Gene-based (genic) microsatellites are a useful tool for plant genetics and simple sequence repeat loci can often be found in coding regions of the genome. While EST sequencing can be used to discover genic microsatellites, direct screening of cDNA libraries for repeat motifs can save on overall sequencing costs. The objective of this research was to screen a large cDNA library from and Andean common bean genotype for six di-nucleotide and tri-nucleotide repeat motifs through a filter hybridization approach and to develop microsatellite markers from positive clones.Robotics were used for high-throughput colony picking and to create a high-density filter of 18,432 double spotted cDNA clones which was followed by hybridization with repeat motif containing probes based on GA, CA, AAT, CAG, CAA and ACG repeats. A total of 1203 positive clones were identified by their addresses and sequenced from 5' ends and if required from 3' ends to confirm repeat motif and length. Out of 886 high quality sequences, 497 had complete microsatellite loci that were not truncated by the sequencing reaction and of these tri-nucleotide repeats were more common than di-nucleotide repeats. Different motifs were found in different frequencies in the 5' and 3' ends of the cDNAs. In a microsatellite development program, primers were designed for 248 SSR loci which were tested on a panel of 18 common bean genotypes to determine their potential as genetic markers finding higher average polymorphism information content for di-nucleotide repeat markers (0.3544) than for tri-nucleotide repeat markers (0.1536).The present study provides a set of validated gene-based markers for common bean that are derived from G19833, an Andean landrace that is an important source of disease and abiotic stress tolerance which has been used for physical map development and as a mapping parent. Gene-based markers appear to be very efficient at separating divergent wild and cultivated accessions as well as Andean and Mesoamerican genepools and therefore will be useful for diversity analyses and for comparative and transcript mapping in common bean. Microsatellites are molecular markers based on simple sequence repeat (SSR) loci that can be isolated from both coding and non-coding regions of the genome . When SSGenic microsatellites are distinguished from genomic microsatellites by being related to the transcriptome . In otheAmong the food legume crops known as pulses, common bean is the most extensive and widely consumed . After oA small number of genic microsatellites have been developed for common bean by searching gene database sequences as was done by Yu et al. ,15 and BIn terms of diversity assessment, Blair et al.  found geThe objective of this research, therefore, was to increase the number of gene-based microsatellites through a cDNA library screening approach followed by germplasm screening. For this, multiple di and tri-nucleotide repeat containing probes were used to screen for SSR containing genes from a large scale cDNA library developed from multiple source tissues of an Andean genepool genotype (G19833 from race Peru). An Andean genepool genotype was also used as they have been less well studied than Mesoamerican beans for which a range of EST collections have been generated. Newly-developed microsatellite markers were evaluated on a diversity panel to determine their utility in diversity assessment.NotI/SalI sites of the plasmid vector, pCMVSport6.0 from Invitrogen Life Technologies  using DNA ligase. The library, now named PV_GEa was transformed into E. coli EMDH12S cells which were plated at a density of 3000 cfu per plate onto Q-plates with Carbenicillin (300 μg/mL), x-gal and IPTG [Library development was as described in Ramírez et al. . Brieflyand IPTG . A Q-Bot20 and (GA)20; and four that targeted tri-nucleotide repeat motifs, namely (AAT)14; (CAG)14; (CAA)14; and (ACG)14, where the sequence within the parenthesis indicates the repeat and the number outside the parenthesis indicates the number of copies of that repeat. Probe preparation and hybridization consisted in end labeling the simple sequence repeat motif probes with T4 DNA Kinase and hybridizing the mixture to the DNA contained on the filters with standard protocols from Edwards et al. [32-ATP in a total reaction volume of 20 ul that was incubated at 37°C for 80 minutes and stopped at 65°C for 20 minutes. The labeled oligo-nucleotide was added directly to the pre-hybridizing filters and incubated at 60°C for 12 hours.Six simple sequence repeat motif oligonucleotide probes were used to screen the cDNA library filters. The probes included two that targeted di-nucleotides repeat motifs, namely (CA)s et al. . BrieflyBoth the di-nucleotide motif probes were grouped together for filter hybridization as were all the tri-nucleotide motif probes. After the hybridization step, the filters were washed twice at 60°C with 6× SSC/0.1% SDS for 5 minutes each. Longer washes were used when signal was intense . The filters were blotted dry, covered with saran-wrap and arranged face-up in cassettes along with three sheets of X-ray film. The films were taped to each other so that they would not shift during the 0 to 8 day exposure in a -80°C freezer. Films were developed after three days to identify positive clones. Filters were re-used for sequential screening of different oligo-nucleotide repeats by stripping them between each use. Stripping consisted in washing the filters at room temperature in 100 mM NaOH, 10 mM EDTA, 0.1% SDS twice, followed by a 5× SSPE  rinse for 10 min.Positive clones were identified by which filter they were on; which field within the filter they were in, and what address they had within the field. Clones could be identified by their position in the double-replicate 4 × 4 pattern found at each grid axis in the address system. Only double-spotted clones were selected. Any spots for which the replicate did not hybridize were considered false positives and were not selected. Putative simple sequence repeat-containing clones were picked from their appropriate position in the 384-well plate format glycerol stocks. DNA was extracted from an overnight culture by standard alkaline lysis procedures according to Sambrook  and usedhttp://www.gramene.org/ to screen for all possible repeats with sequences containing a minimum of five di-nucleotide, four tri-nucleotide or three tetra-nucleotide motif repeats used for primer design. Primer 3.0 http://www.genome.wi.mit.edu/ was then used to design primer pairs which would produce PCR amplification fragments that were on average 150 bp long. All PCR primers had consistent melting temperatures of 55°C or above, average lengths of 20 nucleotides or more and primer pairs were checked to make sure that they had similar melting temperatures and did not suffer from palindromes or end pairing. The newly-designed Microsatellites were tested against a standard survey of 18 genotypes from Blair et al. [SSRs were found in the sequenced clones using the SSR identification tool (SSRIT) available at r et al. , among wr et al.  and was Standard PCR conditions from Blair et al.  were the6.The cDNA library used for this research consisted of 18,432 clones which were arrayed on filters of 36,864 spots each representing 48 plates of 384 clones for a total of 18,432 unique addresses. The preparation of the high-density filters was made possible by the high-throughput and automated nature of the robot used to pick colonies and array the final clones from the plate system to the filters. In addition, the arraying and screening of the library was facilitated by the high quality of the cDNA library itself which was reflected in all the clones (100%) having inserts, in the average insert size of 1.3 kb and in the transformation efficiency of 3.85 × 10Hybridization of the cDNA library with SSR-containing probes detected a total of 1203 positive clones of which 886 were successfully sequenced. These represented 6.5 and 4.8% of the total number of clones in the library, respectively. The read length for the sequences was on average over 700 nucleotides. However, where sequences were short or of low quality it was usually because of difficulty in sequencing through simple sequence repeats from the 5 'end of the cDNA clones or through mononucleotide stretches, such as poly A tails encountered in sequencing from the 3' end. SSR loci were sequenced successfully and clearly identified for a total of 497 clones which represented 2.7% of the full cDNA library and 41.3% of the hybridizing clones. The frequency of individual SSR motifs found in the 5' and 3'sequencing is shown in Table In terms of repeat length, totals of 209 di-nucleotide and 224 tri-nucleotide motif SSRs were found in the successful sequences representing 42.1 and 45.1% of the total number of successfully sequenced clones, respectively with the remainder being tetra- or penta- nucleotide repeats similar to the di-nucleotide repeats we used as probes. Among the di-nucleotide repeats there were approximately three times more AG motif microsatellites (124) than AC motif microsatellites (48), and only a few AT microsatellites were found (24). Interestingly, while the AG and AC motif microsatellites were much more likely to be in the 5' sequences, about half of the AT microsatellites (11 out of 24) were found in the 3' sequences. Meanwhile, no CG microsatellites were found. Among the tri-nucleotide containing repeats, the most common by a factor of almost three was AGC with a total of 83 clones, followed by AAC and AGG with 36 and 31 clones, respectively. More than 20 clones each were found for AAG and ACC motifs; while less than 10 clones were found containing AAT or CCG motifs. The proportion of tri-nucleotide SSR at the 5' end was generally high, between 80 and 96%, except for the AAT motif for which it was 22%.Bean Microsatellites based on cDNA clones and with a consecutive number starting at 121 and ending at 368 to distinguish from previous SSRs analyzed in Ramírez et al. [The most promising positive SSR containing clones were used to design microsatellite primers for the development of new markers for common bean  or ESTs from common bean 210). The number of repeats for each of the repeat types is shown in Figure 0. The nuMost of the microsatellites targeted were simple rather than compound with a total of 203 microsatellites (81.6%) targeting a single motif and 45 microsatellites (18.4%) targeting more than one motif. Several of the microsatellites with a large number of total repeats detected compound SSRs, in the case of the di-nucleotides often this consisted of GA or CA motifs combined with AT motifs as was the case for BMc294 and BMc284. Meanwhile the high repeat number tri-nucleotide markers included compound SSR that had more than one repeat type such as BMc223 with the motif (CAG)10(GCA)6. A somewhat higher proportion (43/112) of dinucleotide markers fit the category of Class I microsatellites (10 or more repeats) compared to the proportion of trinucleotide markers (38/136) fitting this category (7 or more repeats).The diversity values for all the markers that amplified well were estimated based on the evaluation of 18 genotypes from the same mini-core panel evaluated by Blair et al.  that conPolymorphism information content (PIC) ranged from 0.000 to 0.9159, with the most polymorphic markers being BMc238, BMc292, BMc321, BMc171, BMc367, BMc294 and BMc226, all di-nucleotide repeat based markers with PIC values above 0.8000. The most polymorphic tri-nucleotide repeat based markers were BMc349, BMc229 and BMc335 with PIC values of 0.6171, 0.6268 and 0.6270, respectively. The average PIC value was higher for the di-nucleotide based markers (0.3544) than for the tri-nucleotide based markers (0.1563). The high PIC markers tended to be the SSRs with high repeat number and correlation between these values was highly significant . Allele number was even more closely correlated with repeat number . The distributions of PIC values and allele numbers are shown in Figure Among the accessions analyzed the most likely to be polymorphic using the new BMc markers were 1) the wild accession from Colombia G24404); 2) the wild accession from Mexico (G24390); and 3) the race Peru accessions, G19833, G21657 or Radical Cerinza and 4) the Mesoamerican landraces G11350 or G14519. Generally, there