We have previously proposed a theoretical model for studying physical disability and other outcomes in rheumatoid arthritis (RA). The purpose of this paper is to test a model of impairment and functional limitation in (RA), using empirical data from a sample of RA patients. We based the model on the disablement process framework.We posited two distinct types of impairment in RA: 1) Joint inflammation, measured by the tender, painful and swollen joint counts; and 2) Joint deformity, measured by the deformed joint count. We hypothesized direct paths from the two impairments to functional limitation, measured by the shirt-button speed, grip strength and walking velocity. We used structural equation modeling to test the hypothetical relationships, using empirical data from a sample of RA patients recruited from six rheumatology clinics.The RA sample was comprised of 779 RA patients. In the structural equation model, the joint inflammation impairment displayed a strong significant path toward the measured variables of joint pain, tenderness and swelling . The joint deformity impairment likewise displayed significant paths toward the measured upper limb, lower limb, and other deformed joint counts . Both the joint inflammation and joint deformity impairments displayed strong direct paths toward functional limitation , and explained 65% of its variance. Model fit to data was fair to good, as evidenced by a comparative fit index of 0.975, and the root mean square error of approximation = 0.058.This evidence supports the occurrence of two distinct impairments in RA, joint inflammation and joint deformity, that together, contribute strongly to functional limitations in this disease. These findings may have implications for investigators aiming to measure outcome in RA. Physical disability is an important outcome of rheumatoid arthritis (RA) ,2. The Apathology → impairment → functional limitation → physical disability [pathology occurs at the level of molecules, cells, or tissues, and is measured using tests such as the erythrocyte sedimentation rate, the C-reactive protein concentration, cytokine expression patterns, or images of the joints obtained with X-ray or MRI. Impairments are dysfunctions or structural abnormalities that occur at the level of organs or organ systems. They include signs and symptoms of disease such as pain, morning stiffness, joint tenderness, swelling and deformity. Functional limitations are restrictions in basic physical or mental actions, and they involve the whole person. Although they can be measured in a number of different ways, we have chosen to use performance-based functional tests such as the grip strength, walking velocity and the timed shirt-button test to measure functional limitations [Disability involves difficulty with a physical or mental activity, within a social context. The measurement level therefore should include the person and the societal environment. We have used self report measures of physical disability such as the Health Assessment Questionnaire, or the physical function scale of the SF-36, to measure physical disability [Inquiries into physical disability in RA, needing to weigh the influence of numerous variables interacting over time in complex ways, benefit from a conceptual framework, or model . A modelsability . A usefuitations . Disabilsability .functional limitation and physical disability [As noted above, we based our initial model and its proposed measurement strategies on purely theoretical grounds . Since isability -14. In tFrom 1996 to 2000, we enrolled consecutive patients meeting the 1987 RA criteria , into a We recruited patients from six outpatient rheumatology clinics in San Antonio, Texas: 1) An Army Medical Center, 2) An Air Force Medical Center, 3) A private, university-based clinic, 4) A community-based, seven-rheumatologist private practice, 5) A county-funded clinic and, 6) A Veterans Administration clinic. All evaluations were done on location in these facilities.Our study was approved by the Institutional Review Board of each of the clinical facilities were we went to recruit patients, and all patients gave written, informed consent. A physician or a research nurse, assisted by a trained research associate, conducted evaluations at the clinic where the patient was recruited. The evaluation lasted approximately 90 minutes, and consisted of a comprehensive interview, physical examination, review of medical records, and laboratory and X-ray tests. Interviews were conducted in either English or Spanish, as preferred by patients.We measured impairments using self-report and physical examination. We used a validated, one-page joint mannequin for patients to mark the joints that were painful or swollen . This va® Dynamometer . In a sitting position, with the elbow held at 90 degrees, and the forearm supported on a flat horizontal surface, patients were asked to squeeze the handle with as much as strength as possible. Three repetitions from each hand were recorded, in kilograms. The mean value of all repetitions for both hands is shown; 2. Walking velocity. Starting from a standing position, patients were asked to walk at their usual pace, for a distance of 50 feet, or 25 feet if they had difficulty covering the full distance. No effort was made to conceal the stopwatch used to time the patients. Results are expressed in feet per second. Patients unable to walk were assigned a velocity of 0 feet per minute; 3. Timed button test. Patients were asked to don and fasten the front buttons of a standard eight-button, men or women's extra-large, denim shirt . A stop watch was activated when the patient took the shirt as it was offered by the examiner, and stopped when the last button was fastened. This test quantifies the performance and large and small upper extremity joints. Results are expressed as buttons per minute. Patient unable to don the shirt were assigned a value of 0 buttons per minute.We measured functional limitations using the following performance-based rheumatology function tests: 1. Grip strength. We measured grip strength with a hand held JAMARWe began the analysis by inspecting summary statistics and histograms of all study variables. Skewed variables were square root transformed toward normality. We specified a structural equation model (SEM) of impairment and functional limitation in RA . We hypoWe recruited 779 patients from 1996 to 2000. We have described the clinical characteristics of the study sample in earlier publications ,14. BrieA graphical display of the proposed model is shown in Figure The latent variable of joint deformity is measured by the deformed joint count, which we disaggregated into counts for the joints in the upper and lower extremities, and other joints. The path coefficients for upper and lower extremity deformities were stronger than that of the other joints , but all three remained significant Table To obtain a better understanding of the properties of the latent variables, we used the path coefficients from the two impairment latent variables to compute their estimated values, generating a new variable for each one. Figures In the structural equation model, both of the impairment latent variables displayed strong direct paths toward functional limitations. The standardized coefficients were -0.5 or less, representing a change of more than half a standard deviation in the functional limitations for every standard deviation change in the impairments Table . The squpost hoc, we tested a direct path between joint inflammation and joint deformity, but the resulting coefficient was small and did not reach statistical significance. We therefore omitted an inflammation → deformity path from the final model.The initial CFI and NFI of the model were both 0.95, suggesting a close fit of the model to the data. The initial RMSEA was 0.07, suggesting reasonable fit . Modificbody structures and functions in the ICF are analogous to the stages of pathology and impairment, while activities and participation correspond to functional limitations and disability in the disablement process [Several models have been proposed to study disability in the general population ,9,20, an process .We chose the disablement process over other disability models, among other reasons, because it considers the stages in disablement as an explicit sequence of linked events, one leading to the next. Rather than suggesting specific functions or activities to measure, it offers broad definitions of each stage, leaving it up to investigators to find ways to test them. Several of the variables we include in the present analysis are represented in the ICF classification, including joint pain and deformity, and walking. Variables not represented in the ICF, but which we did obtain, include joint tenderness and swelling, and grip strength. Our measurement of the stage of disability, described elsewhere, has considerable homology with that of the ICF . The mosimpairment to be theoretical construct that cannot be directly quantified. We studied it as a latent variable, the articular signs and symptoms listed above serving as the tools we used to tap into the impairment construct.We used the disablement process framework to build a model of impairment and functional limitation in RA . ImpairmThe concept of impairment as a stage in the disablement process is not intended to oversimplify the anatomical or physiological derangements that occur within that stage. In fact, the derangements can be quite complex, depending on the nature of the initial pathology, and the organ system under study. In the case of rheumatoid joints, the initial pathology can be broadly classified into two discrete, but related groups: inflammation and damage . It shouBoth impairment latent variables displayed strong, statistically significant path coefficients toward the measured variables, providing evidence that the measures we chose adequately tap into the proposed impairments. In the diagram of our model, these paths are shown as arrows from the latent to the measured variables, to indicate that it is the joint inflammation or damage (both of them latent variables), that are "causing" the joint signs and symptoms that we are able to measure. Not included in our final model because it did not reach statistical significance, was a path from joint inflammation to deformity. This is likely because we restricted the present analysis to the stages of impairment and functional limitation. Although there is considerable evidence that inflammation leads to damage in RA joints, the link between the two processes occurs during the stage of pathology, not impairment. We expect to find a strong link between inflammation and damage when we extend our analyses to include pathology measures such as the erythrocyte sedimentation rate, C-reactive protein, joint erosions and joint space narrowing.According to disablement theory, impairments lead to functional limitations ,10. We eThe disablement process was proposed as a framework to aid investigators in their efforts to understand the development of disability in aging, and in specific disease states . The fraOne of our goals with this and earlier efforts to model RA's stages -12, is tThe success with which the ACR and similar response measurement systems have been used in clinical trials, does not preclude the possibility that they could be improved . Mixing Certain constraints apply to the interpretation of our findings. The maximum likelihood estimator that we used for SEM assumes multivariate normality, a requirement that is not strictly met by some of the variables we used in this analysis. Non-normality may affect standard errors, and thus significance testing, about the parameter estimates, albeit not the value of the parameters themselves. The overall structure of the model we propose, i.e. two distinct impairments linked to functional limitations, is thus likely to be unaffected by this deviation from assumptions. The patient sample we studied is sufficiently large that the potential deleterious effect of non-normality on the significance of the path coefficients may be offset. It should also be noted that data we used here to test the impairment → functional limitation relationship are cross-sectional. The sequential link between the two stages we propose has face validity in that joint tenderness, swelling and deformity are causes, not consequences, of diminished grip strength, walking velocity and shirt button speed. Nevertheless, confirmation of our findings in a longitudinal dataset would strengthen the evidence for the model.We conclude that two distinct impairments occur in RA, one characterized by signs and symptoms of joint inflammation, the other by joint deformity. Both of these contribute substantially to the functional limitations that occur in this disease.RA = Rheumatoid arthritis;ÓRALE = Outcome of rheumatoid arthritis longitudinal evaluation;CFI = Comparative fit index;NFI = Normed fit index;RMSEA: Root mean square error of approximation;SEM: Structural equation model;ACR = American College of RheumatologyThe author(s) declare that they have no competing interests.AE designed and obtained funding for the study, directed the statistical analysis, and drafted initial and final versions of the manuscript; RWH performed the statistical analysis and edited the manuscript; IDR designed and obtained funding for the study, supervised its implementation, and edited the manuscript.The pre-publication history for this paper can be accessed here:


Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them. Cell cycle checkpoints preserve genome integrity by monitoring the fidelity of DNA replication and segregation. In mammalian somatic cells, the best-characterized checkpoints are the DNA damage/replication checkpoints and the mitotic spindle checkpoint. The DNA damage/replication checkpoints result in cell cycle arrest if DNA is not fully replicated, or is damaged . The mitThere is also evidence that defects in events relating to cell division itself can result in cell cycle arrest. Lanni and Jacks  and CaseOther evidence suggests that mammalian cells are not sensitive to tetraploidy. Rao and Johnson used cell fusion to study the regulation of DNA synthesis and mitosis by fusing cells at different cell cycle stages ,8. BinucHere we investigate whether tetraploidy or other cellular defects in binucleate cells lead to cell cycle arrest. We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause DNA damage that is the likely cause of G1 arrest.Immortalized cell lines often have altered checkpoints, therefore we used early passage primary cells to investigate the tetraploidy checkpoint. All experiments were performed with human diploid fibroblasts (HDF) from infant foreskin and used prior to passage 10. We had previously developed methods for synchronizing these cells , and tesWe first tested HDF cells for the previously described G1 arrest following cytochalasin-induced failure of cytokinesis . Cells wAt 6 h after the removal of cytochalasin, 50% of the untreated control cells had entered S phase, whereas only about 10% of either the mononucleate or binucleate cytochalasin-treated cells had entered S phase Figure ; these nA potential problem with cytochalasin treatment is that depolymerization of the actin cytoskeleton is likely to have effects other than blocking cytokinesis. Indeed, we found that even at 2 μM, cytochalasin had a strong cytotoxic effect, delaying cell cycle progression significantly, with slow recovery after release (data not shown and ). To detHDF cells were synchronized in mitosis by double thymidine block followed by nocodazole treatment, then released into medium containing BrdU, +/- 12.5 μM blebbistatin. By 10 h after the addition of blebbistatin, most cells had exited mitosis; in the presence of blebbistatin approximately 30% of the cells were binucleate and the remaining cells were mononucleate, presumably completing cytokinesis successfully. Blebbistatin was removed, and cells were assayed for S phase entry over time.At 6 h after the removal of blebbistatin, 44% of the untreated control cells and 53% of the mononucleate blebbistatin-treated cells had entered S phase, whereas only 18% of the binucleate blebbistatin-treated cells had entered S phase Figure . By 24 hCells that have failed to divide after mitosis differ from normal cells in that they have two nuclei, two centrosomes, and a decreased surface area to volume ratio. We tested each of these defects individually for an effect on G1 arrest. To test the role of centrosome number, serum-starved G0 cells were fused with enucleated G0 cytoplasts to create cells with two centrosomes, but only one diploid nucleus Figure . The celTo test the role of tetraploidy, serum-starved HDF cells were fused to create binucleate cells. Creating binucleate cells by fusion avoided disruption of the actin cytoskeleton, allowing us to examine the effect of ploidy alone. The binucleate cells resulting from fusion were both tetraploid and had two centrosomes; we showed above that centrosome number was not a factor in the G1 arrest. As above, unfused cells in the population served as an internal control. At 24 h after fusion, 72+/-2% of the unfused cells and 75+/-1% of the fused, binucleate, cells had entered S phase. Therefore, tetraploidy does not cause the observed G1 arrest resulting from cytochalasin-induced failure of cytokinesis.Cells that fail to divide at cytokinesis are larger than normal cells. Larger cells have a decreased surface area to volume ratio, which might affect the response to perturbation of the cytoskeleton. Thus, the apparent sensitivity to cytokinesis failure might derive directly from a difference in size. To test this, we created large binucleate cells by fusing serum-starved G0 cells to each other. The fusion products were released into growth medium for 3 h to allow for reattachment to the culture substrate. We then added 25 μM blebbistatin, 5 μM cytochalasin, or 5 μM AKI-1 to cells for 10 h, followed by release into BrdU-containing medium. Figure If cells were sensitive to failure of cytokinesis, one might expect that the sensitivity would be expressed as a delay in exit from mitosis, a time when a cytokinetic defect could be corrected. This would be similar to the known DNA damage, DNA replication and spindle assembly checkpoints . We testSince we had ruled out most of the cellular defects associated with division failure as being the cause of the G1 arrest, we attempted to further characterize the arrest. Andreassen et al.  reportedp53-p21-dependent G1 arrest often results in either apoptosis or senescence ,19. To dWe have shown that failure of division of synchronized cells results in a p53-dependent arrest, but that the arrest is not due to ploidy, centrosome number, or cell size, and that the arrest is not preceded by a delay in mitotic exit, suggesting that it is not a classical checkpoint. The characteristics of the arrest are similar to those of the G1 arrest caused by the DNA damage checkpoint in HDF cells, which respond to irreparable DNA damage by entering senescence, instead of apoptosis . These sIn the untreated control cells, only 3.4% of cells contained γ-H2AX foci, whereas in the hydrogen peroxide treated cells, 33% of cells contained γ-H2AX foci Figure . RemarkaThe binucleate cells that persisted in culture were arrested in G1, as they did not incorporate BrdU after the previous round of mitosis, and they did not proceed to the next round of mitosis, as evidenced by the preservation of the binucleate phenotype. The presence and persistence of nuclear γ-H2AX foci in the G1-arrested binucleate cells suggested that DNA damage might be the cause of the arrest. However, not all the arrested binuclear cells contained visible γ-H2AX foci, indicating that γ-H2AX-associated DNA damage might not be the only cause of the arrest. The percentage of binucleate cells with nuclear γ-H2AX decreased from 52% to 29% over 8 days of culturing, possibly indicating that some cells were able to correct the DNA damage after being arrested in G1 for several days. In contrast, the percentage of mononucleate cells that displayed γ-H2AX foci decreased dramatically over 8 days of culturing, however this was likely due to proliferation of normal mononucleate cells in the culture rather than a difference in response of the mononucleate and binucleate cells to the treatment.The presence of γ-H2AX foci in the mononucleate cells that successfully completed cytokinesis after blebbistatin treatment suggested that the DNA damage might have been the result of the synchronization treatments, prior to the addition of blebbistatin. Therefore, we tested whether any of the cell synchronization treatments alone had an effect on cell cycle progression. Asynchronous cells were treated with double thymidine block, nocodazole, or blebbistatin individually, following the same protocols used above in multiple treatments. Cells were released from drug and S phase entry was assayed at time points. Figure Since none of the single drug treatments resulted in a cell cycle arrest, we reasoned that some combination of the treatments must be responsible. To determine which combination of treatments caused a G1 arrest, cells were subjected to the treatments in the order double thymidine, nocodazole, blebbistatin. Samples of cells were taken after release from each drug, and S phase entry was assayed Figure . AlthougTo determine whether the thymidine – nocodazole combination caused the DNA damage we observed, cells were treated with both drugs as above and stained for γ-H2AX. At 3 h after release from the drug treatments, about 33% of cells had γ-H2AX foci Figure . Thus thWe have shown that tetraploidy, aberrant centrosome number, increased cell size, and failure of cytokinesis do not lead to G1 arrest in primary human diploid fibroblasts. Rather, we found that the observed G1 arrest in cells that have failed to divide is likely due to cellular damage caused by standard synchronization treatments. We note that all published observations of a G1 arrest in response to division failure involved extensive manipulation of mammalian cells in culture. It seems likely that these manipulations resulted in DNA damage, or in other damage, that resulted in a G1 arrest, but was not directly associated with division failure. For example, Uetake and Sluder  found thHuman diploid fibroblasts (HDFs) were from infant foreskin. Wt, p53 -/- and p21 -/- mouse embryo fibroblasts (MEFs) were the kind gift of Laura Attardi . HDFs and MEFs were cultured in DMEM (Gibco) with 10% fetal bovine serum. HDFs were used prior to passage 10 and MEFs were used prior to passage 5. HDFs were synchronized in G0 by serum starvation  and S phCells were incubated with 20 μM BrdU (Sigma) for indicated times and fixed in -20°C methanol for at least 10 min. Fixed cells were treated with DNase I (Boehringer Mannheim) and exonuclease III (New England Biolabs) to expose the BrdU epitope prior to incubation with anti-BrdU antibodies, as described . Nuclei Cells were fixed with 2% paraformaldehyde at room temperature for 10 min, washed 3× with PBS, then permeabilized with-20°C methanol for 5 min and stained with 27 ng/ml γ-H2AX antibody . Only cells with multiple, clearly labeled foci were counted as being γ-H2AX positive.Cells synchronized in S phase by double thymidine block were released from the block for 6 h to allow completion of S phase. Nocodazole (100 ng/ml)  was then added for 6 h to arrest the cells in mitosis. Cells were released from the mitotic arrest for 30 min, during which time most cells formed a bipolar mitotic spindle Fig . At 30 mCW participated in the design of the study and carried out the experiments. TS conceived of the study and participated in its design.


Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment.Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella using a freely available oligo design software package. 21 ± 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65°C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure.Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65°C, 28 ± 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly. Target molecules  hybridize transiently to the probe oligomers until they form stable double helices with their specific probes. At some point, the rate of on and off reactions reach equilibrium, and the concentration of the target in the sample solution can be calculated. Transcript abundance is assessed by the relative intensity of signal from each spot on the array. This interpretation of array data relies on the assumption that each hybridization reaction goes to completion within the timeframe of the experiment and that the behavior of all pairs of intended reaction partners in the experiment is somewhat uniform.Sequence-specific hybridization of a long single-stranded labeled DNA or RNA target molecule to shorter oligonucleotide probes is the basis of the gene expression microarray experiment. In this type of microarray experiment, gene specific There are three major types of DNA microarrays, which differ in the approach used for probe design: Affymetrix type microarrays , which aA number of oligonucleotide design software packages have been published in recent years, each having design strengths in one of a number of criteria -14. SeveFew of the available array design packages explicitly consider the possible structures of the transcript-derived molecules in the sample solution and their impact on whether the microarray will provide an effective assay, although the OligoDesign web server  does comBrucella suis. We have assessed the stability of structures formed in the transcript and the accessibility of the binding sites of optimal probes generated using commonly applied design criteria. Because random shearing of the full-length target molecule is used in some protocols, we have also modeled the effects of shearing to an average length on the prevalence of secondary structure in selected targets.In order to estimate the prevalence of stable secondary structure in long target molecules, and thus the impact such structures might have on the analysis of microarray data, we have modeled secondary structure formation in mRNA transcripts of the intracellular pathogen Brucella suis  is near 70% of the average for Brucella, while the average ΔG for transcripts from the GC-poor genome (L. lactis) are even lower (30% at 52°C). However, even in the most GC-poor genome, stable target secondary structure in the single-stranded target is widespread.Our modeling results obtained for the genome-wide set of intact single-stranded DNA or RNA targets demonstrate that stable secondary structures are widespread in target mixtures from s Figure  and in rOur results demonstrate that a significant fraction of nucleotide sites in the average target mixture, whether single stranded DNA or RNA, will be found in stable secondary structure under the hybridization conditions used in oligonucleotide microarray experiments, and will be relatively inaccessible for intermolecular interactions. Figure Figure Figure in silico shearing, revealed the likely presence of stable secondary structures in both full-length target and sheared target mixtures. These structures do not convert completely to random coil with either increasing hybridization temperature, more extensive shearing, or both. These secondary structures may therefore compete with the intended target for effective probe annealing in a microarray experiment, resulting in a misinterpretation of the amount of target present in the sample.Lack of bioinformatics tools that incorporate experimentally validated biophysical properties of nucleic acids as a criterion for synthetic oligomer probe design is a major challenge for do-it-yourself microarray designers. One biophysical characteristic, which we predict will reduce the binding efficiency of microarray probes to their targets, is the propensity of long single-stranded DNA or RNA molecules to form stable secondary structure. 3-D structures such as hairpins and stacked regions have the potential to pre-empt target nucleotides, thus blocking regions of the target molecules from hybridizing to their intended probes. Prediction and thermodynamic analysis of secondary structure at a range of temperatures in full length target sequences, as well as in subsequences formed by in silico experiment, secondary structure prediction in the target is being used to develop a new criterion for oligonucleotide probe design. Our results from this modeling experiment demonstrate that the implicit assumption used until now – that eliminating probe secondary structure by avoiding self-complementarity eliminates target secondary structure as well – is valid only when the target and probe are of the same length. Use of target secondary structure as an explicit criterion will allow for masking or preferentially avoiding the regions of the target sequence in which base pairs are directly involved in secondary structure formation, to eliminate these regions from the sequence for the purpose of the search for the optimal probe.Based on the results of this In this study we have assigned accessibility scores to sites in the target sequence based only on the fraction of predicted structures within 5% of the energy optimum, in which a residue is found in a single-stranded conformation. While this measure is not too computationally intensive to compute, and can be applied to genome-scale problems using readily available software (Mfold), it is not the most physically rigorous definition of accessibility. By equally weighting each possible structure in the ensemble of optimal and suboptimal structures that a molecule can form, it is possible that secondary structure at some positions in the molecule is overcounted; bonds which form only in rare conformations are considered equal to bonds which are present in the lowest-energy structure. The program Sfold -18 assigWhen we compared MFold-based accessibility predictions for an individual transcript to those generated by SFold and RNAFold, we found that the difference in average predicted accessibility over an entire transcript is small. We computed accessibility for the transcript of human 1CAM-1, which has been mapped experimentally to determine its accessibility . The aveIn this study, we focused specifically on the DNA/RNA base pairs that are actively involved in hydrogen bond formation. We realize that other accessibility considerations will have to be added to the scoring scheme in practice. The structure of a long single stranded DNA or RNA molecule can contain many nucleotides that, while not part of a double-helical stem, remain inaccessible to hybridization due to their location inside small loops within the target secondary structure. A loop is a somewhat constrained structure as well, and the length at which it presents accessible sequence that favors hybridization has been shown to be on the order of 10 nucleotides and longer , while nB. suis example, use of a low annealing temperature, e.g. 42°C which is the temperature used in some published 70-mer array experiments [Development of a target secondary structure criterion for oligonucleotide array design is expected to impose restrictions on the probe selection beyond the sequence similarity and melting temperature criteria that are currently used, especially in cases where short probe length restricts the annealing temperature used in the hybridization protocol to 22–37°. In the eriments , would rWe have shown here that while shearing reduces overall ΔG of secondary structure formation for individual molecules in the target solution, shearing does not in itself eliminate formation of secondary structure in single-stranded DNA or RNA. The question of whether shearing should be used for long oligomer arrays is still an open one. While some signal may be gained by reducing the stability of secondary structure in the target molecule, random shearing by its nature creates a mixture of targets that may have substantially different affinities. For instance, in a 300 nt transcript that is targeted by a 70mer oligonucleotide, there is nearly a one in four chance that a random break in the sequence will occur within the target site for which the probe is designed. Short fragments may present a substantially different binding site, and therefore have a different binding affinity, than the full-length transcript that is considered when the probe is designed. This is illustrated in Figure 8d, where binding of a 50mer sheared fragment to a 70mer probe leaves a dangling end in the probe. A break very close to one end or the other of the target site may create a target that still binds to the probe, though with reduced affinity; a break closer to the middle of the target site may produce fragments that bind partially to the probe, competing for binding with perfect matches.In other experimental contexts where hybridization is critical to success, the impact of secondary structure in single stranded polynucleotides on results has been recognized and is now being systematically studied (18–21). Intramolecular folding of mRNAs is so extensive that only 5–10% of most transcripts is accessible to binding of complementary nucleic acids; however the modeling of long molecules has not proven to give very accurate binding predictions -25. In fBrucella had binding sites in the target which were predicted to contain a stretch of unpaired bases of at least 14 nt in length; at 65°C, that fraction increased to 93%. Based on these findings we would expect that at 52°C only 57% of our probes would encounter optimal conditions for hybridization and therefore would demonstrate the expected behavior in the experiment, where intensity is expected to scale with target concentration. We predict that the remaining probes, which have shorter, or no, accessible sequences, will exhibit modified binding behavior, and we plan to conduct experiments to characterize this behavior. We have shown conclusively that avoiding self-complementarity in the probe when designing an oligonucleotide array is insufficient to eliminate secondary structure from the binding site in the target. By combining the procedure for systematic computational assessment of transcript accessibility described in this study with selective experimental validation of the impact of predicted accessibility on hybridization, we will develop a useful criterion for avoiding troublesome secondary structure when designing microarray targets.The results of the current study suggest a significant role for target secondary structure in hybridization to oligonucleotide arrays, which will warrant further investigation. Oligonucleotide probe binding sites in a significant fraction of transcripts are found in double-stranded conformations even in cases where self-complementarity was avoided during the probe design process. We find that