) n = 44 men (64%) were confirmed as having quit using the CO monitor, representing 58% of those who attended the first session, 76% of those who set a quit date and 64% of those who gave data at baseline and week 7.A further 3 men reported by telephone that they had quit smoking but did not attend the final session to give clinical data to verify. Nine men (13%) reported not having stopped smoking, n = 6 men (9%) set a quit date at week 3 and did not return to group, n = 7 men (10%) attended the first session only. For the purposes of this analysis, these 25 men were coded as not having quit in the following modelling.This analysis considers those 44 men confirmed as having ceased compared to those 25 categorised as not having quit. Following univariate analysis (see Table This pilot intervention has targeted a hitherto overlooked high smoking prevalence group, and has adapted a Government-approved intervention to meet the specific needs of gay men in an appropriate and acceptable setting. The success rate of 76% of men who had set a quit date being confirmed as having quit at week 7 compares extremely favourably to national monitoring data, which reports a success rate nationally 2001–2002 for smoking cessation services as 53% .Public health targets must consider the needs of high prevalence communities, and this may be achieved through innovative development of existing effective services. However, this study has highlighted the lack of targeted interventions for gay men, and the evidence demonstrates further elevated health needs compared to the general population in the fields of alcohol and drug use  mental hFurther research may identify the factors which contributed to the effectiveness of this pilot complex participative intervention, including offering recruitment and delivery outside of community settings, measuring success rates for gay men in non-gay specific or tailored groups, and the usefulness of "quit cells". Longer-term follow-up data and increasing dosage to include a follow-up session would also provide further useful data. In order to refine the intervention for trial testing, qualitative data regarding the utility, acceptability and preferences for the content of specific sessions would be illuminating. Further, the non-randomised design without comparison group limits presents a limitation to the generalisability of findings, yet still offers cessation outcomes much better than standard national cessation data quoted above which were collected without quasi-experimental design using the same follow-up period. Data were not available on the 29 men who registered for the course but did not attend or complete baseline data, and so it is not possible to compare their demographics or smoking behaviours to those who took up the intervention. Certainly, replication of this first pilot would be necessary in other settings, e.g. non-metropolitan communities, where issues of feasibility and uptake should be addressed. Commissioners may consider the purchase of existing facilitators from cities to deliver in non-metropolitan areas where demand is likely to be lower, as smoking cessation service recommendations state that group leaders need to keep up to date with their skills and to use them on a regular basis .In order to meet the smoking cessation needs of this hitherto overlooked population, and to meet public health policy targets, a rigorous research agenda must be established. While the use of required standard outcome monitoring must be continued, rigorous experimental trials using longer term follow up and commonly reported measures are required. Complex participative interventions must be developed, as in this pilot, from evidence-based interventions with full programme description to ensure replication. The development of appropriate interventions must first pilot services to ensure that they are appropriately adapted to maximise acceptability and uptake among target communities.Lastly, provision of the service by skilled volunteer facilitators has ensured an acceptable, low-cost intervention with a rate of effectiveness in these four pilot groups that compares favourably to national non-targeted interventions outcomes calculated using standard assessment formula. Acceptability of the model appears high with respect to the low number of missed sessions. Voluntary sector provision and delivery should be considered as a low-cost and highly acceptable point of delivery for effective community-level smoking cessation interventions.The authors declare that they have no competing interests.JB and NH managed the pilot study. RH was responsible for data management and analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: