The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon.rbcL, matK and rps2) show significant rate heterogeneity among lineages and among genes. Many of the non-photosynthetic plants have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not always immediate upon loss of photosynthesis. Overall there is a poor correlation of synonymous and nonsynonymous rates. There is, however, a strong correlation of synonymous rates across the 3 genes studied and the lineage-speccific pattern for each gene is strikingly similar. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is a weaker correlation across genes for nonsynonymous rates. Here the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene.Phylogenetic reconstructions of relative rates of sequence evolution for three plastid genes  non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors. Rates of DNA sequence evolution vary among taxa and among genes, and the causes of this variation are many. In some cases, generation time has been shown to be correlated with rates in plants. For example, annual plants can sometimes have higher rates of DNA evolution than perennials . In one rN) and synonymous rates (rS). For example, when rN increases relative to rS, relaxation of purifying selection is a possible explanation. However, when rS increases, but the rN/rS ratio is not greatly affected, then an increase in the mutation rate is a possibility. An example of this is Plantago mitochondrial DNA [e) can increase the fixation rate of neutral and slightly deleterious mutations. Thus, if slightly deleterious mutations are common, both rS and rN are expected to be higher when Ne is low [A useful method for distinguishing among the potential causes of rate variation is to separately examine nonsynonymous rates  and nonsynonymous (dN) substitutions can be measured and are used to compare rates and calculate rate ratios.Rates can also vary if the underlying mutation rate varies ,11 or ifEpifagus virginiana, a nonphotosynthetic plant, has an increased rate of sequence evolution for plastid DNA in general [rps2 gene indicate a significant increase for both dN and dS. This suggests that purifying selection is at least partially relaxed and that there has been an increase in the rate at which mutations are entering the population in this species, due to increased mutation rate or lax DNA repair. MatK, another plastid gene, is characterized by a partial relaxation of purifying selection in the clade containing Epifagus, Orobanche and Boschniakia [E. virginiana and 38 of its relatives for three plastid genes: rps2, matK, and rbcL. Each of these genes is present in photosynthetic relatives of Epifagus, is accelerated (or even lost) in Epifagus or related parasitic plants. Although plastid encoded, the three genes encode proteins that participate in different processes in the plastid. rps2 encodes the ribosomal protein S2 in small subunit ribosome, matK is an intron maturase, and rbcL encodes the large subunit in the CO2-fixing enzyme RUBISCO. We ask several questions: When does the rate increase observed in Epifagus begin, relative to the evolutionary loss of photosynthesis? What are the causes? Relaxation of constraint? More mutations entering the population? Are these patterns consistent across multiple plastid genes? general -17 and rchniakia . In thisSchwalbea relative to the Alectra-Orobanche clade, the Bartsia-Melampyrum clade and the Castilleja-Pedicularis clade. It was also unresolved concerning the relationships among the outgroups Mimulus, Kigelia, Hemimeris, Verbascum, Antirrhinum and Veronica.Phylogenies of the Orobanchaceae and relatives were constructed using maximum parsimony (MP) and maximum likelihood (ML). The MP analysis discovered four most parsimonious trees of 3817 steps, with CI = 0.6275, CI (excluding uninformative characters) = 0.5118, and RC = 0.3888. The strict consensus tree was unresolved as to the position of Cistanche-Epifagus clade. When the MP consensus and the ML consensus were combined into a semistrict consensus tree, a completely resolved tree resulted. This tree is shown in Fig. The ML analysis found two trees, with -ln likelihood values of 24525.88663. The strict consensus of these trees was unresolved, but in a different place, regarding the position of the All three gene trees exhibited statistically significant rate heterogeneity (p < 0.0005), as assessed by the Kishino-Hasegawa (K-H) test . Synonymrps2 and rbcL synonymous and nonsynonymous evolution is poorly correlated, but in matK, the correlation is better  have increases in both synonymous and nonsynonymous rates. Rates are not, however, increased in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides.Some of the non-photosynthetic plants,  usually show an identical pattern of who is faster than whom. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way.Synonymous rates vary markedly among taxa. For example, the branches leading to lar Fig. . For exaMatK is much more rapidly evolving than the other two genes for nearly all taxa, but Epifagus' rbcL pseudogene has a similar rate. When looking at taxa across genes, there is much less consistency than with the synonymous rates. There are some big differences, such as the branch lengths for Striga, Cycnium and Schwalbea, and the Euphrasia species. Overall the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene.There is even more extensive variation in nonsynonymous rates, both among taxa and among genes. This is not surprising because these genes have different functions and some of the taxa are photosynthetic while others are not. The scale bars in Figs. matK rate variation is very similar in the synonymous and nonsynonymous figures. This fits well with the fact that it is less constrained overall, as can be seen by comparing the scale bars in Figs. The pattern of Harveya, Hyobanche and Boschniakia do not have higher synonymous rates.Rates were compared by using two categories at a time and testing for significant differences using likelihood ratio tests. These tests are summarized in Table matK: 2×LR = 10.67, df = 1, p = 0.001; rbcL: 2×LR = 31.3, df = 1, p = 2.2 × 10-8; rps2: 2×LR = 8.56, df = 1, p = 0.00343.Purifying selection is relaxed in the nonphotosynthetic plants for all three genes. The test values are as follows. Epifagus pseudogene has been excluded. Its unconstrained evolution is not typical of "nonsynonymous" change and its position on the plot made it an extreme outlier with an enormous influence on the regression line.Another way to describe the difference in the pattern of synonymous and non-synonymous rates is to say that the former are more correlated across genes. This can be seen in Fig. rps2 and rbcL, the synonymous plots are more highly correlated, whereas for matK, which is relatively unconstrained, they are about the same.For Epifagus [Epifagus, Cistanche, and the Orobanche species, have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not immediate upon loss of photosynthesis, since we do not see increases in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides. This pattern is similar to that found using smaller data sets [The dramatic rate increase observed in Epifagus , with brata sets ,20. Separs (and therefore larger ds) than a species-poor sister group. This was suggested as a cause of rate variation in non-coding DNA in the Lentibulareaceae [Orobanche is not). Thus, accurate numbers of species cannot be assigned to individual branches or clades. However, a few things can be noted. Euphrasia, with ~170 spp., is clearly more speciose than its sister group, with 3 spp. It has a somewhat faster ds. However, the Schwalbea lineage, with a single species, has a fairly high ds. Its position is not certain, but its sister group is probably the Bartsia – Melampyrum clade (>300 spp.), the Castilleja – Pedicularis clade, (>700 spp.), or the union of the two. These groups have do not have dramatically higher ds values; in fact the Castilleja – Pedicularis clade's value is slightly lower.The speciation rate hypothesis predicts that more speciose clades should have a faster areaceae . In thisBartsia, Euphrasia and Melampyrum contains mostly annuals [ds values, as one would expect from a generation time effect. However, both Euphrasia and Melampyrum contain almost exclusively annuals and have very different rates. Likewise, the large clade containing Boschniakia and Epifagus contains mostly annuals and has an overall high ds. The perennials Boschniakia and Cistanche have lower ds than their sister taxa, which also supports the generation time hypothesis, but there is as much variation among categories  as between categories.Differences in generation time may play some role in the observed rate variation. However, as was found previously , the pat annuals  and has The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many  non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors. Clearly, generating additional data from nuclear and mitochondrial genes would help us to more clearly distinguish among these hypotheses. Some of the above-mentioned hypotheses  would be expected to affect nuclear and mitochondrial genomes in a similar fashion, whereas factors affecting mutation rate or efficiency of DNA repair would not, as these process involve different, though perhaps overlapping, sets of enzymes in each of the three genomes -24.We sampled 15 photosynthetic and 16 nonphotosynthetic Orobanchaceae, and eight outgroup taxa. The specimens used and their GenBank accession numbers are given in Table rps2 as in [matK as in [rbcL as in [rps2, 7 matK, and 5 rbcL sequences.We amplified and sequenced s2 as in , matK astK as in , and rbccL as in . A totalrps2 alignment was simple, containing only two small indels. For matK and rbcL, a search for the best alignment was conducted using Clustal X and a variety of alignment parameters. Alignments were evaluated according to the following optimality criterion: whichever alignment yields the MP tree(s) with the highest consistency is considered the best alignment[), which uses the simple indel coding method of Simmons and Ochoterena [rps2 had just one small indel and was aligned by eye. For matK, the optimal computer alignment was generated using GOP = 5 and gap extension penalty (GEP) = 1. Transitions were weighed the same as transitions. The RC from the analysis with indel characters included was 0.3693. The RC without indel characters was 0.3759. The alignment was then adjusted by eye. This final alignment yielded RC values of 0.3878 (indel characters included) and 0.3763 (indel characters excluded).The alignment. For alialignment and 0.4499 (indel characters excluded).For rps2, positions homologous to positions 48–660 of the Nicotiana gene were used. For matK, the entire gene was used. For rbcL, positions homologous to Nicotiana gene positions 5–1325 were used. The three genes were then combined into a single data set. PAUP* 4.0b8 [Nicotiana tabacum as the outgroup taxon, TBR branch swapping and 100 random addition replicates. Bootstrap analyses were conducted with the same settings, except with only 40 random addition orders. 500 bootstrap replicates were performed.For P* 4.0b8  was usedML analyses excluded indels. Using the hLRT method of the program ModelTest 3.06 , the ML . At least nine of the rbcL "genes" are probably pseudogenes. These are indicated in Figure Orobanche caryophyllaceae and Cistanche phelypaea "genes" have internal stop codons and thus are probably also pseudogenes. There may also be other pseudogenes with intact ORFs, making their pseudogene status less obvious [Overall rate heterogeneity was assessed using the K-H test as implemented in PAUP, using the same ML analyses, except that the starting tree was a neighbor-joining tree and the analysis was limited to 40 rearrangements. Nonsysnonymous and synonymous changes were reconstructed on branches using the codon-based likelihood model of Muse and Gaut , as impl obvious . Once a dN, dS, and the dN/dS ratio (ω) in likelihood ratio tests [Orobanche, Epifagus and Cistanche branches and (2) Harveya, Hyobanche and Boschniakia branches. Each of these tests used the data set from a single gene and compared two nested hypotheses: H1: the photosynthetic and non-photosynthetic branches share a single value . H2: the photosynthetic and non-photosynthetic branches have two separate values. If the tree has a significantly higher likelihood under H2, that is taken as evidence that the nonphotosynthetic branches have higher rates. Scatter plots and correlation tests were used to examine the degree of correlation between synonymous and nonsynonymous sites within a gene, and also to see if either class of sites was correlated between genes.Rate increases were compared among categories of taxa (such as photosynthetic and non-photosynthetic), using io tests . These tio tests  indicateNDY and CWD conceived of and designed the study together. NDY did the sequencing, data analyses and drafted the manuscript. CWD provided the genomic DNA samples and provided the conducive laboratory environment, both physical and intellectual, as well as many suggestions for the manuscript.


