Whether a stimulus excites predominantly Aδ or C fibers is a commonly asked question in basic pain research but a quick test was lacking so far.Aδ fiber mediated (punctate pressure produced by means of von Frey hairs) and C fiber mediated (blunt pressure) pain, applied to healthy volunteers in experiment 1. The sensation was assigned to Aδ fibers when “pricking” but neither “dull” nor “pressing” were chosen, and to C fibers when the sum of the selections of “dull” or “pressing” was greater than that of the selection of “pricking”. In experiment 2, with an independent cohort, the three-descriptor questionnaire achieved sensitivity and specificity above 0.95 for distinguishing fiber preferential non-mechanical induced pain .Of 77 verbal descriptors of pain sensations, “pricking”, “dull” and “pressing” distinguished best (95% cases correctly) between Aδ or via C fibers. It meets the criteria of a screening test by being easy to administer, taking little time, being comfortable in handling, and inexpensive while providing high specificity for relevant information.A three-item verbal rating test using the words “pricking”, “dull”, and “pressing” may provide sufficient information to characterize a pain sensation evoked by a physical stimulus as transmitted via A For example, central sensitization to sensory input from Aδ fibers likely explains the response observed in the secondary zone of hyperalgesia δ fiber input δ and non-myelinated C fibers respectively In most medical settings, pain has a high prevalence with 61% in emergency medical care δ fibers and pain transmitted via C fibers. The development of the test originated from the McGill pain questionnaire The differentiation of the nerve fiber systems transmitting these pain sensations is possible most sensitively by invasive methods such as microneurography. However, these sophisticated tests often cannot be carried out. A quick test providing fiber discrimination in experiments or at the patient bed to assess nerve fiber involvement in painful diseases is not available. Therefore, the objective of this study was to create a valid pain questionnaire that allows to discriminate quickly between pain transmitted via Aδ or C fiber mediated pain respectively In a single-blinded study design pain stimuli were applied by a single investigator on the arm or hand of the subjects, who were comfortably seated behind a black curtain shielding the subjects' view on the stimulus application. Subjects were informed about the purpose of the study. The questionnaire was developed from the 77 descriptors  of the validated German version of the McGill pain questionnaire δ and C fiber mediated pain were assessed with the the ratings of a new cohort of 20 healthy men and women  receiving noxious laser heat and electrical stimuli, known to excite preferentially AδIn a second study (experiment 2), descriptors identified to distinguish best between AThe subjects' actual health had been checked by medical questioning. Medications except contraceptives were prohibited for one week, and alcohol for 24 h before the assessments. The study was conducted following the Declaration of Helsinki on Biomedical Research Involving Human Subjects. The University of Frankfurt Medical Faculty Ethics Review Board approved the study protocol. Informed written consent was obtained from all subjects.δ and unmyelinated C fibers respectively 2). It was placed perpendicularly onto the dorsal side of mid-phalanx of the right middle finger. The pressure was increased manually by the operator at a rate of approximately 9 N/cm2 per second until the desired pressure was obtained.Neurophysiological and psychophysical studies in humans suggest that pain evoked by applying punctate and blunt stimuli is transmitted via small myelinated Aδ and C fibers δ fiber elicited first pain precedes the C fiber elicited second pain due to the different conduction velocities of the two afferents (∼10 m/s for Aδ and ∼1 m/s for C fibers), it can be easily distinguished when the stimulus is applied at a remote location such as the back of the hand δ component.Infrared Laser stimuli were administered at the back of the left hand using a thulium solid-state laser  at a wavelength of 1.96 µm. The stimuli were short (1 ms) pulses with a power of 150–600 mJ and a beam diameter of 5 mm. The high power of a laser stimulus produces a very fast heat ramp, which generally activates the terminals of both Aδ fibers Electrical stimuli excite smaller fibers at lower frequencies because the maximum firing frequency is lowest for these fibers c) to data obtained after administration of 10 mechanical stimuli at 10 different strengths  immediately before the actual rating task. Specifically, for each stimulus quality, a parameter set was obtained consisting of Stimuli were administered in duplicates at intervals of approximately 30 s. As different quantitative stimulus intensities may confound the ratings of different stimulus qualities, all pain stimuli were applied at an equal intensity rated of  by the respective subject. Two thirds (67/100) of maximum pain ensured that the stimuli were clearly painful and avoided that measurements were undertaken close to the maximum of the scale. The strength evoking this pain was calculated from the individual relationship between the pain intensity and the physical strength of the stimulus. This was established by fitting a power model  was calculated separately for each descriptor as (A · D)/(B · C), where the capital letters have the following meaning. A: number of selections of the descriptor for C fiber pain. B: total number of presentations of C fiber pain minus A. C: number of selection of the descriptor for Aδ fiber pain. D: total number of presentations of Aδ pain minus C. Descriptors of stimuli distinguishing between Aδ and C fiber mediated pain were identified by submitting the averages of the two verbal ratings of each mechanical stimulus to discriminant analysis. Variables were chosen stepwise for entry into the analysis by how much they lowered Wilk's lambda using F-statistics as the statistical criterion at an α level set at 0.05 . The goodness of the discriminant function was estimated by leave-one-out cross-validated classification. Subsequently, the positive and negative predictive values, PPV and NPV respectively δ and C fiber mediated pain sensations were assessed using the ratings of laser heat and 5-Hz electrical stimulation. This also served to calculate test sensitivity and specificity using standard equations The similarity in pain intensities across stimuli was assessed by means of analysis of variance. For each verbal descriptor of the pain stimuli, the odds ratio of the number of its selections for C fiber mediated pain  and the number of its selections for A2), laser heat (590.5±45.3 mJ) and electrical stimuli (3±0.78 mA) evoked pain at mean intensities of 64.2±15.8, 66.2±16.4, 54.2±18.7 and 68.8±16.4 mm VAS respectively (p>0.05).Due to technical problems, experiment 2 failed to deliver data in one subject and results are therefore reported from n = 19 subjects. Punctate pressure (2.2±1.9 log g von Frey hairs), blunt pressure  and C fiber mediated (blunt pressure) pain sensations   resultedδ mediated pain by pooling the ratings of punctate mechanical pressure and laser heat  were calculated for the number of selections of the descriptors for C fiber mediated pain by pooling the ratings of blunt mechanical pressure and 5-Hz electrical stimuli. The same was done for the descriptors for Aser heat .δ and “dull”, “drawing” and “pressing” indicating C fiber predominance  δ fiber stimulation, additionally activated the frontal operculum and anterior insular cortex, which was interpreted as C fibers being engaged in homeostatic and interoceptive functions in another manner than Aδ fibers. High-frequency stimulation induced potentiation of pain evoked by electrical stimuli was described mainly with “hot” and “burning” or mainly with “piercing” and “stinging” depending on whether the facilitation affected either the conditioned cutaneous C-fiber or Aδ fiber pathway, which agrees well with the present results. Further supporting evidence for the presented finding that fiber selectivity can be identified using verbal descriptors is the dissimilarity of the sensory descriptor choices for tonic and phasic experimentally induced pain Whether a stimulus excites predominantly Aδ fiber origin of this disease. It is noteworthy that many common questionnaires for the screening of neuropathic pain include “burning” as a descriptor for C-fiber involvement Another area, where the involvement of pain fiber types could not yet be fully clarified is visceral pain, where the exact mechanism of convergence of visceral and somatic afferents is still poorly understood δ fibers because the pin prick sensation often elicited on needle insertion δ fibers. The other words were either not suitable for discrimination  or not part of the McGill questionnaire .A special case are studies on the underlying mechanisms of acupuncture. Early experiments focused on AThe present test fulfills the criteria for a rapid screening test in a clinical or experimental setting. It is easily comprehensible, takes little time to administer, is comfortable in handling, is inexpensive, and is reliable as it rarely produces false positive results. Reliability is important as a less specific test would lead to many follow-up investigations with more extensive pain tests. This is difficult to handle when nerve conduction preferences are not the primary focus of the experiment but may prove important for interpreting and discussing its results.δ fibers by electrical stimulation with 5 Hz pulses.However, compared with more extensive non-invasive tests such as the McGill questionnaire δ or via C fibers. It meets the criteria of a screening test by being easy to administer, taking little time, being comfortable in handling, and inexpensive while providing high specificity for relevant information. Apart from these practical aspects, however, it has to be acknowledged that this short test is limited to its purpose of quick fiber distinction in a pain context while it cannot replace a comprehensive phenotyping. It may nevertheless provide a major improvement of the experimental design when information about fiber preference is frequently not gathered at all.Results from this study suggest that a simple three-item verbal rating test using the words “pricking”, “dull”, and “pressing” may provide sufficient information to characterize a pain sensation evoked by a physical stimulus as transmitted via A


Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by typical absence seizures manifested by transitory loss of awareness with 2.5–4 Hz spike-wave complexes on ictal EEG. A genetic component to the aetiology is well recognised but the mechanism of inheritance and the genes involved are yet to be fully established.Zmean = 3.9, p < 0.0001; HLOD = 3.3, α = 0.7). The linked region harbours the functional candidate genes TRAK1 and CACNA2D2. Fine-mapping using a tagSNP approach demonstrated disease association with variants in TRAK1.A genome wide single nucleotide polymorphism (SNP)-based high density linkage scan was carried out using 41 nuclear pedigrees with at least two affected members. Multipoint parametric and non-parametric linkage analyses were performed using MERLIN 1.1.1 and a susceptibility locus was identified on chromosome 3p23-p14 ( The absence epilepsies are a group of idiopathic generalised epilepsies (IGEs) which differ in their seizure frequency, age of onset and pattern of evolution. A typical absence seizure manifests as a transitory loss of awareness with 2.5–4 Hz spike-wave complexes on ictal EEG. Many patients also have generalised tonic–clonic seizures (GTCS), myoclonic seizures or febrile seizures in addition and a variety of ‘atypical’ absence seizures are recognised. The International League Against Epilepsy (ILAE) classification recognises a number of distinct absence epilepsy syndromes including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), epilepsy with myoclonic absences, juvenile myoclonic epilepsy (JME) and eyelid myoclonia with absences as a seizure type . HoweverTwin studies have demonstrated that the IGEs, including those in which absence seizures occur, have a significant heritability , with reECA1), 5q31.1 (ECA2) and 3q26 (ECA3) (CACNG3 (CACNA1A (chromosome 19p13.2-p13.1) and IGE including CAE (CACNA1H (chromosome 16p13.3) have been found in 14 sporadic Chinese Han patients with CAE but not in any of 230 unrelated controls  . We have : tottering  Cacna1a ; letharg, Cacnb4 ; stargaz, CACNG2 ; ducky dCacna2d2 . The du This body of evidence indicates that the majority of idiopathic human and animal epilepsies are channelopathies, with implicated genes likely to be either ion channel genes or genes for related molecules . A SNP-based approach provides increased genomic coverage and overall provided a greater information content in comparison to microsatellites . A “modeTRAK1 and CACNA2D2. Evidence for disease association was shown for variants in TRAK1 and a putative causal variant has been investigated.We report here the identification of a susceptibility locus for CAE on chromosome 3 and further analysis of two positional and functional candidate genes, 63 nuclear pedigrees  and 296 trios (affected child and both parents) were ascertained from populations with European ancestry including the UK, France, Germany, Austria, Greece, The Netherlands, Denmark, Sweden, Finland, Italy and North America. Appropriate informed consent was obtained from all participants. Full protocol and consent form approval was obtained from local research ethics committees and/or participating institutions as appropriate.Details of the clinical data on subjects categorised as affected have been published previously . Inclusi® kit.Genomic DNA was extracted from cheek swab, saliva or blood samples according to standard kits and protocols (Oragene™ and Simhelix DNA Isolation kits). 237 of the 304 samples used in the genome-wide screen were whole genome amplified (WGA) as required using the REPLI-gControl samples were obtained from the European Collection of Cell Cultures (ECACC) human random control panels 1, 2 and 3 . The ECACC HRC panels include DNA from blood donors who are all UK Caucasians and are characterised by gender and age at venesection. All donors have given written informed consent for their blood to be used for research purposes. Conformation to Hardy–Weinberg Equilibrium (HWE) was verified in all SNPs typed in controls.Data for linkage analysis was obtained from a successful genome-wide screen in 240 samples from 41 of the original nuclear pedigrees  and University of California-Santa Cruz (UCSC) Browsers (genome build NCBI36.1), in addition to the PubMed and Unigene databases. By searching through these databases, we identified the most probable functional candidate genes.http://www.polymorphicdna.com).All coding and UTR exons, as well as exon–intron junctions (sequencing 100 bp either side of each exon), in the best functional candidates, were re-sequenced in 48 affected individuals from the multiple case pedigrees, using one case from each pedigree. This re-sequencing work was performed by Polymorphic DNA Technologies Inc., using standard Sanger dideoxy sequencing protocols (r2 ≥ 0.8 between unassayed and assayed SNPs in the HapMap (release 21 CEU genotype data), identified the SNPs which would capture > 99% of all haplotypes. These tagSNPs were typed in the entire resource of nuclear pedigrees and trios and the genotypes were used to construct LD blocks with Haploview 3.2 .Association analysis of the tagSNPs allowed the identification of gene regions for re-sequencing which would not have been re-sequenced initially, e.g. intronic areas. The limits for re-sequencing were determined by the LD structure of the tagSNPs, i.e. re-sequencing was performed around any associated SNP as far as the first SNPs either side to which it was not in LD. This re-sequencing was performed in the same 48 cases as exonic re-sequencing was performed in, by Polymorphic DNA Technologies.in silico methods, such as VisualSNP and FastSNP . The critical region is arbitrarily defined as extending to where the Z score falls below 3; a region of approximately 20 cM  of 3.3 (α = 0.7) occurring at 3p21  is an idiopathic generalised epilepsy (IGE) with a well defined and homogeneous phenotype that displays a clear electrophysiological hallmark and clear evidence of a genetic aetiology. The clinical criteria adopted here provide the reasonable expectation that the patients ascertained represent a homogenous clinical phenotype. It is has been shown that IGE phenotypes may cluster in families with a proband with absence epilepsy, but analysis reveals an increased clustering of CAE and JAE  suggestiZmean = 3.9, p < 0.0001; HLOD = 3.3, α = 0.7). This is the only region of the genome for which there is a significant linkage score in our analysis. The linkage peaks on chromosomes 9 and 17 which are close to being statistically suggestive of linkage might warrant some attention in future in context of the genetically complex architecture of IGE syndromes.We have identified a novel susceptibility locus for CAE/JAE on chromosome 3p23-p14 through a SNP-based genome-wide linkage scan  are consistent with linkage to chromosome 3p23-p14. We have previously demonstrated evidence for linkage to 16p12-p13.1, a region containing the calcium channel gene families . CAE is gle gene . This isTRAK1  and CACNA2D2  as the most plausible functional candidate genes. Both genes were investigated with tagSNPs but only variants in TRAK1 demonstrated significant evidence for transmission disequilibrium. This does not however definitively exclude CACNA2D2 as tagSNPs will only detect disease-associated alleles present at a comparable frequency. It is possible that rare variants present in a subset of the study population will not be detected using this methodology.Data-mining of this linked region identified TRAK1 encodes a trafficking protein originally known as OIP106 that was initially identified as a binding partner for the enzyme β-O-linked N-acetylglucosamine (O-GlcNAc) transferase (n-butyric acid A (GABAA) receptors observed in Trak1 mutant mice suggested that Trak1 may also participate in the regulation of GABAA receptor endosomal trafficking. The protein has been shown to interact with the GABAA receptor α1 subunit and it has been hypothesised that Trak1 has a crucial role in regulating the endocytic trafficking of these receptors and may facilitate the targeting of endocytosed GABAA receptors back to the cell surface or block them from degradation (A receptors (GABAARs) on target neurones either in a phasic or a tonic fashion , it would simultaneously have increased the risk of type II errors (missing any true associations). Thus the association evidence described must be considered as tentative but requiring independent replication.CXCR3 is associated with gene expression levels and asthma risk (A second explanation is that the associated tagSNPs may be in long-range LD with causal variants not identified by the re-sequencing effort . Finallyhma risk .TRAK1. Definitive evidence to confirm or exclude this gene will require re-sequencing across an extended genomic region encompassing TRAK1 in a larger number of patients and a better understanding of the role that non-exonic SNPs play in regulation. We have identified a large number of novel intronic and 3′ UTR SNPs, the possible function of which it is currently extremely difficult to predict. The latter, in particular, may be located within target sites for miRNA molecules, but information is lacking in this area and it is not something which we have assessed in detail. Replication studies in similar resources of CAE patients are required, and future studies into the biological mechanisms of CAE could suggest other candidate genes in the linked region which would need to be re-sequenced to identify putative causal variants.In conclusion, these observations provide genetic evidence of a novel susceptibility locus for childhood absence epilepsy on chromosome 3p23-p14. Common variants showing transmission disequilibrium have been identified in the candidate gene


Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated.3H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls).Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism. Sudden infant death syndrome (SIDS) is poorly understood and unpredictable. By definition, it refers to death in an infant less than 1 year of age when rigorous postmortem analysis fails to reveal an apparent cause. It remains the leading cause of death among infants aged between 1 month and 1 year To date, no biologic abnormality of the peripheral vago-cardiac system has been demonstrated n = 5), infection (n = 7), dehydration (n = 3), metabolic disease (n = 1), shaken baby syndrome (n = 2) and sub-dural hematoma (n = 1). Groups were matched for gender  and age . Autopsies were carried out in the Service d'Anatomie Pathologique et Médecine Légale, Hôpital Raymond Poincaré . All autopsies, cardiac tissue examinations and tissue collections were performed by the same pathologist (CR) betweeen 1997 and 2007. Procedures were conducted in accordance with the French law  and the recommendations of the Haute Autorité de Santé on SIDS handling. Informed parental consent for post-mortem examination and tissue collection for research purposes was obtained in all cases. All samples were analysed following a blinded procedure.Altogether 18 SIDS and 19 control infants were included in this study. Left ventricular samples (50–100 mg)  and blood samples  were obtained from children who died when aged between 1 and 9 months of age. SIDS was diagnosed according to the American Academy of Pediatrics definition: infant death that remains unexplained after a thorough case investigation, including performance of a complete autopsy, examination of the death scene, and review of the clinical history max; fmol mg−1 protein) and the affinity constant . Protein content was measured according to the method of Bradford Methods were adapted from Gies et al. 3H]NMS; 81.0 Ci mmol−1) added in 12 concentrations ranging from 40 to 2000 pmol L−1. Non-specific binding was determined in the presence of 1 µmol L−1 atropine.The density of total muscarinic receptors was assessed using the tritiated non-selective muscarinic receptor antagonist N-methylscopolamine . Unpaired −1 protein and AchE activity ranged from 2.9 to 5.7 units mL−1 erythrocytes. In the control group, both parameters were homogenous whatever the cause of death. The total muscarinic receptor density in the heart of children who died from SIDS was more than double that found in samples from children who died from other causes . Average erythrocyte AchE activity was also significantly increased in SIDS victims . In the latter (control group), cardiac muscarinic receptor density ranged from 46.1 to 100.5 fmol mgmax individual values obtained from SIDS hearts compared with controls (1/9), while there was a quite large overlap for AchE activity (4/9). However, since cardiac and blood samples were obtained from different sets of SIDS, cardiac muscarinic receptor density could not be correlated with AchE activity. In SIDS victims, the compensatory mechanism could be not efficient enough to restore cardiac autonomic balance and prevent from death.In this study, we were seeking possible abnormalities in the intra-cardiac part of the vagal system associated with SIDS. To date, cholinergic abnormalities have been reported only in the central nervous system of SIDS victims: reduced muscarinic binding has been shown in the arcuate nucleus In conclusion, we showed an overexpression of muscarinic receptors in cardiac tissues from SIDS. It remains to be demonstrated whether or not increased number of binding sites reflects functional differences and whether or not such an abnormality associated with vagal excessive activity might contribute to SIDS. In addition, AchE activity level was above control level in SIDS. Cardiac muscarinic receptor overexpression may represent the biological vulnerability to SIDS evoked by Malloy


Phocoena phocoena) in European waters. Combining genetic inferences with palaeo-oceanographic and historical records provides strong evidence that populations of harbour porpoises have responded markedly to the recent climate-driven reorganization in the eastern North Atlantic food web. This response includes the isolation of porpoises in Iberian waters from those further north only approximately 300 years ago with a predominant northward migration, contemporaneous with the warming trend underway since the ‘Little Ice Age’ period and with the ongoing retreat of cold-water fishes from the Bay of Biscay. The extinction or exodus of harbour porpoises from the Mediterranean Sea (leaving an isolated relict population in the Black Sea) has lacked a coherent explanation. The present results suggest that the fragmentation of harbour distribution range in the Mediterranean Sea was triggered during the warm ‘Mid-Holocene Optimum’ period (approx. 5000 years ago), by the end of the post-glacial nutrient-rich ‘Sapropel’ conditions that prevailed before that time.Recent climate change has triggered profound reorganization in northeast Atlantic ecosystems, with substantial impact on the distribution of marine assemblages from plankton to fishes. However, assessing the repercussions on apex marine predators remains a challenging issue, especially for pelagic species. In this study, we use Bayesian coalescent modelling of microsatellite variation to track the population demographic history of one of the smallest temperate cetaceans, the harbour porpoise ( Populations can respond to changing habitats by adapting , moving , by adjusting population size or some combination of the above. Both natural selection and genetic drift can shape populations as they evolve in this context.et al. In the North Atlantic, studies have increasingly reported strong shifts in plankton and fish assemblages with the contemporaneous climate and ocean warming  waters . There hIn order to investigate the splitting process among the three porpoise populations identified in the Northeast Atlantic, we extracted historical information from genetic polymorphism data using model-based Bayesian coalescent approaches, and correlated this genetic inference with palaeo-oceanographic and historical records. Specifically, we tested whether the genetic divergence observed among harbour porpoise populations in the Northeast Atlantic is compatible with the recent changes reported in the seascape and marine assemblages, or is more likely owing to ancient vicariance processes.2.(a)Individual genotypes at 10 microsatellite loci were from (b)μ: the time since population-splitting (T = μt ), measures of neutral population genetic diversity of the two current and one ancestral population  proportional to their effective population sizes Ne (θ = 4Ne μ) and bidirectional migration rates   model of population divergence . The mod = m2/μ) .We analysed population-splitting first comparing the IB with the NBB population. Then, we compared the two ESUs, i.e. harbour porpoises from the Black Sea (BS) with those in the Atlantic . This procedure was adopted because previous works suggested that the Black Sea harbour porpoises separated from Atlantic populations long before IB porpoises separated from the NBB populations. This is supported by complete lineage sorting of mtDNA haplotypes between porpoises from the Black Sea and the North Atlantic . On the We conducted Markov Chain Monte Carlo (MCMC) simulations using the IM program , assumin7 iterations, see the electronic supplementary material, table S1, for details regarding runs). Mixing properties of the MCMC were assessed by visual inspection of the parameter trend plots and by examining the level of autocorrelation between initial and final parameter values . Analyses were considered to have converged upon the stationary distribution if independent runs generated similar posterior distributions, with each having a lowest ESS of 50 for each estimated parameter as recommended in Convergence of such a kind of analysis and dataset is a tricky issue. To ensure reliable convergence towards the stationary distribution, we monitored multiple independent runs for the two datasets, each with 50–100 independent heated chains under Metropolis coupling to improve mixing . Each ruμ (i.e. T and θ) into demographic units (i.e. t and Ne), we used a value of μ = 5.0 × 10−4 mutation per generation, which is considered as the average mutation rate over many species (G) for harbour porpoise ranging between 5   can be converted to calendar years (t) and estimates of population mutation rates  can be converted into estimates of effective population size parameters  as described in M1 and M2) can be used to obtain population migration rates  using the MLE estimates of current θ and of M (2N1m1 = θ1M1/2 and 2N2m2= θ2M2/2)   and 7 ye θ2M2/2) .3.(a)After several steps of trimming, the replicated IM simulations displayed good mixing properties of the coupled MCMCs as suggested by the parameter trend line plots (data not shown) with an ESS of at least 71 for the IB–NBB analyses and 53 for AT–BS analyses . IM replicates provided consistent similar posterior probability densities for all parameters . All these indications suggested that we have sufficiently explored the parameter space. We selected runs that best converged (i.e. those displaying the highest ESS estimates) to convert them into demographic units  and to i(b)a–c and summarized in figure 2b and NA) of ca 20 000 individuals (HPDI: 13 313–30 113), split into two smaller populations: a very small population off the Iberian Atlantic coasts with an effective size of ca 80 individuals (HPDI: 22–328), and a larger population in the northern Bay of Biscay with an effective size of ca 350 individuals (HPDI: 148–2243) (figure 2a) and a MLE between 175 and 245 yr BP (HPDI: 55–2079), depending on the generation time used (5 or 7 years). Migration rate distributions (figure 2c) show a clear signal of migration in both directions with, however, 14 times higher gene flow from Iberia towards the northern Bay of Biscay after population-splitting (Results from the IM model for the isolation of IB porpoises from those of the NBB are shown in figure 248–2243) . The splplitting .Clupea harengus) as far south as in the Bay of Biscay and the southward retreat of warm-water species (e.g. sardine) during the LIA (Ammoditidae sp.), are a major component of porpoise diet throughout its current range d–f and summarized in NA) of ca 30 000 individuals (HPDI: 16 875–45 225). According to the model, this ancestral population split into much smaller populations (ca 360 (HPDI: 148–573) individuals in the Black Sea and ca 1400 (HPDI: 543–2453) individuals in the Atlantic). The population split is estimated to have occurred 1325–1855 years ago , depending on the generation time used. Migration rate posterior distributions (figure 2f) show a clear signal of migration, though very low, from the Atlantic to the Black Sea, but no evidence of gene flow in the reverse direction (figure 2f).Results from the IM model for the isolation of Black Sea porpoises from those in the Atlantic are shown in figures 2−8 substitutions per site and per year (s.s.yr−1) (−4 mutations per year (considering a generation time of 5 years), the mtDNA CR substitution rate can be estimated from our combined analysis at 1.3 × 10−6 s.s.yr−1 (HPDI: 8.4 × 10−7–2.3 × 10−6). This is at least two orders of magnitude faster than ‘traditional’ substitution rate estimates derived from interspecific phylogenetic datasets or the fossil record (−7 s.s.yr−1) (Pygoscelis adeliae (9.6 × 10−7 s.s.yr−1) (Mirounga leonina 9.8 × 10−7 s.s.yr−1 HPDI: 1.7 × 10−9–2.1 × 10−6)  . Provideθ) from mtDNA CR alone. mtDNA θ estimates showed a diametrically opposed pattern to that of nuclear microsatellite loci, with estimates of ancestral population genetic diversity being much smaller than the current population diversity , which necessarily simplifies the population demographic history, and on a specific mutation rate. Departures from the model, such as departure from panmixia within population or change in the effective size of diverging populations, may induce some bias in our estimates. However, the model appeared quite robust to these departures . The bioet al. It seems inevitable that marine predators will need to adapt to a changing spatial distribution of primary and secondary production within pelagic marine ecosystems as they did in the past . This re


At the molecular level, PPARα regulates the transcription of a number of genes critical for lipid and lipoprotein metabolism. PPARα activators are further shown to reduce body weight gain and adiposity, at least in part, due to the increase of hepatic fatty acid oxidation and the decrease in levels of circulating triglycerides responsible for adipose cell hypertrophy and hyperplasia. However, these effects of the PPARα ligand fenofibrate on obesity are regulated with sexual dimorphism and seem to be influenced by the presence of functioning ovaries, suggesting the involvement of ovarian steroids in the control of obesity by PPARα. In female ovariectomized mice, 17β-estradiol inhibits the actions of fenofibrate on obesity through its suppressive effects on the expression of PPARα target genes, and these processes may be mediated by inhibiting the coactivator recruitment of PPARα. Thus, it is likely that PPARα functions on obesity may be enhanced in estrogen-deficient states.Peroxisome proliferator-activated receptor  Obesity is the result of an energy imbalance caused by an increased ratio of caloric intake to energy expenditure. In conjunction with obesity, related metabolic disorders such as dyslipidemia, atherosclerosis, and type 2 diabetes have become global health problems. The peroxisome proliferator-activated receptors (PPARs) have been the subject of intense investigation and considerable pharmacological research due to the fact that they are involved in the improvement of these chronic diseases. Three PPAR isotypes have been identified: PPARα is expressed predominantly in tissues that have a high level of fatty acid (FA) catabolism such as liver, heart, and muscle .–31.α andα and ERs functions is similar . Bo. Bo23]. α although these results were obtained using male animal models [Fenofibrate reportably acts as a weight-stabilizer through PPARl models –11, 22. Fenofibrate seems to differentially affect body weight and adiposity among OVX and Sham mice by a mechanism other than the modulation of leptin gene expression. Although leptin is produced only in adipose tissue and elicits satiety responses by binding to leptin receptors in the brain , 90, chaα target gene expression seem to explain the different effects of fenofibrate on gonad-dependent weight gain in females  whereas a PPRE is a direct repeat with one or two intervening sequences . Nonetheless, these sequences contain an AGGTCA half site, which could be recognized by either ERs or PPARα. Signal cross-talk between PPAR/RXR and ERs has been reported to occur through competitive binding to ERE [α transactivation by ERs may also have been due to their competition for PPRE. PPARor PPARα , 101. Ang to ERE . Therefoα on obesity through its effects on hepatic PPARα -dependent regulation of target genes and that these processes are mediated by inhibition of PPARα recruitment of coactivators by E2-activated ERs (α ligands fibrates may act as efficient weight controllers under estrogen-free conditions. Although E2 alone decreases body weight gain and WAT mass, E2 may impair PPARα actions on obesity. Thus, these results provide a rationale for the use of fenofibrate in men and postmenopausal women with obesity and lipid disorder, but not for premenopausal women with functioning ovaries. In conclusion, in vivo and in vitro studies demonstrate that E2 inhibits the actions of PPARated ERs . PPARα lα has been the subject of intense academic and pharmaceutical research because of its ability to improve obesity-related metabolic disorders. The PPARα ligand fenofibrate seems to exhibit an antiobesity effect through FA β-oxidation in animal models although such an effect of PPARα activators has not yet been reported in humans. However, this idea is supported by several human studies showing that obese patients with impaired fat oxidation failed to lose weight, suggesting that elevated fat oxidation leads to weight loss. Interestingly, there is a sex difference in the control of obesity by fenofibrate. Fenofibrate regulates body weight and adiposity with sexual dimorphism in nutritionally induced obese male mice. Moreover, fenofibrate-induced reductions in body weight gain and WAT mass in male mice were also shown by female OVX mice, but these effects were absent in female Sham mice, suggesting the involvement of ovarian hormones in the differential regulation of obesity among these groups. In OVX mice, E2 inhibited the actions of fenofibrate-activated PPARα on obesity, due in part to reductions in hepatic expression of PPARα-mediated FA β-oxidizing enzymes by E2, a process mediated through the inhibition of PPARα coactivator recruitment by E2. These results provide a mechanism to explain why fenofibrate reduces body weight gain and adiposity in males and OVX female mice but does not regulate obesity in female mice with functioning ovaries.Obesity is the leading cause of the metabolic diseases including type 2 diabetes, atherosclerosis, and hypertension. PPAR


Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity  and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study.The outer layer of the  A crucial feature of this microorganism is its ability to survive in the host and to infect several anatomically distinct sites. This fungus has elaborated numerous mechanisms to adapt to a diversity of niches  and resides in a healthy host without causing overt disease. As an opportunistic pathogen, C. albicans can invade immunocompromised hosts and cause life threatening diseases that may arise as hematogenously disseminated infections or as localized primary diseases of deep organs -anchored proteins, including an N-terminal signal sequence and a C-terminus containing a GPI anchor attachment site (ω-site). GPI anchored proteins (GpiPs) are linked to the plasma membrane via a preformed GPI anchor that is added to the protein in the endoplasmic reticulum by a transamidase enzyme complex  , a Ura+, Arg+, His− derivative of BWP17  . In all of BWP17 , unless of BWP17  and lackthem see . For thethem see  was used2.2−1 (Amp) and 20 μg kanamycin mL−1 (Kan) as required. C. albicans strains were grown in YPD plus uridine  at 30 °C. Following transformation, clones were selected on synthetic medium .DH5α was grown at 37 °C in Luria–Bertani medium supplemented with 100 μg ampicillin mL2.3C. albicans sequences were obtained from the Candida DataBase and the Candida Genome Database web sites (http://genolist.pasteur.fr/CandidaDB/ and http://www.candidagenome.org/). The open reading frame (ORF) of all genes to be disrupted was amplified by PCR and ligated into the vector pGEM-T  to generate the target plasmid for subsequent in vitro transposition using the GPS-M mutagenesis kit . In order to map the integration site of the Tn7-UAU1 in the target plasmid, we digested the mutagenized plasmid with NotI and PmeI. One target plasmid, which had integrated the Tn7-UAU1 transposon near the middle of the ORF was selected for each gene . Plates were monitored over 2 days for altered growth.The phenotypes of the 45 GpiP mutants were monitored using drop tests. All media were supplemented with histidine. Strains were grown overnight in YPD at 30 °C and diluted with H2.6in vitro susceptibility of the mutants was tested with caspofungin. The Minimal Inhibitory Concentration (MIC) was determined using the microbroth dilution method M27-A2  and washed in 1 ml cold lysis buffer . Cells were collected by centrifugation  and resuspended in 250 μl cold lysis buffer. Cells were broken using a FastPrep machine in the presence of acid-washed glass beads (4 × 15 s bursts at speed 6.5 with 1 min on ice between bursts). The extracts were clarified by centrifugation . Protein concentration in the cleared lysate was estimated using the method described by Bradford with BSA as a standard  with NuPAGE®Novex Bis–Tris 4–12% pre-cast gels (Invitrogen) in NuPAGE® MOPS-SDS Running Buffer (Invitrogen) containing NuPAGE® Antioxidant (Invitrogen) as per the manufacturer’s instructions. Approximately 15 μg of protein was loaded in each lane. The proteins were transferred to Invitrolon™ PVDF Membranes (Invitrogen) in NuPAGE® Transfer Buffer containing methanol using the XCell II™ Blot Module (Invitrogen) following the manufacturer’s instructions.Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using the XCell Following transfer, the membranes were rinsed in PBS and blocked in PBS-T+10% BSA  BSA, 50 mM NaF) for 30 min at RT. The membranes were then incubated overnight at 4 °C in PBS-T+5% BSA  BSA, 50 mM NaF) containing a 1:1000 dilution of Phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody . The membranes were washed five times for 5 min in PBS−T  and then incubated for 1 h at RT in PBS−T+5% BSA containing a 1:2000 dilution of Anti-rabbit IgG, HRP-linked antibody . The membranes were washed three times for 5 min in PBS−T and the signal was detected using LumiGLO™ Reagent and Peroxide  as per the manufacturer’s instructions.2.92O and cells were then embedded in TAAB resin before ultrathin silver or gold defracting sections were cut on a Leica UC6 ultramicrotome and stained with uranyl acetate and lead citrate and examined with Philips CM10 transmission microscope  and the images recorded with a Gatan Bioscan 792 . Average cell wall thickness was measured manually for at least 20 individual yeast cells.Mid-exponential phase yeast cells were grown in YPD harvested by centrifugation and the pellets were fixed in 3% glutaraldehyde (v/v) in 0.1 M sodium phosphate buffer pH 7.4. Secondary fixation was performed in 1% osmium tetroxide in dH2.10The β-glucan content was determined by hydrolysis of this polymer and quantification of glucose. Yeast cells were grown in YPD medium at 30 °C, broken and hydrolyzed as described . Hydroly33.1ura3δ3′-ARG4-ura3δ5′) includes a functional ARG4 gene that can be excised by recombination between flanking repeats of an interrupted URA3 gene to regenerate a functional URA3 gene. This cassette, present in the Tn7-UAU1 transposon , was inserted by in vitro transposition into each of the 39 target ORFs encoding putative GpiPs ; (iii) when strains obtained by the UAU1 method and the classical Ura-Blaster method were compared  identical phenotypes were observed.One concern with this approach relates to the mitotic recombination or gene conversion step. The selection for homozygosity of the UAU1 insertion may result in homozygosing of part or the entire chromosome carrying the cassette. This may unmask recessive alleles and generate phenotypes not linked to the interruption of the target gene. We believe nevertheless that this bias might be limited since: (i) we tested at least two independent mutants, (ii) in the case of previously studied genes, the phenotypes observed in our strains matched the published ones  a fluorescent optical brightener with chitin-binding properties and caspofungin a drug inhibiting the synthesis of β-1,3-glucan, the main component of the cell wall. In addition we measured growth in the presence of SDS a detergent that compromises the integrity of the cell membrane, this tests the accessibility of SDS to the membrane through the cell wall.rbt1−/−, hwp1−/−, pga31−/−, pga62−/−, phr1−/− and