Vitis vinifera L.) allowed the identification of many putative conserved microRNA precursors, grouped into multiple gene families.MicroRNAs are short (~21 base) single stranded RNAs that, in plants, are generally coded by specific genes and cleaved specifically from hairpin precursors. MicroRNAs are critical for the regulation of multiple developmental, stress related and other physiological processes in plants. The recent annotation of the genome of the grapevine (Here we use oligonucleotide arrays to provide the first indication that many of these microRNAs show differential expression patterns between tissues and during the maturation of fruit in the grapevine. Furthermore we demonstrate that whole transcriptome sequencing and deep-sequencing of small RNA fractions can be used both to identify which microRNA precursors are expressed in different tissues and to estimate genomic coordinates and patterns of splicing and alternative splicing for many primary miRNA transcripts.Our results show that many microRNAs are differentially expressed in different tissues and during fruit maturation in the grapevine. Furthermore, the demonstration that whole transcriptome sequencing can be used to identify candidate splicing events and approximate primary microRNA transcript coordinates represents a significant step towards the large-scale elucidation of mechanisms regulating the expression of microRNAs at the transcriptional and post-transcriptional levels. MicroRNAs (miRNAs) are small 19-24 nt) non-coding RNAs that play important roles in the regulation of various cellular processes by inhibiting gene expression at the post-transcriptional level 9-24 nt n. Many miab-initio approaches to the prediction of non-conserved (lineage-specific) miRNAs have also yielded some notable successes . Purelyes (e.g. ), althoues .Once mature miRNA sequences have been identified, their expression in different tissues, developmental stages or environmental conditions can be studied through experimental approaches such as northern blotting, oligonucleotide arrays or deep-sequencing of isolated small RNA fractions. However, the fact that identical or highly similar mature miRNAs can derive from multiple loci within a single genome limits the capacity of such approaches to determine which genomic precursor loci are truly expressed.in-silico studies of the regulation of transcription of miRNAs require accurate definition of primary transcript coordinates on a genomic sequence, or at the very least, fairly accurate estimates of transcriptional start sites. Owing to their typically short physiological half-life, relatively few pri-miRNAs have been found in EST and large-scale full-length cDNA sequencing projects in plants. For the same reasons, manual cloning and characterization of primary miRNAs is a laborious process, and has been performed for relatively few plant miRNAs [Meaningful insight into the regulation of miRNA expression at the transcriptional and other levels is undoubtedly desirable in the context of post-genomic and systems biology initiatives. However, large scale and t miRNAs ,22-24.Vitis vinifera L.) [To date, large-scale analysis of expression of plant miRNAs and associated prediction of precursor sequences have been restricted to relatively few species and the paucity of complete plant genome sequences limits the possibilities for studies incorporating extensive genomic information. The recently published genome sequence of the grapevine (fera L.)  providesWe have previously used comparative methods to predict 140 putative pre-miRNAs (representing 28 conserved miRNA families) in the grapevine genome . Here we+ transcriptome permits the precise identification of which candidate precursors are expressed in different tissues and shows that, in many cases, fairly precise estimates of primary transcript coordinates may be inferred from such data. Finally, we show that patterns of splicing and alternative splicing of pri-miRNAs may be elucidated from whole transcriptome deep sequencing data and confirm that a significant proportion of grapevine pri-miRNAs are subjected to such processes, consistent with the suggestion that post-transcriptional regulation might play a widespread role in the regulation of plant miRNA maturation.Additionally, we demonstrate that deep sequencing of the polyAVitis vinifera L.) genome [In the previous description of 8.4 fold coverage assembly of the grapevine  genome  we identVitis vinifera L.) [We previously used the MicroHarvester software  with allfera L.) . Manual Arabidopsis thaliana, Populus trichocarpa, Oryza sativa and Physcomitrella patens). Of the 28 families for which we identified putative precursor sequences in Vitis vinifera  [For the most part, we confirm existing patterns of miRNA family conservation with respect to completely sequenced plant genomes for which extensive analyses of miRNAs have been performed  . The onlVitis vinifera genome, 127 putative pre-miRNAs were intergenic in location (17 overlapped with annotated genes but on the non-coding strand). Four precursor predictions fell within or overlapped annotated coding or UTR exons although homology searches and transcriptomics data generated subsequently to the initial annotations call into question the validity of all but two of these exon annotations. miRNA 156 h is probably an incorrect prediction derived from a coincidentally plausible hairpin structure formed by the opposite strand to the presumed target (a Squamosa-promoter Binding Protein (SBP) box gene). A similar situation is observed for miR171 g which falls on the opposite strand to to a GRAS domain transcription factor gene. Nine precursor predictions were apparently intronic in location. Manual checks of the automated annotations suggested that all of the introns putatively containing pre-miRNAs were likely to be erroneous predictions, being atypically long (over 13 kb) and interrupting putative retroelement derived genes or obvious fusion gene predictions (not shown).With respect to the reference annotation of protein coding genes in the Vitis vinifera L. clone PN40024 leaves. 15,261,352 individual small RNA reads of 19-24 bases  representing 2,626,822 unique sequences were mapped to the draft genome after removal of adapter sequences. Over 40% of the total mapped sequences were of length 21 bases and accounted for 26.3% of the genomic loci represented by the mapped data (mean redundancy of 9.25 reads/locus). 34% of loci represented were of length 24 (13.4% of tags sequenced) with a mean redundancy of 2.27 reads/locus, suggesting, in accord with other studies [We generated 37,219,323 reads with Illumina sequencing of small RNA isolated from  studies ,29, thatMapping of the short tags onto the genome sequence revealed that of the 28 families predicted by our comparative analysis, 22 showed at least one sequence tag either in exact or very close correspondence to the position of one of the predicted mature sequences . For some families, thousands or even hundreds of thousands of short RNA sequences were recovered , while less than 1000 sequences corresponded to each of the remaining represented families. In some cases, the most commonly observed sequences were identical to at least one of the predicted mature sequences  while for other families, the predominant mature miRNA sequenced exhibited small variations (shifts or differences of length of one or two bases) with respect to the predicted mature sequences . This finding was not unexpected given the variation in mature miRNA lengths within families observed in other plant species and the nature of the comparative method used to generate the initial predictions. For three families  the predominant species sequenced showed greater shifts with respect to the prediction. While in the case of miR171 and miR397, shifts of up to three bases within or between organisms are registered in miRBase Figure , the graWe recovered a number of reads that include an additional 3' base that does not correspond to any genomic locus, this tendency has been observed in other species (e.g. ). FurtheDue to identity or similarity among mature miRNAs belonging to the same family, deep sequencing of small RNAs does not allow consistent unambiguous assignment of mature miRNAs to their genomic loci of origin. Nevertheless our analysis provided indications of the presence and abundance of 47 distinct mature miRNAs from conserved families in leaves - corresponding to up to 97 distinct precursor loci  tissues: ripening berries , roots, leaves and young inflorescences. Each hybridization and LMW-RNA extraction was performed twice.Slides were hybridized with low molecular weight RNA (LMW-RNA), extracted from six grapevine In addition to the mature miRNA sequences, the probe set included probes shifted 5 or 10 bases 3' or 5' with respect to the central base of the corresponding mature miRNAs as well as probes derived from regions of the stem not predicted to overlap with the mature miRNA sequence and controls containing maximally destabilizing substitutions with respect to probe sequences Figure . Except Other than for 26 out of 140 pre-miRNAs , showed significant expression in at least one tissue tested  yield significantly higher signals in young inflorescences than both leaves and roots (between 2 and 7.2 fold higher levels in this tissue)Figure . Only onFollowing the widespread assumption that many miRNA/target interactions are conserved between related species ,2, our dA. thaliana. In this light, the preponderance of miR169 family members in the group of miRNAs upregulated in mature berries is striking and might reflect a mechanism to protect maturing fruit from dehydration. We also note that miR535 family, identified so far only in O. sativa and P. patens [Growth Regulating Factor (GRF) genes in Arabidopsis [GRF genes encode putative transcription factors associated with cell expansion in leaf and other tissues in A. thaliana and O. sativa [Apetala 2 (AP2) -like transcription factors, regulators of flowering time, organ identity and of vegetative phase change [Li et al.  recently. patens  is upregbidopsis . GRF gen. sativa . A poten. sativa , again se change . In grape change  and putae change .laccase genes in Arabidopsis are root specific (for example AtLAC15) or mostly expressed in roots [V. vinifera, 17 in Arabidopsis).miR397a, miR398b and miR408 which are extremely highly expressed in root tissues target various copper proteins: plantacyanin, laccases and a superoxide dismutase, all putatively involved in stress responses and lignification ,15,40,41in roots  and are in roots . Given tThe complementary approaches utilized in the current work allowed us to uncover relevant shortcomings of both oligoarray and small RNA deep sequencing in the characterization of miRNA expression patterns. In particular, oligonucleotide arrays, for the most part, do not yield sufficient specificity to discriminate between family members. Our custom array, in which sequences of various length outside the predicted mature miRNA were included, clearly indicates that cross-hybridization is likely to present a confounding factor. For small RNA sequencing by Illumina technology, sequence features and/or post-transcriptional modification may be important features causing unforeseen biases determining an underestimate of abundance of specific miRNA families. This is suggested by the fact that some families, such as miR162, miR535, mirR403, miR482 generated few or no leaf smallRNA sequence reads, even if they can be clearly detected by northern blotting  or oligonucleotide arrays  in the same tissues. Our data suggest that this anomaly is not caused by the presence of unprocessed precursors in the samples used for oligonucleotide array analyses, although we cannot exclude the possibility of environmentally influenced or clone-specific expression patterns of these microRNAs influencing our results. However, it is interesting to note that some of these sequences  have characteristics known to be associated with high levels of sequencing errors with Illumina technology .The majority of plant miRNA genes are transcribed by RNA polymerase II and result in the production of polyadenylated primary transcripts . A stric+ transcriptome data generated with the Illumina Solexa technology [To investigate this hypothesis, we have analyzed whole polyAchnology  and Roch+ RNA isolated from 4 tissues , in vitro cultivated juvenile stem , in vitro cultivated juvenile root  and embryonic callus  were mapped to the grapevine genome and coordinates compared to those for predicted pre-miRNAs.A total of 135,047,735 Illumina sequences (33-35 bases in length) derived from polyAThe statistical significance of the