The mining of information from scientific literature using computational tools has tremendous potential for knowledge discovery, but how close are we to realizing this potential? Biological databases offer access to formalized facts about many aspects of biology—genes and gene products, protein structure, metabolic pathways, diseases, organisms, and so on. These databases are becoming increasingly important to researchers. The information that populates databases is generated by research teams and is usually published in peer-reviewed journals. As part of the publication process, some authors deposit data into a database but, more often, it is extracted from the published literature and deposited into the databases by human curators, a painstaking process.www.ebi.ac.uk/embl/) or ArrayExpress (www.ebi.ac.uk/arrayexpress/), nobody receives credit for the submission of a fact to a database without an associated publication. As long as this practice continues, curation will be necessary to add the (re)formalised facts to biological databases.Research literature and scientific databases fulfil different needs. Literature provides ideas and new hypotheses, but is not constrained to provide facts in formats suitable for use in databases. By contrast, databases efficiently provide large quantities of data and information in a standardised schema representing a predefined interpretation of the data. While the acceptance of a paper can enforce the submission of data to a central data repository, such as EMBL , Unified Medical Language System (www.nlm.nih.gov/research/umls/), and MedDRA (www.meddramsso.com/NewWeb2003/index.htm) [Curators fulfil a second important task: they know how to define standards for data consistency, in particular, the most relevant terminology, which has led to the design of standardised ontologies and controlled vocabularies (see dex.htm) . These tControlled vocabulary: A set of terms, to standardise input to a database.F-measure: A statistic that is used to score the success of NE recognition by text mining tools. The F-measure is an average parameter based on precision (how many of the entities found by the tool are correct identifications of an entity) and recall (how many of the entities existing in the text did the tool find).Machine learning: The technology and study of algorithms through which machines (computers) can “learn”, or automatically improve their systems through data gathered in the past (experience).Ontology: A set of terms with clear semantics (language), clear motivations for distinction between the terms, and strict rules for how the terms relate to each other.The problem with curation of data is that it is time consuming and costly, and therefore has to focus on the most relevant facts. This compromises the completeness of the curated data, and curation teams are doomed to stay behind the latest publications. So, is it possible for curation and text mining to work together for rapid retrieval and analysis of facts with precise postprocessing and standardisation of the extracted information?www.gene.ucl.ac.uk/hugo; 20,000 gene and protein names), GO , Uniprot/Swiss-Prot , and other databases. In addition, terms describing diseases, syndromes, and drugs are available from the Unified Medical Language System. Altogether, about 500,000 terms constitute the basis of domain knowledge in life sciences. To gain some perspective of this figure: an average individual handles 2,000 to 20,000 terms in his or her daily language, and Merriam-Webster's Collegiate Dictionary provides definitions for 225,000 terms (www.merriam-webstercollegiate.com/).There are several software tools that perform well in the identification of standardised terms from the literature. Examples include Textpresso and Whatizit ,6,7,8. EThe identification of all terms by a text mining system still sets challenging demands. All variants of a term have to be taken into account, including syntactical variants and synonyms. In the case of ambiguities, relevant findings have to be distinguished from other findings—a process referred to as disambiguation. Depending on the curation task, it might therefore be advantageous to select only part of the terminological resources and thus restrict the domain of the terminology to the curators' needs .Available text mining solutions are concerned with named entity (NE) recognition , with identification of relationships between NEs (such as protein interactions), and with the classification of text subphrases according to annotation schemata in general (thyroid receptor is a thyroid hormone receptor) ,13,14,15Not all terms used in the literature (NEs) can actually be found in some kind of database . Text mining methods therefore have to detect new terms and map the term to known terminology . If sevewww.pdg.cnb.uam.es/BioLINK/BioCreative.eval.html, and JNLPBA, www.genisis.ch/~natlang/JNLPBA04/) [Over the past several years text mining research teams have presented various approaches that train a software tool to locate representations of gene or protein names  ,18. ThesLPBA04/) ), which www.cs.cornell.edu/projects/kddcup/) had to select documents from a given corpus that contained relevant experimental results about Drosophila [Additional information-extraction methods have been proposed, for example, for the documentation of mutations in specific genes and for the extraction of the subcellular location of proteins ,13. An eosophila .For all automated information-extraction methods, it is obvious that access to literature is crucial. Electronic access has, of course, already had a huge impact, but the structure and organisation of manuscripts could also be improved. For example, semantic tags could be integrated into the text. The markup would not appear on web pages or when the document is printed, but it would help software to deal with semantic aspects of the document. Inserting tags, for example, to mark protein names would allow retrieval software to find documents about proteins even if they look like common English words, such as “you” or “and”. Retrieval engines currently often ignore such terms. In addition, explicit tags would enable text mining methods, for example, when looking for protein–protein interactions, to use the correct semantic interpretation.Text mining systems already available today, such as Whatizit, can integrate semantic tags during submission, which have to be verified by the author. Text mining is ready to deliver tools whereby information is passed back to the authors about the proper use of terminology within their documents. If the use of a term raises conflicts or ambiguities or if the use of a term is wrong, the author is asked to provide feedback. The curation effort is resolved at the earliest possible time-point. Author, publisher, reviewer, and reader profit from consistent information representation, which leads to better dissemination of documents and journals and easily offsets the additional cost in the generation of an article. Publishers and authors have to agree on standards though.Text mining solutions have found their way into daily work, wherever fast and precise extraction of details from a large volume of text is needed. We have to keep in mind, however, that any text mining tool, just like other bioinformatics resources, will only be suitable for a limited number of tasks. For example, the same text may serve curators from different communities who extract different types of facts, depending on their domain knowledge. Furthermore, different communities have different expectations for accuracy. For example, curators dealing with a small set of proteins prefer tools with high recall, whereas curators dealing with a large number of proteins prefer tools with high precision.Although text mining cannot dissect English sentences completely, and cannot extract the meaning and put the facts into a database, text mining tools are becoming increasingly used and valued. Text mining is ready to deliver handling of complex terminology and nomenclature as a mature service. It is only a matter of time and effort before we are able to extract facts automatically. The consequences are likely to be profound. Not only will we have a more effective approach for the mining of knowledge from the literature, our approach to the publication process itself might change. If a fact is clear enough for automatic extraction, it could be reported in a fact database instead of a publication. As methods improve, authors will see more and more of their text being analysed and formalised in a database. If appropriate quality control is provided, and if authors receive due credit for their deposition of facts into databases, we might well see a shift towards original papers describing new creative ideas and visions rather than just listing facts.