ssr1−/− using serial dilutions on plates containing different concentrations of CFW (pga31−/− and hwp1−/− (data not shown).Hypersensitivity to CFW was observed with six mutants s of CFW . In the in vitro susceptibility of the GpiP mutants to caspofungin, MICs of caspofungin were determined using the CLSI (former NCCLS) microbroth dilution method M27-A2  the overall cell wall architecture using electron microscopic imaging; (ii) activation of the PKC signalling pathway by monitoring the phosphorylation of Mkc1 and (iii) the cell wall chemical composition by quantifying the glucan and chitin content.3.4phr1−/− mutant, with a significantly thicker cell wall; the TEM images show an increase of the central low density layer, but not of the peripheral high density layers (mainly composed of mannoproteins) on the outer face of the cell wall. This implies that the chitin/glucan core is affected by the lack of Phr1. This is compatible with the predicted β-1,3-glucanosyl transferase function for Phr1.Transmission electron microscopy (TEM) was used to look for gross changes in the cell wall structure of the mutants with altered caspofungin sensitivities. Although subtle changes in cell wall architecture may be missed by this approach significant changes in the thickness of the cell wall layer were observed. Strains that were less susceptible to caspofungin had thicker walls  than the3.5C. albicans and S. cerevisiae respond to caspofungin by activating the protein kinase cell integrity pathway (PKC)  . Mkc1 wan DAY286 . The antn DAY286 . Cek1 ph3.6phr1−/− and pga62−/− mutants have a significant increase in cell wall chitin content  mutants had a significant increase in cell wall chitin content. However, ssr1−/−  had wild type chitin levels. pga31−/− had a significantly decreased chitin content. Cell wall salvage mechanisms have been well-characterised in S. cerevisiae   . This inpga62−/− mutant was also hypersensitive to CFW. PGA62 and its paralog PGA59, both encode small GPI-proteins of unknown function. This result suggests that Pga62 is involved in cell wall integrity and that Pga59 cannot compensate for the absence of Pga62 under the conditions tested. Pga59 and Pga62 may be capable of functional redundancy but they may be expressed under completely different conditions thus disabling any functional compensation as is the case for Phr1 and Phr2. The overexpression of Pga59 in a pga62−/− mutant background would be an interesting experiment to question the redundancy of function in this small family. The pga62−/− mutant in our screen had increased chitin levels and decreased glucan levels, probably as a consequence of cell wall remodelling. Therefore, these results suggest that Pga62 is likely to play a role in cell wall biosynthesis but we cannot define a specific function from this analysis.The ssr1−/− mutant was hypersensitive to CFW , which is unusual knowing the normal course of events during cell wall remodelling. This suggests alternative hypotheses, which are not mutually exclusive, that Pga31 is part of the cell wall salvage pathway or that Pga31 is involved in the regulation or assembly of chitin. In the latter case the decrease of chitin would be a direct consequence of the absence of Pga31, and the hypersensitivity to CFW could be explained by a weakened cell wall in which the low residual chitin content is essential for integrity. The pleiotropic phenotypes of pga31−/−  mutant was hypersensitive to CFW but not to any other cell wall perturbing agent . This suggests that the two paralogs perform related, but non-redundant functions. C. albicans hyphae to human buccal epithelium cells by acting as a substrate for mammalian transglutaminase. The SDS and CFW hypersensitivity of hwp1−/− imply a marked defect in cell wall composition and structure, which may also explain the filamentation defect. In conclusion, there are reasons to suggest that the role of Hwp1 is not only to serve as a substrate for transglutaminase, but also to participate in cell wall architecture and in hyphal differentiation.The ng agent . Rbt1 haocalized  and prioirulence . Interesaminases . Both ofaminases  and reguaminases , and bot4.2S. cerevisiae and C. albicans  that were more resistant to caspofungin may have triggered a cell wall salvage mechanism that resulted in elevated chitin levels. Increased chitin synthesis has already been associated with reducing caspofungin susceptibility  surprisingly very little correlation can be found between cell wall glucan content or PKC pathway activation and the sensitivity to caspofungin treatment. These observations cannot be generalized for all strains but give new insights in the study of caspofungin resistance.Our results highlight three aspects related to caspofungin sensitivity: (i) chitin levels greatly influence the sensitivity to this drug, high chitin content confers reduced susceptibility while low chitin content confers hypersensitivity; (ii) in the majority of the cases a link can be drawn between thickness of the cell wall, chitin level and sensitivity to caspofungin treatment: thick cell wall have a higher concentration of chitin and confers resistance and C. albicans cell wall integrity Pga31 and Pga62. Pga31 mutant strain shows pleiotropic phenotypes with large modifications in cell wall composition and caspofungin sensitivity, suggesting a function in the early steps of cell wall biosynthesis or in cell wall salvage pathways. The absence of Pga62 strongly alters the cell structure and composition in the opposite way to Pga31 and consequently renders the strain more resistant to caspofungin. The less pronounced phenotypes suggest a function in the late stage of cell wall assembly. This library allowed the identification of three genes that when deleted lead to increased caspofungin sensitivity SSR1, MID1 and PGA31. We also identified four genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. In the literature, no other examples of deletion mutants resistant to caspofungin have been found in C. albicans apart from strains with point mutations in the FKS1 target gene (In terms of gene discovery, this work emphasizes the importance of two new proteins for get gene . This wo


Dmpk, Six5 or Mbnl1 are inactivated were therefore studied to determine the relative contribution of each gene to these cognitive functions. The open field and elevated plus maze tasks were used to examine anxiety, sucrose consumption was used to assess motivation, whereas the water maze and context fear conditioning were used to examine spatial learning and memory. Cognitive and behavioral abnormalities were observed only in Mbnl1 deficient mice, which demonstrate behavior consistent with motivational deficits in the Morris water maze, a complex visuo-spatial task and in the sucrose consumption test for anhedonia. All three models of cDM1 exhibit normal spatial learning and memory. These data identify MBNL1 as a potential regulator of emotional state with decreased MBNL1 levels underlying the motivational deficits observed in cDM1.Assessment of molecular defects that underlie cognitive deficits observed in mendelian disorders provides a unique opportunity to identify key regulators of human cognition. Congenital Myotonic Dystrophy 1 (cDM1), a multi-system disorder is characterized by both cognitive deficits and a spectrum of behavioral abnormalities, which include visuo-spatial memory deficits, anxiety and apathy. Decreased levels of DMPK (Dystrophia Myotonica-protein kinase), SIX5, a transcription factor or MBNL1 (Muscleblind-like 1), an RNA splice regulator have been demonstrated to contribute to distinct features of cDM1. Mouse strains in which either  DMPK gene and located immediately 5′ of the SIX5 gene on chromosome 19 DMPK RNA within the nucleus, DMPK levels are decreased in DM1 cells SIX5 allele leading to diminished levels of SIX5 Myotonic dystrophy (DM1) is a multi-system disorder characterized by muscle weakness, skeletal myotonia, cardiac conduction defects, ocular cataracts and cognitive and behavioral abnormalities Patients with the myotonic dystrophy type 1 exhibit cognitive and behavioral abnormalities including mental retardation, visuo-spatial memory deficits, apathy, anxiety, hypersomnolence, autism, depression and attention deficit hyperactivity disorders Six5, Dmpk and Mbnl1 on three separate behavioral tasks designed to test learning and memory, anxiety and motivation. These three mouse models of DM1 show various physical phenotypes pertaining to myotonic dystrophy. −/−Dmpk mice display muscle weakness and heart disease −/−Six5 mice display cataracts and heart disease −/−Mbnl1 mice display skeletal myotonia, muscle weakness, cataracts and possibly heart disease −/−Mbnl1 mice have deficits consistent with decreased motivation.To investigate the contribution of these genes to cognitive symptoms observed in DM1 patients, we studied the effect of deletion of −/−Six5 and −/−Dmpk mutant mice compared to their wild-type littermate controls  and general activity using the Open Field task. In this task, mice are placed in an open arena and allowed to explore. No differences in Open Field thigmotaxis  when returned to the same context.−/−, Dmpk−/−Six5 and −/−Mbnl1 mice show no deficits in hippocampus-dependent context fear conditioning  were observed in activity  and a deactivity , consistTo further examine hippocampal function as it relates to visuo-spatial learning and memory, we performed the Morris water maze task. In this task, mice are trained daily to find a hidden platform in a circular pool, using external spatial cues to create a map of the environment and assist in navigation. The Water Maze has non-spatial components such as swim velocity, latency or pathlength during acquisition and thigmotaxis as well as spatial components, such as spatially-selective search strategies during probe trials in which the hidden platform is removed −/−Six5 . This is unlikely to be caused by muscular fatigue or myotonia for the following reasons. First, the −/−Mbnl1 mice perform normally in the visible watermaze  or a simple task (sucrose consumption).Neither −/−Mbnl1 mice is difficult to assess. However, −/−Mbnl1 mice show unmotivated behavior in the water maze, failing to switch from passive strategies to a spatially selective search as exhibited by increased floating in the watermaze task −/−Mbnl1 mice may become unmotivated if they fail to learn the platform location expediently. A number of −/−Mbnl1 mice also exhibited such pronounced thigmotaxis that they were excluded from final analysis. The remaining −/−Mbnl1 mice showed normal spatial learning and memory when tested without the platform present, however, the −/−Mbnl1 mice that demonstrated pronounced thigmotaxis did not show any spatial learning and memory. This demonstrates that Mbnl1 deficit in these mice results in a range of severity of the observed phenotypes.Due to their physical symptoms, the cognitive and emotional phenotype of the −/−Mbnl1 mice for apathy towards sucrose. The mice were tested in their home cage with the sucrose and water bottles presented in their normal location. Therefore, physical complications of this task are minimal as the physical demands are the same as drinking water in their home cage: these mutants do not experience dehydration from an inability to reach the water bottle. In this test for anhedonia, −/−Mbnl1 mice show preference for sucrose over water at a much reduced rate than their wild-type counterparts. The −/−Mbnl1 mice appear to require a higher reward as a 16% solution was equally appealing to the −/−Mbnl1 mice as a 4% solution was to the wild-type mice. This suggests that the mutant mice do not have an innate aversion to sucrose but instead that they have a decreased interest in seeking a sweet reward. This finding may be particularly relevant as apathy is a striking feature in myotonic dystrophy patients and has been shown to be independent of both clinical depression and peripheral muscular weakness To substantiate a phenotype of decreased motivation, we tested −/−Mbnl1 mice exhibited high levels of thigmotaxis, a behavior indicative of anxiety. Although −/−Dmpk and −/−Six5 showed no overt anxiety phenotype in the open field, we performed only one test of anxiety and thus subtle phenotypes may exist that can be detected using other tests. In a second test of anxiety, the elevated plus maze, the −/−Mbnl1 mice showed conflicting signs of increased and decreased anxiety. Given these ambiguous results, it is difficult to determine if these mice are good models of anxiety observed in myotonic dystrophy patients. Furthermore, it would be difficult to determine the basis for their altered performance in either the open field and elevated plus maze tasks. The underlying cause of altered performance could be decreased motivation to explore, increased anxiety or musculoskeletal effect, or a combination of all three factors, all three of which are consistent with the human disease.In addition to lack of motivation, myotonic dystrophy patients have increased anxiety. We tested all three mutant mouse strains for alterations in anxiety levels and only −/−Mbnl1 mice. Since DM patients do not show remote memory deficits, we did not test memory at later times. Specifical alterations in 24-hour memory consolidated and remote memory were not tested however, based on a normal memory phenotype at seven days, which is it is indicative of protein-synthesis dependent memory and the transition to a remote memory −/−Mbnl1 mice.Finally, contextual associative learning was normal in the −/−Mbnl1 mice with normal musculoskeletal function. Since these are separable in patients with DM1, it is likely that the transcripts controlled by Mbnl1 processing may play a specific role in apathy and motivation. The specific transcripts that undergo altered splicing due to Mbnl1 are not all known. Deep sequencing of the different mutant models of DM1 will be very useful in generating and further refining genotype/phenotype relationships. Mbnl1 belongs to a three-member family, and the role of specific Mbnl family members may further refine our understanding of the transcriptional misregulation that leads to specific symptoms −/−Dmpk and −/−Six5 mice suggests that these genes do not play a role in motivation when they are specifically deleted but their dysfunction, as caused by the DM1 trinucleotide repeat expansion, may still participate in generating cognitive effects in patients. Defining the specific regions of the brain that show functional impairment in Mbln1 knockout mice and identification of Mbnl1 target RNAs in these areas will be critical to understanding this complex phenotype. These lines of inquiry should allow key insights into the molecular targets and circuits that regulate motivation, and spatial learning and memory in humans.Important questions to address in the future will be to assess motivational drive in Six5−/−, −/−Dmpk, and −/−Mbnl1 mice have been previously described −/−Six5 mice ranged from 8–12 months, −/−Dmpk mice were 8–10 months and −/−Mbnl1 mice were 8–14 months at the time of testing. Mice were used for multiple tests in this order: open field, watermaze, fear conditioning or open field, elevated plus maze, sucrose consumption, watermaze with a minimum of two weeks rest before commencing a new test.All experiments were performed in accordance with the institutional guidelines of the University of California at Los Angeles. For all tasks, male and female mice in the 129B6 genetic background were generated from heterozygous matings and all tests used mutant mice with their respective wild type littermates as controls. All mice were group-housed, maintained in a 12∶12 light/dark cycle and had food and water ad libitum. Since physical deficits are present, we looked at specific measures during the behavioral tasks to minimize the influence of these impairments.Six5 mutant strain, 6 wild-type and 7 mutant mice were tested. For the Dmpk mutant strain, 9 wild-type and 9 mutant mice were tested. For the Mbnl1 mutant strain, 23 wild-type and 19 mutant mice were tested.Open Field activity, the first behavioral test performed, was assessed to examine generalized activity and thigmotaxis . Animals were placed in an open 30.5 cm by 30.5 cm space and allowed to explore for ten minutes under dim light conditions. Horizontal locomotor acivity was measured as beam breaks by paired sets of photo beams using the Activity Monitor systems . We measured thigmotaxis (amount of time in the outer versus inner zone), and general activity levels . For the −/−Mbnl1 mice.In the elevated plus maze, a second anxiety test, mice were placed in the center of a four-arm maze and their movement on the maze was recorded for five minutes in dim light. The maze was 60 cm off the floor. Two arms were sheltered with 16.5 cm opaque walls and the other two arms were open. We measured the amount of time in the closed and open arms and the center of the maze, as well as the number of entries into each arm. The time in a stretch approach position and the number of times the mice dipped their heads over the edge of the maze was also scored. We tested 14 wild-type and 14 −/− mice, 10 wild-type and 6 Dmpk−/− mice, and 12 wild-type and 5 −/−Mbnl1 mice were tested in this task.Mice were trained in context fear conditioning. Animals were placed in the training chamber during which time baseline activity and freezing levels were obtained, followed by a single shock, delivered at 4 minutes. After seven days, mice were returned to the training chamber and tested for long-term memory of the context by measuring activity and freezing levels. Context fear conditioning was performed as previously described −/−Dmpk, days 4 and 7 for −/−Six5 and days 4, 7, 10 and 13 for −/−Mbnl1 mice. Results for the last probe trial are shown. Pathlengths to find the hidden platform during acquisition were used in addition to latency as latencies can be influenced by slow swim speeds. Upon completion of the hidden Water Maze task, all mice were tested in the visible water maze using a single session with three training trials in which the location of the submerged platform is marked by a local cue. This test further controls for performance, including visual capabilities and muscle deficits. −/−Mbnl1 mice were analysed in two groups, data shown excludes individual mice that showed >10% thigmotaxis at the end of training as these mice may have visual complications that affect distant vision. Analysis of all −/−Mbnl1mice is shown in the supplemental data. For the Dmpk mutant strain, we tested 10 wild- type and 6 mutant mice, which were trained with two trials per day. For the Six5 mutant strain, we tested 13 wild-type and 12 mutant mice with two trials per day with probe trials on days 4 and 7. For the Mbnl1 mutant strain, we tested 25 wild type and 21 mutant mice with two trials per day with probe trials. Analysis of −/−Mbnl1 mice was performed both excluding and including mice that exhibited more than 10% thigmotaxis at the end of training. This resulted in exclusion of 1 wild type and 8 −/−Mbnl1 mice.Morris Water Maze training was commenced to examine spatial learning and memory of the mutant mice, as previously described −/−Mbnl1 mice were tested.Mice were tested for motivation/anhedonia using the sucrose consumption test. Animals were water restricted overnight on each day of habituation and testing. Mice were habituated to testing cages with two drinking spouts, both of which contained water after which, one solution was replaced with sucrose. Mice were given 2%, 4%, 8% and 16% sucrose for three days each, the first day was used as habituation to the new solution. The volume of either water or sucrose was averaged for the remaining two days, in which the presentation of water and sucrose was counterbalanced. Fourteen wild-type and 14 The following software was used for analysis of raw data: Open Activity from Med Associates for the Open Field, custom designed software for fear conditioning described Figure S1Mbnl1−/− mutant mice have altered performance in the Morris Water Maze. All mice, including Mbnl1−/− mice that demonstrate pronounced thigmotaxis are included. A) No statistical differences were found in the latency (seconds) to find the platform during training (panel i)  = 1.29, p = 0.26) for Mbnl1−/− mice  compared to their Mbnl1+/+ wild-type littermate controls . A inset: The pathlength (m) to reach the target platform of Mbnl1−/− (white) was significantly different during acquisition compared to Mbnl1+/+ mice (black)  = 2.81, p = 0.012; main effect of genotype F(1.44) = 7.43, p = 0.0092). Mbnl1−/− mutant mice (white) had a slower average velocity during all training days   = 52.7, p = <0.0001) and increased thigmotaxis   = 12.2, p = 0.0011) compared to their wild-type littermate controls, Mbnl+/+ mice (black). B) The % time spent swimming slowly (floating behavior) of Mbnl1−/− (white) increased compared to Mbnl1+/+ mice (black) during acquisition  = 16.4, p = 0.0002; effect of genotype x latency F = 5.25, p = <0.0001) (panel i). Mbnl1−/− mice showed increased floating behavior in all training blocks except block 3. C) Mbnl1−/− mice displayed normal acquisition in the visible water maze task (pathlength (m),  = 0.694, p = 0.50)). Training blocks represent two days  for Mbnl1 mutant mice. D) The percent time spent in the Target Quadrant (TQ), Adjacent Left (AL), Adjacent Right (AR) or Opposite Quadrant (OP) is shown. A score of 25% reflects random searching. Probe test results after training are shown. Statistically significant differences were found in the percent time spent in searching in each quadrant on day 13 as shown for wild-type (black) and Mbnl1−/− (white) mice  = 5.66, p = 0.0011), main effect of genotype  = 0.041, p = 0.84). The Mbnl1−/− mice spent significantly less time searching in the target quadrant  = 7.48, p = 0.0089) and more time in the opposite quadrant  = 8.84,p = 0.0048).(0.17 MB TIF)Click here for additional data file.


Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment. Wisdom of the Body, “when a factor is known which can shift a homeostatic state in one direction it is reasonable to look for a factor or factors having an opposing effect.”In the cell's repertoire of mechanical responses to imposed stretch – mechanoresponsiveness – the newly discovered existence of cytoskeletal fluidizationTo answer these questions, here we used a novel approach that combines cell stretch with traction force microscopy.We plated the isolated human airway smooth muscle cell on a gel substrate and then applied stretch using a punch-indentation system . When anTractions are the local physical forces that an adherent cell exerts upon its substrate, expressed as force per unit area (stress). We begin by posing a simple but important question, how do cell traction forces develop in space and time in response to a rapid transient isotropic biaxial cell stretch? One might reason that cell traction is similar to a first order strain energy derivative and cell stiffness is a second order derivative, but while this statement is true for passive materials it is not true for active biological materials wherein molecular motors and active polymerization responses can generate active stresses and traction forces that can be uncoupled from strain energy derivatives.t test). No changes in focal adhesion area were noted, however  have been well documented. Inhibition of myosin light chain kinase with ML7 or depolymerization of F-actin with Latrunculin-A reduced the pre-stretch tractions to levels far below those observed in untreated cells , inset at test). Despite these static differences, dynamic traction responses within these three stiffness groups were similar  and characterized corresponding cell tractions and their changes. As reported previously in cells that are not subjected to stretch similar . Taken t similar . We haveThe fluidization response stands in contrast to the reinforcement response associated with local cell stretch applied through an attached microbead or microneedle, in which case local stiffness and force are seen to increase and to do so on the time scale of seconds.We then questioned whether reinforcement might alternatively be a response that is peculiar to stretch non-homogeneity. To explore this possibility we used a microneedle punch to induce a single transient non-homogeneous cell deformation  much as To assess further the generality of these responses, we subjected the cell to a transient stretch of longer duration .Taken together, results reported here indicate that in response to repetitive load transients the reinforcement response in the HASM cell is peculiar to non-homogeneous cell deformations, as would occur during repetitive bead pulling or needle poking, but fails to scale up to the case of repetitive homogeneous cell stretch, whether isotropic or anisotropic. The reinforcement response and the fluidization response differ in sign, and this difference suggests either that reinforcement is not triggered by homogeneous cell stretch, or that reinforcement is somehow overwhelmed or blocked by the fluidization response. As regards the important issue of mechano-protection, the reinforcement response vis-à-vis the fluidization response both seem logical protective strategies – either brace for the storm or go with the flow. Nonetheless, in loading conditions that would be expected in most physiological circumstances the reinforcement response in the HASM cell was suppressed and the fluidization response prevailed. Given the complexity of signaling cascades that are triggered during reinforcementTransient stretch causes prompt detachment of the motor protein myosin from actin and a profound reduction in the myosin duty cycleProtein dynamics in the complex functional system that define the cytoskeleton more generally are dominated by multiple weak interactions operating within a noisy thermal microenvironment but far from thermodynamic equilibriumWe do not speculate here on mechanisms that might account for the strange, unexpected, and altogether decisive role of nonhomogeneous versus homogeneous cell stretch. But as regards the latter, which is the more physiological case as regards mechanoresponsiveness, we suggest that the universality, the robustness, and, especially, the rapidity of the fluidization response, when taken together, lead to a remarkably simple physical picture: the cytoskeleton belongs to the special class of materials that are soft and fragile.This physical picture of the cytoskeleton as a fragile material does not at all rule out highly specific mediator-dependent cascades of signaling responses to cell stretch, including both reinforcement Biaxial deformation was imposed on an elastic polyacrylamide substrate using a novel punch indentation system . The ind−1 penicillin, 100 mgml−1 streptomycin, 200 mgml−1 amphotericin B, 12 mM NaOH, 1.7 mM CaCl2, 2 mM L-glutamine, and 25 mM HEPES. Once the cells in passage 4–6 reached confluence, they were serum deprived for 42 hours to arrest the cell growth cycle in the G1/G0 phases. The cells were then plated very sparsely  in serum-free medium on type I collagen-coated (0.1 mg/ml) polyacrylamide gel dishes for 4–8 hours before being tested. The following pharmacological interventions were used to modulate the CSK filaments and baseline contractility: Latrunculin-A , ML7 , Phenylarsine oxide , EGTA  and Gadolinium Chloride .Human Airway Smooth Muscle (HASM) cells were isolated from tracheal muscle of lung transplant donors using a previously described method.2 until the day of the experiment.Polyacrylamide gel substrates were prepared according to a previously described protocol.Single adherent HASM cells were subjected to the testing protocol illustrated in Cell tractions were computed using constrained Fourier transform traction microscopy (FTTM).At the earliest time point following a 10% uniform transient stretch, cells were fixed with 5% formalin for 10 min, permeabilized for 5 min with 0.5% Triton X-100 (Sigma-Aldrich), blocked with a solution containing 5% goat serum and 1% BSA and subsequently stored overnight with a primary antibody directed against vinculin . After three washes with PBS, cells were incubated at room temperature in the presence of a secondary antibody . After two more washes, cells were counterstained with 1 μg/ml Hoechst 33342 (Sigma-Aldrich), mounted, and imaged with a 40× oil immersion lens. Stretched cells in each dish were compared with unstretched cells located outside the region of punch indentation. Focal adhesion area was computed by tracing the vinculin stained regions.The Fluo-4 NW Calcium Assay Kit  was used according to manufacturers' instructions. HASM cells were incubated at 37°C for 30 minutes in the dye loading solution dissolved in assay buffer . Following incubation, the cells were washed twice with assay buffer, equilibrated at room temperature for an additional 30 minutes, and then stretched. Fluorescent measurements were recorded at several time points before and after stretch with a 20× objective lens.Figure S1Experimental protocol for dynamic cell traction measurements.(0.02 MB PDF)Click here for additional data file.Figure S2The fluidization response does not depend upon stretch isotropy. a–b, Anisotropic strains were imposed using two parallel plates. By modifying the ratio of plate separation to plate length, the major/minor strain ratio can be varied. For example, in a, the strain ratio = 30, which is nearly uniaxial whereas in b, the strain ratio = 2. c, In response to a transient stretch, the contractile moment T relative to the unstretched baseline value T0 promptly decreases followed by a slow recovery Click here for additional data file.Figure S3Applied biaxial stretch magnitudes are scalable within the physiological range and highly repeatable. a, Fluorescent bead marker positions embedded within a polyacrylamide gel (thickness = 700 Âμm) were obtained before and after a prescribed indentation with an annular punch with an inner and outer diameter of 2 mm and 3 mm, respectively. b–d, Gel displacement field in the central region (200×200 Âμm) corresponding to three different indentation depths of 150, 200 and 400 Âμm, respectively. The displacement field was calculated based on relative changes in embedded fluorescent bead marker positions. The arrows have been scaled by a factor of 4 for clarity. e, Despite different maximum displacement magnitudes in (b–d), the corresponding strain field is homogenous and uniform in the plane . f, When forty closely spaced transient stretches (indentation depth = 400 Âμm) were applied consecutively, the applied strain was found to be highly reproducible between loading cycles. For all cycles, the displacement field was computed by comparing unloaded images at the end of a loading cycle with a loaded image taken at the start of the experiment.(0.94 MB PDF)Click here for additional data file.Figure S4Applied stretch can be tensile or compressive depending on indenter size. a–b, Finite element analysis of an elastic substrate  indented to different depths (0.05 to 0.15 mm) with an annular punch of various cross-sectional diameters. The resulting radial strain field at the substrate surface is largely isotropic over the central region, scales with indentation depth and changes from a tensile to a compressive field for large diameter indenters. c, Displacement field measured experimentally in the central region (200×200 μm) for an annular indenter with inner and outer diameters of 8×9 mm, respectively. Resulting radial displacements are compressive (Arrows point inwards).(3.10 MB PDF)Click here for additional data file.Figure S5Focal adhesion area does not change immediately after a transient homogeneous stretch. a, Immunohistochemical staining for vinculin in an un-stretched cell. b, Immunohistochemical staining for vinculin immediately after a single transient biaxial stretch . c, No n(0.70 MB PDF)Click here for additional data file.Figure S6The time course of the fluidization response is not mediated by calcium signaling. Also see (0.02 MB PDF)Click here for additional data file.Table S1Sample database for (0.02 MB PDF)Click here for additional data file.Text S1Traction, glassy dynamics and the origin of eukaryotes(0.02 MB PDF)Click here for additional data file.Movie S1Traction dynamics following a single homogeneous biaxial stretch. The time lapse covers a period of 1000 seconds, with a single transient biaxial stretch applied between t = 5.6 seconds and t = 15.6 seconds.(2.12 MB MOV)Click here for additional data file.Movie S2Traction dynamics following a single non-homogeneous stretch. The time lapse covers a period of 600 seconds, with a single transient stretch applied between t = 2 seconds and t = 11 seconds.(0.69 MB AVI)Click here for additional data file.Movie S3Calcium imaging following a single transient homogeneous stretch (10% stretch magnitude) applied between t = 1 second and t = 11 seconds.(6.14 MB AVI)Click here for additional data file.Movie S4Calcium imaging following a single transient non-homogeneous stretch applied between t = 1 second and t = 11 seconds.(0.85 MB AVI)Click here for additional data file.