at 52°C, for example, approximately 57% of probes designed for Brucella suis 1330 genome. Brucella suis has a relatively high (57%) genomic GC content. Brucella suis was chosen for this experiment because our collaborators have previously acquired a custom synthetic oligomer microarray for this organism, developed using standard oligo array design software, and we have access to both target sequences and to a set of unique probe sequences that define the interaction sites for which expression results have been obtained by the laboratory.Prediction and thermodynamic analysis of secondary structure was performed for all protein-coding gene transcripts predicted from 3264 CDSs in the Escherichia coli), and 50 from the GC-poor genome of the nonpathogenic AT-rich gram-positive bacterium Lactococcus lactis (35% genomic GC content). The Brucella suis genes ranged in length from 90 to 4,803 bp, with an average transcript length of 851 bp. The E. coli genes ranged in length from 140 to 2,660 bp, with an average transcript length of 792 bp. The range of GC content in the genes chosen was 37% to 57% with an average value of 50%, which is reasonably representative of the E. coli genome. The L. lactis genes chosen ranged in length from 140 to 2,730 bp, with an average transcript length of 765 bp., and ranged in GC content range from 30% to 42% with an average value of 35%.In order to determine whether Brucella sequences form atypical structures we randomly picked and analyzed 50 gene coding sequences from a compositionally balanced genome  using ArrayOligoSelector (pick70) [70-mer probes for each (pick70) . ArrayOlProbe and transcript secondary structure were predicted using the Mfold 3.1 software package ,29. MfolAccessibility in folded single-stranded DNA or RNA has recently begun to be addressed in a few experimental studies, mainly with the goal of targeting appropriate sites for RNAi. Because the structure of single-stranded nucleotide molecules is much more dynamic than that of proteins, with each molecule likely to exist in an ensemble of structures, and because the 3D structure of these molecules is rarely known, there is not yet a consensus representational standard of per-residue accessibility for single-stranded nucleic acids. Ding et al. ,18 impleIn this study, we chose to use the less physically rigorous approximation of probability of single strandedness as a simple fraction of predicted optimal and suboptimal structures in which a residue is found to be part of a single stranded structure, as computed by Mfold. Accessibility scores derived from MFold predictions have been used in limited studies of RNA structure focused on hammerhead ribozymes, antisenB. suis. Secondary structure and thermodynamics were computed for each of these fragments individually.Random shearing of the target mixture is an approach that is often offered as a solution for the problem of target secondary structure. The actual content of a sheared mixture of DNA or RNA fragments is complex. Shearing breaks the molecule not in predictable locations, but in random locations that give rise to a distribution of fragments around an average fragment length. In order to simulate the effects of different degrees of shearing on structure formation and stability in a transcript, we picked fragments of 200, 100, or 50 bases in length, choosing the start position via a sliding window of 10 bases. Secondary structure prediction for all fragments derived from every transcript in the B. suis genome is computationally intensive and produces an extremely large amount of output. Since our initial goal was to determine how much the method would affect the number and type of secondary structures probes would be expected to bind the shearing simulation was performed for fragments derived from the 300 bp Ure-1A gene of VGR participated in the design of the study, carried out the simulations and analysis, and drafted the manuscript. JWW participated in the design of the study and helped to draft the manuscript. CJG conceived of the study, participated in its design, coordinated the research and analysis, and drafted the manuscript.


The aim of this experimental study was to determine the effect of mild hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model with a human melanoma xenograft.A human melanoma xenograft was implanted into the hindlimbs of 30 athymic nude rats. The animals were randomized into five groups: group I: control, group II: sham group, group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP . Tumor response was evaluated by tumor size determination and immunohistochemical analysis 6 weeks postoperatively. Tissue sections were investigated for expression of CD34 and basic fibroblast growth factor (bFGF).3 to 1388 mm3. In the sham operated group (II) tumor volumes were significantly larger than in group I. Tumor volumes in group IV were significantly smaller than in group I and lowest in group V. There were no significant differences in size between group I and group III after six weeks. In group III and IV each, 5 animals showed tumor progression and one had a partial tumor response. In group V only 2 animals showed tumor progression. Immunhistochemical analysis of the tissue sections demonstrated that angioneogenesis was more pronounced in group II than in group I and less pronounced in group IV and V compared with group I.Average tumor volumes of the controls (I) increased from 105 mmOur results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF.External hyperthermia of 41.5°C tissue temperature for 30 minutes only has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion is an established treatment for multiple, locoregional intransit metastases of malignant melanoma and soft tissue sarcomas of the extremities. It was introduced 1957 by Creech and Krementz . Isolatein vivo [in vitro [Angioneogenesis has been recognized for many years to play a central role in the growth of primary tumors and the formation of metastases in general ,6. Melanin vivo  and in vin vitro . Antisenin vitro .Hyperthermia inhibits angioneogenesis. Some of the antineoplastic effects of hyperthermia are caused by ischemia due to obstruction or destruction of the tumor vessels. Eikesdal et al. described by hyperthermic temperatures disrupture of 25–50 % of the vasculature in malignant tumors. The heated tumors (44°C) had a blood flow reduction of 40–60 % after 24 hours .In vitro data indicate that capillary endothelials cells of malignant neoplasms are more thermosensitive than endothelial cells of normal tissues. The vascularization of tumors can be significantly damaged at temperatures which may alter but do not damage the vasculature of normal tissue [l tissue . The extl tissue . The cril tissue .Hyperthermia increases the effects of angioneogenesis inhibitors on tumor growth and tumor angioneogenesis . The aimad libidum. Maintenance and care of all experimental animals were carried out according to the guidelines of the local responsible Animal Protection Commission and carried out in compliance with national guidelines .Thirty inbred male nude athymic rats (Rowett rnu/rnu) weighting 200 to 280 g were used in this study. Animals were kept separately during the experiment with 12 hours of light per day. They were fed a standard laboratory diet and tap water A solid sample (2 × 2 × 2 mm) of a human melanoma xenograft (SK-MEL-3), derived from a lymph node melanoma metastasis, was implanted into the hindlimbs of 30 nude rats. The largest width and the maximum tumor diameter perpendicular to the width were measured with a micrometer. Only tumors with a diameter greater than > 12 mm in the largest diameter were included in the study.2; 0.85% CO2) and warmed in the heat exchanger. The warmed arterialized perfusate is driven by a roller pump with two synchronously running pump-heads on a single axis for the arterial and venous lines (Masterflex®). The outer diameters of the tubes corresponded with the diameter of the femoral vessels of nude rats weighting about 250 g .The details of the perfusion system have been described previously . The equRat hindlimbs were perfused with ringer's solution and sodium heparin  at 37 or 41.5 degrees C for 30 minutes with a flow rate of 4 ml/min. External hyperthermia was applied by an infrared lamp positioned in a fixed distance to the tumor.For temperature measurement during limb perfusion, a nickel-chrom-nickel thermocouple of 0.6 mm in diameter  was placed at the macroscopic tumor margin. The thermocouple was calibrated before use in a high-precision water bath. Baseline temperature was recorded for 5 minutes before treatment. Temperature was continuously measured during application. In group I (control) and group II (sham operated) temperature measurement was not performed.The animals were randomized into a control and four study groups of six animals each: group I: control (no therapy), group II: sham group (skin incision without ILP), group III: external hyperthermia with a tissue temperature of 41.5°C for 30 minutes without ILP, group IV: normothermic ILP . ILP was subsequently performed with the above-mentioned parameters. Antineoplastic agents were not applied.®, 80 mg/kg i.m.; Pearl-Davis, Berlin, Germany) and Xylazin , and fixed in a supine position. For perfusion, the animals underwent a 3-cm incision of the groin. The left femoral artery and vein were isolated using an operation microscope. A vascular clip was placed across the artery and a silicon catheter (outer diameter 0.7 mm) was introduced via a transverse arteriotomy. The femoral vein was cannulated with a silicon catheter of 1 mm outer diameter . The other part was fixed in 0.9 % saline solution, frozen and stored at -80°C until use.Vascular density and tumor morphology were evaluated by immunohistochemistry. Tumor tissue was tested for expression of basic fibroblast growth factor (bFGF) and CD34.20 and counterstained with hemalaun. Negative controls without primary antibody were run for each sample.Tissue specimens were fixed in 10 % formaldehyde and embedded in paraffin according to routine protocols. Sections 4 μm thick were deparaffinized and rehydrated using graded ethanols following routine protocols. For antigen retrieval all sections were microwave treated for 20 minutes in target retrieval solution  or 10 mM citrate buffer (for CD34) at 700 W. The sections were incubated overnight with primary mouse anti-bFGF  and anti-CD34 antibody . After rinsing in Tris buffer detection was carried out with a biotinylated secondary rabbit anti-mouse antibody diluted 1:50  for 30 minutes at room temperature. The sections were washed again as above in Tris buffer and then incubated with streptavidin-biotin-alkaline-phosphatase  in 10 ml Tris). Immunohistochemical staining was visualized with Fast Red  in 0.2 ml N, N-dimethylformamide,  and 9.8 ml 0.1 M Tris-HCl buffer at pH8.6). The sections were rinsed in HTumor response was graded according to the system described by de Wilt et al. . ProgresIn areas of intense neovascularized spots microvessels were counted in a 100× field. A microvessel was defined as a lumen surrounded by a rim of endothelial cells highlighted by immunostaining with anti-CD34 antibodys or anti-bFGF antibodys. Five separate intense neovascularized areas were assessed, and the mean was calculated as microvessel density of each tumor. The score was assessed by two independent observers.Data were analyzed using SPSS/PC+ statistical software. The mean and range of tumor and lesion volumes were calculated for each group. For comparison of tumor volume and microvessel density between the diffent groups a non-parametric test was used . A p value < 0.05 was considered to be significant.During intervention, the required limb tissue temperature was reached within 8–12 minutes in group IV and 10–15 minutes in group III. Stable temperatures were then maintained for a further 30 minutes with a mean of 41.4°C ± 0.5. To reach limb tissue temperature the perfusion fluid maintained 43°C ± 0.4.3 (4.3), 98 mm3 (5.7), 108 mm3 (3.4), 95 mm3 (5.0) and 107 (4.1) mm3 respectively, with no significant difference between the groups. Average tumor volume of the controls (I) increased to 1388 mm3 (101) during six weeks. In the sham operated group (II) tumor volume was significantly larger than in group I (2350 mm3 (198.6), P = 0.021). Tumor volume in group IV was significantly smaller (1009 mm3 (122.5)) than in group I and lowest in group V 405 mm3 (103.6) . There were no significant differences in size between group I and group III (1135 mm3 (99)) after six weeks (P > 0.05) volume of the treated tumors in groups I, II, III, IV and V was 105 mmAfter the perfusion, body weight decreased in all groups during the first 5 days with a maximum of 8 %. In the following weeks, body weight mounted up to 130 % of the preoperative value. In the last week of the experiment, the weight almost reached a plateau. No significant differences between the five experimental groups were found.In the H&E staining the untreated melanoma (group I) presented as a subcutanous unilocular nodule of moderate differentiation. The tumor showed mainly tubular structures with vacuole like lumen formation and was infiltrated with fibrous septa. The spontaneous rate of necrosis was estimated to be 20 %. Numerous mitoses were detected. Tumor morphology did not change with ageing or tumor size. Histology was similar in group II.In group V the tumors showed signs of irreversible cell damage after treatment. Tumor cells displayed clear shrinkage and partial loss of cell contact. Thromboses of the larger adjacent vessels were found on the tumor-skin border. Macrophage infiltration was present in all groups, but was more numerous pronounced in group V. In group III and IV there were less signs of irreversible cell damage after treatment than in group V.Immunhistochemical analysis of the histological sections for bFGF demonstrated that microvessel density of group I Fig  ranged fImmunhistochemical analysis of the histological sections for CD34 showed that microvessel density of group I ranged from 17 to 23 with a mean value of 19. Angioneogenesis was more pronounced in group II than in group I  and less pronounced in group IV and V compared with group I . There were no significant differences in the expression of angiogenetic markers between group I and group III (p > 0.05).Strong expression of bFGF and CD34 was found in the cytoplasm of intimal endothelial and medial smooth muscle cells. Expression of bFGF and CD34 was stronger at the invasion front than in the centre of tumors.Summarized data of the expression intensity of bFGF and CD34 in the tumors are given in table In group I and II tumor progression was observed in all animals macro- and microscopically. In group III and IV 5 animals showed tumor progression and one had a partial tumor response. In group V, 2 animals showed tumor progression, 2 had a complete remission. One had a partial tumor response and one had no change in tumor response  in which tumors grew more rapidly than in the control group (group I). This implies that resection is associated with an accelerated growth in residual tumors. It is assumed that the surgical manipulation induces liberation of growth factors that in addition to their effect on healing also have a stimulating effect on tumor proliferation.This hypothesis is supported by the results of our immunohistochemical investigations, which revealed an enhanced expression of fibroblast growth factor (bFGF) in group II. In an intraperitoneal tumor model, EGGERMONT et al. showed that the surgical intervention of laparotomy induces an increase of carcinomatosis in the entire peritoneal cavity. At the same time, the immunotherapeutic effect of interleukin 2 and natural killer cells is significantly reduced. It must therefore be speculated that surgical trauma induces a temporary immunosuppressive effect that influences tumor growth ,18,19.In our experiments, group V (hyperthermic perfusion) showed a significant slowing of tumor growth in comparison with the control group. This was also confirmed by the immunohistochemical investigations that showed a reduction in the expression of relevant markers of the vascular endothelium. Indeed, two of the six animals occurred a complete remission of their tumor. The observation that two animals in this group experienced tumor progression can probably be explained by the duration of the intervention, since factors affecting the kinetics of cell death during hyperthermia are not only the maximum temperature induced, but also the duration of the elevated temperature.In an overview report BHUYAN noted that, as a function of various tumor cell types, a temperature of 43°C must be applied between 30 and 150 minutes to irreversibly damage the tumor cells. At a temperature of 45°C, the application time required varied between 13 and 85 minutes . In our Although the temperature in group III  was also 41.5°C applied for 30 minutes, no significant changes in the growth pattern of the tumor could be observed. Furthermore, the histological and immunohistochemical evaluations showed a mild hyperthermic damage at best. A comparison of treatment groups III and V revealed no differences in terms of duration of treatment and induced tissue temperatures.In contrast to group III, however, hyperthermia in group V has been mediated directly by the vascular system. In normal tissues subjected to warming, blood perfusion increases reactively via dilatation of the vessels together with an increase in vessel wall permeability. A decisive factor is that the tumor cells themselves, as well as the endothelial cells of tumor vessels are more thermosensitive than normal tissue ,21,22. OHeat transfer between the vascular system and the tissue is determined in particular by the nature of the blood vessels. Important factors are the number, length, and diameter of the vessels, and also the velocity of blood flow . CREZEE On the other hand, tumor vasculature carries away the externally applied heat (group III) which is then no longer available to destroy the tumor cells. In this case, the temperature gradient decreases from the tumor to the vascular system.Furthermore, in the presence of hyperthermia, microcirculation in the tumor tissue is more affected than in normal tissue . In addiOur results have also confirmed that surgical manipulation of the vascular system and perfusion have an influence on the tumor. Overall, the effect of the intravascular application of hyperthermia appears to be mediated by a combination of hypoxia and heat.Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5°C tissue temperature for 30 minutes has no impact on tumor growth and angioneogenesis in vivo. Hyperthermic isolated limb perfusion effectively suppresses tumor angioneogenesis respectively tumor growth.None declared.JP carried out the treatment and drafted the manuscript.MM carried out the treatment.CS carried out the immunohistochemical studies.JG participated in the design of the study.AD carried out the histological studies.WH participiated in the design of the study.TM participiated in the design and coordination of the study.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Postpartum haemorrhage remains an important cause of maternal death despite treatment with conventional therapy. Uncontrolled studies and one randomised comparison with conventional oxytocics have reported dramatic effects with high-dose misoprostol, usually given rectally, for treatment of postpartum haemorrhage, but this has not been evaluated in a placebo-controlled trial.The study was conducted at East London Hospital Complex, Tembisa and Chris Hani Baragwanath Hospitals, South Africa. Routine active management of the third stage of labour was practised. Women with more than usual postpartum bleeding thought to be related to inadequate uterine contraction were invited to participate, and to sign informed consent. All routine treatment was given from a special 'Postpartum Haemorrhage Trolley'. In addition, participants who consented were enrolled by drawing the next in a series of randomised treatment packs containing either misoprostol 5 × 200 μg or similar placebo, which were given 1 orally, 2 sublingually and 2 rectally.With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment measured in a flat plastic 'fracture bedpan', the primary outcome . There was no difference in mean blood loss or haemoglobin level on day 1 after birth < 6 g/dl or blood transfusion. Side-effects were increased, namely shivering  and pyrexia > 38.5°C . In the misoprostol group 3 women underwent hysterectomy of whom 1 died, and there were 2 further maternal deaths.Because of a lower than expected incidence of the primary outcome in the placebo group, the study was underpowered. We could not confirm the dramatic effect of misoprostol reported in several unblinded studies, but the results do not exclude a clinically important effect. Larger studies are needed to assess substantive outcomes and risks before misoprostol enters routine use. Excessive bleeding from the genital tract after birth, or postpartum haemorrhage (PPH) is the major cause of maternal deaths in many low-income countries. The global estimate is 125,000 deaths per year . In the There is good evidence that a policy of active management of the third stage of labour , and oneWhen postpartum haemorrhage occurs, a number of medical and surgical interventions are used to control the bleeding . A cruciMisoprostol is a methyl ester  of natural prostaglandin E1. It is marketed and registered for use in the prevention and treatment of peptic ulcer disease caused by prostanglandin synthetase inhibitors. Administered orally or sublingually, peak plasma concentrations of misoprostolic acid are achieved in less than 30 minutes . It is aAdministered orally or vaginally, it is an effective agent for the induction of abortion  and of lOral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent . In the Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available .treatment of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review  version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat.All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects.Altogether 244 women were enrolled in the trial. The pack numbers for 6 women were incompletely filled in on the data sheets. The group allocation of these women was therefore unknown, and they could not be included in the analysis. Of these 6 women, the highest measured blood loss after enrolment was 220 ml; none had pyrexia >37.5°C, one had moderate shivering, one had a blood transfusion and none had day 1 haemoglobin level below 8 g/dl or other complications. There was no reason to expect that these exclusions occurred in any selective way which would introduce bias into the final samples, nor could their results have materially altered the conclusions of the study. All the remaining 238 women were included in the analyses.The baseline characteristics are shown in table The outcomes are shown in table Other proxy estimations of blood loss showed no significant differences between the groups. The well-known side-effects of misoprostol, pyrexia and shivering, were significantly more frequent in the misoprostol than the placebo group.Serious morbidity or mortality occurred in 5 women, all from the misoprostol group. Two women who continued to bleed despite conservative therapy underwent abdominal hysterectomy and recovered well. There were 3 maternal deaths:A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions.A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml.A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded.Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths.Pyrexia and shivering were common side-effects of misoprostol, as found in previous studies. Three women, all in the misoprostol group, had severe pyrexia >40°C. Previous studies of high-dose misoprostol for the treatment of postpartum haemorrhage have mostly used the rectal route, and none has had sufficient numbers to be likely to detect rare adverse outcomes such as severe pyrexia. These side-effects may be related to the rapid absorption of misoprostol given orally, and the rapid absorption and high bioavailability when given sublingually. Because of these serious side-effects, we would recommend that for future trials the dose be reduced.Several previous unblinded studies have reported dramatic effects of misoprostol when used to treat postpartum haemorrhage. In 6 observational studies -24, 41 wIn our double blind trial we have been unable to confirm the dramatic effects of misoprostol reported previously in unblinded studies. Our results, however, are consistent with anything from a large beneficial effect to a smaller adverse effect . It is important that this possible benefivial effect be assessed by sufficiently powered randomised trials before the treatment enters routine clinical use.PPH: postpartum haemorrhage; SD: standard deviationNone known.GJH prepared the first draft of the protocol and the final manuscript, and oversaw the clinical workCN assisted with the design of the protocol and data collection sheet, initiated enrolment at two sites, supervised some of the data collection and contributed to the manuscriptSF, ZM, LM, MS and ZJ commented on the protocol, conducted the clinical procedures and collected data.SF entered and collated the dataBM undertook the literature search and commented on the protocol.GW contributed to the protocol developmentAMG contributed to the protocol development and undertook the blinded interim analysisAll authors read and approved the final manuscriptAdditional file 1 - Table 2. Notice informing women about the trial, MS Word file: HofmeyrFile1.docAdditional file 2- Table 3: Information and consent form. MS Word file: HofmeyrFile2.docThe pre-publication history for this paper can be accessed here:Table 2. Notice informing women about the trialClick here for fileTable 3: Information and consent formClick here for file


Every sensation begins with the conversion of a sensory stimulus into the response of a receptor neuron. Typically, this involves a sequence of multiple biophysical processes that cannot all be monitored directly. In this work, we present an approach that is based on analyzing different stimuli that cause the same final output, here defined as the probability of the receptor neuron to fire a single action potential. Comparing such iso-response stimuli within the framework of nonlinear cascade models allows us to extract the characteristics of individual signal-processing steps with a temporal resolution much finer than the trial-to-trial variability of the measured output spike times. Applied to insect auditory receptor cells, the technique reveals the sub-millisecond dynamics of the eardrum vibration and of the electrical potential and yields a quantitative four-step cascade model. The model accounts for the tuning properties of this class of neurons and explains their high temporal resolution under natural stimulation. Owing to its simplicity and generality, the presented method is readily applicable to other nonlinear cascades and a large variety of signal-processing systems. Comparing auditory stimuli that give the same neural response within the framework of a computational model, the authors extract intermediary signal-processing steps with sub- millisecond temporal resolution Animals and human beings rely on accurate information about their external environment and internal state for proper behavioral reactions. This vital requirement has led to a large variety of highly sophisticated sensory systems . A commoWe here show that it is possible to extract fine temporal details of individual processes within such signal-processing chains from observing the output activity alone. This progress results from a new method that extends an experimental strategy well known from measuring threshold curves in neurobiology  or applyUnlike many classical approaches of systems identification, the method is not based on temporal correlations between the input and output; hence, the time resolution of the method is not limited by the output precision of the system under study. In a spike-based analysis of neural response properties, this allows us to assess the dynamical features of the involved processes with considerably higher resolution than suggested by the spike jitter.A particularly fine temporal resolution is needed to analyze signal processing in auditory systems that solve complex tasks such as sound localization, echolocation, and acoustic communication ,13,14,15Locusta migratoria). Its anatomy is well characterized [As a suitable model system to study signal processing in the ear, we chose the auditory periphery of the locust , the sets generally lie on straight lines, at least for low-frequency receptor cells. High-frequency receptor cells do not display straight lines even at the smallest Δt used in the experiment (40 μs) for reasons that will become apparent later. For long intervals , the iso-response sets fall onto nearly circular curves. Note that in t = 500 μs deviates from the symmetry between A1 and A2. In t = 120 μs fell in neither of the two regimes discussed above, and the corresponding iso-response set shows a particularly bulged shape. Recordings from a total of eight cells agree with the observations from the three examples displayed in A1+A2, of both click amplitudes determines the spike probability and demonstrates that the sound pressure is most likely the relevant stimulus parameter. Such linear summation of the pressure on short time scales is not surprising, considering the mechanical properties of the eardrum; owing to its mechanical inertia, rapidly following stimuli can be expected to superimpose. This interpretation is in agreement with laser-interferometric and stroboscopic observations of the eardrum, which have demonstrated that it reacts approximately linearly to increases in sound pressure [The two prominent shapes of the iso-response sets—straight lines and circles—reflect two different processing steps in the auditory transduction chain. A straight line implies that the linear sum, pressure ,19.A12+A22, now determines the spike probability. It follows that the sound energy, which is proportional to the squared pressure, is the relevant stimulus parameter on this time scale. This quadratic summation represents a fundamentally different way of stimulus integration from that of the linear summation on short time scales and indicates the involvement of a different biophysical process. A process that can mediate stimulus integration over longer intervals is the accumulation of electrical charge at the neural membrane. According to this explanation, the electrical potential induced by a click is proportional to the click's energy; contributions from consecutive clicks are then summed approximately linearly because of the passive membrane properties. This is in accordance with earlier investigations for stationary sound signals that revealed an energy dependence of the neurons' firing rate [For the longer intervals, on the other hand, the iso-response sets are circles to good approximation, indicating that the quadratic sum, or ing rate . We concA generates a vibration of the tympanum with strength X = c1·A, i.e., linear in the amplitude with a proportionality constant c1. This mechanical vibration leads to a membrane potential, whose effect on the generation of the spike some time T after the click is given by J = c2·X2= c2·(c1·A)2, i.e., quadratic in the amplitude with an additional proportionality constant c2. The square follows from the circular shape of the iso-response sets for longer time scales, which indicated that a quadratic operation must take place before the accumulation of charge at the neural membrane. Finally, the spike probability p is given by a yet unknown function p = g(J). As J is the relevant quantity determining spike probability, we also refer to it as “effective stimulus intensity.” The model contains a freedom of scaling; any proportionality constants in J can be absorbed into the function g(J). To simplify the notation, we thus set c1 = c2 = 1 and obtain X = A for the strength of the mechanical vibration and J = X2= A2 for the effective stimulus intensity in response to a single click.The above findings motivate the following mathematical model, which describes how a stimulus consisting of two sound clicks is transformed into a spike probability. Within the model, a single click of amplitude X as describing the velocity of the mechanical vibration immediately after the click and J as capturing the membrane potential at the time of spike generation.Note that in this picture, the mechanical vibration and the membrane potential are each captured by a single quantity that does not describe the time course of the corresponding processes, but rather their integrated strength in response to a click. In general, the conversion of the mechanical vibration into a membrane potential as well as the spike generation are dynamical processes that do not happen at a single moment in time. For simplicity, however, one may think of A1 and A2, respectively, we choose the first click to be small enough so that it does not lead to a spike by itself. The measured action potential is thus elicited at some time T after the second click. To derive the model equation for this experimental situation, we divide the time