They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.Vacuolar Hin vivo-FRET between subunits fused to variants of green fluorescence protein  in transfected cells.To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) +-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H Plant V-ATPase has been identified at the vacuolar and various other endomembranes, and also at the plasma membrane [1-part), and VHA-a, c, c', c", d and e for membrane-associated subunits (V0-part) [A. thaliana, oat and barley [A. thaliana [Mesembryanthemum crystallinum [Arabidopsis and Mesembryanthemum express a similar set of subunits [0-sector since its subunit composition is not resolved yet. VHA-a is the subunit with the highest molecular mass within the V-ATPase and reveals a bipartite structure. As first shown in yeast, VHA-a consists of a 50 kDa hydrophilic N-terminal domain and a hydrophobic, membrane-spanning C-terminus [o-domain [Vacuolar Hmembrane -3. The tmembrane . V-ATPasV0-part) . In planV0-part) ,7. Follod barley ,7. Howevthaliana . The sectallinum , and a tsubunits . An impoterminus -13. Inteterminus  could noterminus . Anotherterminus . VHA-H io-domain .The aim of the study was to improve the understanding of V-ATPase distribution in plant cells with emphasis on the localization of VHA-a and VHA-H in the V-ATPase structure. Three specific questions were answered using different methodology: (i) Is VHA-a part of the V-ATPase structure? (ii) Where are different subunits localized within plant cells? (iii) Is FRET a suitable method to investigate subunit interaction within the complex, for example between VHA-a and VHA-c, and VHA-H and VHA-B.M. crystallinum was identified (AI822404) with similarity to known genes coding for VHA-a in yeast. Using RACE-PCR a full length cDNA was obtained with a size of 2783 bp. Its open reading frame encoded a hypothetical protein of 93.1 kDa. Database search with the program FASTA revealed the highest similarities to the entries At2g21410 (77%), At4g39080 (76 %) and At2g28520 (60 %) from the A. thaliana genomic database and also significant similarity on the amino acid level to the genes Stv1 (34 %) and Vph1 (38 %) from S. cerevisiae. These two latter genes are coding for isoforms of the yeast 100 kDa subunit whereas all other subunits of the yeast vacuolar ATPase are encoded by one gene each. The sequence alignment between the newly cloned amino acid sequence from M. crystallinum, the three isogenes from A. thaliana and the two isogenes from yeast  a cDNA fragment from A. thaliana and M. crystallinum to the distinct yeast isoforms Vph1 and Stv1, in order to define orthologous genes, was not possible on basis of the amino acid sequence alignment. The comparison of the C-termini revealed a higher degree of sequence conservation interrupted through various short insertions or deletions. The sequence was analysed for secondary structures  and membrane topology . The prediction correlated regions of high sequence conservation with the localisation of putative transmembrane domains , introduced and expressed in E. coli. The 42 kDa and 13 kDa polypeptides were purified to homogeneity by chromatography on Ni-NTA and by preparative SDS-PAGE. The 42 kDa polypeptide was recognized by a purified antibody against yeast Vph1 [N-term (not shown). The presence of VHA-a was investigated in plant endomembranes and soluble fractions rapidly prepared from 5 week old M. crystallinum plants. In the soluble and the membrane-fraction, the serum against McVHA-aN-term identified two polypeptides with apparent molecular masses between 65 and 70 kDa . Without protection from proteolysis, a band at about 50 kDa and a doublet band above 60 kDa were predominant. In the sample with protease inhibitor, the 95 kDa band appeared although as one of five bands of similar intensity if decreasing transfer efficiency of high molecular mass polypeptides from the gel to the membrane is assumed. Apparently, VHA-a is prone to degradation. However, the results also indicate that VHA-a is part of V-ATPase. To further prove that tentative conclusion, immunoprecipitation was performed using anti VHA-aN-termand anti VHA-amemb followed by Western blot analysis with anti VHA-E and anti-VHA-A  and a labelling of the Golgi apparatus significantly stronger than with anti-VHA-a  and excitation spectra (acceptor) and need to be situated in close proximity. Half maximum energy transfer takes place at distance of the Förster radius R0. Cyan and yellow fluorescent proteins constitute such a FRET pair and were fused to the C-termini of various subunits of V-ATPase. Under the assumption of freely rotating fluorophores, R0 is close to 5 nm. The size of the V-ATPase complex is about 15 nm (diameter) × 25 nm (length from lumen side to tip of head). Arabidopsis protoplasts were co-transformed with vector constructs of VHA-a fused to YFP and VHA-c fused to CFP under the control of the 35S promotor. Upon excitation of doubly labelled protoplasts at 458 nm both, CFP and YFP showed strong fluorescence  this corresponds to a distance between VHA-a, and VHA-c of ~5.4 to 7.2 nm (mean 6.3 ± 0.8 nm). Here it has to be pointed out that each ring of the rotor V0 contains 5 VHA-c-subunits and one VHA-c"-subunit. Hence, dependent on the position of the VHA-c/CFP-subunits in the ring different distances between CFP-labelled VHA-c subunits and the YFP-labelled VHA-a subunit will result.FRET allows to investigate protein-protein interaction nce Fig. . Fluoresnce Fig. . This faSimilar experiments were performed with onion epidermis cells Fig.  co-transArabidopsis gene products contain all charged amino acids that have been shown to be essential for proton-translocation at conserved positions in the membrane spanning region of the C-terminus  have previously indicated a distribution of V-ATPase among nearly all endomembranes of the secretory pathway including the plasmalemma -3, 33-3-333-35. 1-structure of V-ATPase. The presence of both subunits on all these membranes shows the presence of fully assembled V-ATPase.In our examination the antisera against VHA-A and -E showed a staining of the tonoplast and to a lesser extent also of the ER and Golgi-Apparatus (data not shown). VHA-A and E are part of the cytoplasmically exposed VN-term on the other hand. Immuno-labelling indicates that VHA-aN-term antiserum exclusively labels the ER with some rare association on Golgi stacks. These results are surprising since the sequence features of McVHA-a suggest an essential involvement of VHA-a in proton transport. A possible explanation might be the presence of three different isoforms in A. thaliana and O. sativa which all share the localisation of the charged amino acids responsible for proton-translocation  is exclusively associated with V-ATPase localised on the ER. The hypothesis of a compartment-specific localisation of VHA-a subunits is supported through several findings in plants and yeast. In yeast all known subunits and chaperones of the V-ATPase are encoded by one gene. Only the 100 kDa VHA-a is encoded by two different isoforms (Vph1 and Stv1) [S. cerevisiae and A. thaliana  . In genend Stv1) ,39. In aficiency . The N-tIn plants, Matsuoka et al.  were ablIn vivo-FRET in protoplasts expressing VHA-B/CFP and VHA-H-YFP confirms the orientation of the C-termini of VHA-B and H in close vicinity. It should be noted that co-expression of other pairs of chimeric donors and acceptors such as VHA-E/CFP and VHA-c/YFP did not elicit FRET after excitation of CFP (not shown). The assumed location of the C-terminus of VHA-c in the lumen of the endomembrane compartments and the C-terminus of VHA-E most likely in the vicinity of the head is in agreement with the negative result, i.e. the absence of FRET between VHA-E/CFP and VHA-c/YFP. The studies exemplify the suitability of FRET to analyse structural features of V-ATPase in vivo. The efficient but variable FRET in cells expressing VHA-c/CFP and VHA-a/YFP allows two conclusions: First, the C-termini of VHA-c and VHA-a are likely to be located on the same, i.e. luminal, side of the endomembrane compartments supporting the topological model with nine transmembrane-domains of VHA-a in plants as previously suggested for yeast [Our results from FRET analysis also allows to make some structural assignments: From crystal structure of F-ATP synthase, the C-termini of subunit β, homologous to VHA-A, and subunit α, homologous to VHA-B, are located in close vicinity and oriented to the membrane . Occurreor yeast ,23 , 4 mM dithiothreitol and a few crystals of phenylmethylsulfonylfluoride [® cocktail  was added throughout the procedure. Following differential sedimentation and gradient centrifugation, tonoplast enriched membranes were recovered from a 30%/35% sucrose interphase, sedimented, frozen in liquid nitrogen and stored at -80°C.Leaves (50 g) of fluoride . As indi® system according to the supplier .Membrane proteins were separated on 12.5% sodium dodecylsulfate polyacrylamide gels, transferred to nitrocellulose and probed with anti-VHA-E , anti-VHFor immunoprecipitation, membranes were solubilised in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% (v/v) Triton X-100, 5 μl anti VHA-a antiserum was added, and the samples were shaken at room temperature for 45 min. Then 150 μl protein A-sepharose equilibrated in the same buffer was added. After 15 min, the suspension was placed on a cushion of 1 ml of 40% sucrose and spun at 10,000 × g for 1 min. The sediment was washed thrice with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1 % (w/v) SDS, and finally once in 125 mM Tris-Cl, pH 6.8. The sediment was boiled in loading buffer and analysed by Western blot using rabbit antisera raised against VHA-E or A.N-term and anti-VHA-aMemb. For both the corresponding cDNA fragments of Mc-VHA-a were amplified by PCR using primer combinations a-nterm-f (ATG CGA TCG GAG CCG ATG CAA) and a-nterm-r (TTC ACC CAA CTC ATC GGT GG) encoding the 42 N-terminally located fragment, and a-memb-f (CTT CCA AAG CCC TTT ATT ATG) and a-memb-r (TCA CTC ATG TCC ACC ATG TCA ATC) encoding the polypeptide loop of about 13 kDa located between transmembrane domain 3 and 4 according to the topological model of Vph1p of S. cerevisiae [E. coli JM109. The 6x-his-tagged proteins were expressed, purified by chromatography on Ni-nitrilotriacetate columns, separated by preparative SDS-PAGE, excised as protein bands, eluted and used for immunization . In addition, antisera against subunits VHA-A , VHA-E, calreticulin  in a site-directed manner after amplification from cDNA  using th2, 4 mM morpholinoethane sulfonic acid, KOH , phl 5.7) and checked for sufficient intactness in the microscope. In short, 1 mm leaf slices of 3- to 5-week-old Arabidopsis plants were vacuum-infiltrated and cell walls were digested in media containing 1.5 % (w/v) cellulase R10 and 0.4 % (w/v) macerozyme R10. Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium  and checked for sufficient intactness in the microscope. 110 μl PEG-medium (4 % (w/v) polyethylene glycol 4000, 0.2 M mannitol, 0.1 M CaCl2) and 20 μl plasmid DNA (3 μg/μl) were added to 100 μl protoplast suspension. The samples were incubated at room temperature for 15 min and then consecutively diluted with 0.5, 1, 2 and 4 ml W5-medium with 15 min incubation steps in between . Following 24 h incubation at 25°C, sedimented protoplasts were used for analysis.Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium . The distance between macrocarrier and tissue was 12 cm. The epidermis tissue was incubated for about 20 h at room temperature in the dark prior to analysis.Cells of onion epidermis were placed on filter paper soaked with one-strength MS basal medium in petri dishes and were transiently transformed with a biolistic approach. Gold particles  were suspended in 50 % glycerol. 8.33 μl of the suspension were mixed with 8.33 μl plasmid DNA (1 μg/ μl), 8.33 μl 2.5 M CaCl4, 10 mM EGTA, 1 × phosphate buffered saline, pH 6.8), washed, permeabilised in 0.5% Triton X-100 and washed again. Following blocking of non-specific binding sites with 1 % bovine serum albumin, primary antibody was added for over night at 4°C. Washed samples were incubated with secondary antibody labelled with Cy3, Cy5 or FITC for 1 h. Double labelling was performed by combined application of primary antibodies from rabbit and guinea pig. Slides were mounted with Citifluor Mounting Medium. Fluorescence analysis was performed with a confocal laser scanning microscope Leica TCS-SP2  equipped with three lasers and excitation wavelengths of 458, 476, 488, 514, 568 and 633 nm. The double dichroic mirror DD488/543 was used for fluorescein isothiocyanate (FITC), and for Cy5 the triple dichroic mirror TD488/543/633 was used. Background was controlled and photomultiplier voltage (800 V) selected for maximum sensitivity in the linear range.Immuno-labelling was performed according to . In brie2PO4, 50 mM NaH2PO4, pH 7.0) for 45 min, washed with EM buffer and dehydrated with a series of increasing concentration of acetone. Samples were embedded in epoxyresin , cut into ultra-thin cross-sections of 60–70 nm and immobilized on 200 mesh gold nets. Immuno-decoration was performed with antibody diluted in Tris-buffered saline  NaN3) for an hour. Samples were washed five times and incubated with secondary antibody conjugated to 15 nm gold particles. The samples were stained with 0.1 % (w/v) uranyl acetate for 5 s and afterwards with 2 % lead citrate. The samples were analysed with an electron microscope  at 75 kV.Cells were fixed in 2.5% glutaraldehyde in EM buffer (50 mM KHo = 5 nm [CFP/bleached - ICFP/unbleached)/ICFP/unbleached and R = ((Ro6/E)-Ro6)1/6, where E is the transfer efficiency, and ICFP the fluorescence emission intensity in the CFP peak.Transformed protoplasts and onion epidermis cells were examined for the localisation of the CFP/YFP-fused proteins using the same CLSM set-up as mentioned above. Autofluorescence of 10 protoplasts, as well as reference spectra of YFP and CFP-derived fluorescence were recorded in the spectral range of 480 to 700 nm, averaged and used for corrections. Excitation was recorded at 458 nm (CFP and FRET) and 514 nm (YFP), respectively. Scan speed was 800 Hz. Acceptor dye was bleached with 100 % laser intensity. Emission spectra were recorded and averaged from 20 transformed protoplasts. For a first estimate of transfer efficiency, a Foerster radius for green fluorescence protein variants of Ro = 5 nm  was used+-ATPase; Vph1: VHA-a subunit isogene in yeast; YFP: yellow fluorescence proteinCFP: cyan fluorescence protein; CLSM: confocal laser scanning microscope; FRET: (Förster) fluorescence resonance energy transfer; PAGE: polyacrylamide gel electrophoresis; Stv1: VHA-a subunit isogene in yeast; VHA: vacuolar HN-term; TS: co-transfection of protoplasts and epidermis cells, CLSM analysis; SB: immuno-co-localisation and discussion; SS: co-immuno-precipitation; MH: preparation of anti VHA-aMemb; BS-J: immuno-co-localisation and discussion; JR and MS: FRET analysis; DG: transformation and construct design, KJD: project design and supervision.CK: VHA-a sequence analysis, immuno-cytochemistry, transient expression systems, preparation of anti VHA-a


Only controlled blocks are capable of confirming the zygapophysial joints (ZJ) as the pain generator in LBP patients. However, previous workers have found that a cluster of clinical signs ("Revel's criteria"), may be valuable in predicting the results of an initial screening ZJ block. It was suggested that these clinical findings are unsuitable for diagnosis, but may be of value in selecting patients for diagnostic blocks of the lumbar ZJ's. To constitute evidence in favour of a clinical management strategy, these results need confirmation. This study evaluates the utility of 'Revel's criteria' as a screening tool for selection of chronic low back pain patients for controlled ZJ diagnostic blocks.This study utilized a prospective blinded concurrent reference standard related validity design. Consecutive chronic LBP patients completed pain drawings, psychosocial distress and disability questionnaires, received a clinical examination and lumbar zygapophysial blocks. Two reference standards were evaluated simultaneously: 1. 75% reduction of pain on a visual analogue scale (replication of previous work), and 2. abolition of the dominant or primary pain. Using "Revel's criteria" as predictors, logistic regression analyses were used to test the model. Estimates of sensitivity, specificity, predictive values and likelihood ratios for selected variables were calculated for the two proposed clinical strategies.Earlier results were not replicated. Sensitivity of "Revel's criteria" was low sensitivity (<17%), and specificity high (approximately 90%). Absence of pain with cough or sneeze just reached significance (p = 0.05) within one model."Revel's criteria" are unsuitable as a clinical screening test to select chronic LBP patients for initial ZJ blocks. However, the criteria may have use in identifying a small subset (11%) of patients likely to respond to the initial block (specificity 93%). It is estimated that 15 – 40% of chronic low back pain patients have pain arising from the lumbar zygapophysial joints (ZJ). PreviouBecause prognosis for acute low back is good, invasive and expensive and invasive diagnostic testing cannot be justified. However, persistent disabling pain needs more intensive investigation in order to determine appropriate management strategies. Back pain of ZJ origin may be treated using intra-articular steroid injection,10., or The current study objectives were to estimate the predictive value of the two clinical strategies of Revel et al (1998), using similar measurement parameters (75% reduction in pain VAS after ZJ block), and apply alternative analytic methods to explore any potential utility of the variables used.This study utilized a prospective, blinded, concurrent, reference standard-related validity design with intra-articular ZJ or medial branch blocks as the reference standard against which clinical variables were compared. Local Institutional Review Board approval was granted at the beginning of the study. A 75% or more reduction in pain following ZJ block on pain VAS was designated as reference standard A, the same standard used by Revel et al (1998). Based on pain drawings and patient self-report, complete abolition of the patient's primary or dominant pain was designated as reference standard B. Dominant pain location was acquired by pain drawing and direct questioning, and documented prior to clinical examination and diagnostic injection in a prospective manner. Post injection dominant pain location was acquired by reference to pain drawings and direct questioning also.Patients with low back pain with or without lower extremity symptoms, referred to a private radiology practice in New Orleans, USA specializing in the diagnosis of spinal pain, were invited to participate in the study. Patients receiving ZJ blocks were either referred specifically for that procedure or had the procedure included in their radiology examination based on pre-injection clinical evaluation by the injectionist (CA). Between May 2001 and October 2002, physical therapists attended the clinic in blocks that ranged from 4 to 8 weeks (ML) and examined patients. Normal scheduling was not affected by the presence of the visiting therapists, so patients were consecutive during these periods. All patients had undergone imaging studies prior to referral from a variety of medical and paramedical practitioners. Some were self-referred.Patients were excluded from the study if they were unwilling to participate, were too frail to tolerate a physical examination, or were deemed by any member of the clinic team to be unable to comprehend the study procedure. Prior to the formal clinical examination, clinic staff recorded basic demographic and medical data.100 mm visual analog scales (VAS) scales for current, best and worst pain. A current pain VAS was repeated after the clinical examination and following ZJ blocks. The 23-point Roland-Morris Disability Questionnaire  was compHistory taking and a structured physical examination were carried out by a physical therapist with 30 years of clinical experience as a manipulative therapist (ML). Some patients were examined by a physical therapist with 17 years experience (SBY). The clinical examination occupied 30 to 60 minutes and included many tests besides those necessary for the current analysis, as part of a larger project. Inconclusive findings or incomplete examinations were documented. The physical examination included a visual assessment of range of motion, recording anatomical location of dominant pain, nerve tension tests, key muscle strength tests, tendon reflex tests, light touch sensitivity, a McKenzie styled ePrior to ZJ blocks, the radiologist reviewed case notes and imaging studies, and conducted a physical examination that guided the type of diagnostic procedure to be employed and the target structures. Intra-articular ZJ joint injection or MBB using standard technique was carrPhysical therapists conducting the clinical examination were blinded to the results of previous imaging studies and diagnostic injections, the Roland, Zung and MSPQ questionnaires. The injectionist was blinded to the results of the physical therapy examination and diagnostic conclusions.Basic statistical values for demographic variables and regression analyses were calculated using statistics software (Minitab version 13.31 © Minitab Inc 2000). Differences between included and excluded