Relevance of PKCα expression in clinical specimens was determined by comparing PKCα expression in tumours from patients exhibiting disease recurrence with patients remaining disease-free following TAM treatment. Our results suggest that PKCα expression may predict TAM treatment failure.We previously reported that stable transfection of protein kinase C alpha (PKC Identification of the key factors involved in the molecular mechanism of TAM resistance will undoubtedly lead to the development of logical therapeutic targets. It is well documented that the oestrogen receptor (ER) and protein kinase C (PKC) activity and abundance are inversely related in breast cancer cell lines and that PKC is elevated in malignant but not normal breast tissue . The sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (H+L), followed by incubation for 30 min with HRP–Streptavidin . Sections were rinsed with PBS for 5 min between each reaction. 3,3′-Diaminobenzidine (DAB+) solution  was used as a chromogen. Finally, sections were counterstained with haematoxylin. Stained sections were photographed at × 40 magnification using an Olympus BX40 microscope attached to a SONY DP10 digital camera. Analysis of the entire slide was performed to score the intensity of staining. Intensity was evaluated semiquantitatively by assigning the score of either negative (0), weak positive , moderate , or intense . The specificity of the staining was ascertained by several methods, including the use of PBS or isotypic nonspecific antibodies in lieu of the specific anti-PKC-α antibody. Dilution experiments using the primary antibody to assess specificity and observe extinction of signal were also performed. In addition, staining with the PKCα antibody was repeated in the presence of a specific PKCα blocking peptide . There was no staining of the breast epithelial cells under these conditions.Sections, 4 α overexpression with the acquisition of TAM resistance, PKCα expression was assessed by immunohistochemical staining in two patient populations treated with TAM: patients remaining disease-free following TAM treatment and patients exhibiting disease recurrence. Representative immunohistochemical staining of patient tumours demonstrates the cytoplasmic localisation of PKCα  and have tumours of higher grade compared with the disease-free patients (mean age 54 years).To determine the possible association of PKCα overexpression as a predictive tool for improved therapeutic options are based on our T47D/PKCα xenograft model system demonstrating that E2 induces tumour regression   vs TAM for the treatment of postmenopausal metastatic breast cancer indicates that there is survival advantage for women on DES compared to women on TAM (α may improve upon the outcome of treatment with DES. We are in the process of expanding these studies in a larger population of patients who are matched by age and stage to substantiate these preliminary findings. Examination of a larger patient series will also allow us to address the relations between ER status and PKCα expression as well as between the intensity of PKCα staining and time to disease recurrence.These findings, taken together with our previous observation that E2 has a novel antitumour effect on T47D/PKC


In animal groups such as herds, schools, and flocks, a certain distance is maintained between adjacent individuals, allowing them to move as a cohesive unit. Proximate causations of the cohesive and coordinated movement under dynamic conditions, however, have been poorly understood.We established a novel and simple behavioral assay using pairs of small fish (medaka and dwarf pufferfish) by eliciting a simultaneous optomotor response (OMR). We demonstrated that two homospecific fish began to move cohesively and maintained a distance of 2 to 4 cm between them when an OMR was elicited simultaneously in the fish. The coordinated and cohesive movement was not exhibited under a static condition. During the cohesive movement, the relative position of the two fish was not stable. Furthermore, adult medaka exhibited the cohesive movement but larvae did not, despite the fact that an OMR could be elicited in larvae, indicating that this ability to coordinate movement develops during maturation. The cohesive movement was detected in homospecific pairs irrespective of body-color, sex, or albino mutation, but was not detected between heterospecific pairs, suggesting that coordinated movement is based on a conspecific interaction.Our findings demonstrate that coordinated behavior between a pair of animals was elicited by a simultaneous OMR in two small fish. This is the first report to demonstrate induction of a schooling-like movement in a pair of fish by an OMR and to investigate the effect of age, sex, body color, and species on coordination between animals under a dynamic condition. Oryzias latipes), a freshwater teleost native to East Asia. We used medaka for this experiment for the following reasons. (1) Medaka (Oryzias latipes) exhibit prominent shoaling behavior. For example, a single medaka fish tends to approach a conspecific group Animal congregations, such as herds We analyzed interindividual interactions between two fish in a single experiment and demonstrated that coordinated and cohesive behavior (schooling-like behavior) can be induced, even between only two individuals, by eliciting an OMR in two small fish.To examine whether two adult medaka with a simultaneous OMR exhibit schooling-like behavior, we measured the distance between the two fish d in  using a The distance varied within a small range when a simultaneous OMR was elicited in the fish, whereas in the superimposed control, the distance ranged much more widely . SimilarWe then analyzed the relative positions of the two fish. We defined two conditions based on the relative position of the two fish: (1) Fish B seems to go ahead of Fish A (2) Fish A seems to go ahead of Fish B . We counTo evaluate whether early larval medaka have the ability to maintain a coordinated distance, we tested larval medaka 5 days, 7 days, and 10 days after hatching. Larval medaka exhibited an OMR activity, although the abilities were relatively poor compared with those of adult medaka. . We alsoCarinotetraodon travancoricus) called a dwarf puffer, the body length of which is approximately 3 cm, similar to medaka. Although there are no reports on pufferfish shoaling, we demonstrated that a pair of pufferfish maintained 2–4 cm distance between them , saithe (Pollachius virens), and herring (Clupea harengus), the nearest neighbor distance (NND) is maintained within a set range, and the relative positions are not stable These findings represent the first evidence of the ability of small laboratory-fish pairs to maintain a set distance from other conspecific fish upon the elicitation of a simultaneous OMR. Some properties of the coordinated movement are similar to those observed in fish schools. In schools of cod , association preferences for size-matched fish occur both when shoal-mates are conspecific and when they are heterospecific Conspecific interaction in fish has been shown in many studies using a two-way choice test in which test-fish are given the choice between two stimulus fish or shoals presented behind a transparent wall at either end of a test tank Menidia menidia) Phoxinus phoxinus) Pseudocaranx dentex) juveniles and demonstrated that there is a long time-lag between the onset of the OMR (body length 4–6 mm) and that of schooling (body length 16>mm) We also demonstrated that the ability of the schooling-like movement develops during post-hatch growth. In silverside  of drR strain, which contain a red pigment color marker on Y chromosome, (more than 3 months after hatching), Quintet mutant strain (more than 3 months after hatching), T5 strain , and wild-type zebrafish (Danio rerio) TL2E strain, more than 7 months after hatching) were kept in groups in plastic aquaria (12×13×19 cm). The medaka T5 strain is derived from a southern Japanese population wild-type medaka Carinotetraodon travancoricus) were obtained from a local pet shop and housed in a plastic tank (12×13×19 cm). The tank was maintained at a temperature of 28°C and under a 14 h∶10 h light:dark cycle. For behavioral studies using medaka larvae (T5 strain), eggs were placed separately in a 24-well microplate . The walls of the wells were covered with white opaque paper to avoid interindividual visual interaction after hatching. Within 24 hours after hatching, for the “isolation” test each larval medaka was transferred to a 6-well plate  with white opaque walls. For the “group” test, four larval medaka were transferred to a 7-cm diameter glass dish. The 6-well plate and glass dish were maintained at a temperature of 28°C and under a 14 h∶10 h light:dark cycle until the behavioral testing was performed.Medaka (The setup used to measure the optomotor response is shown in A pair of fish (Fish A and B) was tested in each experiment . At the The distance between two individuals in the “simultaneous OMR” experiment of each pair was measured in each frame  Frequency histogram of the distance between two adult female medaka exhibiting an OMR, showing the integration of all the pairs. (n = 10) (B) Adult puffer pairs exhibiting an OMR. (n = 10) (C) Adult male zebrafish pairs. (n = 13) (D) Adult medaka pairs with different body colors . (n = 10) (E) Other adult medaka pairs of different body colors (n = 14) . Error bars indicate standard deviation (SD).(1.52 MB TIF)Click here for additional data file.Figure S2Distance between two conspecific fish exhibiting OMR. (A) Frequency histogram of distance between two adult female medaka exhibiting OMR when the rotation speed of stripes was 80 rpm (n = 10). (B) Frequency histogram of distance between two adult male medaka exhibiting OMR when the rotation speed of stripes was 60 rpm (left) and 80 rpm (right) . (C) Frequency histogram of distance between adult male and female medaka exhibiting OMR when the rotation speed of stripes was 60 rpm (left) and 80 rpm (right) (n = 12). (D) Frequency histogram of distance between adult male zebrafish when the rotation speed of stripes was 80 rpm (n = 13). Error bars indicate standard deviation (SD).(1.59 MB TIF)Click here for additional data file.Figure S3Induction of coordinated movement by the OMR. (A) Six examples of a temporal change in the distance of the two adult female medaka without an OMR (under a static condition). (B) Frequency histogram of the distance between two adult female medaka without an OMR (under a static condition). Error bars indicate standard deviation (SD). (C) Frequency histogram of the distance between two adult female medaka exhibiting an OMR, before the single OMR tests.(1.38 MB TIF)Click here for additional data file.Figure S4Distance between two medaka larvae exhibiting an OMR. The size of the apparatus for the larval fish was smaller than that for the adult fish. The rotation speed of the stripes was 40 rpm. (A) Frequency histogram of the distance between two larvae reared in isolation for 5 days (left), 7 days (middle), and 10 days (right) after hatching (n = 10). (B) Two larvae reared in a group for 5 days (left), 7 days (middle), and 10 days (right) after hatching (n = 10). Error bars indicate standard deviation (SD).(1.22 MB TIF)Click here for additional data file.Figure S5Distance between two adult xenogeneic fish exhibiting a simultaneous OMR. (A) Frequency histogram of the distance between adult medaka and puffer (n = 10). (B) Frequency histogram of the distance between adult medaka and zebrafish (n = 10). Error bars indicate standard deviation (SD). (C) Distance between medaka and zebrafish when the rotation speed of stripes was 80 rpm. (n = 10). Error bars indicate standard deviation (SD).(0.87 MB TIF)Click here for additional data file.Table S1Summary of the OMR tests.(0.04 MB DOC)Click here for additional data file.Movie S1Two adult conspecific fish (Medaka) exhibiting a simultaneous OMR. Blue line indicates distance (d) between the two fish.(3.72 MB MOV)Click here for additional data file.Movie S2Two adult conspecific fish (pufferfish) exhibiting a simultaneous OMR. Blue line indicates distance (d) between the two fish.(2.63 MB MOV)Click here for additional data file.Movie S3Two adult xenogeneic fish (Medaka and pufferfish) exhibiting a simultaneous OMR. Blue line indicates distance (d) between the two fish.(3.80 MB MOV)Click here for additional data file.Movie S4Two adult conspecific fish (Medaka) exhibiting a simultaneous OMR. An example of numerous changes in the relative position of a pair of fish.(1.09 MB MOV)Click here for additional data file.Movie S5Two adult larval Medaka fish (Isolation 7pdf) exhibiting a simultaneous OMR.(6.19 MB MOV)Click here for additional data file.