The visual cortex responds to repeated presentations of the same stimulus with high variability. Because the firing mechanism is remarkably noiseless, the source of this variability is thought to lie in the membrane potential fluctuations that result from summated synaptic input. Here this hypothesis is tested through measurements of membrane potential during visual stimulation. Surprisingly, trial-to-trial variability of membrane potential is found to be low. The ratio of variance to mean is much lower for membrane potential than for firing rate. The high variability of firing rate is explained by the threshold present in the function that converts inputs into firing rates. Given an input with small, constant noise, this function produces a firing rate with a large variance that grows with the mean. This model is validated on responses recorded both intracellularly and extracellularly. In neurons of visual cortex, thus, a simple deterministic mechanism amplifies the low variability of summated synaptic inputs into the large variability of firing rate. The computational advantages provided by this amplification are not known. A simple model accounts for the high variability of firing rates observed in responses of cortical neurons to visual stimuli In the primary visual cortex (V1), different trials of presentation of an identical stimulus yield highly variable firing rates . This trA key property of trial-to-trial variability is that it depends on the strength of the stimulus: Response variance across trials is approximately proportional to response mean . An examBecause the production of firing rates within a neuron introduces remarkably little noise , trial-tFrom traces of membrane potential obtained at high temporal resolution D, I obtamean,V is the “signal” reflecting the stimulus-driven synaptic input to the neuron  whose slope is 0.08 ± 0.01 and whose intercept at restV = −60.4 mV is 3.3 ± 0.1 mV. Similar values were obtained in the rest of the population (e.g., r = 0.40 ± 0.19 (s.d., N = 22), intercept at restV was 3.3 ± 1.4 mV, and mean slope was a shallow 0.14 ± 0.09. In occasional cells . When meanV is higher, however, the tail of the Gaussian that lies above threshold becomes substantially larger, and the distribution of firing rates reaches higher rates (e). The large peak at 0 spikes/s corresponds to the area of the Gaussian that lies below thresh.VIn the Gaussian–rectification model, the stimulus determines the mean of the Gaussian B, and th instant B. The re instant H operate instant A: Each ptly zero A, a. Wheer rates A, e. ThemeanV increases, the distribution of firing rate becomes broader  appears remarkably Gaussian , the firing rate is always zero, whereas larger mean potentials yield a distribution of firing rates that spans values from 0 to 300 spikes/s . Deviations from the predictions are largest where they are least significant, i.e., at high firing rates for the high values of meanV . These high values were achieved seldom; for example, only 21 data points were obtained at meanV = −50 mV (e), compared to 273 at meanV = −54 mV (d) and 1,575 at meanV = −64 mV (a).The model predicts the main features of the distributions of firing rate A. It pre= −50 mV A, e, com= −54 mV A, d and = −64 mV A, a.In fact, the model closely predicts both the firing rate's mean and standard deviation C. It preCrucially, the model closely predicts how firing-rate variance depends on firing-rate mean C and 3D.Overall, the Gaussian–rectification model applied to the trace of mean potential performed as well as the rectification model applied to the individual traces of potential. Both models underestimated the intercept of the lines fitted to the relationship between firing-rate variance and mean: the rectification model by 25 ± 42% A, and thThese results illustrate how the key element in producing the steep growth in firing-rate variance observed with growing stimulus strength is the nonlinear transformation between potential and firing rate H. IndeedThe predictions of the Gaussian–rectification model apply to any neuron that meets minimal criteria: a relationship between synaptic input and firing rate that is monotonic and includes a threshold, and noise in the input that has a Gaussian distribution.As an example, let us consider a neuron that is closer to biological reality than the Gaussian–rectification model, one that receives currents  in its input and produces individual spikes (not continuous firing rates). In particular, consider an enhanced integrate-and-fire neuron, where each spike is accompanied by a temporary increase in spike threshold and by the entry of calcium, which in turn determines an after-hyperpolarization potassium current see .To ensure realism, I fitted the model parameters to responses to injected currents of a regular spiking neuron. This neuron was recorded in vitro in the visual cortex of the guinea pig, in the near