number of reads mapping within a predicted pre-miRNA was evaluated  and 52 predicted precursors show significant expression in at least one tissue  of experimentally validated grapevine canonical splice donor and acceptor contexts  and used these matrices to evaluate all possible canonical splice donors and acceptors from 3 kb upstream to 3 kb downstream of predicted microRNA precursors showing extensive coverage by Illumina RNA-Seq reads. The positions and flanking exonic sequences of all possible splice donor/acceptor pairs were used to combinatorially generate possible splice junctions. RNA-Seq reads which did not map perfectly to the genome sequence were compared to these computationally generated splice junctions and pairs providing perfect matches (with at least 8 bases on either side of the splice junction) were recorded along with the tissue distribution of reads supporting each splice event. Additionally, for each supported splice event, we recorded the ratio of base coverage by RNA-Seq of the flanking 40 putatively exonic bases and the coverage of flanking 40 putatively intronic bases (not including reads previously identified as covering the putative splice junction). Three fold greater coverage of exonic regions was considered as additional support for the presence of a functional splice junction. Introns inferred from mapping of 454 transcriptome reads were also recorded.Visual examination of RNA-Seq coverage of regions upstream and downstream of miRNA precursor loci was used to provide initial estimates of transcript start and end positions. This step was complicated by the known propensity of RNA-Seq to provide uneven coverage of transcript termini - presumably due to the dynamics the nebulization step in sample preparation and to issues associated with differential recovery of fragments during sample preparation. Accordingly, we subjected the 6 kb interval centered on predicted precursors to promoter prediction analysis by TSSP-TCM  in orderFigure Figure The miR162 precursor Figure  is of paFigure Of 25 precursor loci chosen on the basis of extensive RNA-Seq coverage . Thus, evidence of transcription of intergenic pre-miRNAs can reasonably be considered as validation of transcription of the precursor.a-priori gene models are not available. However, the discovery that putative splice junctions for pri-miRNAs can be identified by discontiguous mapping of illumina reads may help to ameliorate this problem for plant pri-miRNAs. The fact that we recovered evidence of alternative splicing of miR162, is consistent with data from A. thaliana [The finding that relative depth of coverage of different regions of primary transcripts is consistent between tissues suggest the presence of systematic biases in either the procedure used to fragment the cDNA, in amplification of fragments for sequencing, or in sequencing efficiency. Dohm et al.  observedthaliana  and valiVitis vinifera including the first evidence of differential expression of miRNAs during fruit maturation in this species. We have shown that, for plants at least, whole transcriptome sequence data can be applied to the detection of differential transcription of putative precursor miRNA loci and to the detection and definition of pri-miRNAs as well as to the tentative definition of patterns of splicing in such precursors. It is probable that similar analyses performed in lines carrying mutations in genes involved in miRNA processing (in particular DCL1- plants) will allow more extensive and accurate definition of miRNA transcripts on a large scale, eventually facilitating detailed analyses of promoter sequences and a deeper understanding of mechanisms of transcriptional regulation of miRNA genes. Our analyses also suggest that splicing  of pri-miRNAs may be widespread and might constitute a general mechanism for the regulation of miRNAs.We have used a combination of high throughput approaches to show highly tissue specific expression of mature miRNAs in the grapevine Grapevine (Vitis vinifera L.) clone PN40024 plants and callus tissue were cultivated in vitro under standard conditions. For oligonucleotide array analyses, fresh tissues, with exception of roots (where in vitro cultivated plants were used), were collected from field-grown V. vinifera L. cv Corvina. Grape berries were harvested 5, 9 and 15 weeks after flowering, while leaves were collected from pre-flowering plants, inflorescence samples were collected 3 weeks before flowering.For Illumina deep sequencing, total RNA from PN40024 was extracted with the Spectrum Plant Total RNA Kit (SIGMA) as directed by the manufacturer. RNA was DNase treated with RQ1 RNAse-Free DNAse (Promega) and RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer.+ RNA was isolated from total RNA fragmented using Ambion RNA fragmentation buffer. cDNA synthesis was performed with Invitrogen random hexamer primers and cDNA was purified using QIAquick PCR spin column (Qiagen). Ends were blunted and 3' A overhangs introduced using T4 DNA polymerase and E. coli DNA polymerase I Klenow fragment. cDNAs were ligated to adapters with a single 'T' base overhang. After selection of 150-200 bp fragments from 2% low-range agarose gel, samples were amplified by 18 PCR cycles to enrich cDNAs with correctly ligated adapters and to amplify the amount of DNA in the library. Samples were loaded on Cluster Station to create flow cells of CSMA  and sequenced on the Illumina platform. RNA-Seq data are available from http://www.genoscope.cns.fr/externe/gmorse/raw_data/.Total RNA samples from leaf, root, stem and callus were processed using proprietary kits at Illumina, Inc. in Hayward . Briefly, PolyASmall RNAs (20-30 nt) were isolated from leaf total RNA by a denaturing PAGE gel. Samples were prepared for sequencing using proprietary kits at Illumina, Inc . Briefly, 5' and 3'-adapters were ligated to small RNAs. After reverse transcription, a low number of PCR cycles were used to create a sufficient amount of cDNA constructs. cDNA sample was then loaded on the Cluster Station and sequenced at ultra-high throughput on the Illumina platform at Illumina. Small RNA sequencing data are available from the Short Reads Archive (SRA) under accession number SRS005164.+ RNA was isolated from V. vinifera L. cv Corvina leaf and berry tissues by according to Rezaian and Krake [For 454 transcriptome analysis, polyAnd Krake . After rnd Krake . NormaliV. vinifera L. cv Corvina tissues and size fractioned, following the procedure [For oligonucleotide array analyses, total RNA was extracted from rocedure  with minPredicted grapevine miRNA precursor sequences have been published elsewhere . A CombiMatrix 12K CustomArray was synthesized with 1947 miRNA-specific probes synthesized to test grapevine miRNA expression profiles.For each grapevine miRNA precursor, we designed a set of 20-22 nt probes specific for the mature miRNA, the miRNA* and their complementary sequences, as well as a probe specific for the non mature microRNA stem/loop region and probes designed on the miRNA and miRNA* sequences but shifted of five or ten nt, forward and backward in order to test probe specificity. snRNA U6 and four grapevine tRNA probes were used as positive controls. Fourteen distinct negative and mRNA degradation control probes were included. Additionally, for each specific probe, a mismatch control with 2 maximally destabilizing substitutions was included. Each probe was present on the final array in three replicates. All probe sequences are available in Additional File Slides were hybridized with 3 g of LMW RNA labeled with Cy5 (Mirus LabelIT miRNA labeling Kit (Mirus Bio Corp.)). Hybridization and washing were performed as indicated by CombiMatrix. Slides were scanned with a Perkin Elmer Scanarray 4000 XL raw data was extracted with Scanarray Express 4.0 and Microarray Imager (CombiMatrix) software. After each hybridization, slides were stripped according to manufacturer's instructions and re-used 5 to 6 times.Two hybridizations were performed with independently extracted LMW RNAs, for each sample. Background level was defined as the average signal of the negative and degradation controls plus two times their standard deviation. The ratio between intensities of the perfect match probe and its mismatch probe (referred to as PM/MM) was also used to estimate the reliability of each signal. Probes with a median signal higher than background and with PM/MM value higher than 1.2 were called as present. The normalization between arrays was performed using the quantile normalization method  using thp-value < 0.05). Significant results were further investigated with Scheffè test, a post hoc test to define which tissues showed significant differences.Normalized signals were Log2 transformed and probes with a low PM/MM ratio (<1.2) were discarded. Differentially expressed genes in various tissues were identified with a one-way ANOVA test  of the null-hypothesis that remaining reads should be distributed randomly among intergenic regions. We consider only reads mapping uniquely to a single genomic locus. Given the expected short half-life of most primary miRNA transcripts, we believe that these criteria constitute an extremely conservative test of precursor expression.For the genomic regions containing predicted miRNA precursors, we used ad-hoc PYTHON scripts exploiting the MatPlotLib library to plot, for each base the log of the coverage . Thus only values above zero reflect higher than expected numbers of transcript-derived matches.Python scripts were used to generate PSSMs for U2 splice donors and acceptors  and aggregate log scores were used to assign scores to all possible canonical splice donors and acceptors. Custom PYTHON scripts were used to combinatorially generate putative splice junctions and SOAP was employed to map RNA-Seq reads that did not provide contiguous perfect matches to the genome sequence to such junctions.Vitis vinifera L. genome sequence using preliminary Blast searches and fine mapping of splice junctions was performed using SPIDEY [454 and RACE reads were mapped to the g SPIDEY  with defEM Extracted RNA for oligonucleotide arrays, performed oligonucleotide array experiments and analyses of oligonucleotide array data and contributed to drafting the manuscript. VP designed the oligonucleotide array, performed analyses of deep sequencing data and contributed to drafting the manuscript. MEP conceived the project, provided technical support and contributed to drafting of the manuscript, MD and AF performed oligonucleotide array experiments and data extraction, MP provided plant biomass, CC performed analyses of oligonucleotide data, GV provided 454 sequence data, CDF and AP provided software for mapping of splice junctions, MM provided Illumina short sequence data, GP supervised the project, provided technical support and contributed to drafting the manuscript. DSH provided software for data analyses, performed data analyses, supervised the project and contributed to drafting the manuscript. All authors have read and approved the final manuscript.Vitis vinifera miRNAsExpression data for . Supplemental Table S1: Frequencies of mapped small RNA reads by size and locus. Supplemental Figure S2: Log expression levels of all predicted miRNAs in all tissues as detected by oligonucleotide array. Supplemental Figure S3: Detailed alignments and frequencies of all small RNA reads mapping to miRNA precursors. Supplemental Table S4: transcript data for 25 Vitis vinifera miRNAs. Supplemental Figure S5: Splice junction read coverage for Vvi-miR394b, Vvi-miR162 and Vvi-miR168.Click here for fileOligonucleotide Array probes for analysis of Vitis vinifera miRNA expression. Supplemental Table S6: All oligonucleotide array probe sequences.Click here for file