were more polymorphisms within the Andean germplasm than within the Mesoamerican breeding lines or germplasm accessions. Figure 404; 2) tThe approach taken here for gene-based microsatellite development was based on the targeted identification of SSRs in high-density filter arrayed clones from a large cDNA library. This was facilitated by the robotic picking of a large number of clones (over 18 thousand) and the automated library duplication and filter preparation features of the Q-bot. Meanwhile, the cDNA library we used was made to represent a wide range of vegetative tissue from three week old, greenhouse-grown plants including young and old leaves, stem and vascular tissues, as well as vegetative meristems and therefore as a multi-tissue library was appropriate to upscale with this technology. To picture the scale of the library, a total of nearly 24 MB of non-redundant transcriptome sequences were represented in the filter set based on the number of clones screened, the low redundancy of clones and the average cDNA insert size of 1.3 kb, which compares favorably to other cDNA libraries that have been used in EST sequencing projects .Hybridization of the cDNA library was straightforward in the filter format used. Positive hit addresses were confirmed through double replicate spots and the next product was a large number of SSR candidate loci which were then sequenced to confirm whether the hybridizing clones from each library had true SSR loci or were false positives. In this step, it was possible to discover the SSR motif and number of repetitions that each positive clone contained. It was important to sequence from both the 5' and 3' ends of the positive cDNA clones to obtain the full SSR and flanking sequences and to be able to design microsatellite primer pairs around the repeat motif. This was also important given the size of the cDNA clones, especially for the larger cDNA clones.Phaseolus in their search of the NCBI gene database; and also with those of Hanai et al. [The use of di-nucleotide and tri-nucleotide SSR probes for filter hybridization and the sequencing confirmation of clones allowed us to calculate the frequencies of various types of motifs in the cDNA library. The results showed that the frequencies of di-nucleotide and tri-nucleotide clones were almost equal in the cDNA library although the tri-nucleotide positive clones were somewhat more frequent than the di-nucleotide positive clones. These results contrast with those of Yu et al.  who founi et al.  who founi et al. . Genomici et al. .Our results suggest that tri-nucleotide repeat are more common in coding sequences than across the entire bean genome, which would be interesting given the triplet nature of tri-nucleotide SSR which is more compatible with the conserved nature of most open reading frames . Tri-nucIn terms of the frequency of individual motifs, the results indicate that GA motif microsatellites were more common than CA motif microsatellite in the sequences of the cDNA library used for this study with implications for the common bean transcriptome. The higher frequency found for GA motifs agrees with results of microsatellite development in several other plant species based on ESTs ,9,34. AnWhen comparing our results with those of Hanai et al. (2007) we can observe some similarities, as these authors found that AG motif SSR were four times more likely than AC motif microsatellites, that AT microsatellites were fairly common  and that no CG motif microsatellites were present in a set of 3,126 common bean unigenes. The differences between our study and theirs in terms of precise number of motifs may have been the result of the random nature of EST sequencing versus the hybridization approach we used, or because Hanai et al.  only seaAmong the trinucleotide repeats, we found AGC microsatellites to be very common, representing 37.1% of the total for the tri-nucleotide SSR as might be expected since this was one of the motifs used as a hybridization probe. The next most common tri-nucleotide motifs (AAC and AGG) were also used as hybridization probes, but were less than half as frequent as this previous motif, representing little more than 5% of the total SSRs found. Two fairly common motifs not used as hybridization probes, AAG and ACC were also identified with the library screening procedure, perhaps because they were similar to the di-nucleotide motif probes used. Finally small number of other tri-nucleotide motif SSRs were found as compound repeats with the more common tri-nucleotide repeats or with the di-nucleotide repeats that had been hybridized with. One interesting trend for the ATA tri- nucleotide motif which was also used as a probe was that these SSR were more likely in the 3' end of the cDNA clones than in the 5' end just as we found for the AT di-nucleotide repeats. No other tri-nucleotide motif presented this same situation and in most cases very few of the tri-nucleotide motifs occurred in the 3' end which is similar to results from Thiel et al. [These results differ from the relative tri-nucleotide motif frequencies found by Yu et al.  or HanaiAll together, these results give us a good idea of which motifs are common in cDNA sequences and at which end of the cDNAs they occur. The total percentage of ESTs that might be expected to contain SSRs is at least 6.52% based on the number of cDNA clones that hybridized with SSR probes in this study. If a larger number of hybridization probes of different motifs had been used, an even larger percentage of the cDNA clones would likely have been identified as containing SSR. The large size of the G19833 cDNA library and the comprehensiveness of the filter screening approach would make the discovery of further SSRs very likely and both library hybridization and EST sequencing as described in Ramírez et al.  will be The source of the RNA for the cDNA library was made from G19833, a race Peru landrace from the Andean genepool, which is one parent of a genetic mapping population that has been used to map microsatellite markers ,24, to sThe sequencing from both the 5'and 3'ends of positive cDNAs was especially valuable for discovering the full complement of microsatellites in the selected clones. This is in contrast to most other studies of gene-based microsatellites which in using the more commonly available 5' ESTs, may not give a complete picture of the frequency of microsatellites in expressed sequences, precisely because they rely on partial sequencing of the cDNA (ESTs). On the other hand, an advantage of EST mining of SSRs is the possibility of sampling genes represented in various cDNA libraries or genes expressed in different organs, at different developmental stages or under varying environmental conditions which can give a complete picture of transcribed genes . The useAnother practical result of this study was that the careful analysis of both 5' and 3' end sequences allowed us to develop a large set of new gene-based microsatellites for diversity assessment or gene tagging projects in common bean. In terms of genetic diversity, the markers detected a moderate number of alleles compared to genomic microsatellites we have evaluated on the same set of genotypes ,24, howeP. acutifolius, which belongs to the tertiary genepool of common bean . This suggests that the gene-based SSRs could be candidates for transfer beyond the boundaries of the tertiary genepool (to other Phaseolus species) or potentially to cowpea and other Vigna species. Supporting this hypothesis, some gene-derived microsatellites from common bean have been effective for diversity assessment in cowpea ; and EST-derived SSRs are known to be better for transferability among related species than genomic SSR [In addition to being valuable functional markers, the new microsatellites were highly transferable from cultivated to wild accessions. Testing of cDNA based microsatellites has shown them to also be transferable to tepary bean, omic SSR ,38.The overall success rate of marker development, from the step of sequencing to primer design was 57.3% for the di and tri- nucleotide markers which were targeted in our study. This is very similar to the success rate for SSR primer design from EST sequences from common bean (64.5%) reported by Hanai et al. . In thatPhaseolus species and for the development of a transcript map for common bean.In summary, this study was successful at determining the relative microsatellite motif frequency in cDNA libraries based on a hybridization approach. In addition, the study produced a large set of Andean-derived, gene-based microsatellites for functional diversity analysis which are complementary to previously developed genomic and Mesoamerican derived markers. The markers were of moderate polymorphism but were very efficient at separating divergent wild and cultivated accessions as well as Andean and Mesoamerican genepools. Multiple microsatellite development approaches are important in common bean given the dual genepool nature of genetic diversity in the crop and the fact that ascertainment bias is a common problem with microsatellites. Gene-based markers from common bean are predicted to be useful for further phylogenetic analysis of the crop, for transfer to related species especially other cultivated MWB designed the study, analyzed data and wrote the paper. MM performed sequencing and primer design and analyzed microsatellite frequency, MCG conducted genotype polymorphism survey and FP hybridized and selected cDNA clones for sequencing. All authors read and approved the final version of the manuscript.Common bean EST-SSR markers from the BMc series. Primer list, product size, blastn homology and GenBank entries for newly-developed SSR markers.Click here for file


The lateral entorhinal cortex receives inputs from ventral tegmental area dopamine neurons that are activated by exposure to food-related cues, and exogenously applied dopamine is known to modulate excitatory synaptic responses within the entorhinal cortex.in vivo synaptic field potential recording techniques to determine how exposure to cues associated with food reward modulates synaptic responses in the entorhinal cortex of the awake rat. Chronically implanted electrodes were used to monitor synaptic potentials in the entorhinal cortex evoked by stimulation of the piriform (olfactory) cortex, and to determine how synaptic responses are modulated by food-related cues.The present study used 1-like receptor antagonist SCH23390, or the muscarinic receptor antagonist scopolamine. However, the reduction in synaptic responses was blocked by injection of the D2-like receptor antagonist eticlopride.The amplitudes of evoked synaptic responses were reduced during exposure to cues associated with delivery of chocolate, and during delivery of chocolate for consumption at unpredictable intervals. Reductions in synaptic responses were not well predicted by changes in behavioural mobility, and were not fully blocked by systemic injection of either the D2 receptors.Exposure to cues associated with palatable food results in a suppression of synaptic responses in olfactory inputs to the entorhinal cortex that is mediated in part by activation of dopamine D Dopaminergic projections to the entorhinal cortex are likely to play an important role in modulating the manner in which the entorhinal cortex contributes to sensory integration and mnemonic processing. The entorhinal cortex provides the hippocampus with much of its cortical sensory input and is thought to play roles in sensory integration, memory formation, and spatial navigation-4. The lin vitro generally results in a suppression of glutamate-mediated synaptic responses in neurons in the entorhinal cortex that is mediated by activation of D2 receptors[1 receptor-mediated facilitation of synaptic responses in layer II of the entorhinal cortex in vitro[in vivo[Application of dopamine to acute brain slices eceptors,14-16. Seceptors, but a deceptors, or by receptors-22. In ao[in vivo. This raThe present study used palatable food reward as a stimulus to assess how the strength of synaptic inputs to the entorhinal cortex might be modulated following endogenous release of dopamine in response to anticipation and consumption of a natural reward. Exposure to food and food-conditioned cues activates dopamine neurons and results in increased dopamine in the nucleus accumbens and prefrontal cortex-30 and d2) and placed in a stereotaxic apparatus. A bipolar, twisted wire, Teflon-coated stainless-steel stimulating electrode (125 μm tips) was lowered into the right piriform cortex  and a bipolar recording electrode was lowered into the lateral entorhinal cortex . Vertical positions of electrodes were adjusted to maximize the amplitude of evoked field excitatory postsynaptic potentials (fEPSPs). A jeweler’s screw in the contralateral frontal bone served as a reference electrode, and a screw in the left parietal bone served as ground. Electrode leads were mounted in a plastic 9-pin connector and the assembly was fixed to the skull by embedding the jeweler’s screws, electrodes, and connector in dental cement. Buprenorphine  was administered following surgery. There was a two-week recovery period with free access to food following surgery. Beginning 1 week prior to testing, access to food was restricted[Fifteen male Long-Evans rats  were housed individually and had free access to food and water prior to behavioural testing, and experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care. Animals were anesthetised with isoflurane  was placed on top of the door. A 2.5 cm-diameter opening in the door allowed an aluminum tube (2 cm diameter) to be used to manually deliver chocolate chips (0.11 g) to a removable steel feeding cup  affixed to the floor 4 cm from the door.Each rat underwent five days of preliminary training to associate the feeding cup, aluminum tube, and cue-light with delivery of chocolate. On each day there were two 15-min counterbalanced sessions, one in which they received chocolate in the presence of the cue-light, feeding cup and delivery tube, and one in which these cues were absent and chocolate was not delivered. In order to deliver chips unpredictably with an Field potential recordings were used to assess the effects of exposure to chocolate-related cues and consumption of chocolate on synaptic responses in the entorhinal cortex. A computer digital-analog channel and a stimulus isolation unit  were used to deliver 0.1 ms biphasic constant-current square-wave pulses to the piriform cortex. A stimulus intensity that evoked synaptic responses that were 60 to 75% of maximal was determined for each animal . Evoked synaptic potentials were filtered (0.1 Hz to 5 kHz pass-band), amplified , and digitized at 10 kHz (12-bit) for storage on computer hard disk using the software package Sciworks (Datawave Tech.).1 antagonist SCH23990  or the D2 antagonist eticlopride . A final test was conducted following injection of the muscarinic receptor antagonist scopolamine . Peak amplitudes of averaged evoked field potentials were measured relative to the pre-stimulus baseline using SciWorks software. Planned comparisons (modified Bonferroni)[Beginning the day following the completion of training, testing sessions were conducted every 2 days following systemic injection of either saline or a receptor blocker. On each day, synaptic responses were recorded every 15 sec during four consecutive time periods. In a subset of 9 animals tested, the cumulative time that the animal spent immobile in these periods  was recorded using a stopwatch. There was a 15 min baseline period in the absence of the cue-light and feeding cup. This was followed by the cued period in which the cue-light was on and the feeding cup was introduced for 10 min, but no chocolate was delivered. The 10-min chocolate-consumption period began with delivery of a chocolate chip to the feeding cup, and animals were allowed to consume a total of 10 chips delivered at unpredictable intervals taken from the lagged exponential distribution described above . The cue light was turned off and feeding cup removed during the 15-min duration follow-up period. Tests on the first day were always conducted following a control injection of saline  15 min prior to recordings. The second and third tests were counterbalanced, and were conducted 15 min following administration of either the DAnimals readily consumed the food reward during preliminary training to associate chocolate with the cue-light and feeding cup. Between 2 and 8 of the 15 chips presented during the first testing session were consumed, and all animals consumed all chips presented in subsequent sessions.1). In animals injected with saline, the amplitude of evoked potentials was reduced to 95.6 ± 2.6% of baseline values during the cued period  and to 93.9 ± 1.9% of baseline values during the consumption period . Responses during the follow-up period showed a partial reversal and did not differ significantly from baseline values . The presentation of cues that predict delivery of chocolate, as well intermittent consumption of chocolate, are therefore both associated with a moderate suppression of synaptic strength within the entorhinal cortex.To assess the effects of anticipation or consumption of chocolate on the strength of synaptic inputs to the entorhinal cortex, the amplitudes of synaptic potentials evoked by stimulation of the piriform cortex were contrasted between the baseline period, a "cued" period in which animals were exposed to a light cue and feeding cup associated with chocolate, a chocolate-consumption period, and a follow-up period in which cues associated with feeding were removed Figure A, B1. In1- or D2-like receptor antagonists might block changes in evoked responses associated with exposure to chocolate. When animals were pre-injected with the D1-like receptor blocker SCH23990, the mean amplitude of synaptic responses was suppressed during both the cued period and the consumption period, but only the suppression during the cued period was statistically significant . However, administration of the D2 receptor blocker eticlopride prevented significant reductions in synaptic responses during both the cued and consumption periods , suggesting that activation of D2 receptors is required for the suppression effect. Additional statistical comparison of the saline and eticlopride conditions showed that 2 by 2, drug by session, interactions were significant for the consumption period  but not for the cued period  indicating a more reliable effect of eticlopride during the consumption period, and suggesting in incomplete block of the suppression by eticlopride during exposure to cues. The analagous interaction comparisons were not statistically significant for SCH23390 for either the cued  or consumption  periods.Tests on separate days were used to determine if systemic injection of Din vivo during periods of movement . Tests were therefore repeated following systemic injection of the muscarinic receptor blocker scopolamine. Reductions in synaptic responses associated with exposure to food-related cues were maintained in the presence of scopolamine  but there reduction observed during the consumption period was not statistically significant , suggesting that muscarinic receptors may contribute to the suppression observed during the consumption period .The amplitude of evoked synaptic responses in the entorhinal cortex can be attenuated by activation of muscarinic acetylcholine receptors 1,7 = 28.07, p < 0.01, and F1,7 = 30.02, p < 0.001, n = 8, respectively) suggesting that exposure to cues led animals to anticipate delivery of chocolate. Second, there were significant increases in mobility during the consumption period following injections of either SCH23390 or eticlopride , and smaller changes in movement during the cued period , suggesting that dopamine blockers may have reduced mobility associated with anticipation of reward or that animals had learned by the second and third testing days that chocolate was not immediately forthcoming. Third, there was increased mobility during baseline following injection of scopolamine in comparison to the saline condition  consistent with the behavioural activating effects of that drug[Analysis of the proportion of time spent mobile during each of the testing phases revealed several effects Figure C. First,hat drug.2 dopamine receptors, and this suggests that increases in dopamine release associated with natural rewards can result in a suppression of glutamatergic synaptic inputs[In the experiments described here we have used a palatable food reward to assess the potential modulatory effect of a rewarding stimulus, and reward-related cues, on synaptic transmission in the entorhinal cortex. Results show that both the exposure to cues associated with chocolate, and the intermittent delivery of chocolate chips for consumption, results in a reversible suppression of the strength of synaptic responses in the lateral entorhinal cortex evoked by piriform cortex stimulation. The size of the suppression effect was relatively small - about 5% of baseline values - but the reliability of the effect allowed pharmacological tests of the contributions of dopamine receptor subtypes and muscarinic receptors. The suppression of evoked synaptic responses during exposure to food-related cues and during the consumption period was dependent upon activation of Dc inputs to the ec inputs, and 43%c inputs, but the2 receptor antagonist eticlopride was effective in blocking the suppression of synaptic responses induced by food-related cues and by chocolate, but other neuromodulatory transmitters that modulate synaptic responses in the entorhinal cortex may be active during anticipation and consumption of food reward[4), suggesting that muscarinic receptors may play a role. Increased behavioural activity can increase hippocampal EPSP slope in part due to increased brain temperature[suppression of responses induced here.The Dd reward,38-41. Ad reward-45. Althperature,47 but s2 receptor-dependent reduction in glutamate-mediated synaptic responses induced by chocolate is consistent with previous findings in vitro that have shown that, although low concentrations of dopamine can facilitate synaptic responses via D1 receptors, higher concentrations of dopamine mediate a suppression of synaptic transmission primarily via activation of D2 receptors[2 receptors in the entorhinal cortex but, because eticlopride was administered systemically, the relevant D2 receptors could be elsewhere; dopamine D2 receptor antagonism might also block the suppression effect by reducing reward-induced activation of brain regions by dopamine[1 receptor activation can lead to a suppression of entorhinal synaptic response by affecting cellular input resistance[1 receptors to the synaptic suppression.The finding that the suppression of synaptic responses is associated with the anticipation of food reward, as well as with the intermittent delivery of chocolate chips, is consistent with the ability of reward-predictive cues to activate dopamine neurons and to modulate cortical excitability,48. The eceptors-16. The dopamine. In addi1 and D2 receptor blockers. Suppression effects observed during exposure to cues, in which there were significant reductions in the presence of SCH23390, and in final tests with scopolamine, suggest that carry-over effects may be minimal, but it is possible that the suppression of synaptic responses induced during intermittent consumption of food reward may be reduced over multiple testing sessions.It is possible that the absence of significant reductions in synaptic responses during consumption of chocolate during drug tests might be due to a learning or experience-induced change, rather than due to drug administration, because the saline condition was always tested first prior to counterbalanced tests of the effects of D1 receptors with low concentrations of applied dopamine strengthens synaptic responses in vitro[The synaptic suppression may weaken the functional impact of sensory inputs to the entorhinal cortex, but it might also serve to make active synaptic inputs relatively more salient in comparison to reduced spontaneous synaptic activity, or may  in vitro,16, sugg in vitro,33. TestAdditional factors that modulate dopamine release are likely to affect the suppression of synaptic strength observed here. Avena et al. found thThe authors declare that they have no competing interests.CAC conceived the study and JAH collected the data. Both authors contributed to the design of the study, data analysis, and preparation of the manuscript. Both authors read and approved the final manuscript.