absence of synaptic inputs . The injJust as predicted, this spiking neuron responds to noisy injected currents with a firing rate whose variance grows with the mean . To simuThe Gaussian–rectification model captures the essence of this behavior. Once it is given the standard deviation of the noise and the relationship between injected current and firing rate F, the GaSimilar results were obtained when the stimulus parameters were changed to simulate synaptic inputs to a complex cell, or when the parameters of the spiking neuron were changed to simulate other cells measured in vitro, or even chosen randomly within reasonable bounds. As predicted, as long as the relationship between synaptic input and firing rate involved a threshold and the input noise was Gaussian, the variance grew with the mean for firing rate but not for injected current.thresh,V (3) the gain k of the relationship between firing rate and potential above threshold, and (4) the exponent n of this relationship. For the purpose of studying the model, we can assume, without loss of generality, that potential is unitless and has standard deviation σ = 1. Then, because threshV can only determine the range of firing rates that is achieved, only k and n control the shape of the variance versus mean curves , variations in potential are compressed into proportionally ever smaller variations in firing rate; the opposite occurs if the curves expand (n > 1).The gain ve shape . Increasightward , rows. Tsaturate , columnse (n = 1 , middle, 1 as in , left. Sn = 1.1 ± 0.6). Accordingly, a mild form of saturation is common in the plots of firing-rate variance and mean , measurements in this dataset yield more precise estimates of firing-rate variance over a wider range of firing rates than are available in the intracellular sample.k and exponent n of the transformation of potential into firing rate. I thus first computed the model predictions for a variety of combinations of k and n  show evident saturation in firing-rate variance as mean firing rate increases. The remaining five show a milder saturation, as expected from their higher exponents (n = 1.1 to 1.2). Saturation was common, as the median n was 1.06, with n < 1 in 13/37 cells. Yet to my knowledge, except for an anecdotal account  = k[V−thresh]n+V, with [.]+ indicating rectification, k a proportionality factor, and n an exponent. Fitted parameters were threshV = −55.3 mV, k = 16.7, and n = 1.2 for the simple cell in threshV = −46.6 ± 10.5 mV, k = 12.4 ± 7.9, and n = 1.1 ± 0.6 for the whole intracellular population (N = 22). The distance between threshV and restV was 5.1 mV for the simple cell in The relation between potential  R e.g., H was fit R e.g., , where RVmean in response to a stimulus was defined as the mean across trials of coarse potential.The mean potential r (meanV isIn the Gaussian–rectification model, the probability of observing a firing rate r A given aR(V) is the relation between firing rate and potential V  depends on whether r is zero or positive:where ential V H, and N[otential B, a Gausr > 0, andfor r = 0. The first expression is simply the value of the Gaussian for V = −1R(r). The second expression is the area of the portion of Gaussian that is below threshold (erf is the error function).for R(V) are obtained independently . The required σ was always larger (by 2.1 ± 1.5 mV) than the standard deviations observed when meanV = rest,V but it was comparable (larger by only 0.9 ± 1.6 mV) to the standard deviations observed when meanV = thresh.VThese expressions allow maximum likelihood estimation of model parameters from measured firing rates. When parameters of the relation between firing rate and potential Vmean(t) be the mean potential at time t from stimulus onset. Because the sample rate is 100 Hz, each time sample corresponds to a 10-ms interval. Of course, meanV(t) depends on the stimulus. To simplify the notation, however, consider the case of a single stimulus.Let v ; (2) find the times (kt) when the mean potential meanV(kt) is within 2 mV of v; (3) pooling across trials j, look at the distribution of potential [jV(kt)] (e.g., a).Distributions for potential at a given mean potential B were co)] e.g., B, a.z-scores  of potential ; (2) for each interval i, find the set of times (ikt) when the mean potential is in the i-th bin; (3) pooling across trials j, compute i,σ the standard deviation of jV(ikt); (4) transform each jV(ikt) into a z-score: zijk= [jV(ikt) − iv]/i;σ (5) look at the distribution of (zijk)  e.g., A.The enhanced integrate-and-fire model was derived in collaboration with Davide where the currents are:spiket the time of the last spike. Ca(t) is the (unitless) calcium concentration:with spike < t.twhere the sum extends over all spikes with Nag(t) and Kg(t) derived from Hodgkin–Huxley equations and are scaled to approximate the spikes from the recorded neuron. They occur when mV exceeds a threshold, which depends on the time since the last spike:Spikes result from stereotyped conductances kV = −80 mV and NaV = 55 mV. Passive parameters of the membrane (mC = 120 pF, leakg = 12.4 nS, leakV = −60.3 mV) were obtained by fitting the membrane potential responses to sinusoids. The remaining parameters (AHPg = 23.0 nS, 50Ca = 10, τCa = 200 ms, Vthresh = −43.5 mV, τthresh = 36 ms) were obtained by a search algorithm aimed at maximizing the quality of the predictions for firing rate.The reversal potentials for sodium and potassium were set to


Few studies have focused on the association between the sociodemographic characteristics of a patient with the change in health-related quality of life (HRQOL) following invasive coronary procedures, and the results remain inconclusive. The objective of the present study was to measure the temporal changes in HRQOL of patients with coronary heart disease, and assess how these changes are associated with invasive coronary procedures and sociodemographic characteristics.t-tests and multiple regression analyses were used to assess temporal changes in HRQOL and to identify the associated factors.This was a prospective study of 254 patients with angina pectoris and 90 patients with acute coronary syndrome. HRQOL was assessed with the multi-item scales and summary components of the SF-36, both 6 weeks and 2 years after baseline hospitalization in 1998. Paired Physical components of HRQOL had improved most during the 2 years following invasive coronary procedures. Our findings indicated that patients with angina pectoris who were younger, male, and more educated were most likely to increase their HRQOL following invasive coronary procedures. When adjusting for baseline HRQOL scores, invasive coronary procedures and sociodemographic characteristics did not explain temporal changes in patients with acute coronary syndrome, possibly due to higher comorbidity.Sociodemographic characteristics should be taken into account when comparing and interpreting changes in HRQOL scores in patients with and without invasive coronary procedures. In the medical treatment of patients with coronary heart disease (CHD), invasive coronary procedures – such as percutaneous coronary intervention – are effective in reducing mortality and morbidity ,2. An imMost studies investigating the association between sociodemographic characteristics and HRQOL in patients with CHD focus on cross-sectional group comparisons -11. LongOnly few studies have focused on the association of sociodemographic variables with temporal changes in HRQOL following invasive coronary procedures. A recent observational study indicated that higher income is associated with greater improvement in physical HRQOL following invasive coronary procedures . ImproveIn the present study of patients with CHD, we aimed to (i) describe the effect of invasive coronary procedures on different domains of HRQOL at both 6 weeks and 2 years after baseline hospitalization, (ii) assess the association between sociodemographic characteristics and temporal changes in HRQOL.Baseline data in the present study were derived from the Norwegian study on outcomes research and quality improvement (RESQUA), a cross-sectional postal survey of HRQOL and the experiences of patients receiving hospital care . All patParticipants with CHD discharged from internal medicine wards were selected for a follow-up postal survey approximately 2 years later, in October and November 2000. We used information on primary and secondary diagnoses from the patient-administration systems of the hospitals, and included patients with acute coronary syndrome (ICD-9 410.xx and 411.xx) and angina pectoris (ICD-9 413.xx). Patients with chronic heart failure (ICD-9 428.xx) as the primary diagnosis were classified as angina pectoris or acute coronary syndrome dependent on their secondary diagnoses.HRQOL was measured by the Norwegian version 1.2 of the Short-Form 36 (SF-36), a widely used generic health status measure that enables comparison with normative scores ,29. The To estimate the potential impact of CHD on HRQOL, we compared the SF-36 scores from the patients in our study with normative data from the Norwegian general population . Norm scInvasive coronary procedures referred to diagnostic and therapeutic procedures, such as coronary angiography, which contributes to diagnosis of potential coronary artery disease, and when followed by angioplasty, it can contribute to a relief from chest-pain as well as improve the prognosis in high-risk patients. Procedure codes were derived from the patient-administration systems of the hospitals in 1998 . We defined invasive coronary procedures as a dichotomous variable, differentiating between patients with and without invasive coronary procedures during baseline hospitalization in 1998. The age and sex data were also derived from the administration systems in 1998. Information about the