All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders.w/Kitw-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB.For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice.TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, KitThis is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders. Attesting to the importance of inflammation in disease, a trans-NIH Inflammation Working Group was formed recently to collect input on proposed research areas within the overarching theme of "Inflammation as a Common Mechanism of Disease" . In geneFour PARs have been cloned so far, and all are co-expressed in the mouse bladder urothelium  and humaE. coli lipopolysaccharide (LPS), substance P, and antigen [PAR1- and to a lesser extent PAR2-deficient mice exhibit a reduced response to bladder inflammation induced by  antigen . The lat antigen ,25. In a antigen .Despite the evidence of PAR involvement in inflammation, pain, healing, and cancer in animal models, there seems to be no published clinical study regarding the efficacy of PAR antagonists. In order to search for therapeutic targets other than receptors themselves, we set forth to determine the transcription factors downstream of PAR activation as the next step in elucidating the network of responses at the molecular level to PAR activation. For this purpose, we introduced a new approach for the study of the transcriptional regulation downstream PAR activation Figure . This tow/Kitw-v and normal C57BL/6 mice were purchased from Jackson Laboratories .All animal experimentation described here was performed in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" . Genetically mast cell-deficient WBB6F1-KitAcute cystitis was induced as we described previously ,23,27-29To decrease the complexity of using whole bladder homogenates, the bladder mucosa was separated from the detrusor smooth muscle, as described . BrieflyMinimum Information About Microarray Experiments – MIAME .To determine the transcriptional regulatory elements (TRE) downstream of the activation of PAR receptor. For this purpose, female C57BL6 mice (n = 3 per group) were instilled two times, 30 minutes part, with: control peptide, PAR1-AP, PAR2-AP, or PAR4-AP, every other day for 3 days. All peptides were used at 10 μM concentration. Mice were euthanized 24 hours after the last instillation and the urinary bladders were isolated, and the mucosa was separated from the detrusor muscle, as described above.Nuclear extracts from the bladder mucosa were obtained as detailed below for EMSA assays and analyzed by protein/DNA combo arrays containing 345 unique consensus-binding sequences per array [To investigate the relative binding of TREs to their unique consensus sequences, we used the TransSignal protein/DNA combo array with spin column preparation  from Panomics  . Array aWe used the same methodology developed for analysis of cDNA microarrays ,27,35,3632P ATP  and T4 polynucleotide kinase (New England Biolabs) and then purified using a G-50 column . A second probe corresponding to the μE3 immunoglobulin enhancer site  was also tested but it was found to have a much lower binding affinity as previously described by others [A commercially available double-stranded TFEB probe corresponding to the adenovirus major late promoter core sequence  was constructed by end-labeling a double-stranded probe with γy others  . Reactions were resolved on a 5% native acrylamide gel with 0.5 × TBE at 300 V for 2 hours. Gels were vacuum-dried and visualized on Kodak Biomax MS Film and quantified using ImageJ Software (NIH) [Nuclear extracts (10 μg) from the bladder mucosa and detrusor muscle were placed in 10 mM Tris HCl pH 7.9, 100 mM KCl, 5 mM MgClre (NIH) .®  and the area expressing positive cells for TFEB at 200× magnification was calculated as percent of the total urothelial cell area. Results are presented as average and standard error of the mean.Frozen bladders were processed for routine immunohistochemistry according to published methods . All reaTarget validation was sought for 7 genes known to have an E-box based on the evidence on the literature  or our own observation Eif4ebp2 . Female Taq polymerase . Primer pairs were designed using Primer 3 [Frozen bladders were shipped to Genpathway  for querPrimer 3 . Detailst-test . A nominal p value less than 0.05 was considered statistically significant.Q-PCRs reactions were run in triplicate and the averaged Ct values were transferred into copy numbers of DNA using a standard curve of genomic DNA with known copy numbers. The resulting TFEB binding values for each genomic region were also normalized for primer pair amplification efficiency using the Q-PCR values obtained with Input DNA (unprecipitated genomic DNA). Results are presented as binding events detected per 1000 cells for each genomic region tested and compared to an untranscribed region used as a negative control. The difference between two mean values was analyzed with an unpaired Student's PAR1-AP, PAR2-AP, and PAR4-AP and control peptide were synthesized at the Molecular Biology Resource Facility, William K. Warren Medical Research Institute, OUHSC, as carboxyl-terminal amides, purified by high-pressure liquid chromatography, and characterized by mass spectroscopy. Peptide solutions were made fresh in PBS from powder. PAR3-AP was not used in this research because of lack of specificity.PAR1-activating peptide (AP), PAR2-AP, PAR4-AP, and control peptide were instilled into the mouse bladder at the concentration of 10 μM. This treatment induces a strong and reproducible bladder inflammation, whereas no inflammation was observed in mice instilled with 10 μM control peptide . We usedTo confirm whether the urinary bladder expresses TFEB, we used a commercially available antibody to localize its expression. Figure w/Kitw-v mice. As shown in Figure w/Kitw-v did not develop an inflammatory response to PAR1 activation  and real-time polymerase chain (Q-PCR). For this purpose, we used the same TFEB antibody employed for IHC. CHIP was obtained from the whole bladder isolated from PAR2-AP- and control peptide-treated bladders. We queried the expression of selected genes known to have an E-box on their promoter and compared the results with an un-transcribed region of the DNA. Figure w/Kitw-v mice. Kitw/Kitw-v mice failed to mount an inflammatory response to PAR1-AP, and EMSA confirmed that TFEB is upregulated by all PAR-APs in wild type mice but not in Kitw/Kitw-v mice. Finally, we used a combination of chromatin immunoprecipitation and QPCR to target validate transcripts known to be under the control of TFEB. This assay takes into consideration genes that are actively transcribed, in contrast to cDNA array technologies that queries RNA accumulation. The disadvantage of the CHIP/Q-PCR method is the amount of chromatin necessary for CHIP, which limits this analysis to the whole bladder and not to specific layers (mucosa and detrusor) used for EMSA. Therefore, our CHIP/Q-PCR results have to be taken in the context that migrating inflammatory cells, in addition to resident cells, contributed to the measured values. These results indicate that TFEB is downstream of PAR activation, that this response occurs primarily in the bladder urothelium, and that TFEB mobilization depends on the presence of either mast cells or alternatively c-kit receptors, which are expressed on a variety of tissues, including certain nerves. Given our previous findings demonstrating the importance of mast cells in bladder inflammation and our finding of MiTF expression, we speculate that the mast cell is the key contributor to this response, but further studies will be necessary to delineate the precise mechanism of TFEB mobilization.We used a top-down approach for determination of transcription factors involved in the response of the mouse bladder to pro-inflammatory PAR-APs Figure . First, TFEB belongs to the microphthalmia (MiTF)-TFE subfamily of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors which includes four family members: TFEB, TFE3, TFEC, and MiTF. MiTF family members are required for the proper development of osteoclasts, melanocytes, retinal pigment epithelial cells, mast cells, and natural killer cells . In addiEach protein of the MiTF family forms homo- or heterodimers with other family members  and recoThe only other finding implicating this family in the urinary tract is their involvement in renal cell carcinoma. In this regard, TFE3 is involved in chromosomal translocations recurrent in renal cell carcinoma . Transloex vivo display a number of functional defects [c-kit- and m-Csf-dependent MAPK phosphorylation transcriptionally activates both TFE3 and MiTF, which is necessary for mast cell developmental functions [w/Kitw-v mice do not develop bladder inflammation in response to antigen challenge [w/Kitw-v with mast cells from wild type mice restores the inflammatory response [MiTF mRNA and protein levels are higher in human mast cells than monocytes and granulocytes . MiTF se defects . In addiunctions . Our finhallenge , and subhallenge  stimulatresponse ,8. ThereThrombin and tryptase are endogenous agonists of PARs in humans. Along with serine proteases, it seems that tissue factor and element of the coagulation cascade are also endogenous activators of PARs ,62. In hw/Kitw-v mice. The differences observed were abolished when Kitw/Kitw-v mice had their mast cell deficiency selectively repaired by transplantation of bone marrow-derived mast cells from the normal congenic +/+ mice [Mast cells are strategically located for optimal interaction with the environment and for their putative functions in host defense . Among t+/+ mice ,8. As th+/+ mice ,8.PARs provide an answer to the question of how mast cell products, such as tryptase, produce signals and induce bladder inflammation. Recently, we characterized the bladder responses to PAR-APs and presented evidence of a mandatory role for PARs in experimental cystitis . We wentTo the best of our knowledge, this is the first report describing TFEB expression in response to PAR activation in the bladder and suggest a unifying, mechanistic pathway of bladder inflammation as follows: we hypothesize that mast cells release tryptase resulting in increased PAR activation and, consequently, TFEB/MiTF activity. As a result of increased of MiTF activity in mast cells there is mMCP-6 and mMCP-7 synthesis, leading to additional signals for inflammation. The findings that TFEB is downstream of activation of all PARs, makes this transcriptional factor a novel therapeutic target for the treatment of inflammatory disorders of the bladder.CHIP, chromatin immunoprecipitation; IHC, immunohistochemistry; PAR, protease-activated receptors; PAR-AP, protease-activated receptor-activating peptide; Q-PCR = real-time polymerase chain; TF, transcription factor.The authors declare that they have no competing interests.RS conceived of the study and drafted the manuscript; JM participated on the design of immunohistochemistry protocols and choice of antibodies, performed the immunohistochemistry and confocal microscopy analysis; BF participated on the design of confocal experiments; CAD maintained the genotyping and animal colony; CS helped MRS with animal experiments, performed protein/DNA combo array, EMSAs, and gel shift assays, ID performed the statistical analysis of protein/DNA combo array, MI participated in its design and helped to draft the manuscript, REH participated in its design and helped to draft the manuscript, BW consulted RS regarding the participation of mast cells, MRS participated in its design, carried out all the animal experiments, and removed the tissues.All authors read and approved the final manuscript.