from the first click to spike generation into the period between the two clicks and the period following the second click.For the two-click stimulus with amplitudes X1 = A1. However, how much electrical charge accumulates during the inter-click interval to influence spike generation at time T after the second click depends on the length Δt of the inter-click interval. This effect is incorporated by a Δt-dependent scaling factor Q(Δt) into the model and results in a first contribution from the first click to spike generation given by J1= A12·Q(Δt). Since Q(Δt) denotes the effect of the first click within the inter-click interval only, it should vanish in the limit of very small Δt.Let us start by focusing on the inter-click interval. After the first click, the mechanical vibration has the strength t. This is modeled by a scaling factor L(Δt), i.e., the vibration after the second click has a strength X2 = A1· L(Δt) + A2. Accordingly, the effect of the two-click vibration on the membrane potential at time T after the second click is J2= (X2)2= (A1· L(Δt) + A2)2. For very small Δt, L(Δt) should approach unity to account for the equal contribution of both clicks for vanishing inter-click intervals. The total effective stimulus intensity is then given byLet us now consider the remaining time before spike generation. After the second click, the mechanical vibration is due to a superposition of both clicks. For short inter-click intervals, the straight iso-response lines suggest a simple addition of the two click amplitudes; in general, however, the contribution of the first click to the membrane vibration after the second click will again depend on the inter-click interval Δp via the relation p = g(J).This quantity determines the spike probability L(Δt) = 1 and Q(Δt) = 0 (straight line) and (2) L(Δt) = 0 and Q(Δt) = 1 (circle).How does this model explain the particular shapes of the iso-response sets in t and the second case in some range of larger Δt. In our biophysical interpretation, the first case means that the two clicks are added at the tympanic membrane (L(Δt) ≈ 1), but the short interval between the two clicks prevents a substantial accumulation of charge from the first click alone (Q(Δt) ≈ 0), as already discussed above. The second case may be found for Δt long enough that the mechanical vibration has already decayed (L(Δt) ≈ 0). The two clicks are then individually squared, i.e., they independently lead to two transduction currents. The currents add up if the time constant of the neural membrane is significantly longer than the inter-click interval (Q(Δt) ≈ 1).We can therefore regard A1 and A2, reflecting the symmetry of, e.g., the data in t where neither of the two cases is strictly fulfilled, this symmetry of the iso-response sets will be distorted, as is noticable for the longer Δt in L(Δt) and Q(Δt) may lead to very different iso-response shapes, as in In the two limiting cases, L(Δt) and Q(Δt) are thus filter functions associated with the linear and quadratic summation, respectively.L(Δt) and Q(Δt) can be expected to retain the salient features of the underlying biophysical processes such as frequency content and integration time. In L(Δt) as the velocity of the mechanical vibration and of Q(Δt), at least for large enough Δt, as the time course of the membrane potential following a click. In this generalized model, the input signal is an arbitrary sound pressure wave A(t), and the effective stimulus intensity is a continuous function of time, J(t), which is given byDespite the simple structure of the model, the filters A(t) is first convolved with a temporal filter, l(τ), the result is squared and subsequently convolved with a second filter, q(τ), as depicted in l(τ) and q(τ) have characteristics similar to the click-version filters L(Δt) and Q(Δt), but are not identical to them. Their relations follow from the calculation in L(Δt) and Q(Δt).Here, the input A1, these additional spikes lead to measured values of A2 that are slightly smaller than expected for a circular iso-response set. The data in Note that we interpret t, however, their dynamics may largely overlap. Is it nevertheless possible to design an experiment that directly reveals the whole time course of the mechanical vibration L(Δt) and the electrical integration Q(Δt)? This would provide a parameter-free description of both processes and advance the quantitative understanding of the auditory transduction dynamics. To reach this goal, we again measure iso-response sets. As before, we exploit that for fixed Δt, any pair of click amplitudes  should result in the same spike probability p as the pair  as soon as J = J. It is this straightforward relation that allows us to determine both L(Δt) and Q(Δt) independently of each other. In fact, some appropriate set of measurements that fulfill the iso-response relation is all that is needed to calculate L(Δt) and Q(Δt). Illustrating this concept, we now proceed with a particularly suited choice of stimulus patterns, which keeps the mathematical requirements for the calculation at a minimum. For each Δt, we measure two different iso-response stimuli, and as a key feature, one of these has a “negative” second click, i.e., a sound-pressure pulse pointing in the opposite direction as the first click, as depicted in L(Δt) and Q(Δt), which can be solved explicitly, as explained in t, the whole time course of L(Δt) and Q(Δt) is obtained.The previous experiment showed that the separate effects of the two summation processes can be discerned for short and long time intervals. For intermediate ΔL(Δt) and Q(Δt) for three different cells. L(Δt) displays strong oscillatory components, as was observed for all cells. This property presumably reflects the eardrum's oscillation at the attachment site of the receptor cell. The detailed temporal structure of L(Δt) now allows us to investigate the salient features of this oscillation. To quantify our findings, we fit a damped harmonic oscillation to the measured data for L(Δt) and extract the fundamental frequency as well as the decay time constant. We can use these values to predict the neuron's characteristic frequency (the frequency of highest sensitivity) and the width of its frequency-tuning curve. L(Δt). The remarkable agreement confirms that the new analysis faithfully extracts the relevant, cell-specific properties of the transduction sequence. The correspondence between the tuning characteristics and the filter L(Δt) also explains why high-frequency receptor cells do not feature straight lines for their iso-response sets even at the shortest inter-click interval (40 μs) used in the experiment. For those cells, L(Δt) decays rapidly, thus not allowing access to the region where L(Δt) ≈ 1.Q(Δt) in The short initial rise phase of the measured L(Δt) was considerably smaller than that of Q(Δt). This usually leads to a region around a Δt of 400–800 μs, depending on the specific cell, where L(Δt) ≈ 0 and Q(Δt) is still near unity. These findings correspond to the circular iso-response sets of the initial experiment.In most of our recordings, the temporal extent of the filter t, on the other hand, the data show that Q(Δt) usually decreases strongly. As explained earlier, this is expected from the linear iso-response sets, and it is observed exemplarily in the data shown in Q(Δt) for some recordings, as in Q(Δt). (1) The electrical potential might be shaped by further dynamics in addition to the low-pass properties of the neural membrane, such as inactivation of the transduction channels or electrical resonances as found in some hair cells [Q(Δt)—though too small to be picked up reliably by the present experiments—can be observed in simulations of the processing cascade, see Towards very small Δir cells . (2) TheL(Δt) and Q(Δt), completes the model. In order to test its validity and suitability to make quantitative predictions, we investigated the model's performance on a different class of stimuli, namely combinations of three short clicks. Having measured the required values for L(Δt) and Q(Δt) with two-click stimuli as in the previous experiment (see L(Δt) and Q(Δt) to calculate these predictions and experimentally test them by performing a series of three-click iso-response measurements. This experiment was performed on three different cells; one cell featured an unusually high response variability, and results from the other two cells are shown in Measuring the mechanical and electrical response dynamics, ment see , we now We have presented a novel technique to disambiguate single processing steps within a larger sensory transduction sequence and to analyze their detailed temporal structures. Our approach is based on measuring particular iso-response sets, i.e., sets of stimuli that yield the same final output, and on specific quantitative comparisons of such stimuli to dissociate the individual processes. For the investigated auditory transduction chain in the locust ear, this strategy led to a precise characterization of two consecutive temporal integration processes, which we interpret as the mechanical resonance of the eardrum and the electrical integration of the attached receptor neuron. The method revealed new details of these processes with a resolution far below 1 ms. The results for the time course of the mechanical resonance agree with traditional measurements of tuning curves and show the decay of the oscillation with a temporal precision much higher than expected from the jitter of the measured output signal, the spikes. The time constants of the electrical integration that were extracted from the data had not been accessible by other means.t and is in accordance with the energy-integration model that was found to capture the sound-intensity encoding of stationary sound signals in these cells [The analysis resulted in a four-step model of auditory transduction in locusts. The model comprises a series of two linear filters and two nonlinear transformations. The quadratic nonlinearity that separates the two linear filters suggests that the mechanosensory transduction can be described by an energy-integration mechanism, as the squared amplitude corresponds to the oscillation energy of the tympanum. This quadratic form was derived from the circular shape of the iso-response sets for longer time scales Δse cells . Furtherse cells , and in se cells ,24,25,26se cells . This efAlthough the quadratic nonlinearity is fully consistent with our data, there is a second possibility within the general cascade model, L(Δt) and Q(Δt) were interpreted as the mechanical oscillation of the tympanum and the electrical integration at the neural membrane. Their oscillatory and exponential decay characteristics, respectively, support this view. In principle, however, other processes may well contribute to these characteristics, e.g., electrical resonances as seen in hair cells of the turtle and bullfrog [Q(Δt), but our data generally provide little evidence for such effects. Deviations from the exponential decay characteristics in Q(Δt) may in part be attributable to the oscillatory influx of charge resulting from the tympanic vibration. This may lead to a small oscillatory