highest level of educational attainment was obtained from self-reported data in the 1998 postal survey. This variable was heavily positively skewed, and we therefore created two groups: (1) below and equal to, and (2) above the median value in our cohort. As a crude estimate of the degree of comorbidity for each patient, we used the total number of secondary diagnoses registered in the administration databases during baseline hospitalization in 1998.2-statistics or the t-test for independent samples to analyze the extent of selective attrition, and differences in the use of invasive coronary procedures across characteristics of respondents.Changes in HRQOL were only assessed in patients who had valid scores on all multi-item scales both in 1998 and 2000. We used χt-tests. As a measure of the minimally important difference in intra-individual scores, we calculated the standardized response mean, a distribution-based approach that compares temporal change by the standard deviation of change [Temporal changes in HRQOL were analyzed with paired-samples f change . Standarf change .Additionally, we applied multivariate linear regression analyses to determine the association of invasive coronary procedures and sociodemographic factors with PCS and MCS scores 2 years after baseline hospitalization. By including baseline PCS and MCS scores in the regression model, the regression coefficients of invasive coronary procedures and sociodemographic factors indicate the one unit increase in 2-year PCS and MCS scores, provided that baseline scores are held constant.All analyses were performed separately in patients with angina pectoris and acute coronary syndrome. We chose a 5% statistical significance level. The Regional Medical Research Ethics Committee, the Data Inspectorate, and the Norwegian Board of Health approved the study.A total of 1,534 patients with CHD were sent a questionnaire in 1998, and 1,059 (69%) responded. In 2000, 700 patients with valid HRQOL scores were sent a follow-up questionnaire (and, where necessary, one reminder), and 473 patients responded. After excluding 38 patients who had recently died and 9 patients with an unknown address, the adjusted response rate in the follow-up study was 72% . Among patients with valid HRQOL scores both in 1998 and 2000, women, elderly patients, and patients with higher comorbidity had fewer invasive coronary procedures during the baseline hospitalization  or percutaneous coronary intervention (N = 45). Twenty-one patients underwent coronary bypass surgery and the remaining patients (N = 4) underwent other medical procedures related to the cardiovascular system. Compared to the original cohort of patients, patients with valid HRQOL scores both in 1998 and 2000, more often had undergone invasive coronary procedures, were male, younger, and had lower comorbidity . PatienP = 0.001) and Social Functioning (P = 0.003) improved in angina pectoris patients without invasive coronary procedures, corresponding to a small effect size (Table 3). Physical Functioning (P = 0.03), Physical Role Limitations (P < 0.001), and Bodily Pain (P = 0.03) improved in patients with angina pectoris undergoing invasive coronary procedures. The change in Physical Role Limitations corresponded to a moderate effect size. A significant deterioration was found in General Health Perceptions (P = 0.04).Over the 2 years analyzed, Physical Role Limitations (P = 0.003), Social Functioning (P = 0.005), and Emotional Role Limitations (P = 0.009) significantly improved. Physical Role Limitations (P = 0.001) improved in patients with acute coronary syndrome undergoing invasive coronary procedures.In patients with acute coronary syndrome without invasive coronary procedures, Physical Role Limitations (P = 0.034 for angina pectoris and P = 0.015 for patients with acute coronary syndrome). MCS scores remained stable during the 2 years of follow-up; only patients with angina pectoris without an invasive coronary procedure experienced a small improvement in MCS scores (P = 0.007).Patients with invasive coronary procedures showed a small improvement in PCS scores . For thIn the present study, most improvement was found in the physical components of HRQOL 2 years following invasive coronary procedures. These results support the findings of Krumholz et. al.  that theWhen adjusting for baseline scores, invasive coronary procedures and sociodemographic characteristics did not explain any additional variation in PCS and MCS scores 2 years after hospitalization in patients with acute coronary syndrome. This may be due to the relatively small sample size. An alternative explanation is that patients with acute coronary syndrome exhibited higher comorbidity that could limit the effect of invasive coronary procedures on HRQOL, and accordingly, the sensitivity of the SF-36 in detecting differences .Our results demonstrated that invasive coronary procedures and