Whether listening to background music enhances verbal learning performance is still disputed. In this study we investigated the influence of listening to background music on verbal learning performance and the associated brain activations.Musical excerpts were composed for this study to ensure that they were unknown to the subjects and designed to vary in tempo (fast vs. slow) and consonance (in-tune vs. out-of-tune). Noise was used as control stimulus. 75 subjects were randomly assigned to one of five groups and learned the presented verbal material (non-words with and without semantic connotation) with and without background music. Each group was exposed to one of five different background stimuli . As dependent variable, the number of learned words was used. In addition, event-related desynchronization (ERD) and event-related synchronization (ERS) of the EEG alpha-band were calculated as a measure for cortical activation.We did not find any substantial and consistent influence of background music on verbal learning. There was neither an enhancement nor a decrease in verbal learning performance during the background stimulation conditions. We found however a stronger event-related desynchronization around 800 - 1200 ms after word presentation for the group exposed to in-tune fast music while they learned the verbal material. There was also a stronger event-related synchronization for the group exposed to out-of-tune fast music around 1600 - 2000 ms after word presentation.Verbal learning during the exposure to different background music varying in tempo and consonance did not influence learning of verbal material. There was neither an enhancing nor a detrimental effect on verbal learning performance. The EEG data suggest that the different acoustic background conditions evoke different cortical activations. The reason for these different cortical activations is unclear. The most plausible reason is that when background music draws more attention verbal learning performance is kept constant by the recruitment of compensatory mechanisms. Whether background music influences performance in various tasks is a long-standing issue that has not yet been adequately addressed. Most published studies have concentrated on typical occupational tasks like office work, labour at the conveyer belt, or while driving a car -13. ThesWhether background music influences performance of academic and school-related skills has also been investigated. A broad range of skills have been considered, including the impact of background music on learning mathematics, reading texts, solving problems, perceiving visual or auditory information, learning verbal material (vocabulary or poems), or during decision making ,8,15-40.The present study was designed to readdress the question of whether background music enhances verbal learning. There are a number of reasons for this renewed interest: Firstly, only a few of the preceding studies have examined the effects of background music on verbal learning in particular ,41-45, r1. We used musical pieces unknown to the subjects. For this, we composed new musical pieces, avoiding any resemblance to well-known and familiar tunes. In doing this, we circumvented the well-known effect that particular contents of episodic and semantic memory are associated with musical pieces -50. Thus2. A further step in avoiding activation of a semantic or episodic network was to use meaningless words. In combination with using unfamiliar musical pieces, this strategy ensures that established (or easy to establish) associations between musical pieces and particular words are not activated.3. The musical pieces were designed in order to evoke pleasantness and activation to different degrees. Based on the mood-activation hypothesis proposed by Glenn Schellenberg and colleagues, we anticipated that music that evokes more pleasant affect might influence verbal learning more positively than music that evokes negative emotions -53.changing state effects  and with acoustic background [background+]). We also measured the electroencephalogram (EEG) during the different verbal learning conditions to explore whether the different learning conditions are associated with particular cortical activation patterns. The order of background stimulation  was counterbalanced across all subjects. There was an intermittent period of 12-14 minutes between the two learning sessions during which the subjects rated the quality of the background stimuli and rested for approximately 8 minutes.Several studies have shown that tempo and the level of consonance/dissonance of musical excerpts strongly determine the level of arousal and emotional feelings ,65,66. WIn a pilot study, 21 subjects (who did not take part in the main experiment) evaluated these stimuli according to the experienced arousal and valence on a 5-point Likert scale (ranging from 0 to 4). In-tune music was generally rated as more pleasant than both out-of-tune music and noise . In terms of arousal, the slow musical excerpts were rated as less arousing than the fast excerpts and the noise stimulus . These five acoustic stimuli were used as background stimuli for the main verbal learning experiment. In this experiment, background stimuli were binaurally presented via headphones (Sennheiser HD 25.1) at approximately 60 dB.Verbal learning was examined using a standard verbal learning test, which is frequently used for investigations with German-speaking subjects . This test has been shown to validly measure verbal long-term memory ,63. The Several psychological measures were obtained after each experimental condition. First, the participants rated their subjective mood state using the MDBF questionnaire  . The MDBThe electroencephalogram (EEG) was recorded from 30 scalp electrodes (Ag/AgCl) using a Brain Vision amplifier system . Electrodes were placed according to the 10-20 system. Two additional channels were placed below the outer canthi of each eye to record electro-oculograms (EOG). All channels were recorded against a reference electrode located at FCz. EEG and EOG were analogue filtered (0.1-100 Hz) and recorded with a sampling rate of 500 Hz. During recording impedances on all electrodes were kept below 5 k.EEG data were preprocessed and analysed by using BrainVision Analyzer  and Matlab . EEG data were off-line filtered (1-45 Hz), and re-referenced to a common average reference. Artefacts were rejected using an amplitude threshold criterion of ± 100 μV. Independent component analysis was applied to remove ocular artefacts ,72. EEG task - band powerbaseline) * 100/band powerbaseline). Note that negative values indicate a relative decrease in the alpha-band (event-related desynchronization) during the experimental condition compared to the baseline condition, while positive values indicate an increase of alpha-band power during the experimental condition (event-related synchronization). In order to avoid multiple comparisons, event-related synchronization/desynchronization values were averaged over 10 time windows with a duration of 400 ms, and were collapsed for the frontal , central-temporal , and parieto-occipital  electrode locations [Analysis of the time course of event-related desynchronization and event-related synchronization was performed according to the classical method described elsewhere ,74. We iocations  . Asymmetric brain activation patterns during music perception have been reported by some studies ,77. DiffFor the main analysis of the event-related synchronization/desynchronization, a four-way ANOVA with the repeated measurements on the following factors was applied: Time Course (5 epochs after word stimulus presentation), Brain Area , Background , and the grouping factor Group . Following this, we computed a four-way repeated measurements ANOVA, including the event-related synchronization/desynchronization data obtained for the left- and right-sided electrodes of interest, to examine whether hemispheric differences might influence the overall effect. For this, we used the multivariate approach to handle with the problem of heteroscedasticity . ResultsIn order to assess whether there are between hemispheric differences in the cortical activations during listening to background music, we subjected the event-related synchronization/desynchronization data of the frontal, central-temporal, and parietal-occipital ROIs separately to a four-way ANOVA with Hemisphere (left vs. right), Group, Background, and Time as factors . If background music and especially background music of different valence would evoke different lateralization patterns than the interaction between Hemisphere, Group or Hemisphere, Group, and Background should become significant. Thus, we were only interested in these interactions.2 = 0.001; Group: F = 1.42,p = 0.235, eta2 = 0.075) nor a significant interaction  = 0.90, p = 0.47, eta2 = 0.049)  and the grouping factor  revealed no significant main effect  = 0.073, p = 0.788, etaThe valence and arousal ratings for the three background conditions were subjected to separate repeated measurements ANOVAs. The valence measures were significantly different for the five background conditions  = 4.75, p < 0.002). Subsequently performed Bonferroni corrected t-tests revealed significant differences between the in-tune and out-of-tune conditions . For the arousal rating we obtained no significant difference between the five conditions  = 1.07, p = 0.378).2 = 0.42). There was also a trend for interaction between Background and Group  = 2.40, p = 0.06, eta2 = 0.12), which is qualified by reduced pleasantness ratings for the in-tune melodies for the condition in which the subjects were learning. For the sadness scale we obtained a main effect for Background  = 8.14, p = 0.006, eta2 = 0.10) qualified by lower sadness ratings for the music heard while the subjects were learning.The MEQ rating data were subjected to three 2-way ANOVAs with one repeated measurements factor (Background: background+ vs. background-) and the grouping factor (Group). There was a significant main effect for Group with respect to pleasantness, with the in-tune melodies receiving the highest pleasantness ratings and the noise stimulus the lowest  = 12.5, p < 0.001 eta2 = 0.26), with less pleasantness during learning while acoustic background stimulation was present. The subjective feeling of arousal and sadness did not change as a function of different acoustic background conditions.The kind of acoustic background also influenced the subjective experience of pleasantness  = 24.6, p < 0.001, etaThe event-related synchronization/desynchronization data of the alpha band were first subjected to a 4-way ANOVA with repeated measurements on three factors  and one grouping factor . Where possible we used the multivariate approach to test the within-subjects effects, since this test is robust against violations of heteroscedasticity . Figure 2 = 0.86). The time courses of event-related synchronization/desynchronization are different for the different brain areas with larger event-related desynchronization for the parieto-occipital leads at the second time segment and larger event-related synchronization also at the parieto-occipital leads for the 5th time segment after word presentation  = 34.50, p < 0001, eta2 = 0.81) . In order to delineate this three-way interaction we conducted two-way ANOVAs with Background and Group as factors separately for each time segment. There were only significant Background × Group interactions for the time segments 3 and 5  = 2.6, p = 0.038, eta2 = 0.13; T5: F = 2.7, p = 0.036, eta2 = 0.13). The interaction at time segment 3 (800 - 1200 ms after word presentation) was qualified by a larger event-related desynchronization for verbal learning with background music (background+) for the in-tune-fast group  = 9.2, p = 0.009, eta2 = 0.397 significant after Bonferroni-Holm correction at the level of p = 0.05). The interaction at time segment 5 (1600 - 2000 ms after word presentation) was qualified by larger event-related synchronization for the out-of-tune-fast group during background+  = 6.3, p = 0.025, eta2 = 0.13, marginally significant after Bonferroni-Holm correction at the level of p = 0.05). Figure This complex ANOVA revealed several main effects and interactions. The event-related synchronization/desynchronization data showed a typical time course with a strong event-related desynchronization peaking at the second time segment (400 - 800 ms after word presentation). After reaching the maximum event-related desynchronization, the alpha-band synchronises again with the strongest event-related synchronization at the 5th time segment (1600 - 2000 ms after word presentation). This time course is highly significant  = 106.2, p < 0.001, etaThe ANOVA analysis conducted to examine potential between-hemisphere differences in terms of event-related synchronization/desynchronization revealed that none of the interactions of interest  turned out to be significant, even if the statistical threshold was lowered to p = 0.10.Arousal-Emotion hypothesis, we anticipated that consonant and arousing musical excerpts would influence verbal learning positively, while dissonant musical excerpts would have a detrimental effect on learning. Drawing on the theory of changing state effects, we anticipated that rapidly changing auditory information would distract verbal learning more seriously than slowly changing music. Thus, slower musical pieces were expected to exert less detrimental effects on verbal learning than faster music. In order to control for the global influence of familiarity with and preference for specific music styles, we designed novel in-house musical excerpts that were unknown to the subjects. Using these excerpts, we did not uncover any substantial and consistent influence of background music on verbal learning.The present study examined the impact of background music on verbal learning performance. For this, we presented musical excerpts that were systematically varied in consonance and tempo. According to the Arousal-Emotion hypothesis, we hypothesised that positive background music arouses the perceiver and evokes positive affect. However, this hypothesis was not supported by our data since there was no beneficial effect of positive background music on verbal learning. In addition, according to the theory of changing state effects we anticipated that rapidly changing auditory information would distract verbal learning more seriously than slowly changing music. This theory also was not supported by our data.The effect of passive music listening on cognitive performance is a long-standing matter of research. The findings of studies on the effects of background music on cognitive tasks are highly inconsistent, reporting either no effect, declines or improvements in performance (see the relevant literature mentioned in the introduction). The difference between the studies published to date and the present study is that we used novel musical excerpts and applied experimental conditions controlling for different levels of tempo and consonance. According to the As mentioned above, the findings of previously published studies examining the influence of background music on verbal learning and other cognitive processes are inconsistent, with more studies reporting no influence on verbal learning and other cognitive tasks. But before we argue too strongly about non-existing effects of background music on verbal learning we will discuss the differences between our study and the previous studies in this research area. Firstly, it is possible that the musical excerpts used may not have induced sufficiently strong arousal and emotional feelings to exert beneficial or detrimental effects on verbal learning. Although the four different musical excerpts significantly differed in terms of valence and arousal, the differences were a little smaller in this experiment than in the pilot experiment. Had we used musical excerpts that induce stronger emotional and arousal reactions, the effect on verbal learning may have been stronger. In addition, the difference between the slow and fast music in terms of changing auditory cues might not have been strong enough to influence verbal learning. There is however no available data to date to indicate "optimal" or more "optimal" levels of arousal and/or emotion and of changes in auditory cues for facilitating verbal learning.Arousal-Emotion hypothesis. However, the specific mechanisms responsible for improving memory functions while listening to music (if indeed present) are still unclear. It is conceivable that the unknown musical pieces used in our study did not activate the memory system, thus, exerting no influence on the verbal learning system.A further aspect that distinguishes our study from previous studies is that the musical pieces were unknown to the subjects. It has been shown that music has an important role in autobiographical memory formation such that familiar and personally enjoyable and arousing music can elicit or more easily facilitate the retrieval of autobiographical memories (and possible other memory aspects) -50. It iAlthough verbal learning performance did not differ between the different background stimulation conditions, there were some interesting differences in the underlying cortical activations. Before discussing them, we will outline the similarities in cortical activations observed for different background conditions. During learning, there was a general increase of event-related desynchronization 400-1200 ms after word presentation followed by an event-related synchronization in a fronto-parietal network. This time course indicates that this network is cortically more activated during encoding and retrieval in the first 1200 ms. After this, the activation pattern changes to event-related synchronization, which most likely reflects top-down inhibition processes supporting the consolidation of learned material .Although the general pattern of cortical activation was quite similar across the different background conditions, we also identified some considerable differences. In the time window between 800 - 1200 ms after word presentation, we found stronger event-related desynchronization at frontal and parietal-occipital areas for the in-tune-fast group only. Clearly, frontal and parieto-occipital areas are stronger activated during verbal learning in the ambient setting of in-tune-fast music but not in the other conditions. Frontal and parietal areas are strongly involved in different stages of learning. The frontal cortex is involved in encoding and retrieval of information, while the parieto-occipital regions are part of a network involved in storing information -87. One A further finding is the stronger event-related synchronization in the fronto-parietal network at time segment 5 (1600 - 2000 ms after word presentation) for the out-of-tune groups and especially for the out-of-tune-fast group. Event-related synchronization is considered to reflect the action of inhibitory processes after a phase of cortical activation. For example, Klimesch et al.  propose We did not find different lateralisation patterns for event-related synchronization/desynchronization in the context of the different background music conditions. This is in contrast to some EEG studies reporting between hemispheric differences in terms of neurophysiological activation during listening of music of different valence. Two studies identified left-sided increase of activation especially when the subjects listened to positively valenced music opposed to negative music evoking a preponderance of activation on the right hemisphere  ,77,88. TFuture experiments should use musical pieces with which subjects are familiar and which evoke strong emotional feelings. Such musical pieces may influence memory performance and the associated cortical activations entirely differently. Future experiments should also seek to examine systematically the effect of pre-experimentally present or experimentally induced personal belief in the ability of background music to enhance performance and, depending on the findings, this should be controlled for in other subsequent studies. There may also be as yet undocumented, strong inter-individual differences in the modulatory impact of music on various psychological functions. Interestingly, subjects have different attention and empathy performance styles, and this may influence the performance of different cognitive functions.A methodological limitation of this study is the use of artificial musical stimuli, which are unknown to the subjects. In general we listen to music we really like when we have the opportunity to deliberately chose music. Thus, it might be that in case of listening to music we really appreciate to listen to while we learn the results might be entirely different. However, this has to be shown in future experiments.Using different background music varying in tempo and consonance, we found no influence of background music on verbal learning. There were only changes in cortical activation in a fronto-parietal network (as measured with event-related desynchronization) around 400 - 800 ms after word presentation for the in-tune-fast-group, most likely reflecting a larger recruitment of cortical resources devoted to the control of memory processes. For the out-of-tune groups we found stronger event-related synchronization around 1600 - 2000 ms in a fronto-parietal network after word presentation, this thought to reflect stronger top-down inhibitory influences on the memory system. We suggest that this top-down inhibitory influence is at least in part a response to the slightly more distracting out-of-tune music that enables the memory system to adequately reengage in processing the verbal material.The authors declare that they have no competing interests.LJ and PS both designed the experimental paradigm, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.