Fibrocytes are circulating precursors for fibroblasts. Blood fibrocytes are increased in patients with idiopathic pulmonary fibrosis (IPF). The aim of this study was to determine whether alveolar fibrocytes are detected in broncho-alveolar lavage (BAL), to identify their prognostic value, and their potential association with culture of fibroblasts from BAL.We quantified fibrocytes in BAL from 26 patients with IPF, 9 patients with Systemic Sclerosis(SSc)-interstitial lung disease (ILD), and 11 controls. BAL cells were cultured to isolate alveolar fibroblasts.Fibrocytes were detected in BAL in 14/26 IPF (54%) and 5/9 SSc patients (55%), and never in controls. Fibrocytes were in median 2.5% [0.4–19.7] and 3.0% [2.7–3.7] of BAL cells in IPF and SSc-ILD patients respectively. In IPF patients, the number of alveolar fibrocytes was correlated with the number of alveolar macrophages and was associated with a less severe disease but not with a better outcome. Fibroblasts were cultured from BAL in 12/26 IPF (46%), 5/9 SSc-ILD (65%) and never in controls. The detection of BAL fibrocytes did not predict a positive culture of fibroblasts.Fibrocytes were detected in BAL fluid in about half of the patients with IPF and SSc-ILD. Their number was associated with less severe disease in IPF patients and did not associate with the capacity to grow fibroblasts from BAL fluid. Lung fibrosis is a common trait of different diseases with different etiologies and different pathophysiological pathways. Lung fibrotic disorders are characterized by accumulation of fibroblasts, myofibroblasts and extra-cellular matrix leading to chronic respiratory failure. The origin of the fibroblasts remains debated and is probably multiple, the respective role of the proliferation of resident fibroblasts or mesenchymal precursors, of the transition of epithelial, endothelial or mesothelial cells toward a mesenchymal phenotype, or the recruitment to the lung of circulating blood mesenchymal precursors is debated In experimental models of acute lung injury (ALI), bone-marrow derived cells have been shown to be recruited to the lung and to contribute to normal and pathologic repair We asked whether fibrocytes could be recruited to the alveolar space in IPF and SSc-ILD and if the detection of fibrocytes in BAL would indicate a worse prognosis in those patients. Furthermore, we determined wether fibrocytes detection was associated with the capacity to grow fibroblasts from BAL.The study was approved by the Institutional Review Board of the French learned society for respiratory medicine -Société de Pneumologie de Langue Française. Oral informed consent was obtained from the participants to the study at the time of bronchoscopy and recorded in the clinical charts. Clinical and functional data were collected on a standardized and anonymous collection form.A BAL was performed for a diagnostic purpose when a diagnosis of IPF or SSc-ILD was suspected from March 2006 to August 2008, with standard procedures Pulmonary hypertension was defined by PAPs>40 mm Hg assessed by echocardiography. CT-scan scoring of ground-glass opacities and fibrosis was made according to Kazerooni score The BAL was performed and processed as previously described Fibrocytes  were analyzed by flow cytometry analysis as described elsewhere Flow Cytometry was performed utilizing FACScalibur flow cytometer (BD-Biosciences). For each sample at least 10,000 events were collected. Data were analyzed using FlowJo® software . The negative threshold for all markers was set using the matched IgG isotype control. Fibrocytes numbers were expressed as absolute number per milliliter of BAL fluid.To grow fibroblasts, the BAL cells were cultured for at least 28 days and characterized as previously described The alveolar cells were fixed with acetone. Anti-human antibodies (Ab) directed against collagen 1 , vimentin , prolyl-4-hydroxylase (α-4H), desmin, pancytokeratin, CD31, α-SMA, CD45 , smooth muscle myosin 1 , CD14, CD34  or isotype-matched control Ab were used. Positive cells were revealed using the Vectastain ABC-alkaline phosphatase kit mouse immunoglobulin G  and the fast red substrate (Dako SA).Cells cultured on Lab-Tek slides were fixed after 21 days of culture with acetone and saturated with 50 mM NH4Cl and permeabilised with 0.1% Triton X100. After saturation with normal horse serum, slides were incubated with anti-collagen 1 monoclonal Abs (1∶290) (Sigma Aldrich) prior to the addition of fluorescent-labelled Abs (biotinilated horse antimouse (1∶300) plus Alexa Fluor 568-conjugated streptavidin (1∶100); Invitrogen, Carlsbad, CA, USA). After washing, the cells were incubated with anti-CD45-Alexa Fluor 488 Abs (1∶25) . Cell nuclei were localised by DNA staining with thiazole orange protein 3 . Slides were mounted with Prolong Gold anti-fade reagent (Invitrogen) and examined by confocal laser scanning microscopy . To test for the specificity of immunostaining, Abs were replaced by an isotype-matched control Ab.CCL2 and CXCL12 concentrations were measured in BAL fluid supernatant by ELISA . The detection threshold of the assays were 15 and 16 pg/ml for CCL2 and CXCL12 respectively. When a chemokine concentration was below the detection level of the assay, it was attributed a value of 0.As the distribution of the % of fibrocytes in the population studied was not normal, and as the number of patients studied in every group was below 30, non parametric tests were used for statistical analysis. Continuous variables were expressed as the median (range) and compared by the Mann-Whitney U test. Categorical variables were expressed as counts and proportions and compared by the Fisher’s exact test. Correlations were assessed with the Spearman correlation test Survival was assessed using the Kaplan–Meier estimator. All tests were two-sided, with p<0.05 indicating statistical significance. All the analyses involved use of the software GraphPad Prism.Twenty-six patients with IPF, 9 patients with SSc-ILD (cutaneous involvement was diffuse in 8 patients and limited in one) and 11 control patients were included  and 2. T3) and were absent in two BAL. In view of the limited number of SSc BAL evaluated, further analyses were limited to IPF patients.Alveolar fibrocytes, defined as cells expressing CD45 and collagen 1, were detected in 14/26 patients in the IPF group (53%), in 5/9 patients in the SSc-ILD group (55%) and in 0/11 controls . In IPF patients, the number of fibrocytes in BAL positively correlated with BAL macrophage counts , and negatively with the BAL neutrophil counts , but did not correlate with lymphocyte or eosinophil counts. We identified no link with infection as no pathogen grew from these BAL fluids at significant concentrations.IPF patients with detection of fibrocytes in BAL did not differ from patients without fibrocytes in any of the clinical, biological or radiological criteria evaluated , table 3We had the opportunity to study the evolution of BAL fibrocytes count in three IPF patients who required a second BAL procedure , and in one IPF patient who required two BAL procedures (respectively 260 and 642 days after the first BAL). In all cases, the BAL were performed for a clinical purpose. Alveolar fibrocytes were detectable in the first BAL in 2/4 patients. The alveolar fibrocytes were no more detectable in the second lavage for one of them, diminished in the second and were no more detectable in the third lavage .The morphological and immunohistochemical characteristics of cells grown from BAL are shown in The IPF patients were divided according to the cell culture results. The clinical, functional and radiographical parameters and survival were similar in the two groups . BAL cytThis study demonstrates that fibrocytes are detected in BAL in almost 50% of IPF or SSc-ILD patients whereas these cells are always absent from control BAL, and that in IPF patients, the number of alveolar fibrocytes correlates with the number of alveolar macrophages, and with markers of less severe disease : DLCO, CT-scan score of fibrosis and PaO2. However, our results do not support the presence or absence of BAL fibrocytes as a prognostic marker in those patients.This is the first study to identify fibrocytes in the alveolar milieu in patients with fibrotic lung disorders. Fibrocytes are monocyte-derived cells that have features of macrophages and fibroblasts The origin of BAL fibrocytes is not unequivocal. Circulating fibrocytes could directly be attracted to the alveolar space because of the local expression of chemokines for fibrocytes (such as CCL2 or CXCL12) already demonstrated in lung fibrosis It is also probable that blood monocytes penetrating the alveolar space in patients with ongoing lung fibrosis could differentiate into fibrocytes, since profibrotic mediators such as IL-4, IL-13, TGF-β and endothelin-1 are present in the fibrotic alveolar milieu and have been shown to induce the differentiation of monocytes to fibrocytes in vitro We observed that the number of alveolar fibrocytes was positively correlated with the number of alveolar macrophages and negatively correlated with the number of alveolar neutrophils. As most fibrocytes arise from the differentiation of blood monocytes in tissue, the correlation with monocyte/macrophage count was not unexpected, as we observed the same correlation in patients with ALI/ARDS We observed that the number of alveolar fibrocytes inversely correlated with the severity of lung disease as assessed by DLCO, score of fibrosis and PaO2. Most importantly, fibrocytes counts were not increased in patients with AE of IPF, and when BAL were repeated we observed a decrease of the number of alveolar fibrocytes. Altogether, these observations suggest that alveolar fibrocytes expressing CD45 and collagen-1 could be a marker of early fibrotic disease, and that the differentiation of fibrocytes evolve with time.Fibrocytes differentiate into fibroblast in vitro when appropriately stimulated Our study has some limitations. As we did not collect blood at the time of BAL, we were not able to measure circulating fibrocytes although it would be interesting to compare circulating and alveolar fibrocytes in the same patient. The second limit is the relatively low number of patients included.In summary, we were able to detect fibrocytes in BAL fluid in about half of the patients with IPF and SSc-ILD. Fibrocytes detection was not associated witht the capacity to grow fibroblast from BAL fluid. Further studies are needed to investigate the role of alveolar fibrocytes and their interaction with circulating fibrocytes.