patients were evaluated with the student's t, chi square, and Kruskal-Wallis tests where appropriate. Significance for differences was set at p < 0.05.Calculations of sensitivity, specificity, predictive values and likelihood ratios with 95% confidence intervals were performed using Confidence Interval Analysis software © Bryant T.N. 2000.Initial ZJ blocks were carried out on 151 chronic low back pain patients. Thirty-four patients were excluded from analysis as they received another intervention in the same procedure session and did not return for differentiating and confirmatory blocks. One case was excluded through incomplete data on Revel's criteria. Following ZJ block, 27 of 116 patients satisfied reference standard A. Data required for determination of Reference standard B were missing for five of the 116 cases. Eighteen of these 111 patients satisfied reference standard B. Table In specifically evaluating Revel's criteria against reference standard A, logistic regression failed to achieve significance as a model . Two variables; "absence of pain with coughing and sneezing" and "no exacerbation of pain rising from flexion", showed a trend towards significance as predictors within the model (p = 0.07).Using Revel's criteria within a logistic regression with reference standard B as the response variable, a strong trend towards significance was reached . One component variable (age over 65) individually reached significance within the model: . In the whole sample 12.5% were aged over 65 years whereas of the 19 positive responders six were over 65 years (31.6%).The same patients satisfied both strategies of Revel et al. Estimated sensitivity, specificity, predictive values and positive likelihood ratios for both strategies of Revel et al are presented in Table Of the 27 patients satisfying reference standard A, 13 (48%) returned for confirmatory ZJ blocks. Three of these reported 75% or more reduction in pain following the confirmatory block. None of the three patients with confirmed ZJ pain satisfied Revel's criteria.The current data produces results that are in stark contrast to those of Revel et al (1998) with sensitivity low and specificity high. However, 'no pain with cough and sneeze' and 'no exacerbation of pain rising from flexion' approached statistical significance in relation to a 75% reduction pain after ZJ block (reference standard A). These variables are in line with Revel's results. Age over 65 is associated with reference standard B (abolition of primary pain). Likelihood ratios for the criteria are lower in the current data also. Some of the differences in results may be explained by a number of factors:1. Revel et al's study was a placebo controlled design whereas we did not routinely utilize a similar or equivalent control.2. The patients in Revel et al's sample were older (mean 58 versus 43 years), and had a shorter duration of symptoms (mean 78 versus 160 weeks).3. It is also possible that the high level of standardization when acquiring the criteria data in the current study, and the prospective methodology might have contributed to the differences in results.Following anaesthetic blocks, patients frequently state that the pain prompting consultation is abolished, yet a post-procedure pain VAS still registers more than 0/100. In this study reference standard B was evaluated as an alternative to the usual standard, so that pain in areas above the lumbar spine or pain directly attributable to the needle insertion site do not result in inappropriate post procedure VAS scores. In the interests of developing more precise instruments documenting results of diagnostic blocks, we propose that in future studies, the VAS scale should specifically refer to the primary pain complaint for which the diagnostic block is undertaken.Based on the current data, Revel's criteria are not suitable as a screening test to select patients suitable or unsuitable for an initial screening ZJ block. Such a screening device should have high sensitivity to ensure that most patients likely to respond are included in the initial diagnostic block. Our study suggests that, at best, Revel's criteria, might identify a small subset (11%) of patients likely to respond to a screening block (specificity 93%).Neither strategy utilizing Revel's criteria is suitable as a clinical screening device for selection of chronic LBP patients for initial diagnostic ZJ blocks. In contrast to Revel's findings, the current data demonstrated low sensitivity and high specificity for these clinical criteria. The high specificity of the criteria reported in this study relates to a single uncontrolled screening block. Consequently these criteria can not considered diagnostic of painful lumbar ZJ. Only placebo controlled or double ZJ blocks are able to diagnose this source of low back pain.The author(s) declare that they have no competing interests.ML conceived the design of the study, examined all but 10 patients, carried out data analysis and prepared the manuscriptBÖ assisted in project design and manuscript preparationCNA assisted in project design, provided facilities and conducted fluoroscopically guided injectionsBMcD carried out data analysis and assisted in manuscript preparation.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Male suicide rates continued to increase in Scotland when rates in England and Wales declined. Female rates decreased, but at a slower rate than in England and Wales. Previous work has suggested higher than average rates in some rural areas of Scotland. This paper describes trends in suicide and undetermined death in Scotland by age, gender, geographical area and method for 1981 – 1999.Deaths from suicide and undetermined cause in Scotland from 1981 – 1999 were identified using the records of the General Registrar Office. The deaths of people not resident in Scotland were excluded from the analysis. Death rates were calculated by area of residence, age group, gender, and method. Standardised Mortality Ratios (SMRs) and 95% confidence intervals were calculated for rates by geographical area.Male rates of death by suicide and undetermined death increased by 35% between 1981 – 1985 and 1996 – 1999. The largest increases were in the youngest age groups. All age female rates decreased by 7% in the same period, although there were increases in younger female age groups.The commonest methods of suicide in men were hanging, self-poisoning and car exhaust fumes. Hanging in males increased by 96.8% from 45 per million to 89 per million, compared to a 30.7% increase for self-poisoning deaths. In females, the commonest method of suicide was self-poisoning. Female hanging death rates increased in the time period.Male SMRs for 1981 – 1999 were significantly elevated in Western Isles , Highland , and Greater Glasgow . The female SMR was significantly high only in Greater Glasgow .All age suicide rates increased in men and decreased in women in Scotland in 1981 – 1999. Previous findings of higher than expected male rates in some rural areas were supported. Rates were also high in Greater Glasgow, one of the most deprived areas of Scotland. There were changes in the methods used, with an increase in hanging deaths in men, and a smaller increase in hanging in women. Altered choice of method may have contributed to the increased male deaths. Compared to the adjacent countries of England and Wales, Scotland had a low suicide rate through most of the twentieth century . This diSeveral authors have noted the importance of suicide in Scotland as a public health problem ,10. TherWe used anonymised information on deaths by suicide and undetermined deaths provided by the General Register Office for Scotland (GROS). Deaths registered during 1981 – 1999 were included if the cause of death was recorded as suicide or as undetermined cause (ICD-9 E950-E959 and E980-E989 respectively). Undetermined deaths were included as suicide deaths may be misattributed ,14.Population figures were taken from the GROS annual reports for the mid-year of each period. For analyses by area, if a death was registered away from the person's home address, the death was allocated to their area of residence, rather than the area in which they died. Deaths of people resident outside Scotland were identified using country codes, and were excluded. As far as possible, therefore, results reflect the rates of suicide and undetermined deaths of people resident in each area of Scotland. Standardised Mortality Ratios were calculated for National Health Service administrative areas, with 95% confidence intervals. In time period descriptions, the periods 1981 – 1985, 1986 – 1990, 1991 – 1995, and 1996 – 1999 were used. Data were analysed using Excel and SPSS.There were 14502 deaths recorded as suicide or undetermined cause in the time period. Of these deaths, 28.5% occurred in females (n = 4137) and 71.5% in males (n = 10365).Table Examining changes by age group in males Table , there aIn women, there were increases in the three youngest age groups, with a 76% increase in rates in the under 15 year old group, 150% in the 15 – 24 year group and 37% in 25 – 34 year olds  (Table The epidemiology of suicide in Scotland has changed greatly between 1981 and 1999. Male suicide rates have increased in all age groups up to and including 35 – 44 years. The highest male suicide and undetermined death rates in 1996 – 1999 were in the 25 – 34 year age group. In women, rates dropped in age groups from 35 – 44 years up to and including 75 years and over. Rates increased in younger women.There is limited information on the factors underlying individual deaths from suicide in Scotland. Squires and Gorman  reviewedSome methods of self-harm have higher case fatality rates . FirearmThe difference between areas was also of note. The lower rates of increase in the areas with the highest initial rates may reflect to regression to the mean. Method availability  may be iAvailability of method would not explain the differences between apparently similar rural areas. Previous work has found that deprived areas of Scotland tend to have higher suicide rates ,28. DeprUsing routine information allowed a large number of suicide and undetermined deaths to be included in this series. There are, however, limitations to the use of anonymised routine data. No qualitative information was available, and our exploration of the data was limited to trends with no examination of possible underlying causes. The increase in the rate of deaths recorded as suicide or undetermined cause of death, but where no detail on method was included, could conceal recent trends. The increase as a percentage of relevant registrations was small, however, increasing from 7.5% in the first period to 9.1% in the last period studied. The classification of deaths as suicide is often difficult, but the inclusion of undetermined deaths as well as deaths recorded as suicide should have helped to minimise bias from under identification ,30. SquiA divergence between male rates in England and Wales and in Scotland, and in male and female rates within Scotland, had been identified for the first part of the time period described here. This work found that male rates of suicide and undetermined death continued to increase in Scotland, but also identifies increases in younger female age groups. Examination of changes in method by male and female age group will help to establish whether changes in case fatality because of altered method choice  may be pSome rural areas of Scotland had significantly elevated male suicide rates. We have suggested that access to lethal means of suicide may be one contributing mechanism for this, and have also noted the higher than expected suicide numbers in some rural occupations . Rural aThe authors declare that they have no competing interests.CS had the idea for the study, wrote the grant application, contributed to the design and interpretation and drafted the paper. Diane Gibbs and Tracey Rapson analysed the data. Paddy Hopkins contributed to the design, undertook part of the analysis, and commented on the interpretation of the results. Alan Belbin and Alistair Hay contributed to the design and helped interpret the results. All authors read and approved the final draft of the paper.The pre-publication history for this paper can be accessed here:


Batrachochytrium dendrobatidis was identified from sick and dead frogs. Since then, several lines of evidence suggest that B. dendrobatidis may be involved in frog declines: the fungus has been found on frogs in afflicted areas; lab studies show that it's highly pathogenic to some frog species; and pathological evidence links it to host mortality. But with little information about the prevalence of this fungal infection in wild frogs, or how the disease impacts frogs in the wild, the causal role of this chytrid fungus remains unclear.Amphibian declines have reached crisis proportions in various parts of the world. In many areas, habitat loss is the likely culprit. But when mass die-offs suddenly occurred in relatively undisturbed habitats, the cause was far less obvious. Fourteen species suffered either extinctions or major declines in the pristine rainforests of Queensland, Australia, between 1979 and 1993. It was suggested in 1996 that some unknown disease had spread through the populations, but no pathogen was discovered until 1998, when the fungus B. dendrobatidis on frogs in their natural habitat, Richard Retallick et al. focused on six species living in the high-elevation rainforest streams of Eungella National Park in Queensland, Australia, where frog losses were “particularly catastrophic.” Two species vanished between 1985 and 1986: the Eungella Gastric-Brooding Frog (Rheobatrachus vitellinus), which is now thought extinct, and the Eungella Torrent Frog (Taudactylus eungellensis), which later reappeared in a few small populations. Other local frog species escaped this period relatively unscathed.To evaluate the effects of B. dendrobatidis had yet to be identified, but Retallick retained the toe tips, and the authors tested the toes for disease in 2002–2003. Fungal infections were found in two species—T. eungellensis and Litoria wilcoxii/jungguy ; the other four species were infection-free. L. wilcoxii/jungguy did not decline to any great extent during the 1985–1986 die-off.Retallick captured frogs from six sites from 1994 to 1998, clipped one or two toe tips from each frog to age and identify them, and then released the frogs back into the wild. At the time, T. eungellensis frogs was greatest at three particular sites, which showed peak infections during cooler months. Prevalence of infection was highest during winter and spring, but did not vary from year to year, suggesting that the infection is now endemic. Fungal infections were found in 27.7% of L. wilcoxii/jungguy frogs, with no evidence that prevalence differed among sites, seasons, or individuals . The probability of recapture was significantly lower for frogs that were already infected when first captured. While this might suggest a correlation between infection and death, it's impossible to distinguish death from simple failure to recapture the animal. On further analysis, McCallum and colleagues found no evidence that survival differed between infected and uninfected frogs, suggesting that this potentially devastating amphibian disease now coexists with the frogs, with little effect on their populations.The proportion of infected T. eungellensis, a rainforest frog listed as endangered, “now persist with stable infections of B. dendrobatidis.” While these findings do not exonerate the fungus as the agent of mass declines, they do rule out the possibility that the fungus caused the decline, then vanished from the area, allowing frog populations to recover. The authors allow that it's possible that B. dendrobatidis did not cause the initial T. eungellensis declines. Or alternately, the fungus could have emerged as a novel pathogen in the ecosystem, causing massive casualties before some form of evolutionary response took hold. Surviving frog populations may have evolved resistance to the pathogen, for example, or less virulent strains of the fungus may have evolved. If it turns out that frog populations can develop resistance to the chytrid fungus, the researchers point out, then a conservation program of captive breeding and selecting for resistance could potentially thwart the extinction of these, and other, critically endangered frogs. A critical next step, then, is to determine whether frogs and fungus do coevolve.These results, the authors conclude, “show unequivocally” that remaining populations of


The purpose of this study was to evaluate the role of study quality assessment of primary studies in cancer practice guidelines.Reliable and valid study quality assessment scales were sought and applied to published reports of trials included in systematic reviews of cancer guidelines. Sensitivity analyses were performed to evaluate the relationship between quality scores and pooled odds ratios (OR) for mortality and need for blood transfusion.Results found that that whether trials were classified as high or low quality depended on the scale used to assess them. Although the results of the sensitivity analyses found some variation in the ORs observed, the confidence intervals (CIs) of the pooled effects from each of the analyses of high quality trials overlapped with the CI of the pooled odds of all trials. Quality score was not predictive of pooled ORs studied here.Had sensitivity analyses based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been found or that different clinical recommendations would have emerged in the guidelines. Quality assessment of trials included in systematic reviews of evidence is a resource intensive and scientifically controversial endeavour. On the one hand, the routine use of quality assessment in the development of systematic reviews is encouraged by the Evidence-based Practice Center Program of the Agency for Healthcare Research & Quality (AHRQ) and the Cochrane Collaboration, two well respected groups that coordinate a substantial number of systematic reviews -3. IndeeThe concept of incorporating study quality assessment into systematic review methodology has also found empirical support. There is evidence that studies of lower methodological quality tend to report larger treatment effects than high quality studies -7. For eAlthough this seminal work yields compelling results, these findings are not universal and the issue is not without detractors -14. SomeTogether these results suggest the study quality issue is controversial and that the merits of this methodological step in systematic review requires thoughtful analysis. Indeed, West et al conclude with recommendations advocating for research dedicated to comparing quality rating systems and the role of quality assessment within individual clinical contexts and for studies targeted at determining specific quality factors that make a difference in final quality scores .The Practice Guidelines Initiative of the Cancer Care Ontario's Program in Evidence-based Care (PEBC) uses the Guidelines Development Cycle to create cancer practice guidelines comprised of a systematic review of the research literature, an interpretation and consensus of the evidence by members of the guideline development team, clinical recommendations informed by the evidence, and an external review process by Ontario clinicians -19. We f1. What valid and reliable quality assessment instrument would be most appropriate for our context?2. How is study quality currently being used in published systematic reviews of cancer trials and what is the relationship between effect size and study quality in this disease area?3. What impact would study quality assessment have on the clinical recommendations made in evidence-based practice guidelines developed by the PEBC?For a comprehensive review of the strengths and weaknesses of quality assessment instruments, readers are referred to the 2002 West et al. evidence report commissioned by the AHRQ . For ourTo locate systematic reviews on oncology topics, the strategy suggested by Moher et al for finding systematic reviews  was combThe validated scales were applied by two methodologists (MJ and MC) to studies reported in any our practice guidelines that included a pooled analysis based on at least ten randomized trials related to the main guideline question. Intraclass correlation coefficients with 95% confidence intervals (CI) were calculated using a random sample of the RCTs to assess inter-rater reliability, one coefficient calculated for each of the scales used. Because of budgetary limitations for staff time, the analysis was conducted on 18 randomly selected studies rather on the whole group of articles. This is a methodological limitation as fewer studies result in larger confidence intervals and less precise estimates. This may account for the difference in reliability ratings we found for the Sindu scale compared to published norms  based on total quality score. Where the scale developer suggested a cut-off point for low versus high quality, this was used. Where no cut point was specified, the observed median study quality score was used as the dividing point between low and high quality. Meta-analyses were repeated with the high quality studies. Because there would never be a situation in which guideline developers would consider low quality studies only, a meta-analysis using this sample of the studies was not conducted.Four scales meeting our criteria were found; two instruments, Jadad et al  