sociodemographic characteristics were weakly associated with MCS scores and indicated small deviations from the population norm, which corresponds to previous findings in patients with CHD ,38. ThisOther factors may have influenced our results, for example selective attrition. In our study, the respondents to both surveys represent a survivor cohort, and hence attrition may have reduced the temporal changes in SF-36 scores and possibly lead to underestimation of the associations with invasive coronary procedures and sociodemographic factors. Moreover, the use of self-administered and postal questionnaires may have contributed to missing SF-36 items, especially in elderly subjects who are associated with a higher frequency of missing values for items used to score physical and emotional role functioning . The appAnother limitation of our study is the lack of HRQOL data before the baseline hospitalization in 1998, which prevented us from assessing the full impact of invasive coronary procedures on subsequent HRQOL and its association with sociodemographic characteristics. We also did not examine the influence of use of medical services after the baseline hospitalization. Finally, coronary patients and invasive procedures were defined by registry data from the patient-administration systems of hospitals, which might be inaccurate and mask some of the underlying clinical differences that could influence the HRQOL results.Our findings indicated that patients with angina pectoris who were younger, male, and more educated were most likely to increase their HRQOL following invasive coronary procedures. In patients hospitalized for acute coronary syndrome, temporal change in HRQOL was not associated with invasive coronary procedures and sociodemographic characteristics, possibly due to higher comorbidity. In a usual care setting the occurrence of invasive coronary procedures varies with sociodemographic characteristics ,43. The MV carried out the follow-up study, analyzed the data, and drafted the manuscript. KIP performed the baseline survey. AR participated in the design of the study. KS participated in the design and coordination of the study. All authors read and approved the final manuscript.Table 3: SF-36 multi-item and summary scales 6 weeks and 2 years after baseline hospitalization in patients with angina pectoris and acute coronary syndrome according to invasive coronary procedures (ICP). Norwegian norm data and 2-year change scoresClick here for fileB) and 95% Confidence Intervals (CI)Table 4 Predictors of Physical Component Summary (PCS) and Mental Component Summary (MCS) scores 2 years following the baseline hospitalization in patients with angina pectoris and acute coronary syndrome. Multivariate linear regression analysis; unstandardized regression coefficients (Click here for file


Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity.Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 [Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex. Endosomal compartments receive membrane bound cargo from both the biosynthetic and the endocytic pathways. Receptor downregulation by endocytosis includes transport to early endosomes and either recycling or sorting into late endosomes. The latter have the morphological characteristics of multivesicular bodies (MVB)  that canvacuolar protein sorting (VPS) into the class E compartment and are recruited to endosomal membranes from the cytosol in order to assemble into three ESCRT  complexes that function in MVB formation [Gene deletion and inactivation studies in yeast have identified 17 proteins that directly affect MVB formation (yeast class E compartment) by resulting in aberrant endosomal/vacuolar morphology . All proormation -11. Receormation . Initialormation , which iormation . ESCRT-Iormation ,9. Assemormation .Two recent crystal structures of a core of the ESCRT-II complex reveal a trilobal complex, containing two copies of Vps25, one copy of Vps22 and the C-terminal region of Vps36. Each subunit is composed of two winged helix domains and an N-terminal region of Vps25 interacts with Vps22 and Vps36 ,2.ELL Associating Protein; Vps22/EAP30; Vps25/EAP20; Vps36/EAP45), associated with the RNA polymerase II elongation factor ELL in the nucleus [Although ESCRT-II is essential for the MVB pathway, since cells missing ESCRT-II components fail to localize ESCRT-III to late endosomes ,9 the co nucleus ,15. Cons nucleus . To date that are arranged in tandem  [The structure of Vps25 was solved by single wavelength anomalous diffraction (SAD) using selenomethionine-derivatized crystals. Vps25 consists of two homologous winged helix domains as detected by the program GRATH m Figure . Winged ptional) , which fptional) ,2, folloptional) . Strand Two recent crystal structures of the ESCRT-II core reveal trilobal structures with head to tail interactions of one copy of Vps25 with Vps22 and the other copy of Vps25 