Self-reported limitations in physical function often have only weak associations with measured performance on physical tests, suggesting that factors other than performance commonly influence self-reports. We tested if personal or health characteristics influenced self-reported limitations in three tasks, controlling for measured performance on these tasks.N = 5396) from the Third National Health and Nutrition Examination Survey to examine the association between the repeated chair rise test and self-reported difficulty rising from a chair. We then tested if personal characteristics, health indicators, body composition, and performance on unrelated tasks were associated with self-reported limitations in this task. We used the same approach to examine associations between personal and health characteristics and self-reported difficulty walking between rooms, controlling for timed 8-foot walk, and self-reported difficulty getting out of bed, controlling for repeated chair rise test results.We used cross-sectional data on adults aged ≥ 60 years (In multivariate analyses, participants who performed worse on the repeated chair rise test were more likely to report difficulty with chair rise. However, older age, lower education level, lower serum albumin, comorbidities, knee pain, and being underweight were also significantly associated with self-reported limitations with chair rise. Results were similar for difficulty walking between rooms and getting out of bed.Self-reports of limitations in physical function are influenced by personal and health characteristics that reflect frailty, and should not be interpreted solely as measured difficulty performing the task. Physical functioning is a key component of health-related quality of life (HRQL) . AttentiA gold standard method to measure physical functioning does not exist. Self-report questionnaires have been adopted as easily administered instruments that can capture limitations in a wide spectrum of tasks ,12. HoweAlthough self-reported functioning and performance on objective physical tests are correlated, these associations are generally weak -17. ThesAn alternative explanation for the modest association between self-report and physical performance tests may be that factors other than performance affect self-reports of limitations. Personal and health characteristics may influence how different patients appraise the limitations they have.Despite extensive literature on the use of self-reports to measure physical functioning, few studies to date have examined whether factors other than measured performance on the same task influence self-reports. Our primary objective was to determine if self-reported limitations in physical functions were associated with personal and health characteristics, after accounting for measured performance on the same task. To address the problem of compensability, we analyzed three self-report tasks  for which there were corresponding physical performance tests . This design provided a unique method with which to assess influences on, and the meaning of, self-reported limitations.We analyzed data from the Third National Health and Nutrition Examination Survey (NHANES III), a national population-based sample of non-institutionalized individuals in the United States . In thisTo maximize the specificity of the association between physical performance tests and self-reported physical function, we studied performance tests in relation to their corresponding self-reported functions: 1) repeated chair rise test and its relationship with self-reported difficulty rising from a chair; 2) repeated chair rise test and its relationship with self-reported difficulty getting in or out of bed; and 3) 8-foot walk test and its relationship with self-reported difficulty walking between rooms on the same level. Limitations on eight additional physical functions were asked, but were not included in the analysis because they did not have a corresponding physical performance test administered in NHANES III.Ability to perform the three self-reported physical functions of interest was assessed by the following: "Please tell me if you have no difficulty, some difficulty, much difficulty or are unable to do these activities at all when you are by yourself and without the use of aids." 1) "Standing up from an armless straight chair?", 2) "Getting in or out of bed?", and 3) "Walking from one room to another on the same level?".Data on six physical performance tests were collected in NHANES III by trained assessors: repeated chair rise test, 8-foot walk, lock and key test, shoulder range of motion, active hip and knee flexion, and timed tandem stand test ,22-25. TThe lock and key test, a test of eye-hand coordination and fine motor skills, was a timed test of unlocking a lock with a key. Internal and external rotations of both shoulders were scored as full, partial, or unable to perform. Hip and knee flexion were scored similarly. For analysis, results on the lock and key test were categorized into quartiles, and range of motion of the shoulders and flexion of the hips and knees were dichotomized as either full or not, with full as the reference group. We did not include the timed tandem stand test because almost all participants attained the maximum allotted time. Reliability of these physical performance tests has been reported to be good ,22-25.We included covariates available in the data set and known to be associated with physical function. Demographic characteristics included age, gender, race-ethnicity, and education level. We categorized age into five groups  to allow for non-linear relationships, and categorized education level, recorded as highest grade attained, into three groups .The health indicators were current cigarette smoking, hemoglobin level, serum albumin concentration, knee pain, and comorbidities. We included current cigarette smoking, hemoglobin, and serum albumin because they are indicators of general health -29. Hemo2), normal weight (18.5 - 24.9 kg/m2), overweight (25.0 - 29.9 kg/m2), and obesity (≥ 30.0 kg/m2) because of its non-linear relationship with physical function [Body composition was assessed by body mass index (BMI) and skeletal muscle mass, which are prognostic indicators of physical function. BMI, measured as weight in kilograms/height in meters squared, was grouped using World Health Organization categories of underweight  were coded as "blank but applicable". After extensive review by survey analysts, data believed to be extreme or illogical and viewed as virtually impossible were also coded as "blank but applicable". We treated data coded as blank but applicable as missing at random. We used the multiple imputation method with the Markov Chain Monte Carlo algorithm to impute missing values . This alTable Participants had a mean (± standard error of the mean) age of 70.7 ± 0.2 years Table . ArthritCompared to participants included in the study, those who were excluded due to lack of assessment of physical function by physical performance tests had similar demographic characteristics. They had a mean age of 71.6 ± 0.6 years (vs. 70.7 ± 0.2 years), were mostly women (58.9% vs. 57.3%), and non-Hispanic white (81.0% vs. 84.7%). They generally had more comorbidities, with a higher proportion reporting ever been diagnosed with arthritis (46.7% vs. 44.7%), stroke (11.7% vs. 6.8%), diabetes mellitus (14.5% vs. 12.6%), and chronic bronchitis (12.6% vs. 9.3%).Table In the first set of analyses that tested the association of the repeated chair rise test and self-reported limitations rising from a chair, worse performance on the chair rise test was significantly associated with the odds of reporting worse limitations Table . In adjuTable rd and 4th quartiles on the chair rise test were more likely to report worse limitations than those in the best-performing quartile. In the adjusted model, older age, lower education level, arthritis, stroke, congestive heart failure, cancer, lower serum albumin level, bilateral knee pain, and lower BMI were also significantly associated with an increased odds of worse self-reported functioning, independent of measured performance on the chair rise test.Results of models predicting self-reported ability to get in or out of bed were similar Table . ParticiTable In the third set of models examining self-reported ability to walk between rooms, the 8-foot walk test was significantly associated with the odds of a worse level of self-reported limitation Table . In the Table Self-reported limitation in rising from a chair was associated not only with performance on the chair rise test, but also with performance on the 8-foot walk and with limitation in hip and knee flexion, when these physical performance tests were included in the model Table . AssociaResults of complete case analysis were similar to those of the main analysis that used multiple imputation of missing values. In the complete case analyses, self-reported limitations rising from a chair were associated not only with worse performance on the repeated chair rise test, but also with older age, current cigarette smoking, arthritis, stroke, myocardial infarction, congestive heart failure, lower BMI, knee pain, and lower serum albumin level. Self-reported limitations getting in or out of bed were associated with worse performance on the chair rise test, arthritis, stroke, chronic bronchitis, congestive heart failure, and bilateral knee pain. Worse performance on the 8-foot walk test was associated with higher odds of self-reported limitations walking between rooms. Additional significant covariates included arthritis, stroke, chronic bronchitis, congestive heart failure, and knee pain. These results indicate that self-reports were influenced by personal and health characteristics and not exclusively by the measured difficulty in performing the task.Our findings indicate that self-reported limitations in physical function were associated with measured performance on the task being assessed. Nonetheless, self-reported physical functioning was influenced also by personal and health characteristics and not solely by the measured difficulty in performing the task. These findings indicate that self-report captures information above and beyond performance on the specific task itself.Functional limitations were strongly associated with physical performance tests, particularly for participants in the worst-performing quartiles. Despite the importance of physical performance tests, other factors were independently associated with self-reported physical functioning. Advanced age showed strong graded associations with limitations in each of the three functions. Participants with comorbid conditions were more likely to report worse limitations, consistent with prior reports ,37. For Despite extensive literature on this topic, the nature of the association between physical performance tests and self-reported limitations has remained incompletely characterized. Most prior studies compared a group of physical performance tests  with multi-item self-report functions ,18-20,39Physical performance tests that were not specifically paired to the physical functions studied were also significantly associated with self-reported limitations. For example, poor performance on the 8-foot walk test, a test of gait, was associated with worse self-reported functioning in rising from a chair and getting in or out of bed, which are measures of changes in body position. These associations demonstrate performance tests were not exclusive correlates of their specifically paired self-reported limitations, and that tests of other lower extremity functions (but not of upper extremity functions) also influence self-reports.In contrast to many previous studies, we used highly specific pairings to provide a more valid test of the relationship between physical performance tests and self-reported physical function, thereby minimizing problems of compensability and the risk of confounding across different domains of physical functioning. We also tested a broad set of personal and health characteristics as correlates. Moreover, the population-based national sample increases the generalizability of our results.Our study also has some limitations. Because we wanted to examine self-reported functions for which there were corresponding physical performance tests, we were able to examine only two physical performance tests. Although we do not know if our results apply to other physical performance tests, the consistency of results suggests that the findings may be relevant for other physical functions. We did not have data on depressive symptoms, personality measures, fatigue, and cognitive functioning, each of which can affect physical functioning . HoweverOur results support the validity of self-reported physical function by finding associations between self-reports and measured performance on similar tasks. More important, however, were our findings that these associations were neither specific nor exclusive. Personal and health characteristics of respondents also influenced self-reported physical function. Our findings caution against a narrow or strict interpretation of self-reported limitations in individual tasks. Self-reported limitations represent a gestalt rather than an appraisal isolated from its context. Self-reports capture information beyond task difficulty.The authors declare that they have no competing interests.GL and MW conceived of the study, designed the study, performed the statistical analysis, interpreted the results, and drafted the manuscript. All authors read and approved the final manuscript.


Retiform hemangioendothelioma (RH) is an infrequently encountered vascular neoplasm of intermediate or borderline malignancy. Treatment of RH is controversial. We present a case of a 44-year-old Asian male presenting with an unresectable RH of the pelvis. The patient was treated with concurrent low-dose Cisplatin and External beam Radiation (4140cGy in 180cGy per fraction). This is the first report of a clinical complete response and a long-term local control of this rare tumor. This has significant clinical implication, since it gives the first evidence of treatment of this rare tumor using concurrent low-dose chemotherapy and radiation. Retiform hemangioendothelioma (RH) is an infrequently encountered vascular neoplasm of intermediate or borderline malignancy. Treatment of RH is controversial though most reports have described surgical resection followed by adjuvant radiation (RT) with or without chemotherapy. We describe a case report of a 44-year-old Asian male presenting with an unresectable RH of the pelvis and successful treated with concurrent chemotherapy and radiation.2) was given concomitantly with radiation. The patient tolerated the treatment well and had an excellent clinical response  is an infrequently encountered vascular neoplasm of intermediate or borderline malignancy that has been classified as a distinct type of low-grade cutaneous angiosarcoma. Up to date, a total of 25 cases were described in the literature –11. FirsMorphologically, RH presents as exophytic mass or nodule or a plaque, dermal or subcutaneous, that grows slowly. Most tumors present in young to middle-aged adults Range 9–78 years) . Most le8 years .Ulex earopaeus agglutinin (UEA) (7). The spindle cells are positive for UEA-1 and CD 31 but not for FVIII-Rag and CD34 [Clinically, RH lesions could be located in the dermis (predominantly) with extension into the subcutaneous tissues or striated muscle as was also reported as in our case . A charaand CD34 . In summand CD34 .Other vascular tumors that morphologically can present similarly to RH and need to be differentiated from RH include Dabska's tumor, lymphoma, dermatofibrosarcoma protuberans, hemangioma, bacillary angiomatosis, cutaneous metastases, blue-rubber bleb nevus syndrome, Kaposi's sarcoma, targetoid hemosiderotic hemangioma, hobnail hemangioma, epitheloid hemangioendothelioma, and polymorphous hemangioendothelioma of lymph node , 2, 7, 9 Treatment of choice for RH has been surgical excision with histopathologically tumor-free margins. In the cases reported up to date, patients were treated initially by surgical excision. Approximately 50% of the cases recurred –13. Ampu


Investigators frequently quantify and evaluate the location and size of stroke lesions to help uncover cerebral anatomical correlates of deficits observed after first-ever stroke. However, it is common to discover silent infarcts such as lacunes in patients identified clinically as 'first-ever' stroke, and it is unclear if including these incidental findings may impact lesion-based investigations of brain-behaviour relationships. There is also debate concerning how to best define the boundaries of necrotic stroke lesions that blend in an ill-defined way into surrounding tissue, as it is unclear whether including this altered peri-necrotic tissue region may influence studies of brain-behaviour relationships. Therefore, for patients with clinically overt stroke, we examined whether including altered peri-necrotic tissue and incidental silent strokes influenced either lesion volume correlations with a measure of sensorimotor impairment or the anatomical localization of this impairment established using subtraction lesion analysis.Chronic stroke lesions of 41 patients were manually traced from digital T1-MRI to sequentially include the: necrotic lesion core, altered peri-necrotic tissue, silent lesions in the same hemisphere as the index lesion, and silent lesions in the opposite hemisphere. Lesion volumes for each region were examined for correlation with motor impairment scores, and subtraction analysis was used to highlight anatomical lesion loci associated with this deficit.p = 0.025). In a sub-analysis of patients with small subcortical index lesions, adding silent lesions in the opposite hemisphere to the volume of the index stroke strengthened the volume-impairment association.For subtraction lesion analysis, including peri-necrotic tissue resulted in a larger region of more frequent damage being seen in the basal ganglia. For correlational analysis, only the volume of the lesion core was significantly associated with motor impairment scores (r = -0.35, Including peri-necrotic tissue strengthened lesion localization analysis, but the influence of peri-necrotic tissue and incidental lesions on lesion volume correlations with motor impairment was negligible barring a small index lesion. Overall, the potential influence of incidental lesions and peri-necrotic tissue on brain-behaviour relationships may depend on the characteristics of the index stroke and on whether one is examining the relationship between lesion volume and impairment or lesion location and impairment. The size and anatomical location of stroke lesions is frequently evaluated to help advance our understanding of links between brain structure and human behaviour -4. AccorWhen manually tracing lesions, defining the lesion boundary is challenging for infarcts that exhibit a well-defined necrotic core with poorly-defined bordering zones of peri-necrotic damage  as demo. In chroBrain-behaviour studies may be further complicated when remote, asymptomatic stroke is noted in addition to the primary lesion of interest. A recent study of incidental MRI findings in two-thousand persons from the general population (mean age of 63.3 years) found silent brain infarcts in 7.2% of participants, and found that the prevalence of such infarcts increased with age . Also, tOverall, the extent to which computer-based lesion analyses of chronic stroke may be affected by the inclusion of remote strokes, lacunes or ill-defined peri-necrotic tissue is not known. We believe that how one defines the boundaries of the primary stroke and how one addresses secondary brain lesions will have an important impact on brain-behaviour relationships and lesion volume correlations with clinical measures after stroke. Therefore, using manual lesion tracing on 3D T1 MRI for a sample of chronic stroke patients with first-ever clinically overt stroke, the aim of the present study was to examine how subtraction lesion analysis and lesion volume correlations with a clinical measure were influenced by the inclusion of 1) ill-defined peri-necrotic tissue, 2) silent stroke lesions that were present in the same hemisphere as the clinically-overt stroke, and 3) silent lesions in the hemisphere opposite to the overt stroke. This investigation may help to guide neuroimaging analysts' decisions regarding the extent of stroke-related tissue damage to include in lesion analysis procedures for future lesion-based studies. Upper and lower extremity post-stroke impairment was chosen as our measure of interest, as limb hemiparesis is readily measureable and has been previously associated with injury in certain regions of the brain.A cross-sectional sample of consecutive participants in a research database who were identified clinically as first-ever hemispheric stroke, who presented with clinically-significant unilateral hemiparesis, and who underwent MRI scanning at 3 months post-stroke were analyzed in this study. Participants provided written informed consent to participate in research studies that were approved by the Research Ethics Board at Sunnybrook Health Sciences Centre. Each patient in our sample was drawn from the pool of participants from the Subacute Therapy with Amphetamine and Rehabilitation for Stroke (STARS) study . Given tThe National Institutes of Health Stroke Scale (NIHSS), which is a reliable and valid measurement tool , was useAnatomical imaging was performed at 3 months post-ictus on a dedicated research MRI scanner . High-resolution T1-weighted images were obtained using a standard three-dimensional fast spoiled gradient-echo anatomical imaging sequence .Manual image alignment, lesion tracing and lesion volume calculations were completed using ANALYZE 6.0 software. Tracing was performed with knowledge of patients' side of hemiparesis. Stroke lesions were visually identified, by a trained image analyst, as being hypointense compared to homologous contralateral tissue. The stroke lesions also did not exhibit mass effect, and MRI images were AC-PC aligned in order to facilitate contralateral tissue comparison. An experienced research neuroradiologist, who was blind to patient data and to the study's purpose, confirmed that the regions identified by the image analyst were in fact stroke-related lesions. The image analyst then traced the lesions from digital T1 images. Traced lesions were labelled as one of four regions:(a) Region 1: Necrotic lesion core (b) Region 2: Altered peri-necrotic tissue (c) Region 3: Lesions in the same hemisphere as the index stroke(d) Region 4: Stroke lesions in the other hemisphereExamples of each Region are shown in Figure p = 0.71).Spatial normalization to the Montreal Neurological Institute (MNI) brain template was performed with lesion cost-function masking  and 16 np-values listed are two-tailed.Statistical analyses were performed using SPSS version 15.0. Each variable was tested for normality using the Shapiro-Wilks test. Correlations between lesion volumes and Fugl-Meyer scores were determined using Spearman rank correlation coefficients. All A total of 41 patients were identified for analysis. Demographic data and stroke characteristics for included patients are available in Table 3, SD 30.7) Region 2 for 19.6% , Region 3 for 0.28%  and Region 4 for 3.06% . Lacunes in the same hemisphere as the index stroke were noted in 29% of patients. Three patients with severe hemiparesis exhibited incidental lacunes. Also, 49% of patients had a lesion in the hemisphere opposite to the index stroke (5 of these patients were in the severe motor impairment group), and 24% of our 41 patients had both a lacune (Region 3) and a lesion in the hemisphere opposite to the index stroke (Region 4) in addition to their index stroke (Region 1). Lacunes accounted for 35% of the strokes in the hemisphere opposite to the index stroke (Region 4), while the lesions in 5% of these patients were relatively large and accounted for more than half of the total lesion volume.The majority of patients exhibited relatively large lesions involving both cortical white matter and subcortical structures, and all but 3 patients exhibited some degree of tissue that was