Hereditary angioedema (HAE) is a term coined for a familial form of potentially life-threatening angioedema first described by William Osler in 1888. It is inherited as an autosomal dominant and the older literature often employs the term angioneurotic edema because it was believed that swelling could be a consequence of an emotional disorder. This type of angioedema, in particular, often included family histories of death by asphyxiation so that family members appeared "neurotic," that is, overly fearful, anxious, depressed, and so on. It also turns out that episodes of angioedema may well be triggered by emotional events in addition to other known triggers such as trauma, infection, and estrogen-containing hormones, but the biochemical explanation for this is not yet known. However, the conception of the disorder changed in 1953 when Donaldson and Evans discovered that it is due to a mutation in a plasma enzyme inhibitor known as C1 inhibitor (C1 INH) [The next era dealt with attempts to identify this vasoactive factor. It then became clear that his inhibitor could inactivate enzymes that are not part of the complement cascade. The first discovery was that it inhibited plasma kallikrein , an enzyDuring the years 1972-1990 I was studying the mechanism of activation of the plasma bradykinin cascade. Two forms of activated Factor XII were described, that is, Factor XIIa at about 80 kd and Factor XII fragment (XIIf) at 28.5-30 kd ,7; soon Nevertheless, in 1983 I decided to try to reproduce these experiments demonstrating plasmin cleavage of C2a or C2b to generate a kinin-like molecule. We activated C1 by immune complexes in the presence of C4 and C2, added plasmin, and could not demonstrate any kinin . By contBecause C2-kinin and bradykinin were the only 2 possibilities considered as possible mediators of the swelling of HAE and the data upon which C2-kinin was based could not be reproduced, bradykinin would be the "answer." But not being mathematicians (who accept disproving 1 of 2 possible choices as answering the question), this conclusion was not accepted until an additional 15 years passed, even after the nonexistence of C2-kinin was confirmed by a second study . FinallyThe notion that bradykinin is the mediator of HAE has lead to therapeutic advances that specifically target the Factor XII-prekallikrein HK-bradykinin cascade. Of course, repleting C1 INH by infusion can be employed to control acute episodes of swelling and for prophylaxis . When prNew observations requiring more work in terms of their relationship to HAE include the following : Prekall


Nautilus (Nautiloids) has a pinhole eye that lacks lens and cornea. Since pygmy squid and Nautilus are closely related species they are excellent model organisms to study eye evolution. Having being able to collect Nautilus embryos, we employed next-generation RNA sequencing using Nautilus and pygmy squid developing eyes. Their transcriptomes were compared and analyzed. Enrichment analysis of Gene Ontology revealed that contigs related to nucleic acid binding were largely up-regulated in squid, while the ones related to metabolic processes and extracellular matrix-related genes were up-regulated in Nautilus. These differences are most likely correlated with the complexity of tissue organization in these species. Moreover, when the analysis focused on the eye-related contigs several interesting patterns emerged. First, contigs from both species related to eye tissue differentiation and morphogenesis as well as to cilia showed best hits with their Human counterparts, while contigs related to rabdomeric photoreceptors showed the best hit with their Drosophila counterparts. This bolsters the idea that eye morphogenesis genes have been generally conserved in evolution, and compliments other studies showing that genes involved in photoreceptor differentiation clearly follow the diversification of invertebrate (rabdomeric) and vertebrate (ciliated) photoreceptors. Interestingly some contigs showed as good a hit with Drosophila and Human homologues in Nautilus and squid samples. One of them, capt/CAP1, is known to be preferentially expressed in Drosophila developing eye and in vertebrate lens. Importantly our analysis also provided evidence of gene duplication and diversification of their function in both species. One of these genes is the Neurofibromatosis 1 (NF1/Nf1), which in mice has been implicated in lens formation, suggesting a hitherto unsuspected role in the evolution of the lens in molluscs.Coleoid cephalopods like squids have a camera-type eye similar to vertebrates. On the other hand,  The eye has been one of the wonders of evolution. That the common ancestor of all eyes must have been a cell (or few cells) that responded to light is more or less an accepted notion. During evolution though many different types of eyes have appeared including mirror, pinhole, camera-type and compound eyes. It is, however, very intriguing that the camera-type eye, a feature of all vertebrates, can be also found in invertebrates. Have all eyes evolved from a prototype eye or have they been evolved independently many times? The ideas on the monophyletic origin of eyes have been bolstered by regulation of their morphogenesis by pax-6 (and its homologues in invertebrates), thus heralded as the eye master gene in all species . HoweverNautilus has a pinhole eye, lacking lens and cornea. Thus the retina of Nautilus is basically open to the environment that is seawater. The small opening is required in order for the vision not to be blurry, however, this reduces the visual information [Nautilus is probably due to the fact that Nautilus evolved to be a scavenger and opportunistic predator with a shell, actually keeping the ancestral characteristics of the cephalopod lineage. Nautilus can withdraw inside the shell for protection from predators and a hard lens might not have been the best tissue for this task. On the other hand, a camera-type eye provides significant perception of the surroundings which can be utilized for predation. This is hypothesized to be the driving force for positive selection of camera-type eyes. An interesting case in eye evolution can be found in molluscs. All cephalopods, except Nautiloids, have a lens-baring camera-type eye. ormation –6. The lNautilus and its relative, the pygmy squid must provide important and crucial information pertaining to the mechanisms that led to loss of lens in Nautilus. Recently the transcriptome of both species has been established and assembled de novo [Thus, examination of gene expression in the developing eye of  de novo . In the Idiosepius paradoxus (pygmy squid) embryos at stage 24 and Nautilus pompilius embryos 30 days in development [Raw sequencing reads were obtained from the DNA Data Bank of Japan [Nautilus and pygmy squid (mentioned as squid in the rest of the text) contigs were annotated using BLAST2GO program against the non-redundant (nr) database with e-value cutoff of 1E-10 [Nautilus and squid contigs (FDR < 0.05).De novo assembly was performed using Phred/Phrap. Read alignments to the reference were performed with Novocraft novoalign v2.06.09 package and expression of contigs was represented as normalized, based on samples’ reads, average coverage. De novo assembly and expression calculation was performed by Cofactor Genomics Inc. and as reported by us . Only coof 1E-10 . Gene Onof 1E-10 . Custom http://www.uniprot.org/)[Homo sapiens” or “Drosophila melanogaster”. All the protein results from these searches were downloaded as a fasta file. Human and Drosophila fasta files were compared using the BLAST tool[Nautilus and squid transcriptomes making the raw eye-related contigs for each of the organism. Next the complete Human and Drosophila proteomes were downloaded from UniProt and were combined to a single file. This file was compared using the BLAST tool to the Nautilus and squid transcriptomes finding the best hits of each of the contigs. These results were compared to the results of the raw eye-related contigs to ensure that they are the best eye-related hits. Comparisons between the groups were performed using custom perl scripts.Human and fly eye-related genes were found from UniProt (rot.org/) by searcLAST tool to the NNautilus or squid transcriptome and share the same best hit then it is probable that there is a duplication of this gene. Evaluation of the candidates also included the length of the contigs (>100bp) and the similarity between the two contigs (<95%). For the present study all the candidates were tested manually using the BLAST tool. The analysis of differential selection of genes was based on the fact that if there are two contigs, one expressed in the Nautilus transcriptome and one expressed in the Squid transcriptome, and they share the same hit in the Drosophila and Human homologues then this gene is a candidate for differential selection after the diversification of the Nautilus and the squid. All the candidates were tested manually using the BLAST tool. Candidates needed to share a hit in a region that is not conserved between Human and Drosophila. A general scheme of these comparisons is presented in Nautilus and squid transcriptomes, and Human and Drosophila proteomes. Drosophila and Human homologues were found with HomoloGene (http://www.ncbi.nlm.nih.gov/homologene) and GeneCards [Nautilus NF1 contig (comp83316_c0_seq1), Nautilus Nf1 contig (comp63708_c0_seq1), squid NF1 contig (comp475299_c0_seq1), squid Nf1 contig (comp304995_c0_seq1), Human NF1 (NP_001035957.1), Human NF2 (CAG30416.1), Drosophila Nf1 (AAB58976.1) and Drosophila Merlin (AAB08449.1).The analysis of gene duplication was based on the fact that if there are two or more contigs in eneCards . SequencNautilus and squid developing eye samples, respectively. From those reads, 36,392,640 (45.9%) and 10,926,326 (12%) were aligned to the de-novo assembled reference transcriptome. Sequences that were more than 100bp were further used. Expression of contigs was calculated as normalized average coverage, and a cutoff of 10 was used in order to obtain genes that were adequately expressed and to rule out false positives. Contigs expressed at least two-fold more between the Nautilus and squid transcriptome were considered to be differentially expressed. These sequences were annotated using the BLAST2GO program. From the total 64,335 and 38,148, 7,219 (11.2%) and 3,941 (10.3%) contigs were annotated and associated with Gene Ontology terms in the Nautilus and squid samples, respectively. These data are comparable to the assembly created before [Nautilus sample but these reads failed to contribute to the