However, the lack of formally well defined methods to identify randomness in sequence alignments has prevented a routine application of alignment masking. In this study, we compared the effects on tree reconstructions of the most commonly used profiling method (GBLOCKS) which uses a predefined set of rules in combination with alignment masking, with a new profiling approach (ALISCORE) based on Monte Carlo resampling within a sliding window, using different data sets and alignment methods. While the GBLOCKS approach excludes variable sections above a certain threshold which choice is left arbitrary, the ALISCORE algorithm is free of ALISCORE was successfully extended to amino acids using a proportional model and empirical substitution matrices to score randomness in multiple sequence alignments. A complex bootstrap resampling leads to an even distribution of scores of randomly similar sequences to assess randomness of the observed sequence similarity. Testing performance on real data, both masking methods, GBLOCKS and ALISCORE, helped to improve tree resolution. The sliding window approach was less sensitive to different alignments of identical data sets and performed equally well on all data sets. Concurrently, ALISCORE is capable of dealing with different substitution patterns and heterogeneous base composition. ALISCORE and the most relaxed GBLOCKS gap parameter setting performed best on all data sets. Correspondingly, Neighbor-Net analyses showed the most decrease in conflict.Alignment masking improves signal-to-noise ratio in multiple sequence alignments prior to phylogenetic reconstruction. Given the robust performance of alignment profiling, alignment masking should routinely be used to improve tree reconstructions. Parametric methods of alignment profiling can be easily extended to more complex likelihood based models of sequence evolution which opens the possibility of further improvements. Multiple sequence alignments are an essential prerequisite in alignment based phylogenetic reconstructions, because they establish fundamental homology assessments of primary sequence characters. In consequence, alignment errors can influence the correctness of tree reconstructions -3. To deTo improve the signal-to-noise ratio, a selection of unambiguous alignment sections can be used. It has been shown that a selection of unambiguously aligned sections, or alignment masking , improvehttp://aliscore.zfmk.de. In short, within a sliding window an expected similarity score of randomized sequences is generated using a simple match/mismatch scoring for nucleotide or an empirical scoring matrix for amino acid sequences (see Methods), actual base composition, and an adapted Poisson model of site mutation. The observed similarity score is subsequently compared with the expected range of similarity scores of randomized sequences. Like GBLOCKS it is independent of tree reconstruction methods, but also independent of a priori rating of sequence variation within a multiple sequence alignment. Because of its explicit use of, although rather simple, models of sequence evolution, ALISCORE can be called a parametric method of alignment masking.The recently introduced alternative profiling method, ALISCORE , identifIt has been demonstrated that both methods correctly identify randomness in sequence alignments, although to a very different extent -12. A coQ,  to score differences between amino acids, Qij. Pairs containing indels and any amino acid are defined by using the value of a comparison of stop codons and any amino acid defined within Q. The observed score within a window of pairwise comparisons is generated by summing scores of single site comparisons. Starting from a multiple sequence alignment of length L, sequence pairs  are selected for which the following procedure is executed: In a sliding window of size w at position k, a similarity score S(k) is calculated comparing positions (i(k), j(k)), ∀ k ∈ , using the following simple objective function:As for nucleotide sequences , ALISCORObserved scores are compared to a frequency distribution of scores of randomly similar amino acid sequences with length given by the window size. The generation of randomly similar sequences follows the Proportional model , which iPij (t) as the probability of change from amino acid i to j, πj the frequency of amino acid j, μ the instantaneous rate of change, and t the branch length/time. Different to the algorithm used with nucleotide sequences in which scores are adapted to varying base composition along sequences and among sequences, the frequency distribution of scores of randomly similar sequences is only produced once for amino acid data. The frequency distribution is generated by: 1) collecting frequencies of amino acids of the complete observed data set, 2) generating 100 bootstrap resamples of this amino acid frequency distribution and 100 delete-half bootstrap resamples of each of the 100 complete bootstrap resamples, and 3) by using these 10,000 delete-half bootstrap resamples to generate 1,000,000 scores of randomly similar amino acid sequences with length given by the window size. This complex resampling leads to an even distribution of scores of randomly similar sequences. The frequency distribution of randomly similar sequences is used to define a cutoff c(α = 0.95) to assess randomness of the observed sequence similarity within the sliding window. Matching indels are defined as Qij = c/w. The principle of the complete scoring process is described in [with ribed in .Compared to GBLOCKS, using ALISCORE resulted in the exclusion of fewer positions in most data sets Figure . GBLOCKSResulting ML trees and Neighbor-Net graphs were examined under two different aspects: 1) We compared trees of all unmasked alignments with trees of differently masked alignments per data set to analyze the influence of each masking method on data structure and presence/absence of selected clades Figure . 2) We cIn general, ALISCORE masked alignments resulted in consistent ML topologies among identical but differently aligned sequence data. The ALISCORE algorithm performed in most cases better or at least equal well than the best GBLOCKS settings , GBLOCKS). Application of GBLOCKS(none) yielded less congruent trees.While plants, fungi, metazoans, and included subtaxa were fully resolved in unmasked trees of data set mtI, sister group relationships between major clades  could not be resolved without alignment masking. If alignments were masked according to the ALISCORE profile, all ML trees showed a sister group relationship between fungi and metazoans. In the case of the T-COFFEE alignment, the Amoebozoa were placed as sister group to Fungi + Metazoa. The alignment masking of GBLOCKS and GBLOCKS led to comparatively resolved topologies. Masking the alignments with the GBLOCKS(none) option reduced signal in the data Figure . BootstrFor the mtII data set we were not able to recover monophyletic plants and Amoebozoa as sister group to Fungi + Metazoa. The sister group relationship between Fungi and Metazoa was fully resolved in all ALISCORE, GBLOCKS, and GBLOCKS(none) masked data sets. GBLOCKS(none) and GBLOCKS masked alignments supported in several instances implausible clades Figure .Bootstrap support values only marginally increased after alignment masking Table . NeighboAgain, ALISCORE and GBLOCKS masking improved tree-likeness of the 12S + 16S nucleotide alignments at the taxonomically ordinal level. ALISCORE outperformed GBLOCKS and GBLOCKS in all instances. GBLOCKS(none) clearly performed worst  option tended to result in suboptimal node resolution and support values Figure , 3. In tThe ALISCORE and GBLOCKS approach performed quite similar and best on all data sets. This demonstrates that even a predefined set of rules suffices to extract randomness within sequence alignments. Talavera & Castresana  showed tALISCORE optionally uses a BLOSUM62 or various PAM matrices to score differences between amino acid sequences, or a simple match/mismatch score for differences between nucleotide or amino acid sequences. It uses a simple modified Poisson model of character state change  approach is independent of It will be a matter of further analyses, whether an extension of the sliding window approach to more realistic likelihood models of change and Monte Carlo resampling will further improve the performance. However, it would be conceivable to implement a more explicit model based approach in GBLOCKS as well. The advantage of improved parameterizing GBLOCKS could be a significant gain in speed compared to the sliding window approach. The best approach should be the most efficient one in terms of computational time and increased reliability of trees, the latter one admittedly hard to assess.COII, COIII, ND2, ND3, ND4L, ND6) show high sequence variability compared to the less variable genes . The first mt data set (mtI) included protein sequences of all chosen mitochondrial genes of 17 taxa. The second mt data set (mtII) comprised the five less variable genes out of data set mtI but with 24 taxa, corresponding to Talavera & Castresana [We used four different types of real data sets in combination with different alignment approaches, three mitochondrial (mt) and one nuclear (nu) data set Fig. . Completstresana . The thiw = 6. Within its scoring function gaps were treated like ambiguous characters on nucleotide level. On amino acid level we used the BLOSSUM62 substitution matrix. Positions identified by ALISCORE or suggested by GBLOCKS as randomly similar were removed and single genes were concatenated for each data set and each approach. Percentage of remaining positions after masking was plotted for each alignment and masking approach  trees were estimated with RAxML 7.0.0  and the The authors declare that they have no competing interests.PK and BM conceived the study, PK designed the setup and performed all analyses. PK and BM wrote the paper with comments and revisions from KM, BT, BMvR and JWW. Beta testing of ALISCORE and processing of ESTs was conducted by KM. Sequence data was provided by KM , JD (12S + 16S rRNA), and BMvR . All authors read and approved the final manuscript.Table S1. Detailed analyses results. Detailed results including lists of the percentage of remained positions after alignment masking per data set and alignment method. Given are all considered clades and corresponding bootstrap values (>50%) per data set, alignment method and (un)masked approach as well as all Neighbor-Net graphs.Click here for file


Localized anterior arthrofibrosis (cyclops lesion) is the second most common cause of extension loss after anterior cruciate ligament (ACL) reconstruction. We present and discuss two patients with prior ACL reconstructions, who presented with pain and loss of extension following surgery. MRI and arthroscopy of the knee revealed typical features of a cyclops lesion. The patients showed significant symptomatic improvement following arthroscopic resection of these lesions. Cyclops lesion or localized anterior arthrofibrosis, an arthroscopically treatable complication of anterior cruciate ligament (ACL) reconstruction, is a fibrous nodule located in the intercondylar notch anterior to the ACL graft. The lesiAn 18-year-old boy had undergone left ACL reconstruction 8 months back. He presented with pain and stiffness of the left knee for the last 6 months. MRI Figure –C was peA 52-year-old man presented with pain and extension loss of the left knee for 3 months after ACL reconstruction surgery performed 6 months back. MRI Figure –C revealMRI is the primary post-operative investigative tool in patients with failed ACL reconstruction and is used to evaluate complications, the cause of graft failure, post-operative re-injury and pre-operative planning for repeat surgery. Important complications of ACL reconstruction include graft instability, disruption, extension loss, hardware failure (screw displacement and bone plug dislodgment) and patellar fracture (unique to the use of the patellar tendon autograft).Loss of motion, particularly loss of knee extension, is a frequent cause of morbidity in patients with prior ACL reconstructions. Localized anterior arthrofibrosis, also called the cyclops lesion, is the second most common cause of extension loss after ACL reconstruction, with a frequency of 1–9.8%, the most common being graft impingement, which develops due to anterior placement of the tibial tunnel. Other leOn histopathology, the lesion consists of central granulation tissue surrounded by dense fibrous tissue. The cyclThe nodule is located in the intercondylar notch anterior to the ACL graft. It may be attached to the graft fibers via a pedicle. The nodule gets pinched between the tibia and femur, which then causes a mechanical block to terminal extension. On MRI, the lesion has signal characteristics consistent with fibrous tissue. On T1W images, it may be indistinguishable from the adjacent joint fluid due to its low signal intensity. However, on T2W images, it has a heterogenous low signal intensity, enabling clear differentiation from high signal intensity joint fluid. On MRI arthrography, it can be outlined against the intra-articular contrast. On arthr237The cyclops syndrome was first described by Jackson and Schaefer in patients with ACL reconstruction as a condition presenting with loss of full extension, with development of an audible and palpable “clunk” in terminal extension. The cyclIn conclusion, it is important to recognize a cyclops nodule as a possible cause of extension loss in any patient with ACL injury because it is readily amenable to arthroscopic resection and good patient outcome.


Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) and spinal cord dorsal horn, as a primary anatomical pathway mediating opioid-based analgesia. Since these initial studies, the PAG-RVM-spinal cord pathway has been characterized anatomically and physiologically in a wide range of vertebrate species. Remarkably, the majority of these studies were conducted exclusively in males with the implicit assumption that the anatomy and physiology of this circuit were the same in females; however, this is not the case. It is well established that morphine administration produces greater antinociception in males compared to females. Recent studies indicate that the PAG-RVM pathway contributes to the sexually dimorphic actions of morphine. This manuscript will review our anatomical, physiological, and behavioral data identifying sex differences in the PAG-RVM pathway, focusing on its role in pain modulation and morphine analgesia. It was first reported that electricalstimulation of the midbrain periaqueductal gray (PAG) produced profoundanalgesia in the male rat in 1969 –9, cat –18, primWhile it iswell established that the PAG-RVM-spinal cord pathway is essential for theanalgesic actions of both systemic and intra-PAG morphine, these early studieswere conducted exclusively in male subjects. Only recently have studies begunincluding “sex” as an independent variable, and it is becoming increasinglyclear that morphine does not produce the same degree of antinociception in malesand females, especially following the induction of persistent pain. Sexdifferences in morphine potency were first reported in rodents in the late1980s, when it was shown that systemic morphine administration produced asignificantly greater degree of antinociception in males using acute painassays –42. ThisRecently,clinical studies in humans have also reported sex differences in morphineanalgesia. Of the limited number of studies that examined “gender” or “sex” asan independent variable, it has been reported that males experience greatermorphine analgesia compared to females –55. In fSex differencesin opioid analgesia are not limited to mu opioid agonists. In both human andanimal studies, sex differences in the analgesic effects of kappa or deltaopioid agonists have also been reported, although again, not withoutcontroversy –65. Seve50 forfemales is twice the ED50 of males. Similarly, morphine isapproximately 5-fold more potent in producing antihyperalgesia in arthriticmales compared to arthritic females [While it isclear that sex differences in opioid analgesia are not a simple and straightforwardphenomenon, when sex differences are reported, they are not trivial inmagnitude. In our persistent inflammatory , 46 and  females . Sex dif females  or rodenGiven that thePAG and its descending projections to the RVM and dorsal horn of the spinalcord provide a primary pathway for the actions of opiates in pain modulation,inherent differences in this pathway could contribute to the sexually dimorphicactions of morphine. Thus, we tested three hypotheses: (1) are there sexdifferences in the anatomical organization of the PAG-RVM pathway? (2) is therea sexually dimorphic response of the PAG-RVM output neurons to persistent pain?(3) does the administration of morphine differentially engage the PAG-RVMpathway in male and female rats?We usedneuroanatomical tract-tracing techniques to examine whether there werequalitative and/or quantitative differences in the neural projection from thePAG to the RVM in male and female rats. Consistent with previous anatomicalstudies , 80, 81,Inflammatory pain results in theactivation of descending modulatory circuits , 85 andWe have recently begun exploring thispossibility using combinatorial anterograde and retrograde tract-tracing incombination with persistent pain-induced Fos labeling. The results of thesestudies suggest that there are indeed sex differences in both the efferent andafferent projections of the PAG. Specifically, the amygdala, ventromedialhypothalamus, and periventricular nucleus project more heavily to the PAG infemales than males. In contrast, the medial preoptic area, parabrachialnucleus, and locus coeruleus project more heavily to the PAG in males thanfemales . In addipopulations of PAG-RVM neurons in the PAG of males and females.Although sexdifferences in PAG-RVM output neuron activation do not appear to contribute tosex differences in pain, they do appear to contribute to sex differences inmorphine analgesia. Until recently, all studies examining the mechanisms ofmorphine action in the PAG were conducted exclusively in males; therefore itwas unknown whether morphine administration has the same physiological effecton PAG neurons in females. Electrophysiological studies of PAG neurons arelimited because they examine the response of a single neuron , 101–10950 for systemic morphineis approximately twofold higher in females compared to males whether administered systemically [Systemicmorphine administration attenuates the persistent pain-induced Fos expressionwithin the PAG of male but not female rats  and iscemically  or direcemically . Intereemically . No sex emically . Indeed,emically , 111. ThGiven that more PAG neuronsproject to the RVM in female compared to male rats, it is possible that painmodulation in females is less dependent on opioids. If this is the case, thendirect activation of PAG output neurons should produce greater antinociceptionin females, not males. Microinjection of the GABA antagonist bicuculline intothe PAG produces antinociception , 112 byRepeated orcontinuous administration of morphine into the ventrolateral PAG of male ratshas been shown to result in the development of tolerance , 114–11850 for males. Repeated administration of systemic morphine induced tolerance in males to asignificantly greater extent than in females [Morphine was administered once or twice a dayfor three days in rats that had previously received retrograde tracerinjections into the RVM. To examine the activation of PAG-RVM neurons duringthe development of tolerance, males and females were both administered 5 mg/kg of morphine, the ED females , consist females . The hal females . While t females ).male ED50 dose of morphine. While a singleadministration of this dose of morphine resulted in comparable activation ofthe PAG in males and females, it was suboptimal in producing behaviorally definedantinociception in females and may account for why females did not developtolerance to the same degree as males. Future studies employing sex-specific ED50 doses areclearly warranted.Whiletogether, these data provide compelling support for a central role of the PAGin the development of morphine tolerance; these studies administered the Studies inrodents indicate that sex differences in the organizational and activationaleffects of the gonadal hormones estradiol and testosterone influence morphineanalgesia. For example, male rats castrated at birth demonstrate decreasedmorphine potency in adulthood, while female rats masculinized at birthdemonstrate greater morphine potency in adulthood , 122. Siα (ERα) have been localized in the PAG in the malerat [The anatomical substrate(s) wherebygonadal steroids influence pain and analgesia is unknown. Both androgen (AR) and estrogenreceptorsmalerat . Althougmalerat , goldenmalerat , guinea malerat , and themalerat , 136. Toα in the PAG-RVM pathway of male and female rats[α in the PAG was comparable between the sexes (seeFigures α (We havecombined neuroanatomical tract-tracing techniques and steroid receptorimmunohistochemistry to characterize the expression of AR and ERale rats. In thesigures α  with theigures α . Thehigα is required for estradiol-induced MORinternalization [α in the PAG-RVM output neurons may provide a painmodulatory mechanism. Interestingly, administration of estradiol togonadectomized males reinstates morphine analgesia while dihydrotestosteronedoes not [There areseveral mechanisms whereby gonadal steroids may modulate opioid-sensitivePAG-RVM output neurons, thereby potentially resulting in a dimorphic responseto morphine. First, estradiol has been shown touncouple the MOR from G protein-gated inwardly rectifyingpotassium channels  resultinlization  supportidoes not , suggestdoes not .Researchspanning for fourdecades has shown that the PAG and its descending projections to the RVM andspinal cord dorsal horn constitute an essential neural circuit for opioid-basedanalgesia. During the last half of that period, numerous rodent and humanstudies have established sex differences in the antinociceptive and analgesiceffects of morphine; however, the neural mechanisms underlying the sexuallydimorphic actions of morphine remain poorly understood. It is now clear thatthe anatomical and physiological characteristics of the PAG and its descendingprojections to the RVM are sexually dimorphic, with clear biological consequencesin terms of morphine potency. Our studies, as well as those of others, haveshown that morphine is less potent in females compared to males in thealleviation of persistent pain. Future research efforts utilizing femalesubjects in both the investigation of persistent pain mechanisms andidentification of both effective and potent pain therapeutics are clearlywarranted.