classified as Region 2. On average for the 41 patients, Region 1 accounted for 77.2% of total lesion volume (mean volume 15.9 cmp = 0.10). The association between motor impairment scores and the mean volume of lacunes did not reach statistical significance , nor did the association between motor impairment scores and the mean volume of lesions in the opposite hemisphere . As also indicated in Table p = 0.028), Regions 1+2+3 , and Regions 1+2+3+4 .As indicated in Table 3), we performed a sub-analysis to examine if the observations from our whole-group analyses were also seen in a subset of patients with small index lesions. Patients with 'small' lesions were defined as those with a core lesion volume that fell within the lower quartile (0.12 - 0.92 cm3) of core lesion volumes from whole-group data (n = 41). From our sample, we analysed 9 patients with small subcortical lesions where the mean volumes of Region 1 and Region 2 were 0.40 cm3 and 0.34 cm3 respectively. In other words, the mean total volume of the index stroke in this subset was ≤ 0.8 cm3. Of these 9 patients, 3 exhibited lacunar infarcts. The mean volumes of Region 3 and Region 4 were 0.038 cm3 and 0.92 cm3 respectively (see Table p = 0.05); Region 2, r = -0.22 (p = 0.3); Region 3, r = -0.64 (p = 0.03); and Region 4, r = -0.25 (p = 0.3). The association between FM scores and the cumulative lesion volumes in this group was: Region 1: r = -0.58, p = 0.05; Regions 1+2: r = -0.55, p = 0.06; Regions 1+2+3: r = -0.55, p = 0.06; and Regions 1+2+3+4: r = -0.62, p = 0.04.Since the average volume of the lesion core was relatively large in this sample 15.9 cm, we perfAs seen in Figure There is a need to clarify the influence of lesion boundary definitions and of the inclusion of incidental infarcts on lesion-based brain-behaviour studies. In general, stroke neuroimaging analysts lack standardized guidelines for how to address incidental infarcts and define lesion borders when manually tracing lesions . While iOur results showed that the volume of the necrotic lesion core (Region 1) was most closely associated with motor impairment scores. Although the volume of the peri-necrotic region showed a weak trend toward significant association with motor impairment scores, analysis of cumulative lesion volumes showed that the addition of the peri-necrotic tissue to the volume of the necrotic lesion core neither strengthened nor weakened the association between lesion volume and motor impairment scores for whole group or subgroup (n = 9) analyses. With regard to our subtraction lesion analysis, previous research suggests that the posterior putamen is involved in motor control -27. InclIn general, these results suggest that the correlation between lesion volume and post-stroke impairment may not be strongly influenced by the volume of the peri-necrotic region. However, it may be the location more so than the volume of the peri-necrotic tissue that is important in behavioural-anatomical relationships, since accounting for the volume of this region had an equivocal influence on lesion volume correlations with motor impairment, while accounting for peri-necrotic tissue notably strengthened the anatomical localization of motor deficits in our subtraction analysis. This finding regarding volume-impairment correlations may have been due to the fact that the average volume of the necrotic lesion core was 4 times larger than the volume of the peri-necrotic region, which could have dampened the influence of the peri-necrotic region on correlational analyses; however, including the peri-necrotic region may have impacted our lesion subtraction analysis because increasing the size of lesion tracings may increase the probability that overlap will be observed for lesions that fall within close proximity to one another.A recommendation that peri-necrotic tissue be included in lesion tracings for lesion-based studies that involve procedures such as subtraction analysis is supported by previous literature, which suggests that this region may in fact remain dysfunctional in chronic stroke patients. For instance, in a functional MRI (fMRI) study of the activation observed in the cortical peri-infarct region during finger tapping, Cramer and colleagues  noted thThe inclusion of pre-existing lacunes in the same hemisphere as the index stroke did not notably influence our subtraction lesion analysis - most likely because of their small volume - and the volume of lacunes was not significantly correlated to motor impairment scores, either alone or when added to cumulative lesion volumes. However, this latter finding may stem from the observations that only 29% of patients in our full sample (n = 41) exhibited lacunes, few patients in our sample had small index lesion volumes, and the average volume of the lesion core (Region 1) was substantially larger than Region 3, which may have dampened the potential association between lacunar lesion volumes and clinical measures. Previous investigations suggest that lacunes, which result from the occlusion of a penetrating cerebral artery , can havLastly, the volume of lesions in the hemisphere opposite to that of the index stroke (Region 4) was not significantly associated with motor impairment scores for whole-group (n = 41) analysis and showed no appreciable overlap for lesion location on subtraction lesion analysis. This latter finding may be due, in part, to the relatively small size of these lesions in patients with severe hemiparesis in our sample. This suggestion highlights a shortcoming of the subtraction lesion analysis technique used in this study. Since patients are dichotomized into 'affected' and 'unaffected' groups for this analysis rather than being evaluated along a behavioural continuum, which is possible when using analysis techniques such as voxel-based lesion symptom mapping (VLSM) , data reIn view of the increasing number of studies that use computer-based tools for analyzing the size and location of lesions underlying post-stroke deficits in the many domains of brain function such as memory, learning, perception and sensorimotor function, it is important to better understand how what one defines as 'lesion' could influence the findings of such studies. Although exploratory, the results from this study suggest that the influence of peri-necrotic tissue and incidental lesions may depend on whether one is examining the relationship between lesion volume and impairment or the relationship between lesion location and impairment. The characteristics of the index stroke may also be an important determinant of the magnitude of influence associated with additional regions of stroke-related ischemic damage. It should be noted that our results can only be generalized to patients with hemiparesis; the behaviour or impairment under study could also be an important determining factor underlying the relationship between lesion characteristics and measures of behaviour, as other behaviours may be more heavily influenced by lacunes or contralateral lesions. A degree of caution is also important when ascribing pathological significance to areas of MRI signal abnormality, as the underlying etiology of these abnormalities cannot be determined for certain from MRI alone. Careful imaging-pathological comparisons are needed to understand this, and such studies have described gliosis and demyelination in the peri-infarct region. However, it is often not possible to infer whether any neural activity could still be supported by this perinfarct region. Accordingly, careful MRI analysis and quantification of the clinically relevant signs are essential when conducting lesion-based analyses of brain-behaviour relationships. Finally, for lesion-based studies involving computerized manual or automated lesion tracing, we also wish to emphasize the need for authors to explicitly describe lesion identification and analysis procedures  in future study methodologies in order to facilitate comparisons between investigations.The authors declare that they have no competing interests.LDA co-conceived of the research question and study design, performed data analysis, interpretation and statistical analysis, and was the primary manuscript author. SEB was involved in formulating the research question and study design, and assisted with data interpretation and manuscript authorship. FG was involved in neuroimaging analysis and the interpretation of neuroanatomical data. GS devised our lesion analysis procedures. CJD assisted in data collection, interpretation, and manuscript review. WEM was involved in formulating the research question and study design, data analysis and interpretation, and assisted with authoring the manuscript. All authors read and approved the final manuscript.


The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development.Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys  during their pubertal development, classified into five stages: prepuberty , early , mid , late-1  and late-2 . Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L.Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5–95 percentiles: <4 – 7) pmol/L) to early puberty (6 (<4 – 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 – 17) pmol/L) and late-1 (21 (12 – 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 – 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h.With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys. During the last 10–15 years, the role played by estrogens in male physiology has been highlighted. Apart from normal physiology processes involving the reproductive system, growth spurt, epiphyseal closure and bone mineralization , estrogeDetermination of serum estradiol concentrations in boys has been fraught with problems of analytical insensitivity, lack of specificity  and has In the present study, we present data on the 24-hours secretion of serum estradiol in healthy boys throughout puberty in relation to their testosterone secretory dynamics, determined by a validated ultra-sensitive extraction estradiol RIA with detection limit 4 pmol/L and functional sensitivity 6 pmol/L.The study group consisted of 44 healthy Swedish boys derived from a group described in a previous report . Twenty-Assent to inclusion in the study was obtained from each boy and informed consent from parents. The protocol was approved by the Ethics Committee of the Medical Faculty at the Sahlgrenska Academy at University of Gothenburg.A total of sixty-two 24-hour serum profiles were taken from the 44 boys. Serum estradiol concentrations in mid-puberty and late puberty have in part been reported previously for semi-longitudinally followed participants . The 44 During each visit to the clinic, height was measured by stadiometer, testicular volumes were determined by