with Vps36 at the center. In both cases a conserved proline rich N-terminal region of Vps25  and members of the CHMP protein family  are also found to act in the nucleus as well as in the cytosol and at endosomal membranes [Ste2 is downregulated via the endosomal pathway [Clear evidence suggests that ESCRT-II recruitment is involved in MVB formation leading to plasma membrane receptor downregulation . On the embranes -23. Inteembranes  and pher pathway . Similar pathway ,25. Prot pathway . Therefo pathway  a signalE. coli BL21 codon+ cells. For purification, cell pellets from 6 liter cultures were lysed in 150 mls of buffer A (50 mM Tris-HCl, pH 8.5, 200 mM NaCl, 0.2 mM DNaseI, 2 mM β-ME, 2 complete EDTA-free protease inhibitor tablets (Pierce)) and 0.1 mg/ml lysozyme for one hour on ice. The cell lysate was cleared by centrifugation and loaded onto a GST-sepharose (Pharmacia) column. The column was extensively washed with buffer B (50 mM Tris pH 8.5, 200 mM NaCl) and Vps25 fusion protein was eluted with buffer B containing 5 mM reduced glutathione. GST was then removed by TEV cleavage  at 4°C overnight. His-tagged GST and TEV were subsequently both removed on a Ni2+ chelating sepharose column. Vps25 was further purified on a superdex75 column (Pharmacia) in buffer C (50 mM Tris 8.5, 200 mM NaCl, 2 mM βME). Selenomethione-labeled Vps25 was produced using standard procedures and purified as described above.Full length yeast Vps25 DNA (gene bank #CAA89632) was cloned into expression vector pETM30 (EMBL, Protein Expression Facility) and the Vps25 GST fusion protein was expressed in Crystallization conditions for Vps25 (7 mg/ml) were first determined by screening 600 conditions using a Cartesian crystallization robot. Initial conditions were refined using the hanging drop method, and the final crystallization condition (100 mM Na cacodylate pH 6.5, 200 mM Mg or Ca acetate, 5–7% glycerol, and 15–18% polyethylene glycol 8000) produced rectangular- and wedge-shaped selenomethionine-labeled Vps25 crystals in the same drop. Native Vps25 crystallized initially only with rectangular morphology and wedge-shaped crystals were produced by microseeding with the original SeMet crystals. For cryogenic data collection, the crystals were equilibrated in 25% glycerol and flash cooled in a gaseous nitrogen stream at 100 K.12121 with unit cell dimensions as indicated  beamline ID14-EH3 and data from SeMet-labeled crystals were collected at the ESRF beam line ID29 at three wavelengths . Accordingly, mol D is poorly defined (43 residues missing out of 204). The final R factor and R free (0.275/0.327) reflect missing residues and the poor model for molecule D. The model exhibits otherwise overall good stereochemistry with no outliers in the Ramachandran plot as defined in PROCHECK (table . Sequences were aligned using Clustalx [Figures were generated using coordinates of molecule C with programs MOLSCRIPT , Raster Clustalx . SecondaClustalx . The burClustalx  and the Clustalx .W.W. conceived of the study, and participated in its design, coordination and writing of the manuscript. W.W. expressed, purified and established initial crystallization conditions and participated in data collection. A.K.W. carried out data collection, structure solution and refinement and participated in writing of the manuscript. All authors read and approved the final manuscript.


In addition, the cranial muscles that normally attach to the affected cartilage region and their associated nerve are secondarily reduced in −integrinα5 animals. Earlier in development, integrinα5 mutants also have specific defects in the formation of the first pouch, an outpocketing of the pharyngeal endoderm. By fate mapping, we show that the cartilage regions that are lost in integrinα5 mutants develop from neural crest cells directly adjacent to the first pouch in wild-type animals. Furthermore, we demonstrate that Integrinα5 functions in the endoderm to control pouch formation and cartilage development. Time-lapse recordings suggest that the first pouch promotes region-specific cartilage development by regulating the local compaction and survival of skeletogenic neural crest cells. Thus, our results reveal a hierarchy of tissue interactions, at the top of which is the first endodermal pouch, which locally coordinates the development of multiple tissues in a specific region of the vertebrate face. Lastly, we discuss the implications of a mosaic assembly of the facial skeleton for the evolution of ray-finned fish.Pharyngeal endoderm is essential for and can reprogram development of the head skeleton. Here we investigate the roles of specific endodermal structures in regulating craniofacial development. We have isolated an  A developmental mutant of the zebrafish is helping to uncover the processes by which the facial skeleton is patterned in vertebrates The skeletal elements that form and support the vertebrate