Stramenopiles constitute a large and diverse eukaryotic clade that is currently poorly characterized from both phylogenetic and temporal perspectives at deeper taxonomic levels. To better understand this group, and in particular the photosynthetic stramenopiles (Ochrophyta), we analyzed sequence data from 135 taxa representing most major lineages. Our analytical approach utilized several recently developed methods that more realistically model the temporal evolutionary process.Phylogenetic reconstruction employed a Bayesian joint rate- and pattern-heterogeneity model to reconstruct the evolutionary history of these taxa. Inferred phylogenetic resolution was generally high at all taxonomic levels, sister-class relationships in particular receiving good statistical support. A signal for heterotachy was detected in clustered portions of the tree, although this does not seem to have had a major influence on topological inference. Divergence time estimates, assuming a lognormally-distributed relaxed molecular clock while accommodating topological uncertainty, were broadly congruent over alternative temporal prior distributions. These data suggest that Ochrophyta originated near the Proterozoic-Phanerozoic boundary, diverging from their sister-taxon Oomycota. The evolution of the major ochrophyte lineages appears to have proceeded gradually thereafter, with most lineages coming into existence by ∼200 million years ago.The evolutionary timescale of the autotrophic stramenopiles reconstructed here is generally older than previously inferred from molecular clocks. However, this more ancient timescale nevertheless casts serious doubt on the taxonomic validity of putative xanthophyte/phaeophyte fossils from the Proterozoic, which predate by as much as a half billion years or more the age suggested by our molecular genetic data. If these fossils truly represent crown stramenopile lineages, then this would imply that molecular rate evolution in this group proceeds in a fashion that is fundamentally incompatible with the relaxed molecular clock model employed here. A more likely scenario is that there is considerable convergent morphological evolution within Heterokonta, and that these fossils have been taxonomically misdiagnosed. The photosynthetic stramenopiles (Ochrophyta) The major ochrophyte lineages, often considered as different classes, include Aurearenophyceae, Bacillariophyceae, Eustigmatophyceae, Dictyochophyceae, Synchromophyceae, Chrysophyceae, Chrysomerophyceae, Bolidophyceae, Xanthophyceae, Synurophyceae, Schizocladiophyceae, Raphidophyceae, Pinguiophyceae, Phaeothamniophyceae, Phaeophyceae, Picophagea and Pelagophyceae. Due to inferred paraphyly, the status of Picophagea Palaeovaucheria; Xanthophyceae) suggest that the photosynthetic stramenopiles had appeared by 1000 million years ago (Ma) Jacutianema . Moreover, scales, similar to those seen in modern chrysophytes and structures resembling centric diatom valves, have been recovered from 811.5–717.4 Ma deposits in northwestern Canada The earliest fossil remains  heterogeneity and heterotachy The nuclear-encoded SSU rRNA was chosen as the molecular marker for inferring phylogenetic relationships among the major ochrophyte lineages and the timeframes within which the photosynthetic heterokonts originated and diversified. Utilization of this gene allowed for the most expansive taxonomic sampling of the autotrophic stramenopile classes, including the non-photosynthetic oomycetes which are thought to be the closest living relatives of the ochrophytes The software package DAMBE v4.5.55 http://www.evolution.rdg.ac.uk/BayesPhy.html). A reversible-jump Markov chain Monte Carlo (rjMCMC) algorithm was used to determine how many distinct rate-variation patterns among sites and branch length parameters (with a maximum of two parameters for each branch) were required to optimally describe the empirical data matrix. In addition to potentially indentifying regions of the tree where phylogenetic reconstruction might be misled , an initial well-resolved tree was required to guide the placement of fossil calibrations in the divergence time analyses (below). A General Time Reversible (GTR) model of nucleotide substitution with discretized gamma-distributed rate variability  was employed throughout. This is slightly simpler than the model implemented in divergence time estimation , as the authors of BayesPhylogenies recommended against estimating the proportion of invariant sites. Five independent MCMC analyses  were conducted to approximate the posterior distribution of phylogenetic trees, and post-burnin samples (with burnin set to 10%) from all analyses were combined for parameter summary. Convergence of the MCMC runs was assessed graphically by examining the cumulative posterior and between-run variation in split frequencies In an attempt to reduce bias in phylogenetic inference, we employed a joint model that accommodates both rate-  were monitored through the use of Tracer v1.5 Divergence time estimation accommodating topological uncertainty was performed using the relaxed clock model of Drummond et al. 25 HBI alkene producing rhizosolenids and the Corethron lineage was treated as a bounded constraint (91.5+/−1.5 Ma) because the sudden rise of the C25 HBI alkene in the geological record has been dated to have occurred between 90 and 93 Ma BangiomorphaPrior age calibration distributions are given in 8 generations) for the purpose of exploring joint-prior space . A comparison of posterior and joint-prior estimates gives an indication of the information content in the data; if these estimates are identical then the empirical data contain no information regarding the relevant parameters .The constraints above were used in all divergence time analyses. For nodes of less precise ages  we opted to assess the sensitivity of inferred results through considering two distinct calibration procedures . In the In trees derived from the BayesPhylogenies and BEAST analyses, the pigmented heterokonts formed a monophyletic group and a sister-lineage relationship with the oomycetes with high support values .Ceratium and Alexandrium lineages (Dinophyceae) which had a posterior probability of 0.7  and Xanthophyceae . However, more notable instances exist. For example, in all trees Antarctosaccion applanatum is removed from the remaining ‘Phaeothamniophyceae’ species (Phaeothamnion confervicola and Stichogloea doederleinii), instead forming a sister-relationship (with posterior probability 1.0) with Chrysomerophyceae representative Giraudyopsis stellifera, a result that has independently been inferred from 18S rDNA Botrydiopsis pyrenoidosa with Xanthophyceae Chrysosphaerella lineage (Synurophyceae) forming a sister-relationship with Chrysamoeba mikrokonta (Chrysophyceae) with posterior probability 1.0 in all analyses.All trees reconstructed herein agree on inferred instances of paraphyly. Minor examples include genera in Bacillariophyceae (rbcL sequences and combined data sets (both molecular and non-molecular data), are generally poorly resolved, and thus few consensus relationships exist across studies n = 35) showed improved phylogenetic resolution among some of the pigmented heterokont classes relative to previous studies, suggesting that extended gene-sampling may offset difficulties arising from limited taxon-sampling. Nevertheless, the phylogenetic positions of several classes  remained poorly supported  developed from analyses of nuclear 18S rRNA sequences holds that the photosynthetic stramenopiles originated between 498 and 293 Ma Prorocentrum as well as between polycysteine radiozoans and the silicoflagellate genus DictyochaOur results provide evidence for an intermediate Neoproterozoic-Paleozoic timeframe, the divergence between the oomycetes and the pigmented heterokonts inferred to have occurred between the mid-Neoproterozoic and Early Devonian  in an old non-stramenopile group.In general Class Xanthophyceae is characterized by having a poor fossil record There is not much known about the fossil history of the chrysophytes, which include both Chrysophyceae and Synurophyceae Class Dictyochophyceae, which once was considered to be a member of the class Chrysophyceae, includes the silicoflagellates , a group characterized by formation of a silicified skeleton. Due to the silicified structures these organisms possess a fossil record starting in the Early Cretaceous . Diatoms are known from sediments as old as the earliest Cretaceous  and Antarctosaccion applanatum . These are considered to be reliably dated geological events and provide important historical information in lineages with poor or absent fossil records. While we recognize that these alkenes  may have evolved following cladogenesis (rendering our divergence time estimates as overly young), because the nodes involved are all located near the tips of the tree, it appears quite unlikely that a speciation/alkene decoupling at this tree depth would seriously mislead our broader inferences regarding the earliest putative fossils from this group (see above).In the present study we considered alternative (tight) exponential and (broad) lognormal calibration priors to investigate the sensitivity of resulting inferences to choice of temporal prior distributions. Not surprisingly, exponential priors (lending more credence to a strict reading of the fossil record) generated generally younger and more precise inferred divergence times than the considerably broader lognormal priors (which model an expected lag between speciation and diagnosable fossil deposition). Nevertheless, the two sets of estimates are broadly congruent with considerably overlapping 95% CIs, suggesting for the present data set that (reasonable) alternative prior constructs are unlikely to significantly support dissonant temporal evolutionary hypotheses. Some of the calibration dates used here are not based directly on recognizing the first appearance of morphological species in the fossil record but rather on the time of the final closure of the Isthmus of Panama A third issue in molecular dating involves the general modelling of among-lineage rate heterogeneity. For example, typical empirical molecular genetic alignments of non-trivial size are rarely fit by ‘global’ molecular clocks. Relaxed clock approaches offer a break from the unwarranted assumption of a global clock through allowing individual branches within a tree to have unique rates of molecular evolution. However, many of the available relaxed clock models Finally, a general issue for all phylogenetic studies concerns taxon and molecular sampling. In the present study we elected to maximize taxonomic sampling, as this has been demonstrated to be important in divergence time estimation Palaeovaucheria, Jacutianema, Germinosphaera) and phaeophyte (Miaohephyton bifurcatum) fossils, casting doubt on the taxonomic validity of these fossils. Elucidation of their taxonomic affinity is difficult because Vaucheria-like characteristics have most likely evolved in these extinct Proterozoic lineages independently of those seen in the extant forms of the genus Vaucheria (Xanthophyceae). Nevertheless, the degree of dissonance suggested by our results is such that if these fossils truly represent crown stramenopile lineages then we would have to conclude that ochrophyte molecular evolution proceeds in a fashion that is fundamentally incompatible with the uncorrelated relaxed clock model employed here. It is the implausibility of this scenario that we regard convergent morphological evolution as a more likely explanation.The results of the divergence time analyses presented herein indicate that the first pigmented stramenopiles most likely evolved between the mid-Neoproterozoic and the Early Devonian. These estimates suggest that the radiation of the majority of the known heterokont algal classes occurred throughout the Paleozoic and in the Triassic/Jurassic of the Mesozoic. Our uncorrelated relaxed clock investigation gave rise to generally older origination times for most stramenopile classes relative to most previous fossil- and molecular genetic-based estimates. Nevertheless, these results are topologically and temporally incompatible with putative Mesoproterozoic/Neoproterozoic xanthophyte . All included taxa are shown. Nodes are plotted as mean divergence time estimates (Ma), and blue horizontal bars represent 95% posterior credible intervals. Numbers in the tree diagram indicate posterior clade probabilities. Estimates are derived from the summary of post-burnin samples from six independent MCMC analyses.(3.21 MB TIF)Click here for additional data file.Figure S2Maximum clade credibility chronogram. Maximum clade credibility chronogram from BEAST analyses utilizing an uncorrelated lognormal relaxed clock model and exponentially-distributed temporal constraint priors (see main text for explanation). All included taxa are shown. Nodes are plotted as mean divergence time estimates (Ma), and blue horizontal bars represent 95% posterior credible intervals. Numbers in the tree diagram indicate posterior clade probabilities. Estimates are derived from the summary of post-burnin samples from six independent MCMC analyses.(3.14 MB TIF)Click here for additional data file.Table S1GenBank accession numbers of the 135 species used in the study(0.12 MB DOC)Click here for additional data file.