de-novo assembly and, subsequently, to the alignment. This resulted to the better coverage of the Nautilus sample indicated by the better representation in the nr database, however, most of the genes associated with eye development have been found in both the samples  had a better hit with its Human homolog or with its Drosophila homologue. All the sequences (from both species) were compared to proteins that have a GO term related to eye, photoreceptor, retina, lens, R1/R6, R2/R5, R3/R4, R7 or R8 from human (Homo sapiens) or Drosophila (Drosophila melanogaster) origin. We used these two organisms because they have the best annotated genomes of a vertebrate and invertebrate, respectively. Nautilus and squid contigs were compared to human and fly proteins as seen in the work flow in Since there are differences in gene expression between Nautilus and this might indicate their role in the evolution of the pinhole eye in Nautilus [The analysis revealed an interesting pattern. We found that genes involved in eye morphogenesis have the best hit with their Human homologues. These are: PAX6, SOX2, MITF, OTX2, SIX3 NR2E1, PAX2, MAB21L2, VAX1, TFAP2A, MEIS1, and PBX1 . Also, sNautilus .On the other hand, cytoskeletal genes involved in photoreceptors have a very interesting combination of human and fly best hits. Genes involved in cell polarity and planar cell polarity, which mainly involve rearrangement and regulation of cytoskeleton, have a best hit with the Drosophila homologues while genes involved in centrioles and other cytoskeletal components, like the Bardet-Biedl syndrome complex, have a best hit with the Human homologues. These results clearly reflect the morphology of rhabdomeric versus ciliary photoreceptors . Intereseyeless [This analysis clearly indicates that eye morphogenesis genes have not really changed much over the course of evolution and that have been always used in different eye types. On the other hand, photoreceptor-related genes have either been selected for rabdomeric or ciliary photoreceptors, indicating a possible independent evolution. In addition, eye morphogenesis in arthropods seems to have diversified from a conserved group of proteins that could account for a vertebrate camera-type eye. This diversification could have led to changes also in the circuits of gene interactions as found from experiments with fruit flies including the duplication of the ortholog of the human PAX6, eyeless ,16. Nautilus and squid) that have best hit with either Human or Drosophila homologues? To answer this question, we compared the Nautilus transcriptome with the combined Human and Drosophila proteome and we did the same with the squid transcriptome , while other are more related to invertebrates . But are there any contig pairs  and we selected homologous contigs one showing best hit with a Human homologue and the other with the Drosophila homologue. We performed the same analysis with the squid transcriptome (Squid versus Human and Drosophila). Candidate contigs were selected based on the degree of conservation and if the hit was in the same region of the human and fly homologues  of NF1/Nf1 might include the formation of lens in cephalopods. Previous studies have shown that there was duplication of part of the genome in the cephalopod lineage . The newmologues . The anaequences . There aassembly . Sequenctogether . NF1/Nf1feration . The rolNautilus and pygmy squid and exemplifies the importance of whole transcriptome studies in animal models for eye evolution and evolution in general.In all, our study is the first to provide such information at the molecular level related to eye evolution in Table S1GO enrichment analysis of contigs up-regulated at least two-fold in Nautilus and squid samples.(XLSX)Click here for additional data file.Table S2Nautilus and squid eye-related genes as found by comparisons with Human and Drosophila homologues.(XLSX)Click here for additional data file.Table S3Potential candidate gene pairs for lens evolution in squid.(XLSX)Click here for additional data file.Text S1Nautilus contigs that have best hits with the Human and Drosophila NF1/Nf1 protein.Alignment of NF1/Nf1 (TXT)Click here for additional data file.Figure S1Alignment of squid and Nautilus contigs that have best hits with Human and Drosophila CAP1/capt homologues respectively. Asterisk(*) indicates sequence identity.(TIF)Click here for additional data file.Figure S2Alignment of squid and Nautilus contigs that have best hits with Drosophila and Human Gef26/RAPGEF2 homologues respectively. Asterisk(*) indicates sequence identity.(TIF)Click here for additional data file.Figure S3Alignment of squid and Nautilus contigs that have best hits with Drosophila and Human CG5198/CD2BP2 homologues respectively. Asterisk(*) indicates sequence identity.(TIF)Click here for additional data file.


The widespread use of natural health products is also a problem, as they could interact with prescribed drugs in patients. One commonly used product is glucosamine for osteoarthritis and some reports have found increased values of cholesterol and other lipids in patients treated with simvastatin for hypercholesterolemia. The aim of this trial was to investigate the effects of glucosamine on s-cholesterol levels  in primary care patients on treatment with simvastatin or atorvastatin.Controlled, randomized, open, crossover pharmacodynamic study in two primary health care centres. Patients were treated with Artrox® (glucosamine) 625 mg twice daily and control . The study started with a run-in period of four weeks followed by control or active treatment with randomization of sealed envelopes. Each treatment period was four weeks and the treatment with simvastatin or atorvastatin was unchanged during the study (12 weeks). 34 patients were treated with a stable dose of simvastatin (n=21) or atorvastatin (n=13) for at least three months. Assessments of total s-cholesterol, s-HDL, S-LDL and s-triglycerides were performed in the morning with the patients in a fasting condition. T-tests for paired samples were used for statistical analyses and a p-value <0.05 was considered significant. Endpoints were the differences in lipid values at week 8 and week 12..All patients completed the study. No significant changes were seen on any of lipid levels in the simvastatin group.The actual glucosamine product did not change lipid levels of patients treated with simvastatin. Atorvastatin group was too small for safe calculations but was also without changesEUDRACT2006-001458-28 There is a widespread use of natural health products ,2. It isThere were totally 21 patients with hypercholesterolemia on treatment with a stable dose of simvastatin (10–40 mg once daily) and 13 patients with atorvastatin (10–20 mg daily) that were included in the study. The 11 males and 10 females in the simvastatin group had a mean age of 66 years, range 57–77 years. Fourteen patients had hypertension, nine type 2 diabetes and five had suffered from cardiac infarction or stroke . Patients were recruited from the primary health care centres in Mölnlycke and Landvetter in the Western region of Sweden. Eligible patients on simvastatin or atorvastatin treatment were identified by the routinely used data system at the primary health care centres. Patient characteristics are given in Table Exclusion criteria were; unstable angina pectoris, recent myocardial infarction (within a year), recent stroke (within a year), cardiac failure, HIV and dementia. There have not been shown any significant interactions involving glucosamine except case reports to warfarin treated patients. However, we excluded same drugs as in our earlier trials with St John´s Wort as our hypothesis was a stimulation of CYP3A4. Thus, treatment with birth control pills, warfarin, theofylline, cyclosporine, amitryptilin, nortriptilin, digoxin or sertraline was not allowed.The study started with a four week run-in period. At the initial visit (week 0), the patients were informed about the study, inclusion and exclusion criteria were verified and a physical examination was performed. All blood samples were obtained in the fasting condition and analysed at the accredited central laboratory at Sahlgrenska University Hospital in Gothenburg . The laboratory was unaware of patient treatment. Patients were then to return after 4 weeks (week 4) and another physical examination was performed and blood samples as described at week 0 above were collected. At this visit, the treating physician randomised , the order of treatment with active (Artrox®) and control . Thus, 50% started with active treatment and the other 50% started with control treatment and all were crossed- over to the other treatment modality after another 4 weeks (week 8). We considered that the treatment period o four weeks was adequate for possible glucosamine effects to vanish and thus no further wash out period was mandatory. Assessment of total s-cholesterol, s-HDL and s-LDL-cholesterol and s-triglycerides were then performed at the end of each treatment period (week 8 and 12). Compliance was verified by pill counting at each visit. The patients were on their regular dose of simvastatin or atorvastatin and the dose of simvastatin and atorvastatin was kept unchanged during the whole study period until all patients completed the study. The mean dose of simvastatin was 21 mg (range 10 mg – 40 mg) and of atorvastatin 14.6 mg (range 10–20 mg).Glucosamine could have an effect on glucose metabolism as stated in the SPC of the product. In order to exclude negative effects on this variable, HbA1c was followed as a safety measure,. The study was powered to detect a difference of 0.48 mmol/L with a power of 81.2 % with a significance level of 0.05 (two sided) and a standard deviation within the groups of 0.52. Power calculations showed that participation of 20 subjects would give statistical significance. Randomization procedure was performed by a person not involved with the patients.T- test for paired samples was used for statistical analyses and a p-value <0.05 was considered significant. The primary efficacy endpoint was the difference in LDL-cholesterol at week 8 and 12 between active treatment and control. Secondary endpoints were the corresponding difference in total cholesterol, HDL- cholesterol and in triglyceridesBasal measurements were made after four weeks of run-in. There were no significant effects on s-LDL, s-HDL, total s-cholesterol, or s-triglycerides after four weeks treatment with glucosamine. Neither were there any significant changes of p-glucose and HbA1c Table This group (n=13) was too small for safe calculations but also in this