and Cho The literature review located 32 published systematic reviews on oncology-related topics that included some measure of study quality. Five of the reviews examined changes in pooled estimates of effect size of mortality rates when meta-analysis was restricted to high-quality randomized trials -30. As sThree of the PEBC practice guidelines included at least 10 RCTs in their systematic reviews of the evidence and were eligible for inclusion in this evaluation -33: concAt the conclusion of our study, we identified a fourth practice guideline which originally did not meet our 10 RCT inclusion criteria, but later did so after it was updated. The guideline focused on the role of erythropoietin (EPO) in the management of cancer patients with non-hematologic malignancies . Unlike Intraclass correlation coefficients used to established inter-rater reliability were 0.71 for the 3-item Jadad scale , 0.80 for the 6-item Jadad scale , 0.62 for the Sindhu scale , and 0.63 for the Downs & Black Scale . Disagreements were resolved by consensus. Where consensus could not be reached, a third rater (MB) assessed the items and provided the tiebreaker score.While the total quality scores emerging from each of the different scales did all significantly correlate with one another (range r = .35 to r = .73), there was considerable variation in the classification of studies as high quality or low quality as a function of the scale that was applied Table . For exaThe 20 comparisons from the 18 trials included in the head and neck concomitant therapy systematic review yielded 2, 14, 12 and 14 high quality studies, respectively, when the Jadad 3-item, the Jadad 6-item, the Sindhu and the Downs & Black scales was used. Although both Jadad 6-item and Downs & Black scales both assessed 14 comparisons to be from high quality studies, only 11 of these 14 studies were the same. For the 12 comparisons from studies categorized as high quality with the Sindhu scale, 10 of these were also rated high quality by both the Jadad 6-item and the Downs & Black scales, the other 2 were rated as high quality by the Jadad 6-item scale only. There was 1 comparison from a study rated as high quality by the Jadad 6-item scale only and two from studies rated as high quality by the Downs & Black scale only.Mortality data  used for the meta-analysis included in the guideline reports were available for two guidelines and need for blood transfusion data were available for the third ,32,34. FOnly the 6-item Jadad scale was applied to the studies of the EPO guideline and the data were pooled to calculate an overall risk ratio for blood transfusion . The risSeveral conclusions can be drawn from this study and review of the literature. First, there are established methods for assessing the quality of randomized controlled trials in which data on adequate reliability and validity were available. West et al uncovered 32 scales, check lists and component systems concerned with evaluating RCTs ; more thAlthough all of the scales we used have established reliability and validity estimates, we found that the number of trials categorized as high quality or low quality depended specifically on the scale that was applied. For the head and neck cancer systematic review, the number comparisons from high quality studies ranged from 2  to 14 . The range for the colon cancer review was 0 to 9. There was also considerable variability regarding the specific quality category in which each trial was placed. These finding are consistent with those of Juni et al [The lack of consistency of study classification from one scale to the next and the lack of clear cut-off criteria for users to employ when measuring quality of studies, presents a challenge to guideline developers when they need to make choice about which instrument they ought to adopt if the choose to adopt an instrument at all. Rather than clear evidence driving our decisions, we considered other features of the instrument in our decision making. Of the rating scales we examined, our preferred choice would be the Jadad 6-item instrument. In contrast to the others considered in this report, this instrument is relatively easy to implement and interpret and good inter-rater reliability was established. Further, although the 3-item version of the Jadad scale is most commonly used, we found the original 6-item version to be more relevant in our clinical context as it provides greater variation in scores. In the cancer discipline, few trials are placebo-controlled and treatment allocation tends to be poorly reported. In contrast to the pain trials which were profiled in the development phase of the Jadad instrument, the majority of the items in the 3-item version (randomization and blinding items) yield no variation in scores in our context and are, therefore, not useful to discriminate among cancer trials. The 6-item version of the scale more aptly differentiates quality across studies and includes more quality domains for which an empirical basis has been established .Another conclusion that can be drawn from this study is that effect size can be related to study quality but that the nature of the relationship in one clinical area may not generalize to another clinical area. Some of the original work examining the role of study quality reinforces the need to be mindful of the variation among studies included in systematic review -7. HowevWe conducted sensitivity analysis on the systematic reviews comprising the guidelines developed by the PEBC. Only four systematic reviews among 36 eligible practice guidelines included more than 10 trials with data appropriate for pooling; three from which we could extract data. Although there was some variation in the odds ratios observed, the confidence intervals of the pooled effects from each of the analyses of high quality trials overlapped with the confidence intervals of the pooled odds with all of the trials. In no case would the conclusions based on these results be affected by restricting the meta-analysis to only high quality studies; the recommendations remained the same. Had sensitivity analysis based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been drawn from the systematic review or that different clinical practice guidelines would have been formulated.Together, these findings lead to our final conclusion that measuring study quality did not translate into altered conclusions from a systematic review in the oncology domain for the outcomes we used here. Thus, at this time we have decided that measuring study quality using a numerical assessment scale for the purposes of sensitivity analysis will not be a routine part of our guideline development program. We will, however, encourage guideline developers to describe the variation among studies and to point out methodologic flaws. In addition, it will be important for us to repeat this study looking at other outcome measures, such as quality of life and adverse effects, as they become more routinely reported in primary cancer research and incorporated into our practice guidelines. Outcomes other than those studied here may be more sensitive to the issues of study quality.This study highlights a strategy that may be useful for guideline programs to utilize in making decisions regarding the methods employed in their guideline development process. It is important that scientific inquiry be maintained in studying the value and role of study quality assessment rather than accepting its role as convention. By exploring it within a specific clinical context one can identify it's most appropriate application.The author(s) declare that they have no competing interests.MB, MJ and GB conceived of the project idea and developed the protocol. MB, MJ, and MC conducted by study. SH provided statistical advice and AJ provided conceptual advice. The manuscript was drafted initially by MB and MJ. All authors contributed to the final version submitted for publication.The pre-publication history for this paper can be accessed here:


Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC.Western blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 μM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37°C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity.These data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response. Peptides that are similar in sequence to the tethered ligand domains of PAR-2, such as SLIGRL-NH2 (SLI) or tc-LIGRLO-NH2 (tc-LIG) are able to interact directly with the activation site and act as potent agonists [Protease activated receptor-2 (PAR-2) has been identified on a variety of cell types including eosinophils , neutropagonists .A growing number of studies have identified a role for PAR-2 in inflammation. There is a delayed onset of inflammation in PAR-2 knock out mice , and PARin vivo could be MC-mediated. The administration of PAR-2ap or trypsin into the rat hind paw enhanced vascular permeability and caused edema formation, which can be abolished by repeated pre-treatment with compound 48/80, known to deplete the MC of its granular content [et al., 1999 [The ability of serine proteases to activate MC and the observation that MC express PAR-2 ,13, sugg content . By contl., 1999  showed tActivation of PAR-2 on the surface of mast cells could act as part of an autocrine and paracrine positive feedback loop through the release of serine proteases that could activate further PAR-2 on mast cells or other neighboring cells. Therefore, we investigated the direct effects of PAR-2ap on the release of serine proteases from purified PMC and the effects of these released proteases on extracellular protein degradation. In particular we studied the release of rat mast cell protease-1 (RMCP-1), RMCP-5 and carboxypeptidase A (CPA).To identify proteases released by mast cells following PAR-2ap stimulation we activated PMC with tc-LIG (10 μM), and analyzed the supernatants for various mast cell proteases by western blotting, using antisera against the amino-terminal sequences of RMCP-5 and MC-CPA and an antiserum against RMCP-1 protein. In supernatants from tc-LIG-treated PMC one band for RMCP-1 (30 kDa), two bands for RMCP-5 (34 and 35 kDa), and three bands for CPA  were detected Fig. . The PARNippostrongylus brasiliensis antigen (Nippo Ag) (10 We/ml) induced no detectable release of any of the three proteases studied , which however, was associated with protease release , tc-LIG (10 μM), tc-OLR (10 μM) or compound 48/80 (0.5 μg/ml) for 20 min or 8 hr at 37°C and the supernatants were collected. These supernatants or media were then seeded with 150 pg/ml of rat recombinant TNF and incubated for an additional 8 hr. At the end of the incubation TNF was measured by ELISA and the proteolytic activity was calculated as % degraded TNF (as discussed in the methods