Using nicotine replacement therapy (NRT) while still smoking in the lead up to quitting could enhance success at quitting, one of the most cost-effective means of improving health, but little is known about its acceptability and tolerability.To test the acceptability and tolerability of using NRT while smoking for two weeks before quitting, to inform a randomised controlled trial of pre-quitting NRT versus usual NRT-based quitting practice.Prospective pragmatic uncontrolled clinic-based pilot study in which 14 adult smokers recruited from a smoking cessation clinic were prescribed nicotine patches or gum with follow up for two weeks. Data were collected on participants' concerns about smoking while using NRT, importance of quitting, urges to smoke, smoking behaviour, previous NRT use and the length of the pre-quitting period. Urine tests were collected weekly for cotinine, and participants recorded smoking activity and noted experiences and changes in their health in diaries.Only 21% of 14 participants expressed concerns about using NRT while smoking. All of the nine followed up used it as recommended, 56% of these reporting no unpleasant symptoms. Median urine cotinine levels declined over the two weeks. Urges to smoke averaged 1.8 on a 4-point scale. All participants decreased the number of cigarettes per day. Diary records showed wide variation in smoking and NRT use, with an increased sense of control and determination to quit.Smokers using pre-quitting NRT over two weeks appeared to titrate nicotine levels and symptoms of toxicity were uncommon and of low intensity. Quitting smoking using nicotine replacement therapy (NRT) is one of the most cost-effective means of reducing risk and improving current and future health, yet sustained quitting rates are still far from ideal. New apprPrior to embarking on a large randomised controlled trial of pre-quitting NRT versus usual practice on quit rates at six months, we undertook a small pilot study involving adult smokers who attended a cessation clinic. We aimed to investigate the acceptability, toxicity and tolerability of NRT while still smoking, for two weeks prior to the nominated quit day.Adult smokers of at least 10 cigarettes per day who wanted to quit within the next few weeks were recruited into this prospective, uncontrolled non-experimental study between February–March 2005 at a university cessation clinic in Christchurch, New Zealand in response to a local newspaper advertisement. People with unstable medical conditions, or who were pregnant, breastfeeding or on psychotropic medication, were excluded. The study was approved by the local research ethics committee and all participants provided written informed consent.ad libitum for two weeks prior to a nominated Quit Day. Instructions were given to participants that they could continue smoking as they wished over this period. Eight weeks of subsidized NRT treatment combined with support telephone calls then followed Quit Day, in line with standard cessation practice in New Zealand. In this pilot study only the two week pre-quit period was studied.Participants received two weeks supply of NRT, either as patches or gum in doses appropriate to their level of dependence, as determined by discussion between client and a cessation therapist (MW-B), to use while still smoking Brief questionnaires were administered at enrolment and one week later by one of the investigators (MW-B) to collect demographic (enrolment only) and smoking-related data . Participants rated urges to smoke on a 5-point scale (0 = none to 4 = severe), and importance of quitting on a scale of 1 (no importance) to 9 (very important). They provided urine samples for cotinine testing at enrolment, and at seven and fourteen days post-enrolment (Quit Day). Cotinine was measured at a regional hospital laboratory using capillary gas chromatography and nitrogen selective detection. Diaries were given to participants at enrolment to record daily smoking consumption, NRT use and note any toxicity-related symptoms and other relevant experiences. On Quit Day, diaries were collected, final urine cotinine samples taken, a supply of NRT provided for the next 4 weeks and support calls arranged.Fourteen of the fifteen (93%) people responding to the newspaper advertisement met the study inclusion criteria, 67% of them female, with a median age of 44 yrs (range 18–70 yrs) and all but one of European New Zealander ethnicity. Participants smoked on average 21 (range 10–35) cigarettes per day (cpd). All were interested in finding new ways to quit, and rated quitting highly . All participants had used NRT before, nine (64%) using patches, two (14%) gum, two (14%) both patch and gum; one did not answer the question. Most (71%) had no concerns about using NRT while they were still smoking. Of those with concerns, two were worried about overdose and the other about feeling light-headed. Eleven (79%) participants kept diaries and completed the follow-up questionnaireEleven participants (79%) used NRT as recommended; three were unresponsive to follow up calls. Eight (64%) received 15 mg/16 hr NRT patch, two (14%) 10 mg/16 hr NRT patch, three (21%) 2 mg NRT gum, and one 4 mg NRT gum. Excerpts from participants' diaries give insight into their experiences as they embarked on the study. Several participants were optimistic about their chances of success:"I have more success integrating and maintaining new habits when introduced gradually rather than drastic changes" "Patch on at 7.00 am, coffee and felt that I shouldn't smoke." Gum was uniformly regarded as unpleasant to taste whereas patches were well-tolerated. When asked on Day 14 whether the two week period of pre-quitting NRT was adequate to prepare for quitting, 55% preferred at least 3–4 weeks, three felt two weeks was just right while two recommended a shorter period.Considerable day-to-day variation in smoking patterns was evident. Smoking reduced by an average of 4 cpd (range 2–10) in the 11 participants for whom data were available. None stopped smoking completely. Urges to smoke over the pre-quit period were rated mild-moderate, mean score 1.7/4 (range 0–3). Further diary excerpts shed light on the participant's experiences. At the end of the first day, one participant commented on the power of habit despite low level of cravings:"Didn't often feel like smoking, but habituated ...after lunch...after work" "Morning craving not so pronounced – cigarettes are beginning to taste awful" "Three smokes and one gum at 3.30 pm – feeling confident about working towards quit day" "I have noticed I am not so preoccupied with making sure I have cigarettes with me when leaving the car" "For the first time in years did not have cigarette first thing. Felt better. Had three cigarettes later on and felt lousy" "15 cigarettes today. Started buying 20 packs instead of 30." "Patch on when I awoke; had two cigarettes in the morning, two at midday and one in the afternoon. Felt fine today" Participants reported few unpleasant feelings related to use of NRT while smoking. Only four reported experiencing symptoms, including mild nausea, low-grade headaches, sleep disturbance and dizziness, and were most likely due to high nicotine levels rather than withdrawal. Urine co"Initially felt 'racy' but [patch] definitely has helped, inhaling less deeply ...able to delay several hours" Despite being small and uncontrolled, this study nevertheless provides some useful insights into concomitant smoking and NRT use prior to quitting. First, the findings align with those from other studies showing NRT use while smoking to be safe. Few partA second and related finding is that urges to smoke were only mild to moderate in strength, and therefore, we suggest, likely to enable a more positive experience for smokers planning to quit compared to the usually abrupt transition. Past positive experiences are the most influential source of self-efficacy, which is important in making behavior changes. Also, eaad libitum smoking, reported no fall in cpd but a 14% drop in CO together with less satisfaction from cigarettes and fewer and weaker urges to smoke. Our study, however, supports the notion of down-regulation of nicotine intake. Studies examining the effect of nicotine gum on ad-lib smoking have found an inhibitory effect [Thirdly, all participants reduced smoking during the pre-quit phase, two thirds by more than 5 cpd, in line with the finding from a review by Fagerstrom and Hughes of eleven studies with concomitant smoking and NRT use in which the number of cpd declined by 50%. However,y effect  but thisy effect  Using niy effect  but the Fourthly, the two-week pre-quit period appears to a reasonably acceptable pre-quitting period, and likely to be sufficient for effectiveness, based on the experience of Schuurmans et al.Finally, several themes emerged from the analysis of participant's diaries that shed light on possible mechanisms for any effect, if indeed one exists: an increasing sense of personal control over smoking; and a reduction in the stress associated with changing smoking and cigarette purchasing behavior compared with previous quit attempts.Our findings suggest that a short period of around two weeks concomitant smoking and NRT patches or gum at normally prescribed doses may maintain nicotine at high but safe and well-tolerated levels sufficient to minimize cravings. This may facilitate a gradual reduction in smoking through down-regulation. We hypothesize that this, together with a growing sense of personal control over smoking behaviors, may facilitate a more successful and sustained transition to quitting than the usual practice of suddenly stopping the main source of nicotine (i.e. cigarettes) on Quit Day. A large randomized trial is planned to address this key question. The trial will also enquire in detail about smoking activity and NRT use over the pre-quit and immediate post-quit period to further illuminate the various mechanisms that underpin the changes and experiences of smokers undertaking this approach.The authors declare that they have no competing interests.