group there were no effects on the different lipids that could be demonstrated when compared with run-in determinations and after four weeks of active treatment. Values of p-glucose and HbA1c were also unchanged Table All patients completed the study and no significant side effects were reported.The main finding in our study was that we could not demonstrate any significant effect on total s-cholesterol, s-LDL, S-HDL or s-triglycerides after treatment with the glucosamine product Artrox® on patients treated with neither simvastatin nor atorvastatin for increased levels of lipids. However, the atorvastatin group comprised of 13 patients why safe conclusions could not be drawn from these patients. This is valuable knowledge as the use of glucosamine is widespread and a considerable amount of people is now also treated with lipid lowering drugs.Many patients are not aware of possibilities for interaction with increasing lipid values and different natural health products that you can buy on the counter ,5. We haA limitation in our study is that it was not double-blind, why we cannot exclude carry-over effects in the cross-over regimen. However, the half life for glucosamine is about two hours why this seems unlikely in treatment periods of four weeks. Furthermore, the study was randomized, and assessment of the lipids was performed by an accredited central laboratory, blinded to the treatment regimen. We can not with used sample sizes definitely exclude that there may be a type 2 error because our study was dimensioned to detect differences between the groups. Further studies with larger materials could be necessary. The metabolism of glucosamine is not known. This was an explanatory study to investigate if there was a signal that could verify clinical observations of possible interactions. If a signal had been detected further studies on possible mechanism had been justified.We conclude that no increased levels of lipids were seen in patients on lipid lowering drugs as simvastatin if they simultaneously take glucosamine for articulation problems.None of the authors have any relevant conflicts of interest.All authors planned and managed the study, performed the analysis and wrote the main results and final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/13/10/prepub


Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.Molecular typing is an important tool in the investigation of methicillin resistant  This region was named the hypervariable region (HVR) due to DNA length polymorphisms. Ryffel and colleagues sequenced the HVR of one MRSA strain and identified ten direct repeat units (dru) of 40 bp each. Most initial studies compared the gel band sizes or hybridization patterns of dru PCR products dru region dru region of 277 EMRSA-15 and EMRSA-16 led to a universal nomenclature dru types have been reported www.dru-typing.org.In 1991, Ryffel dru region of 302 MRSA isolates of global geographic distribution representing a broad range of genetic backgrounds and the SCCmec types I to VI. The collection included sixty-eight isolates from epidemiologically related Danish patients with t024-ST8-IVa and 26 isolates over a four-year period from the same patient. The purpose of our study was to investigate the diversity of the dru region in relation to different SCCmec cassettes and to evaluate its variability in both an epidemiological outbreak and over time in the same patient.We studied the spa types. The isolates harboured SCCmec types I to VI plus several SCCmec subtypes  and dru types  were named according to the nomenclature published by Goering et alwww.dru-typing.org. For some isolates the dru region was established based on an ongoing whole genome sequencing project on an Illumina HiSeq platform.dru types, the TRST plug-in tool of BioNumerics v7.0 was used for cluster analyses. With this plug-in, sequences are compared and aligned using an algorithm based on the DSI  model for pairwise alignment of repeats, which considers that modification of sequences can occur through duplication of tandem repeats, substitutions, insertions, and deletions (the latter two events are collectively termed indels) (1). A similarity matrix is generated based on the DSI model and used to construct a minimum spanning tree (MST); the type with the greatest number of related types is assigned as the root node, and the other types derive from this node. In the present study, the default parameters were used for alignment of sequences. The software creates groups of certain distance intervals or similarity values (which BioNumerics terms bins) and converts the data into distance units. Because of the highly clonal nature of the MRSA isolates investigated in the present study, the bin distance was set to 0.5%,  on the MST, and the distance between two entries with 99 to <99.5% similarity was 1. Using the MSTs, dru types were deemed to belong to different subgroups if they were separated by an MST distance of ≥2 . Therefore, if two dru types were at an MST distance of <2, they were considered to be closely related .To compare the relatedness between dru type and were all identified by the first primer set. Additionally one isolate was PCR positive by the first primer set but resisted sequencing. Seventeen isolates were dru negative by all three primer sets and one isolate had a double band with the primer set druGF/druGR but when sequenced contained no dru repeats. The 19 dru sequence negative isolates harboured SCCmec I (6 isolates), SCCmec II (11 isolates), SCCmec IV (1 isolate) and one isolate had lost part of SCCmec when retrieved from the −80°C freezer. In one dru PCR negative isolate , the region between the mecA gene and IS431 was sequenced and contained no dru repeats. Among the 283 dru sequenced isolates we found 53 dru types. The dru types contained from two to 14 repeats with the majority of dru types having 10 repeats (13 sequence variants)  of the sequenced isolates and identified in all SCCmec types except type II. In mec non-typeable (NT) isolates, forty-five dru types were restricted to one SCCmec type. However, 36 of these dru types were only found in one or two isolates each. The remaining nine dru types were found in three to 14 isolates with two to five spa types in each group belonging to two or three different CCs (dru types were found in at least two SCCmec types when excluding the non-typeable cassettes (Two-hundred and eighty-three of the 302 isolates (94%) had a ariants) . The mosrent CCs . Seven dassettes .spa types and dru types in five samples taken on the same day: t024-SCCmecV-dt11p (throat and groin), t024-V-dru negative (wound), t024-V-dt8l (axilla) and t292-V-dt11p (axilla). In October 2005 a t024-V-dt11bz was isolated from her groin. Just one month later, t024-IVa-dt10a was found in three samples and continued to be the type cultured from the patient throughout 2006 (seven samples), 2007 (one sample), 2008 (three samples) and 2009 (three samples), except for one sample from January 2006 that was similar to the October 2005 t024-V-dt11bz isolate.The patient with 26 MRSA isolates over a four-year period had her first MRSA isolated in May 2005, where we found different dru region in an outbreak situation, 68 epidemiologically related isolates of spa type t024-IVa from 68 patients were analyzed. These patients had either been admitted to the same hospital at the same time or lived in the same nursing homes. Sixty-one of the 68 isolates had dt10a, while the remaining seven isolates had dt2a (1), 7h (1), 7i (2), 8l (3). Two of the dt 8l and dt7h were isolated in 2004, dt2a in 2005, the two dt7i isolates in 2006 and one dt8l in 2008. Within the same nursing home three different dts were found .To test the variability of the dru types in 281 MRSA isolates suggesting a high diversity in the dru region. Thirty-three new dru types were found. We believe that the main reason we identified so many new dru types was that our collection was selected to be very diverse in regard to genetic background, SCCmec types and geography. Identical SCCmec types were often found to have different dru types and the same dru type was sometimes found in different SCCmec types. A single dru type, dt10a, dominated (53%) both in the Danish and International isolates as has been observed by others dru type is the ancestor dru type for most MRSA lineages and that the other dru types have evolved from it as is depicted in mecII isolates and the strain specific association of some dru-SCCmec types  seen here and elsewhere dru types of human CNS to MRSA would be of interest. The frequent association of dt10a with the different SCCmec types included in this study supports evolution of the dru region after the insertion of the SCCmec into MSSA. However, because the dru region and the insertion sequence IS431 are in close proximity within SCCmec it is possible that they may jointly insert downstream of mecA during the SCCmec assembly process. In addition, the stable association of dru types and SCCmec ccrB sequences has yielded interesting information regarding the potential movement of SCCmec elements in staphylococcal populations and MRSA phylogenetic interrelationships In this study we found 53 dru typing was useful to distinguish between subtypes of the same clone dru types ) other than dt10a among 68 isolates from 68 epidemiologically related patients with MRSA t024-IVa. While these minor dru types are not closely related to dt10a based on MST (mec IVa clone were identified ccrAB4 and the arc gene cluster within J3). One of the patients with a dt8l isolate lived in another nursing home together with a person with a dt10a isolate both of which exhibited an alternative J3 variation (the arc gene cluster but no ccrAB4). The presence of the same J3 variation in isolates with different dru types suggests their derivation from a common ancestor. While the separation of the J3 and dru regions by IS431 suggests independent evolution of the two regions dru repeat structure.In studies on EMRSA-15 and EMRSA-16 d on MST  we beliespa region is the most likely explanation for this difference dru types at the same time in the same genetic background could reflect evolution in the dru sequence in the very rare t024-V clone. An alternative explanation would be multiple acquisitions of t024 MRSA with different dru types which is consistent with the observation, five months later, that the patient had acquired MRSA t024 with a SCCmec IVa instead of SCCmec V. The shift from t024-V to t024-IVa in this patient was probably caused by replacement of one strain with another rather than the excision of SCCmec V and insertion of