orchidometer  and bone® Estradiol RIA, Orion Diagnostica; Espoo, Finland). The extraction estradiol RIA has been meticulously validated for pediatric use [Serum 17β-estradiol concentrations were determined in duplicate by an assay which involves a diethyl ether extraction step prior to quantification by a modified commercial RIA  were stored at -20 C and determination of estradiol concentrations have been done yearly for seven years. It was found that the estradiol concentrations were not affected by long term storage or affected by repeated freeze/thaw cycles.th and 95th percentiles were performed on log-transformed data. A p-value of < 0.05 was considered significant.Non-parametric statistical methods were used for analyses. The Mann-Whitney test was used for comparison in estradiol concentrations between pre-, early and mid-pubertal stages and therefore boys followed with repeated estradiol measurements (n = 2) were excluded. The Wilcoxon signed rank sum test was used for analysis of diurnal variations and the progress of estradiol concentrations between mid, late-1 and late-2 pubertal stages as the progress was mainly based on serum from boys who had participated several times. In statistical analyses and figures, values of estradiol below the detection limits were set to the expected value of 2 pmol/L (the detection limit divided by 2). Mean estradiol levels were calculated as the mean value of six samples drawn over an interval of 24 hours. As the serum estradiol and testosterone levels were not normally distributed, linear regression analyses and 5Serum estradiol levels increased progressively throughout male puberty Figure . The incThe progress of serum estradiol, serum testosterone and serum DHEAS in relation to chronological age and bone age is presented in Figure 2 = 0.56, p < 0.001). When dividing the group into different pubertal stages, the strongest correlation was found for late pubertal boys , where we also found the steepest regression line (Figure Serum testosterone levels associated strongly with total serum estradiol levels (RThis is the most comprehensive study on the 24-hours estradiol levels and the diurnal variation of serum estradiol in healthy boys before puberty and during pubertal development. No diurnal rhythm was identified in pre- or early puberty, but it was discernable in mid-puberty and evident in late pubertal stages with peak values at 0600 to 1000 h and nadir at 2200 to 0200 h. Even if the diurnal rhythm of estradiol was not as marked as for testosterone in mid- and late pubertal boys , the intThe strengths of our estradiol assay is due to the fact that the RIA itself is sensitive as well as specific and that the extraction procedure removes interfering substances . FurtherIt is well established that direct immunoassays do not perform well at low estradiol concentrations, not even those with low analytical detection limit ,11,13. TThe source of estradiol biosynthesis in the growing boy is complex and not fully understood. In adult men however, the major source of estradiol biosynthesis is aromatization of androgens in peripheral tissues e.g. muscles and adipocytes by endocrine or intracrine pathways or by sulfated precursors being hydrolysed and converted into estrogens -31. OnlyWith the use of an ultra-sensitive extraction RIA, designed for measuring low estradiol concentrations in serum, we have established reliable and clinically useful data from a group of healthy prepubertal and pubertal boys. Serum concentrations of estradiol increased from pre- to early puberty but then remained relatively constant until the marked increase between mid and late-1 puberty, and thereafter a slower increase until late-2 puberty. The diurnal rhythm of serum estradiol in boys was not apparent until mid- to late puberty. The slow progress of estradiol from early puberty to mid-puberty despite a marked increase in testosterone levels is most likely due to a limited aromatization capacity in the growing boy.CV: coefficient of variation; DHEAS: dehydroepiandrosterone sulphate; MS: mass spectrometry; PHV: peak height velocity; RIA: radioimmunoassay; TW II: Tanner Whitehouse.The authors declare that they have no competing interests. The manuscript was partly funded by a grant by Pfizer.CAL participated in the design of the study, drafted the manuscript, carried out the radioimmunoassay and validation of the assay, performed analysis and interpretation of the data and the statistical analysis. EN conceived of the study, participated in its design, interpretation of the data and helped to draft the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Successful treatment of ocular tumours by chemotherapy and radiotherapy is sometimes limited by the special nature of the eye. Improvement is needed to avoid enucleation. Previous studies using locally administered antineoplastic agents have given promising results in the treatment of experimental ocular melanoma and spontaneous lymphoma. Another approach is use of radiosensitizers to improve radiotherapeutic effect. For the present study, 5-thio-D-glucose and cis-platin were chosen for evaluation in an ocular system because they exhibit antitumour activity, and interaction with radiation, particularly in hypoxia. Ocular absorption, toxicity, and pharmacokinetics after subconjunctival administration in rabbits were determined, and the effect of drug on tumours was measured using a Greene melanoma model. CHO cells were used for complimentary in vitro studies. 5-thio-D-glucose was readily absorbed into the eye (300 mg resulting in 5 mM in the aqueous) with no observable toxicity. When 5TG (300 mg) was administered at implantation, tumours were approximately half the size of controls. 5 mM 5TG is toxic to extremely hypoxic cells and gives measurable radiosensitization. Cis-platin levels as high as 0.68 microM were attained in the aqueous without local toxicity after 400 micrograms injection. This concentration causes toxicity in vitro. Cis-platin (400 mg) had a larger effect on tumour growth than 5TG given at, or one week after, implantation. Cis-platin may have potential for treatment of ocular tumours by local injection.


Side-alternating vibration training (SAVT) was used for 15 weeks in a patient with Late-onset Pompe disease who had never used enzyme replacement or chaperone therapy. Prior to the use of SAVT, the patient had experienced declining muscle performance and her 6-minute walk distance decreased from 210 to 155 metres in 6 months. After SAVT, her 6-minute walk distance increased 70% from 166 to 282 metres, muscle jumping power increased by 64% from 83 to 166 watts, isometric knee extensor strength increased 17% from 38 to 44 Nm, and she achieved a more normal pattern of ankle, knee, and joint kinematics and kinetics. Her functional ability measured through the Rotterdam 9-item score was unchanged at 19/36. There were no elevations in serum creatine kinase or lactate. This is the first report, to our knowledge, of a performance improvement in a patient with Pompe disease using SAVT. Late-Onset Pompe disease  can lead to progressive muscle weakness and loss of mobility . Many paGAA gene  showed a c.-32-13 T > G and a c.2481 + 102_2646 + 31 deletion of intron 1 and introns 17 to 18, respectively. Her T-cell alpha-glucosidase activity was <2.5 nmol/h/mg protein (controls 28.3–66.2) . She had never used enzyme replacement therapy or any other form of drug therapy targeted to acid alpha-glucosidase deficiency. She was ambulatory but walked slowly, and required a cane in the months prior to using SAVT, had difficulty ascending or descending roadside curbs, and was unable to stand from a sitting position without the use of aids. She reported chronic weakness and fatigue resulting in difficulty using stairs. Additionally, she noted an accelerated decrease in her physical abilities over the last two years with the need to reduce the number of hours she was working. She never required ventilatory support and had normal cardiac function.Our patient was a 34-year-old woman who was diagnosed at 30 years of age with late-onset Pompe disease. Sequencing of the Baseline investigations included the Rotterdam 9-item scale , 6-minutThe SAVT was generally well tolerated. The following symptoms were reported as a percentage of total vibration sessions: muscle discomfort  in the legs (49%), twitching while at rest (31%), cramping (2%), and fatigue (4%). Most often, these symptoms occurred the day following SAVT would be aggravated by longer days at work and caused minor discomfort but did not cause the patient to change her activity level. Other than the muscle twitching, she did not feel that these symptoms had worsened compared to her baseline and generally improved with massage therapy. There was no change in her weight, blood pressure, resting heart rate, serum creatine kinase, or 24 hour urine myoglobin . Venous After showing an initial decline in her 6-minute walk test (6 MWT) prior to commencing SAVT, the patient showed a 70% improvement in the distance traveled, from 166 to 282 metres after using SAVT . Peak loManual muscle testing (MMT) was completed by the same therapist at baseline and post-SAVT  using a During level walking at a self-selected speed, hip, knee, and ankle joint kinematics and kinetics improved dramatically between the pre- and post-SAVT intervention, with a pattern of greater improvement distally (ankle) than proximally (hip) . The ankThere was no change in pulmonary status or forced vital capacity (FVC) in the patient over the course of vibration training.Conventional methods of improving muscle performance use resistance and endurance training. Many patients with late-onset Pompe disease report decreased motivation for exercise because of muscle weakness, generalized fatigue, shortness of breath, and a higher level of effort required. To compound the underlying myopathy, the inactivity further deteriorates muscle function through deconditioning. Even in healthy subjects, deconditioning can lead to loss of muscle bulk and performance , 11. Vibration is a mechanical oscillation characterized by amplitude (mm) and frequency (Hz) which is applied indirectly to muscles through the joints while standing on a vibrating platform . The vibThe 6 MWT is a standard clinical measure of walking performance . The impThe results from our single case report show that SAVT can be tolerated by patients with late-onset Pompe disease and may improve muscle function and walking. We feel there is potential for using SAVT in patients with late-onset Pompe disease and impaired mobility. Whether there is a general benefit to patients and improved performance of activities of daily living will require clinical trials of longer duration involving more subjects.


Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes.Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions.First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest. For example, in early embryogenesis, transcription of the antisense genes Tsix and Air determines the fate of expression of their sense partners Xist and Igf2r, respectively [There is a growing body of evidence that natural antisense transcripts (NATs) play important regulatory roles in various biological processes. NATs are usually transcribed from the opposite strand of a particular gene locus, and they are thought to regulate sense gene expression ,2. One oectively ,4. The aectively -7. Moreoectively ,9.The other primary model of NAT-mediated gene regulation is induction of the production of small RNAs from NAT loci and their subsequent function in RNA interference (RNAi) pathways. Endogenous small interfering RNA (endo-siRNA) molecules, generated from NAT loci, are induced specifically under conditions of salt stress and immune response in plants -15. Recein silico studies are unknown. Computational identification of NATs is based mostly on the analysis of cDNA and EST sequence collections by sequence alignment, and this process has identified several thousand sense-antisense pairs [Although the number of NATs thought to have biological functions has gradually increased, the functions of most NATs discovered in recent large-scale se pairs . Howeverse pairs . This fii.e., known genes), referred to as artificial antisense sequence (AFAS) probes. This approach has the ability to detect antisense expression that cannot be identified by using information from the cDNA and EST collections and has the advantage of compatibility with the computational methodology widely used for sense gene expression analysis [To discover novel NATs expressed under various biological conditions, we proposed a microarray-based technique involving the use of 60-mer oligonucleotide DNA probes selected from the complementary sequences of cDNAs -induced mammary tumors. This technique is applicable to all genes and sample types and can be used for antisense expression identification that is not possible by using conventional cDNA information alone.Tsix, which reflects the abundance of Xist RNA, detected expression in the 11 adult mouse tissues , but not in the testis, as expected , which are known to give rise to antisense transcripts [Myc (myelocytomatosis oncogene) [Tgfb2 (transforming growth factor beta2) [P = 5.1e-12 by Welch's t-test). These data indicate that endogenous NAT expression of known genes is potentially detectable by probes designed for the complementary strand of known genes.To verify whether our methods can detect NAT expression, we initially examined the signal intensities of AFAS probes that targeted previously identified antisense transcripts. For example, AFAS probes designed for nscripts , also gancogene)  and Tgfbr beta2) , were alP < 2.2e-16, Fisher's exact test, see Additional file P < 2.2e-16, Fisher's exact test, see Additional file P < 2.2e-16, Median test).Before screening for novel NATs using AFAS probes, we first analyzed the global tendency of signal intensities from all AFAS probes applied to our custom microarray platform. Because Northern blot analyses for particular gene loci have previously shown that NATs tend to be poly(A)-negative , we checWT1, Sphk1, and Tsix [Several large-scale studies using information in cDNA and EST collections and from genome-wide tiling arrays in yeast previously showed that NATs tend to be transcribed from the 3' region of its counterpart mRNA -27, thusand Tsix -30.To test the ability of AFAS probes to detect novel NATs, we initially applied our microarray approach to the human-mouse orthologous gene set. In many studies, inter-species conservation of NATs is implied by the presence of common cDNA sequences between the two species ,31,32. HWe designed AFAS probes corresponding to 635 mouse orthologous partners, for which the antisense counterpart has been identified in humans, but not in mice (one sense and one antisense probe were designed per gene). We then profiled the expression of these genes to detect antisense expression within 12 normal mouse tissues. We identified 420 (66.1%) probes that gave a signal , at least in a single particular tissue, and 58 of these (9.2%) showed tissue-specific expression. Probes of 120 genes gave signals with a higher than average intensity according to inter-array normalization  by performing Northern and in situ hybridization (ISH) analyses. Whereas human ACAA1 (acetyl-Coenzyme A acyltransferase 1) overlaps with DLEC1  in a tail-to-tail overlapping manner, its orthologous counterpart in the mouse genome (Acaa1b and Dlec1) shows a tail-to-tail relationship but not a reciprocal overlapping relationship, according to the annotated gene structure + or total RNA (data not shown), but quantitative RT-PCR, ISH and microarray analyses were able to detect this transcript within the testis and kidney , which is a functionally uncharacterized gene but is known to be expressed within the adult testis and XY fetal gonad [AARD  overlap with that of an unnamed uterus EST (GenBank: AK093981), whereas mouse Aard has no EST arising from the antisense strand  were detected by ISH within a particular region of the seminiferous tubules . We found that Aard-AS was expressed within the early period of spermatogenesis, whereas the sense transcript appeared at a later phase -negative and nuclear-localized.We also analyzed the expression of al gonad . In humad Figure . Northerc Figure ; howeverc Figure . By comps Figure , thus com Figure . Becausee Figure . This fit Figure , indicatThese data clearly confirm that AFAS probes can detect the expression of antisense transcripts in normal tissues, and that they can also identify transcripts expressed in a tissue- and cell-type-specific manner. Detection of such expression dynamics for antisense transcripts is possible only by using the analytical platform targeting the complementary strand of the annotated genes. Thus, AFAS probes, when used within appropriate biological samples and combined with other analytical modalities, can be used to discover genuine functional NATs; this is an advantage over conventional approaches that depend on publicly available cDNA data.CDKN2B, WT1, and HBA2 [We next checked whether AFAS probes have the ability to detect antisense transcripts in cancerous tissues. Examples of functional antisense transcripts identified in abnormal cells are and HBA2 ,9,29. Thand HBA2 .P ≤ 0.05 by Student's t-test)  and its antisense counterpart [For the probes designed to detect the sense transcripts, we identified 57 genes showing differential expression. Among these, 48 were up-regulated and 9 were down-regulated within tumor regions, compared with in normal regions, according to a set statistical threshold  Figure . Among tThbd is shown as an example in Figure Drd4 is shown as an example in Additional file Thbd (thrombomodulin) , expression of Aard-AS was localized to the nucleus and was detected only in random-primed target samples. In addition, multiple-size hybridized bands pattern was observed especially for total RNA membrane, not for poly(A)+ RNA membrane. This observation is similar to that of previously identified antisense transcripts [Aard-AS transcripts. Because ISH and the microarray data on other antisense transcript examples also show nuclear localization and poly(A)-avoidance (data not shown), it is possible that these features are general characteristics of the antisense transcriptome.By designating AFAS probes to human-mouse orthologous genes, we identified many probes showing positive signals. Two of these probes identified transcripts for which DNA data ,31,32. Onscripts , and thiCDKN2B and CDKN2BAS [We also designed AFAS probes for well-characterized genes and identified several examples of correlated and anti-correlated expression between the NATs and the corresponding sense transcript within MMTV-induced mammary tumors. We observed differentially expressed genes for which expression of the antisense transcript had changed, whereas that of the sense transcript had not. Given that differential antisense expression might induce changes in epigenetic status, for example in CDKN2BAS , antisenAlthough next-generation high-throughput transcriptome sequencing (RNA-seq) might replace microarray-based expression analyses, antisense transcriptome analysis by sequencing is still under development because of the laborious nature of strand-specific library construction . DNA micWe showed here that probes targeting the complementary strand of the annotated genes successfully identify novel NAT expression, including those altered tissue- and tumor-specifically. The results suggest that there are more examples of NATs that cannot not be collected from public cDNA sources. Further functional investigation is required for such dynamically expressed NATs, and the use of microarray platforms targeting both strands of the gene locus will help to narrow down the proper candidates for further functional analyses.The AFAS probes for detecting NATs were designed to detect antisense transcription originating from genes categorized into three groups: (1) 48 genes in which antisense transcription has been previously reported and 87 imprinted genes in mice, (2) 404 selected well-annotated genes, (3) orthologous genes in NAT loci (detailed definition given below), and (4) randomly selected genes for which there were no cDNA, EST, and CAGE tags in the antisense orientation. For categories (1) and (2), the AFAS probes were designed to correspond to every 500 bases of the antisense strand of the exonic regions of each gene. For category (3), the AFAS probes were designed to correspond to a single specific sequence in each transcript. For category (4), two AFAS probes were designed per transcript. Target region selection for the probe design is summarized in Additional file Total RNA for the mouse (C57BL/6J) microarray experiments was isolated from NIH3T3 cells (fibroblast cell line), SL10 cells (fibroblast cell line), brain, heart, intestine, kidney, liver, lung, placenta (d.p.c. 10.5 and 13.5), spleen, stomach, testis, and thymus. Testis was from C57BL/6J males (8 to 10 weeks), placenta was from pregnant mice, and the other tissue was from both male and female mice. Nuclear and cytoplasmic fractionation of NIH3T3 cells was carried out according to the Protein and RNA Isolation System (PARIS) instructions (Ambion Inc.). For the microarray analysis of murine mammary tumors, RNA samples were collected from normal and cancerous mammary glands of dissected GRS/A mice .Numerical processed signal values  of the Agilent Feature Extraction File were obtained as representative expression levels for each probe within the array. If a spot had an intensity value lower than five, or if there was no prominent difference between foreground and background signals, then the intensity value was adjusted to five and the corresponding probe was treated as an "absent probe". To perform normalization of signal intensity distribution between multiple arrays, the whole mean signal of every hybridization experiment was adjusted to that of the data from SL10 cells by oligo-dT priming. Probes with intensity values lower than five, as well as being flagged as "saturated", were discarded for the inter-array-normalization step. Tissue-specificity of the expression signals was evaluated according to τ measurement . The rawin silico identification of sense-antisense pairs by the same procedures as previously published [To identify orthologous genes in NAT loci Figure , we initublished , by usinublished ,48, NCBIublished  and the ublished . This idublished ) was theublished . FinallyAcaa1b (NM_146230), Aard (NM_175503), and Thbd (NM_009378) were amplified by the PCR , and the NIH3T3 was isolated by using Trizol reagent (Invitrogen Corporation). Northern analyses were performed as previously described . Loadinghbd NM_00378 were Acaa1b (NM_146230), Aard (NM_175503), and Thbd (NM_009378) were amplified by the PCR . Probe sequence of negative control experiment was selected from Oryza sativa putative leaf protein (NM_197207)  from selected tissue RNA , then subjected to quantitative RT-PCR. Gene expression level was normalized with Gapdh. Primers are listed in Additional file cDNA was initially synthesized with gene-specific reverse primers , but not in the testis.Click here for fileAntisense expression of mouse imprinted genes. AFAS probes for several imprinted genes , which are known to give rise to antisense transcripts, gave prominent signals.Click here for fileNumbers of valid probes in adult mouse tissue profiling. A significantly higher number of AFAS probes than sense probes detected transcripts only within random-primed samples, but not among the oligo-dT primed targets.Click here for fileHighest signal intensities from expression profiling of the 12 normal adult tissues. Probes of 120 genes gave signals with a higher than average intensity according to inter-array normalization.Click here for fileAcaa1bExpression analyses of sense and antisense transcripts of . Quantitative RT-PCR, ISH and microarray analyses were able to detect this transcript within the testis and kidney.Click here for fileList of genes for which expression of the antisense transcript and sense transcript markedly changed in tumors. Forty-eight were up-regulated and nine were down-regulated within tumor regions, compared with in normal regions.Click here for filePdcd6 and Drd4Changes in expression of .Click here for fileSelection of target regions for microarray probe design. Target region selection for the probe design is summarized.Click here for filein situ hybridizationList of primers for PCR amplification of cDNA fragments to generate probes for Northern blot analysis and . Primers to amplify probes specific for sense and antisense of Acaa1b, Aard, and Thbd are listed.Click here for fileAard in situ hybridizationNegative control experiment of .Click here for filePrimers for real-time quantitative RT-PCR. Primers for real-time quantitative RT-PCR (Gapdh and Acaa1b-AS) are listed.Click here for file