This review provides evidence to suggest a cell-type specific modulation of presynaptic inhibition in cortical regions.Several lines of evidence suggest that the modulation of presynaptic GABA release is mediated by a variety of receptors including; presynaptic AMPA, cannabinoid, GABAB, kainate, metabotropic glutamate, NMDA, and opioid receptors. The evidence supporting presynaptic modulation of inhibition is predominantly obtained from studying stimulus elicited, spontaneous or miniature synaptic events, where the information regarding the identity of the presynaptic cell is lost. This article summarises these findings then focuses on another approach to study the presynaptic modulation of GABA release by comparing the modulation of GABA release at unitary synapses identified morphologically, immunocytochemically and electrophysiologically. To date, evidence for cell-type specific regulation of presynaptic inhibition at identified synapses involving most of the above presynaptic receptors does not exist. Therefore, the key presynaptic modulators that will be focused on here are kainate and cannabinoid receptors and their intracellular signalling cascades that orchestrate GABA release. There will be some discussion on presynaptic modulation  Inhibition is essential in shaping response properties in single cells and assisting co-operativtity in large populations of cells. It is the network of GABAergic interneurones that balances excitability by controlling dendritic electrogenesis and spike generation of pyramidal cells as well as setting and maintaining oscillatory rhythms. In CA1 alone there are currently 22 identified subclasses of interneurones . These iA receptors .  illustrates IPSPs between SCA interneurones which are reduced by a CB receptor agonist, Anandamide. The decrease of GABA release was prevented by the CB1 receptor antagonist AM-251. Fig. (2B) also demonstrates there is a persistent inhibition due to activation of CB1 receptors at these connections that was relieved after the bath application of AM-251, resulting in an enhancement of the unitary IPSPs . This persistent silencing via CB1 receptors probably plays a role in differentiating between certain ensembles of pyramidal cells, allowing ongoing activity at some ensembles, while silencing others. In contrast, the persistent silencing via CB1 receptors observed in hippocampal regions is not always present at neocortical inhibitory connections involving presynaptic regular spiking interneurones , suggesting that persistent silencing via CB1 receptors may be predominantly a hippocampal function.Co-localisation of CCK and CB1 receptors are not restricted to proximally targeting basket cells and are also expressed by dendrite targeting interneurones see , 72, 93.quencies . These squencies . Recentlvia CB1 receptors at synapses that co-localise CCK and CB1 receptors. This probably contributes to short and long term synaptic plasticity throughout the brain. Interestingly, CCK, itself is a modulator at synapses by acting as molecular switch that determines the source of perisomatic inhibition [In summary, there is a consistent observation that GABA release is modulated hibition . The stuhibition  leading 2+ influx into the postsynaptic cell. Thirdly, DSI expression is thought to be presynaptic since it does not affect the quantal size of miniature GABA mediated events [via CB1 receptors usually employ the DSI protocols, however in the neocortex, connections between CB1-positive cells lack DSI [Much of the attention focused on endogenous cannabinoids as a retrograde signal is linked with DSI  due to the following observation; firstly exogenous cannabinoids modulate GABA release , 69, 98.d events  consistelack DSI  suggestivia the inhibition of adenylate cyclase and N and P/Q type calcium channels [When endocannabinoids are released they activate CB1 receptors to modulate neuronal signalling mainly channels , 90, 98 channels , 79, 92.channels , 44, 50.The presynaptic activation of CB1 receptors is most likely linked to an inhibition of N- or P/Q-type voltage gated calcium channels involved in vesicular release , 90. RecOpioids are powerful modulators of inhibition in the hippocampus , 80, 99 This review focuses on the regulatory mechanisms of identified subclasses of basket and other CCK-positive cells and their presynaptic inhibition involving kainate and cannabinoid receptors. The cascade of events leading to presynaptic mechanisms regulating GABA release is probably a dynamic process with postsynaptic mechanisms. These presynaptic modulatory pathways are also strongly correlated with the class of presynaptic interneurones recruited and perhaps there is a selective insertion of presynaptic receptors. Subclasses of basket cells illustrate this well, for example, CCK-positive basket cells have a great array of presynaptic receptors and modulators in comparison to the parvalbumin-positive basket cells, specializing CCK-basket cells to be highly modifiable allowing fine-tuning of perisomatic inhibition. Perhaps parvalbumin basket cells that have a more rigid and precise nature of inhibition in synchronizing the network , 53 do nvia specific presynaptic receptors may change the strength and properties of inhibition in cortical regions. These modulatory pathways may also act in a complementary manner that regulates these two distinct sources of inhibition in a co-ordinated, but opposing manner by amplifying one source and dampening inhibition on another.Thus selective modulation by activity-dependent release of neurotransmitter Future experiments need to focus on revealing how the many presynaptic receptors and modulators regulate synaptic strength at other identified subclasses of interneurones. With the existence of diverse inhibitory circuitry in the cortical regions, it is of interest to dissect how these interneurones and the pre and postsynaptic mechanisms involved in mediating responses determine the overall effect of synaptic inhibition.


The growth of some of the adenocarcinomas is virtually identical to that of malignant mesothelioma, also known as pseudomesotheliomatous adenocarcinoma of lung. Their differentiation on the basis of histopathology can pose diagnostic difficulties; hence immunohistochemistry and electron microscopy may be required for further differentiation. Pseudomesotheliomatous adenocarcinoma is an uncommon variant of peripheral adenocarcinoma first described by Harwood et al in 1976. It usually presents with signs of pleural thickening. Hence it may be a diagnostic challenge in patients in whom malignancy is least suspected. Further, even if a diagnosis of malignancy is made on histopathology, it may be confused with epithelial variety of mesothelioma. This case is being reported to emphasize the presence of this entity and modalities which can be used to make the exact diagnosis.A 25-year-old, non-smoker, male presented with dull aching left sided chest pain, dry cough and exertional dyspnoea for past two months. He was being treated with antituberculosis drugs over past two months for left sided pleural effusion. He denied history of exposure to organic or inorganic dust. There was no evidence of digital clubbing or lymphadenopathy. Examination was unremarkable except loss of volume, dull note and decreased auscultatory sounds over entire left hemithorax. Chest radiograph  shows inHistopathological examination of the biopsy specimen shows  tumor cePeripheral adenocarcinoma of the lung with extensive pleural involvement also called as pseudomesotheliomatous adenocarcinoma of the lung (stage −3b).Peripheral adenocarcinoma typically manifests as a nodule with retraction of overlying pleura and an expansile growth pattern that destroys and displaces adjacent lung parenchyma. The borders of tumor may be rounded, lobulated, or poorly defined. The lobulation reflects the histological heterogeneity of lung cancer whereas ill-defined borders may be related to invasion of adjacent lung, fibrosis or interstitial oedema. The growth of some these adenocarcinomas are virtually identical to that of malignant mesothelioma, also known as pseudomesotheliomatous adenocarcinoma of lung.Distinction between pseudomesotheliomatous adenocarcinoma and epithelial variety of mesothelioma may be difficult. Histochemical stains, immunohistochemical markers and electron microscopy can facilitate the distinction between the two. Histochemical stains include Periodic acid Schiff stain after diastase digestion (PAS-D) and Alcian blue stains. PAS-D stains neutral mucin, which is produced by adenocarcinomas. Alcian blue stains acid mucin that is produced by epithelial mesotheliomas. Further, immunohistochemical staining can be done to differentiate between the two. Adenocarcinomas immunoreact with glycoproteins  like CEA, Ber EP4, B72.3, Cluster of differentiation (CD) 15, and BG-8 whereas mesotheliomas do not react with glycoproteins. Mesotheliomas show a thick, brush border like staining pattern with HBME-1 while adenocarcinoma do not stain with HBME-1 or show a cytoplasmic or thin membrane pattern of staining6Our patient was referred to oncology for further management. Chemotherapy has an established role in the therapy of Stage-3b non-small cell lung carcinoma; randomized clinical trial data demonstrate improved median and long term survival when antineoplastic agents are used as part of multimodality approach. However, the optimal sequence for various modalities and best chemotherapy regimen remains to be defined.7


Endotracheal intubation is an important part of pre-hospital advanced life support that requires training and experience, and should only be performed by specially trained personnel. In Norway, anaesthesiologists serve as Helicopter Emergency Medical Service HEMS physicians. However, little is known about how they themselves evaluate the quality and safety of pre-hospital advanced airway management.Using a semi-structured questionnaire, we interviewed anaesthesiologists working in the three HEMS programs covering Western Norway. We compared answers from specialists and non-specialists as well as full- and part-time HEMS physicians.Of the 17 available respondents, most (88%) felt that their continuous exposure to intubations was not sufficient. Additional training was mainly acquired through other clinical practice and mannequin- or cadaver-based skills training. Of the respondents, 77% and 35% reported having experienced difficult and failed intubations, respectively. Further, 59% reported knowledge of airway management-related deaths in their HEMS program. Significantly more full- than part-time HEMS physicians had experienced these problems. All respondents had airway back-up equipment in their service, but 29% were not familiar with all the equipment.The majority of anaesthesiologists working as HEMS physicians view pre-hospital advanced airway management as a high-risk procedure. Relevant airway management competencies for HEMS physicians in Norway seem to be insufficiently trained and maintained. A better-defined level of competence with better training methods and systems seems warranted. Endotracheal intubation (ETI) plays an important role in pre-hospital advanced life support (ALS) -3. Despi2 region with a total of approximately 970 000 inhabitants and an overall population density of about 22 individuals per km2  and analysed using SPSS .Since the activity data used in this study are officially available in annual reports and all questions were answered voluntarily, it was not deemed necessary to seek approval from the Regional Ethics Committee for this study. The results were compared using Fischer's Exact test. A p-value < 0.05 was considered statistically significant.In 2006, the three HEMS programs overall completed 3451 missions (Table The majority (71%) of the anaesthesiologists working as HEMS physicians were certified specialists Table . Only onAll but two respondents felt they needed a certain volume of ETIs to maintain their airway management skills. The desired number of ETIs per month ranged from five to fifteen (median 5), but only one HEMS physician (6%) said he achieved this goal. The number of actual ETIs encountered per month ranged from one to ten (median 2).Thirteen (77%) of the 17 HEMS physicians reported having experienced a difficult airway situation in the pre-hospital setting, and six respondents 35%) had experienced a failed pre-hospital ETI. More full- than part-time HEMS physicians reported these airway problems  , the Laryngeal Tube (LT) and the Gum Elastic Bougie Table . SignifiWhen asked how they maintained their own advanced airway skills, all but one had some strategy for this. Six respondents relied solely on the experience gained in their work as anaesthesiologists or HEMS physicians, while the other 10 (59%) combined this with training on manikins (n = 7), training on cadavers (n = 5) or