SCCmec IV. We base this assumption on the fact that the patient was living in a nursing home with an outbreak of t024-IVa-dt10a. However, whole genome sequencing of the type IV and type V isolates reveal that they were closely related (data not shown) and therefore an excision/insertion event of the SCCmec cannot be ruled out. Over a four-year period we found 20 t024-IVa isolates from this patient of which 19 had dt10a and one dt11bz suggesting a stable dru region, however, the mutation rate in the dru region is unknown. From each positive sample we only typed one MRSA colony. Typing several colonies from the same specimen could have resulted in the finding of MRSA isolates with different dru types and it is therefore possible that the patient was colonized with a dt10a and dt11bz at the same time. Based on our findings we believe that it is impossible to determine when and if one dru type evolved from the other in cases where different dru types are found in otherwise identical isolates from different patients. Changes in the repeat region of the spa gene has been analyzed spa region during long-term persistence has been reported to be one genetic change every 70 months dru region is similar, it is not surprising that we did not find genetic changes in the dt10a of the patient.We studied 26 isolates over a four-year period from a patient who seemed to have acquired several MRSA types over time. In the first year, the patient had five t024-V isolates with either dt8l, dt11p or dt11bz, and one isolate with a t292-V-11p. t292 is one repeat shorter than t024 and we believe that a deletion in the dru repeats in 18 isolates. dru negative isolates have also been found in other studies dru negative isolates belong to ST225. In a study by Nübel et aldru region was found in all isolates belonging to ST225. This indicates a spread of one MRSA clone rather than several episodes of acquisition of an SCCmec lacking the dru region into the same genetic background.We found no dru type and SCCmec type, spa type or Sequence type. Therefore dru typing is not a first line epidemiological typing method for MRSA. In our study, the dru region seems rather stable in an outbreak situation, although isolates from seven of 68 patients had variations in the dru region. Therefore, a change in dru type in otherwise identical isolates is not enough to separate patients in an outbreak situation, but might be informative for epidemiological subtyping. Furthermore, dru typing can add interesting information on the evolution of SCCmec. Although this study includes 52 dru types it is worth noting that the dru database (www.dru-typing.org) contains 421 dts as of February 2013 and the conclusions drawn in this study are therefore based on a subset of the total dru database.In conclusion, we found no correlation between dru type was retained in 90% of isolates, but the same common dru type, dt10a, was also found in several unrelated isolates. The finding of dt10a in so many different SCCmec types suggests that the dru region and the mecA originate from the same Staphylococcus species, while SCCmec II might have a different evolutionary pathway.Over a seven year outbreak the


There is disagreement in the literature about the importance of patellofemoral joint degeneration and knee pain for the outcome of unicompartmental knee arthroplasty (UKA). We therefore investigated the importance of selected predictors including patellofemoral joint degeneration and the location of preoperative knee pain for the early outcome of UKA.The study group comprised 260 consecutive patients from 5 hospitals who underwent Oxford UKA for anteromedial osteoarthritis. Data were collected at baseline and included pain location, radiologically observed degeneration of the patellofemoral joint including subluxation of the patella, intraoperative cartilage status of the patellofemoral joint, disease-specific knee status, and Oxford knee score (OKS). Outcomes were evaluated after 1 year using the OKS, global patient satisfaction, and global patient result.The average OKS score at baseline was 24 (SD 7), and it was 40 (SD 8) at the 1-year follow-up. 94% of the patients claimed improvement after the operation and 90% were satisfied with the UKA. Lateral subluxation of the patella was a predictor of poor outcome, and the preoperative OKS score was also a predictor of outcome. Full-thickness cartilage loss at any location gave a similar outcome to that with a normal or near-normal joint surface, and likewise, preoperative anterior knee pain was not a predictor of outcome.We conclude that the good early outcome after UKA in this study is in line with the best reported results. Patellofemoral degeneration should not be considered a contraindication to Oxford UKA. Patients with lateral subluxation of the patella have an increased risk of a poor result after UKA and should preferably be offered a total knee replacement. There is no consensus about the indications for choosing unicompartmental knee arthroplasty (UKA) instead of total knee arthroplasty. Here we describe early outcome after Oxford phase-III UKA. We also investigated the importance of selected predictors—including patellofemoral joint degeneration, subluxation of the patella, and the location of preoperative knee pain—for early outcome.The study was a prospective cohort multicenter study, where we consecutively included all patients who were operated with a medial Oxford UKA  between January 2007 and December 2008 at 5 cooperating hospitals in Denmark. The patients were included according to the criteria specified by the Oxford Group . PatientData were collected at baseline before the operation and subdivided into five predictor groups: (1) hospital and patient, (2) location of pain, (3) radiographically observed degeneration including subluxation, (4) cartilage degeneration, and (5) disease-specific knee status.As potential predictors, we included the hospital in which the patient was operated together with gender, age, height, weight, and body mass index (BMI) where we considered high BMI to be a potential predictor of poor outcome.On a knee picture with 3 circles anteriorly and posteriorly, the patients were asked to mark their knee pain anteromedially, anterocentrally, anterolaterally, posteromedially, posterocentrally, or posterolaterally .In all 202 patients tested (202 knees), the radiographic assessment included a skyline view taken with the knee flexed between 30 and 45 degrees . Both meThe standard minimally invasive surgical procedure was used  and no sDisease-specific knee status was assessed by using the summed score from the Oxford knee score (OKS) questionnaire . OKS wasFollow-up was done 1 year after the operation. The OKS summed score was used as primary outcome measure at all time points, and both follow-up score and the change in score from baseline to follow-up were estimated. OKS questionnaires were sent to the patients 1 year after surgery, together with pre-paid reply envelopes. OKS was supplemented with the same pain drawing as used preoperatively, where the patient could make a mark if he or she still had knee pain . The patTo avoid having missing data, we sent a reminder with a second copy of the questionnaire if we had not received the questionnaire within 4 weeks. If we did not receive the second questionnaire within another 4 weeks, the patient was contacted by telephone.2. All the predictor variables that remained in the final model with OKS score at follow-up were always included in the model building with OKS change score from baseline to follow-up.Univariate and multivariate ordinary least-squares regression by stepwise model building according to 268 patients (268 knees) were eligible for the study. Of these, 8 patients were excluded at the 1-year follow-up. There were 3 revision surgeries. 1 knee was revised for early component loosening, 1 for infection, and 1 for severe cartilage injury in the lateral compartment because of retained loose cement. 2 patients were excluded because of cognitive impairment after vascular cerebral insults. 1 patient died in the follow-up period. 2 patients failed to return the OKS questionnaire.260 patients (mean age 66 (SD 9), 51% men) from the five hospitals were included in the study .204 patients (79%) marked the preoperative location of pain on the knee picture. These locations are presented in 202 knees (78%) had a preoperative skyline view of the patellofemoral joint. Only 10 (5%) had severe degenerative changes in the medial part of the patellofemoral joint and 5 (3%) in the lateral part. Lateral subluxation was identified in 7 knees (3%) (congruence angle median 19° (IQR: 17–29), lateral tilt median 9° (IQR: 8–12)). 1 of the patients with lateral subluxation of the patella had severe degenerative changes in the lateral part of the patellofemoral joint .The state of the cartilage in the patellofemoral joint was recorded intraoperatively in 253 patients (97%). Full-thickness cartilage loss on the medial site of the patella was seen in 7 patients (3%), on the lateral site of the patella in 4 patients (2%), on the medial site of the trochlea in 19 patients (8%), and on the lateral site of the trochlea in 12 patients (5%) . 1 of thThe OKS at baseline was 24 (SD 7) . At the Hospital and patient characteristics. No hospital or patient characteristics were found to be predictive of outcome at the 1-year follow-up or the change from baseline to follow-up ); however, it was not found to be a statistically significant predictor in the multivariate analysis. Posterocentral pain was found to be a predictor of good outcome at follow-up (OKS 3.0 (p = 0.04)) in both the univariate and the multivariate analyses ) in both the univariate and the multivariate analyses for OKS score at follow-up and the change in OKS score from baseline to follow-up (ollow-up  and 4.Disease-specific knee status as a predictor. The OKS at baseline was found to be a predictor of outcome at follow-up (OKS 0.4 (p < 0.01)) in both the univariate and multivariate analyses (analyses . The OKSanalyses .In most reports on patients with medial knee osteoarthritis, anterior pain from the patellofemoral joint is not clearly distinguished from pain from the femorotibial joint , and antIn a study by In conclusion, the clinical outcomes in this multicenter study after UKA are in line with the best results reported. Patellofemoral degeneration should not be considered a contraindication to Oxford UKA. Patients with lateral subluxation of the patella have an increased risk of a poor result after Oxford UKA and should preferably be offered a total knee replacement instead.