component in the early phase of the filter , which includes thresholding and saturation. By always comparing stimuli that yield the same output activity, our analysis is independent of the actual shape of g(J). Preceding integration steps may thus be analyzed without any need to model g(J). This feature is independent of the specific output measure and applies to spike probabilities, firing rates, or any other continuous output variable.Finally, in many input–output systems, the last stage of processing can be described by a monotonic nonlinearity. Here, this is the relation between the effective stimulus intensity t. Instead, performing more than the two measurements at each intermediate Δt in the second experiment  cascade, composed of two linear temporal integrations and two nonlinear static transformations . SimilarComplementary to the correlation techniques underlying the parameter estimations in those models, the method presented in this work provides a new way of quantitatively evaluating and testing cascade models. The increased complexity of the LNLN cascade identified in the present case was made accessible by invoking particular iso-response measurements, and a higher temporal resolution was achieved by focusing on how spike probabilities depend on the temporal stimulus structure instead of relying on temporal correlations between stimulus and response.Our experimental technique will be most easily applicable to systems whose signal processing resembles the cascade structure investigated here. The general concept of combining different measurements from within one iso-response set covers, however, a much larger range of systems. With increasingly available high-speed computer power for online analysis and stimulus generation, this framework therefore seems well suited to solve challenging process-identification tasks in many signal-processing systems.Locusta migratoria. Details of the preparation, stimulus presentation, and data acquisition are described elsewhere [We performed intracellular recordings from axons of receptor neurons in the auditory nerve of adult lsewhere . In shorSpikes were detected online from the recorded voltage trace with the custom-made Online Electrophysiology Laboratory software and used for online calculation of spike probabilities and automatic tuning of the sound intensities. The measurement resolution of the timing of spikes was 0.1 ms. During the experiments, the animals were kept at a constant temperature of 30 °C by heating the Peltier element. The experimental protocol complied with German law governing animal care.p of the studied receptor neurons increases monotonically with stimulus intensity, parameters of iso-response stimuli corresponding to the same value of p can be obtained by a simple online algorithm that tunes the absolute stimulus intensity. For fast and reliable data acquisition, we chose p = 70%. The response latency of the neurons varied by 1–2 ms, so that spike probabilities could be assessed by counting spikes over repeated stimulus presentations in a temporal window from 3 to 10 ms after the first click.Since the spike probability A1 and A2, and the tuning was achieved by adjusting the two amplitudes simultaneously. The ratios were chosen so that the angles α in the A1–A2 plane given by tanα = A2/A1 were equally spaced. In the second set of experiments, A1 was kept fixed, and only A2 was adjusted; similarly, in the three-click experiments, only A3 was adjusted. In the following, the intensity I always refers to the peak amplitude Amax of the stimulus pattern, measured in decibel sound pressure level (dB SPL),In the first set of experiments, stimulus patterns were defined by fixed ratios of I70 corresponding to a spike probability of 70% was determined online in the following way. Beginning with a value of 50 dB SPL, the intensity was raised or lowered in steps of 10 dB, depending on whether the previous intensity gave a spike probability lower or higher than 70% from five stimulus repetitions. This was continued until rough upper and lower bounds for I70 were found. From these, a first estimate of I70 was obtained by linear interpolation. Seven intensity values in steps of 1 dB from 3 dB below to 3 dB above this first estimate were then repeated 15 times. From the measured spike probabilities, a refined estimate of I70 was obtained by linear regression. Nine intensities from 4 dB above to 4 dB below this value were repeated 30 times (in some experiments 40 times). The final estimate of I70 was determined offline from fitting a sigmoidal function of the formFor each stimulus, the absolute intensity α and β to these nine intensity-probability pairs. This relation between p and I was then inverted to find the intensity and thus the absolute values of the amplitudes that correspond to p = 0.7.with parameters L(Δt) and Q(Δt) can be obtained independently of each other by combining the results from different measurements within one iso-response set. Here, we derive explicit expressions based on a specific choice of stimuli that are particularly suited for our system. Two measurements are needed to obtain both L(Δt) and Q(Δt) for given time interval Δt. Each stimulus consists of two clicks. The first click has a fixed amplitude A1; the amplitude A2 of the second click at time Δt later is adjusted so that a predefined spike probability p is reached. For the second measurement, the experiment is then repeated with a “negative” second click, i.e., a click with an air-pressure peak in the opposite direction from the first click. The absolute value of this click amplitude is denoted by Ã2. We thus find the two pairs  and  as elements of an iso-response set. Since the spike probability increases with the effective stimulus intensity J, equal spike probability p implies equal J. The two pairs  and  therefore correspond to the same value of J. According to the model, The response functions Setting the two right sides equal to each other, we obtainorÃ2 = − A2, does not correspond to a physical situation as both A2 and Ã2 denote absolute values and are therefore positive. The remaining, second solution readsThe first solution of this mathematical equation, L(Δt), we obtainSolving for L(Δt) from Substituting This yieldsc = J/A12. As we keep A1 and J constant throughout the experiment, this determines Q(Δt) up to the constant c. It can be inferred from an independent measurement with a single click: by setting A1 = 0 in J corresponds to the square of the single-click amplitude that yields the desired spike probability. Alternatively, c can be estimated from the saturation level of Q(Δt) for large Δt, as was done in the present study.with L(Δt) and Q(Δt); see The specific form of the effective stimulus intensity, L(Δt) were fitted with velocity response functions of a damped harmonic oscillatorThe datasets for ω and δ were optimized for minimizing the total squared error. From these, the fundamental frequency f and the decay time constant τdec were determined as f = ω/(2π) and τdec = 1/δ. A simpler fit function of the formwhere f and τdec.led to essentially indistinguishable results for fCF, of the tuning curve, and the tuning width, Δf3dB, can be predicted from the fitted values of ω and δ according to the theory of harmonic oscillators:The resonance frequency, which corresponds to the characteristic frequency, Q(Δt) were fitted with an exponential decayThe datasets for a, τint, and c were adjusted. Here, only data points for Δt > 150 μs were taken into account, as Q(Δt) initially shows a rising phase. The obtained value for c was used to determine the constant J/A12 in where the parameters For comparing these predicted values with measurements, the minimum and width of the tuning curves see A were deA1, A2, and A3 that are separated by time intervals Δt1 and Δt2, respectively  and is augmented by A2. This yields a membrane potential proportional to (A1·L(Δt1) + A2)2. After the third click, the tympanic deflection has evolved to A1·L(Δt1 + Δt2) + A2·L(Δt2) so that the membrane potential is increased by (A1·L(Δt1 + Δt2) + A2·L(Δt2) + A3)2. Summing up the different contributions and approximating the influence of the inter-click intervals on the membrane potential by appropriate factors of Q, we find for the effective stimulus intensityFor stimuli consisting of three clicks with amplitudes vely see A, an appJ for a predefined spike probability can be measured from a single-click experiment by setting A1= A2 = 0 and tuning A3 until the desired spike probability is reached. After having measured L(Δt) and Q(Δt) from two-click experiments, the above equation can be used to predict the amplitude A3 needed to reach this predefined spike probability for any combination of A1, A2, Δt1, and Δt2.The value of Protocol S1(50 KB PDF).Click here for additional data file.Figure S1t = 50 μs, (C) a double click with Δt = 500 μs, and (D) another double click with Δt = 500 μs whose second click points in the oppositve (“negative”) direction. The measurements of air-pressure fluctuations indicate a slight broadening of the click width and some residual vibrations, but they nevertheless present a good approximation of the sharp original pulses.The four panels show different examples of stimuli used in our study. Each panel illustrates the computer-generated pulse signal that drives the loud speaker (upper trace) and the resulting air-pressure fluctuations as measured with a high-precision microphone at the site of the animal's ear (lower trace). The computer-generated clicks are triangular with a total width of 20 μs. The stimuli shown are (A) a single click, (B) a double click with a peak-to-peak interval Δ(10 KB PDF).Click here for additional data file.Figure S2l(t) = sin(2πft)exp(−t/τdec) and q(t) = exp(−t/τint). The parameters were taken from the first two cells presented in detail in the main text: f = 14.5 kHz, τdec = 100 μs, and τint = 300 μs for Cell 1 (left column) and f = 5.1 kHz, τdec = 154 μs, and τint = 590 μs for Cell 2 (right column).The general cascade model, x(t) denotes the signal after application of the linear filter l(t), arbitrary units, for positive second click (solid line) and negative second click (dashed line). Inter-click intervals in these two shown examples were Δt = 80 μs for Cell 1 and Δt = 130 μs for Cell 2.(A and B) Responses of tympanic vibration. J(Δt). The second click was tuned so that the maximum of J(Δt) was equal for positive and negative second clicks. This required click amplitudes of size 1.92 and −2.49 relative to the first click for Cell 1 and 2.09 and −1.27 for Cell 2.(C and D) Corresponding responses of L(Δt) and Q(Δt) extracted according to J(Δt) for many different values of Δt (gray dots). The parameters f, τdec, and τint indicated in the plots were obtained by fitting a damped harmonic oscillator and an exponential function to L(Δt) and Q(Δt), respectively (black lines). The initial part of Q(Δt) shows small fluctuations that result from the oscillatory influx of charge following the tympanic vibrations. In (G), a magnified view of the initial section is shown in the inset.(E–H) Filters (138 KB PDF).Click here for additional data file.