attending airway management courses (n = 3). Only one reported experience with high fidelity patient simulators for this purpose.There seems to be a need for continuous ETI skills maintenance and difficult airway management training among anaesthesiologists working as HEMS physicians in Norway. While airway management problems do occur, not all of the physicians seem prepared or properly trained to handle them. Our survey indicates that, although the HEMS physicians felt they needed training to maintain their advanced airway management proficiency, this was left to the individual physician to organize.One limitation of our survey is that it covered only three HEMS programs in one region of Norway. We still believe that the findings are representative for other HEMS programs in Norway since these other programs are organized in a similar fashion. However, beyond Norway, it is more difficult to generalize based on our findings, because physician-manned HEMS in other countries are organized differently and may have different systems for training and maintaining advanced airway management skills. Despite this, we believe that the problems addressed here are applicable to other HEMS systems manned with anaesthesiologists ,11. FurtRegarding the statistical analysis, the significance tests should be interpreted with caution because the sample set is limited. Still, we believe the differences that were uncovered are valid as indicators of how anaesthesiologists themselves view the risks associated with pre-hospital airway management.There are currently 11 HEMS programs  in NorwaIt is well documented that there is a certain learning curve involved with critical skills in the speciality of anaesthesiology, including ETI ,16. WhenThe frequency of airway management complications is probably lower in physician manned pre-hospital services than in non-physician manned pre-hospital services ,8, but aIt has been reported that more than 80% of HEMS physicians in Norway are specialists in anaesthesiology . AccordiIn recent years, training in full-scale medical simulators has emerged as a new way of training health-professionals. Successful airway management curricula have been created , also foAlso, from a patient safety perspective, we think it is important to better define what competence HEMS physicians should have and establish better routines for training and retaining critical skills like advanced airway management. However, further studies are needed to better quantify the hazards and risks that patients are exposed to in the current system and to tailor future training and continuing educational programs for HEMS physicians.Relevant airway management competencies for HEMS physicians in Norway seem to be insufficiently trained and maintained. A better-defined competency level for HEMS physicians seems warranted. Further studies are needed to determine how new training methods can improve the airway management competence of HEMS physicians and to what extent this will improve outcome.The authors declare that they have no competing interests.SJMS conceived the study and designed the questionnaires, carried out halve of the interviews, managed the data and carried out the statistics and drafted the manuscript. JKH participated in the design of the study and the questionnaires, carried out halve the interviews and helped to draft the manuscript. ES helped conceive the study and helped to draft the manuscript. HML helped conceive the study and helped to draft the manuscript.Translated questionnaire. English translation of the Norwegian questionnaire used during interviews.Click here for file


Mus musculus house mouse, occurs in seal rookeries.Mass mortalities of marine mammals due to infectious agents are increasingly reported. However, in contrast to previous die-offs, which were indiscriminate with respect to sex and age, here we report a land-based mass mortality of Subantarctic fur seals with apparent exclusivity to adult males. An infectious agent with a male-predilection is the most plausible explanation for this die-off. Although pathogens with gender-biased transmission and pathologies are unusual, rodents are known sources of male-biased infectious agents and the invasive Cardiovirus and Leptospirosis genomes in heart and kidney samples, respectively, but identified a novel Streptococcus species with 30% prevalence in mouse kidneys.Molecular screening for male-biased pathogens in this potential rodent reservoir host revealed the absence of Inter-species transmission through environmental contamination with this novel bacterium, whose congenerics display male-bias and have links to infirmity in seals and terrestrial mammals (including humans), highlights the need to further evaluate disease risks posed by alien invasive mice to native species, on this and other islands. Phoca vitulina) which occurred in 1988 during the seal haul-out in the North Sea was, for example, shown to be due to phocine distemper virus (PDV), a morbillivirus with a significant mortality rate Phocarctos hookeri) occurred, with two bacterial genera being implicated as possible causal agents Arctocephalus tropicalis at Fur Seal Peninsula on Subantarctic Marion Island    in JanuaA. tropicalis pupping season, a time when dominant adult males had already been ashore for 3–4 weeks, securing and defending territories to gain exclusive breeding rights to harems of females At least 250–300 individual adult males succumbed within a period of two weeks (02–15 Jan 2007) between observer visits to this area. No additional atypical mortalities were observed at this beach following the mass die-off. Approximately 50 male carcasses were however encountered around the remainder of the island in smaller rookeries and always in pools of water, at sites spaced 1–5 km apart, prior to the die-off. The nearest of these affected rookeries was approximately 2 km from Fur Seal Peninsula. However, it was only after the observed Fur Seal Peninsula die-off that these noticeable, but not markedly anomalous, deaths were considered to have possibly been linked. The mass mortality coincided with the end of the Marion Island A. tropicalis mortalities occurred (<2 weeks), the relative absence of obvious physical injuries on carcasses, the low levels of physical contact during territorial disputes resulting in the virtual absence of fatal confrontations 2007 = 10.8°C) temperatures for this period showed no marked variation exceeding the standard deviations of the means for the last 10 years (Maximum Average±SD = 11.56±0.71). Subantarctic fur seals are more susceptible to higher-than-ordinary temperatures compared to lower temperatures Picornaviridae. In particular, Encephalomyocarditis (EMC) virus, a member of the Cardiovirus genus which is classified within the Picornaviridae, shows a marked bias towards adult male mice Mus musculusLoxodonta africanaTo our knowledge no documented cases of such severe gender-biased natural mass mortality exist for seals, or other marine mammals. A variety of infectious and non-infectious diseases (such as toxins and stress) were considered as a potential cause for the die-off. As no marine mammal deaths in the wild have conclusively been shown to result from organochloride or toxic element exposure, even in heavily polluted waters Mus musculus, linking higher testosterone levels to increased susceptibility. As the mass seal die-off occurred in the breeding season when testosterone levels are significantly higher in adult male Subantarctic fur seals Members of the Cardiovirus genus have a worldwide distribution and an extremely wide host range, with rodents being implicated as the reservoir host and source of infection for other species Mus musculus domesticus, were accidentally introduced to Marion Island by sealers during the early 1800's Alien mice, Mus are reservoir hosts. Mice are abundant on beaches due to the greater availability of seal and seabird faeces, carcasses and related detritus as food items and as centers of high productivity Arctocephalus tropicalis, Arctocephalus seals are known to investigate objects such as rocks, by smelling them and through tactile response elicited by facial vibrissae  and 10U of AMV-RT (SBS Genetech) as described previously for another picornavirus Cardiovirus primer sets.In order to determine whether mice may harbour the EMC virus and/or be shedding bacteria Leptospirosis, a spirochete which is shed in the urine of a wide range of small rodents has recently been found in seals where it was linked to recent, significant die-offs Mus urine thus provide a likely route of transmission of mouse-borne diseases. To determine whether Leptospirosis or any other bacterial species may be shed in mouse urine, total genomic DNA was extracted from kidney samples of the same 46 mice, using the Roche High Pure PCR Template Preparation Kit, according to supplier specifications. Leptospira PCR primers targeting the 16S rDNA bacterial gene rpoB gene Leptospirosis-presence could not be confirmed through amplification and nucleotide sequencing, with either of the primers sets, indicating the absence of this bacterial genus in the Marion Island mice sampled. However, the broad-range bacterial primers amplified a 16S gene fragment in 18 samples. All positive amplicons were purified with the Roche High Pure PCR Template Purification kit (according to manufacturer specifications), and sequenced with each of the PCR primers in separate reactions. Sequences were viewed, edited and aligned in Mega4 Streptococcus bacterial prevalence of at least 30.4 % in the M. musculus kidney samples screened. A Blast nucleotide search (www.ncbi.nlm.nih.gov/blast) of the Marion Island mouse kidney sequence (which has been submitted to Genbank under accession number EU626397) revealed the closest cultured bacterial match to be S. sanguinis (Genbank entry EU189961), a bacterial agent associated with infective endocarditis in humans S. sanguinis 16S gene sequence was 95.6%, and ranged from 91.6% to 95.2% in pairwise comparisons with other species within the Streptococcus genus. To assess phylogenetic affinities among the presently-documented Streptococcus species and the bacterium identified in Marion Island mouse kidneys, a 16S gene dataset was compiled comprising 37 Streptococcus type specimen sequences, the Mus kidney Streptococcus sequence generated in this study, and four Enterococcus sister-taxon sequences. Following sequence alignment in Mega4 Streptococcus genus (100% bootstrap support). However, the bacterial species in Marion Island mice is distinct from all other congenerics, including those previously isolated from murid rodents and pinnipeds (Streptococcus in mouse urine is significant for a number of reasons. Firstly, this was the most prevalent bacterial genus occurring in mixed Arcanobacterium infections linked to male-biased mortalities of antlered white-tailed deer Odocoileus virginianusStreptococcus bacterial species have thus far been identified from seals (Streptococcus susceptibility has been documented for inbred mice In contrast to the gut which contains multiple bacterial species, kidneys generally have markedly lower levels of bacterial species diversity, making them amenable to identification of unknown bacteria using broad-range bacterial PCR primers innipeds  and therom seals  sufferinStreptococcus, members of which have documented adult, male bias in the Mus rodent host via urine to other species is possible. However, in the absence of seal samples from the adult males that succumbed, this hypothesis that mice were the source of the infectious agent remains tenuous. The indirect evidence presented here, indicating that mice may harbour a bacterium with a potential predilection for male mammals, whilst preliminary, highlights the generally overlooked threat that alien invasive mice may pose to the health of island endemics. We propose several future lines of study that would be useful for providing more definitive answers in the event of a future seal die-off:Invasive mice have direct and indirect adverse effects on invertebrates, birds and plants Collection of blood samples from healthy seals to obtain baseline blood profiles as well as obtaining biopsy samples on a routine basis from dead animals.Demographic studies of the increasing Subantarctic-and Antarctic fur seal populations Assessing sex-specific survival and mortality rates of mice on Marion Island, at both spatial and temporal scales in conjunction with monitoring rodent density and reproductive output in relation to changing climatic conditions.Streptococcus infection dynamics in mice populations, which may assist with modeling and predicting inter-species transmission.Longitudinal monitoring of in vitro and in vivo whether susceptibility to the novel Streptococcus bacterium identified from Marion Island mice displays gender-bias in this rodent host.Investigate