section). Proteolytic activity in the supernatants of sham-treated cells was subtracted from that in the supernatants of activated PMC. Supernatants from sham-treated PMC showed significant loss of seeded TNF (17 ± 7 % at 20 min and 22 ± 5 % at 8 hr) as compared to media. At both 20 min and 8 hr of treatment supernatants from tc-LIG-treated MC (10-0.1 μM) showed a greater loss of seeded TNF compared to supernatants of sham-treated MC (p < 0.05), suggesting tc-LIG-mediated activation induced the release of proteolytic activity. Proteolytic activity, following subtraction of spontaneous proteolytic activity released, was 44 ± 5 % at 8 hr and 30 ± 4 % at 20 min following PMC activation with 10 μM of tc-LIG  before seeding with TNF. SBTI inhibited TNF loss from the supernatants of tc-LIG (10 μM) stimulated PMC by 82% Fig. , confirmWe have previously shown that PMC express PAR-2 mRNA, that can be regulated by cytokines and PAR-2ap . We haveNippo Ag.In this study, we provided the first direct evidence for serine protease release from PMC measured by Western blot analysis of the supernatants, in addition to proteolytic activity assays. The sizes of released RMCP-1 (~30 kDa), RMCP-5  and CPA  are similar to the sizes of the stored forms of these proteases that we published previously . The difin vivo and in vitro. The release of RMCP-2 by rat mucosal mast cells has been reported to be induced by antigen challenge in parasitic infections, and during anaphylaxis [2+ ionophore and antigen activation of PMC [Previous studies have shown that activation of MC to release protease activity may be induced by a variety of agents both phylaxis -21. The phylaxis . Furthern of PMC .In our experiments FcεRI-mediated PMC stimulation did not release detectable levels of RMCP-1, RMCP-5 or CPA, or any proteolytic activity with the ability to degrade TNF. It is possible that FcεRI-mediated activation induces low levels of protease release which is undetectable by Western blotting. Furthermore, the lack of demonstrable proteolytic activity in the supernatants does not necessarily indicate that proteases are not released since it may be due to an FcεRI-mediated simultaneous release of protease inhibitors stored in the MC. Indeed, MC produce and release secretory leukocyte protease inhibitor (a chymase inhibitor) and latexin (a CPA inhibitor) ,24. FinaGiven that RMCP-1 and RMCP-5 are present in the supernatants of tc-LIG-stimulated MC, it is likely that these proteases are involved in the TNF degradation. However, antibodies to RMCP-1 inhibited TNF degradation by supernatants of sham treated cells but did not affect the additional degradation of supernatants from tc-LIG activated MC (unpublished observation). We cannot rule out that PAR-2ap activated PMC release other proteases, including the tryptases RMCP-6 and RMCP-7 , which min vivo. It may be that such proteolytic activity directed against TNF, and possibly other cytokines, is an important anti-inflammatory function for mast cell serine proteases.Our data further suggest that MC may regulate TNF function by releasing proteases that can directly degrade this cytokine. Given that both TNF and serine proteases are stored and released from MC, our present findings suggest an important mechanism by which MC may regulate TNF function In vitro, MC tryptase can stimulate histamine release by human tonsillar [The expression of PAR-2 by mast cells and the involvement of MC in PAR-2-mediated inflammation has been controversial. onsillar  and guinonsillar  MC, but onsillar . Tryptasonsillar , indicatonsillar  and rat onsillar  mast celonsillar , but othonsillar . Taken ttrans-cinnamoyl group, which acts to stabilize the peptide and prevent its degradation by aminopeptidases. It is unlikely that the different sensitivity to proteases can explain fully the difference between the effects of the two PAR-2ap peptides. It is also unlikely that the trans-cinnamoyl modification on tc-LIG is solely responsible for tc-LIG-mediated activation of MC, because it is also present on the reverse sequence peptide tc-OLR, but does not have the same effects with tc-LIG on protease release, as shown in this study, or in the release of β-hex, as we showed before.In a previous study we have shown that only one of two PAR-2ap (tc-LIG) activates β-hex release from PMC . The othCompound 48/80 along with other cationic compounds can activate MC by directly interacting with a pertussis toxin sensitive component . Our pre2 has been identified pharmacologically in murine vascular smooth muscle [Recently, a new receptor activated by the PAR-2 activating peptide tc-LIGRLO-NHh muscle . In thatOur study provides evidence that a PAR-2ap, tc-LIG, activates MC to release proteases and proteolytic activity that could potentially have both pro- and anti-inflammatory functions. We further showed that these proteases may degrade extracellular proteins and affect the inflammatory environment in areas of mast cell activation. Although the presence and function of PAR-2 on MC is still controversial, our findings indicate that PAR-2 may be part of an autocrine loop. PAR-2 activation leads to the release of serine proteases which in turn may further activate more PAR-2 receptors on mast cells and also on other cells.2-terminal sequences were synthesized at Zymogenetics Inc, Seattle, WA, and used to immunize rabbits to develop specific polyclonal anti-protease antibodies. Professor H. Miller, Edinburgh, Scotland, kindly provided rabbit antibody to RMCP-1.Compound 48/80, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (β-hexosaminidase (β-hex) substrate) and soybean trypsin inhibitor (SBTI) were purchased from Sigma Chemical Co. . PAR-2ap and PAR-2 control peptides (PAR-2cp) were synthesized by the Peptide Synthesis Facility, Faculty of Medicine, University of Calgary. These peptides were determined to be ≥ 95 % pure by mass spectrometry and HPLC. Polyclonal RMCP-5 and CPA antibodies were produced and characterized as described previously . BrieflyNippostrongylus brasiliensis, by a single subcutaneous injection of 3000 third-stage larvae in 0.5 mL of saline as described previously [Outbred male Sprague-Dawley rats (weight 250–500 g) were purchased from Charles River Canada Inc., . Rats were maintained in an isolation room with filter-topped cages to minimize unwanted infections. For the experiments where MC were activated through their IgE receptor, rats were sensitized to eviously . The expFifteen mL of ice-cold Hepes-buffered  Tyrodes buffer supplemented with 0.1% BSA was injected into the peritoneal cavity of each rat for the isolation of PMC. MC in peritoneal lavage fluids were enriched by centrifugation through a discontinuous density gradient of Percoll, as described previously . Recover6 cells/mL. Cells were placed in 1.5 mL Eppendorf tubes or in 48 well plates, incubated at 37°C for 10 min, and then the same volume of pre-warmed (37°C) PAR-2ap or controls in complete RPMI were added, to give a final cell concentration of 0.5 × 106 cells/mL. The cells were incubated for different times (20 min to 8 hr) depending on the experiment.After isolation and enrichment, PMC were rested in RPMI  supplemented with 5% FBS for 2 hr at 37°C. After incubation, the cells were washed twice by centrifugation (150 g) and resuspended in RPMI at 1 × 10Nippostrongylus brasiliensis Antigen /mL [To measure spontaneous release of mediators by PMC, cells were mixed with media alone. As positive controls, either compound 48/80 (0.5 μg/mL) or  (WE)/mL ) were miSupernatants were concentrated (10×) using Centricon (YM-10) centrifugal filter devices . For Western blot analysis, proteins were transferred electrophoretically  to a polyvinylidene difluoride (PVDF) membrane  using the Semi-Dry Trans Blot System. The membranes were blocked in Tris-buffered saline containing 0.02% Tween, 5% w/vmilk (Bio-Rad Laboratories) and 5 % v/vgoat serum (Invitrogen) for 1 hr. The membranes were probed with 1/1000 dilution of anti-RMCP-1, 1/600 anti-CPA, 1/5000 anti-RMCP-5 and then incubated with donkey anti-rabbit IgG HRP-conjugated antibody (1:5000). Protein bands were detected by enhanced chemiluminescence using ECL Western blotting detection system .β-hex was measured in the supernatants and cell pellets, as described . ResultsTo measure release of proteolytic activity, supernatants from stimulated PMC (containing secreted TNF) were transferred to a 96-well plate. After 2 min incubation, exogenous TNF or medium was added to the supernatants and mixed to give a final concentration of 150 pg/ml. Plates were incubated at 37°C for 8 hr and then TNF content was measured by ELISA. Percent TNF proteolysis was calculated by the following formula:% TNF degraded = 1 - (TNF recovered / (rat recombinant TNF seeded + measured TNF release) × 100)For protease inhibition experiments SBTI (1 mg/ml) was added to the supernatants before seeding with TNF. The supernatants were then processed as above and used to measure TNF degradation.Supernatants from activated PMC were analysed for TNF using a rat TNF ELISA kit , according to manufacturer's instruction. The sensitivity of the TNF assay was < 10 pg/ml. To exclude the possibility that proteases contained in PMC supernatants interfere with ELISA determination of TNF we incubated the TNF antibody coated wells with PMC supernatants washed them and then added specified amounts of TNF for determination. Pre-incubation with PMC supernatants did not affect the ability to measure TNF, indicating that the proteases in PMC supernatants do not degrade the antibodies of the assay.t-test and ANOVA.All values are given as mean ± standard error of mean (SEM) for the numbers of experiments noted and statistical analyses were performed using the Student's β-hex β-hexosaminidaseCPA carboxypeptidase-A2 (PAR2-cp)LSI LSIGRL-NHMC mast cellPAR protease-activated receptorPAR-ap protease-activated receptor-agonist peptidePAR-cp protease-activated receptor-control peptidePMC peritoneal mast cellsRMCP-1,5 rat mast cell protease-1, 5SBTI soybean trypsin inhibitor2 (PAR2-ap)SLI SLIGRL-NHtrans-cinnamoyl-LIGRLO-NH2tc-LIG trans-cinnamoyl-OLRGIL-NH2tc-OLR Nippo Ag Nippostrongylus brasiliensis Antigen.WE worm equivalentHNA carried out the majority of the experiments presented and drafted the manuscript. This work was part of his MSc thesis. GRS assisted with mast cell isolation and some of the activation experiments and participated in the experimental design. JLW participated in the design of the study. MDH participated in the design of the study. ADB participated in the design of the study, supervised the work shown and made substantial contributions in manuscript preparation. HV participated in the design of the study and contributed in the preparation of the final manuscript. All authors read and approved the final manuscript.