jaw and gills are derived from a specialized population of ectomesenchyme cells, the cranial neural crest . In the casanova mutant embryos make no endoderm and pharyngeal cartilages fail to form, and transplantation experiments show that wild-type endoderm is sufficient to rescue cartilage development (tbx1 (vgo) mutant embryos, most pouches fail to develop and posterior pharyngeal cartilages are reduced and fused together  23 between the markers Z5141 and Z20492, a region containing a zebrafish integrinα5 homolog  (integrinα5 mutants, we saw variable reductions of the SY element in addition to aHM (integrinα5 (b926) phenotype, HS was reduced to a rod that variably fused with CH; rarely, the first arch joint was fused as well , the CH, and the hyoid opercle bone remained unaffected. In addition, integrinα5 mutants had variably reduced numbers of ceratobranchial (CB) cartilages and rare fusions of adjacent CBs  B. In othn to aHM C. In wha as well D. Howeveintegrinα5 mutants  transgenic line  and persists as pharyngeal cartilages and bones develop. Pouches are evident as nonexpressing regions between the GFP-labeled crest-derived cells of the arches. We sorted live −integrinα5 animals for first pouch defects at 36 hpf and then grew these animals to 4 d to analyze pharyngeal cartilages. Strikingly, we found a strong correlation between reductions of the first pouch and later hyoid cartilage defects in integrinα5 mutants. −integrinα5 sides that lacked the first pouch early had hyoid cartilage defects 93% of the time (n = 116), whereas only 7% of −integrinα5 sides with a normal first pouch early developed hyoid cartilage defects (n = 108). The correlation was highly significant (p < 0.0001).As both the pouch and cartilage phenotypes were incompletely penetrant in nic line . fli1-GFintegrinα5 mutants, the dorsal first and second arch muscles, do and ah, were selectively reduced, whereas levator arcus palatini, ao, and lo were present but appeared closer together  cranial muscles. Second arch mesoderm gives rise to interhyal and hyohyal muscles, ventrally, and adductor hyomandibulae (ah), adductor operculi (ao), and levator operculi (lo) muscles, dorsally M. In a ftogether . The musintegrinα5 mutants had specific defects in the nerve that innervates second arch muscles. Facial motor neurons send a ventral-directed nerve (VII) that innervates dorsal second arch muscles ah and ao, passes through the foramen of HM, and subsequently branches to innervate ventral muscles interhyal and hyohyal , expressed integrinα5, whereas integrinα5 expression was no longer seen in the first pouch . In conclusion, integrinα5 expression in the pharyngeal endoderm is in a pattern that both spatially and temporally corresponds to regions of pouch formation, and expression of integrinα5 in the crest begins during premigratory stages and later becomes refined to patches of crest derivatives within the pharyngeal arches.In order to understand where Integrinα5 might be acting to control pharyngeal arch development, we examined the expression of ribution A. Matern in frog . By gasttegrinα5 B. At theecursors C. In addecursors . By 5-s tegrinα5 D, and phtegrinα5 E. From 1 placode F and 3G.sections H–3J. In st pouch H. At latst pouch K. At 38 pressing L. In addintegrinα5 expression was observed in both pharyngeal endoderm and neural crest, we used transplantation experiments to determine in which tissues Integrinα5 was required for first pouch and hyoid cartilage development   J. Thus, −integrinα5 cartilage phenotype requires a fate map of pharyngeal cartilages in wild-type animals. Here we focus on the origins of the SY, aHM, and pHM regions of HS, and a more complete mandibular and hyoid fate map will be published elsewhere. We used in vivo microelectroporation . These results are consistent with the first pouch being required for the survival of hyoid crest that contributes to aHM and SY.We next investigated whether the losses of aHM and SY regions in oid arch . At 25 h embryos B, and 6Eo 35 hpf D and 6E.o 35 hpf B and 6D,st cells B′. Inter develop E. In addintegrinα5 mutant embryos. Hyoid crest expresses Hox class 2 genes, whereas mandibular crest is Hox nonexpressing (hoxa2 in the hyoid arches of integrinα5 mutants at 36 hpf (data not shown). In 36-hpf wild-type animals, goosecoid (gsc) expression marks dorsal and ventral domains within the hyoid arch  that has variably penetrant and expressive reductions of the first pouch and hyoid aHM and SY cartilage regions. By positional mapping, allele segregation, and morpholino phenocopy, we identified the genetic basis of the lesion as a missense mutation in the ligand-binding domain of Integrinα5. As similar pharyngeal phenotypes were observed in mutant and morpholino-treated animals, we conclude that b926 is a loss-of-function allele of integrinα5. However, we do not know if Integrinα5 activity is completely eliminated in b926. In addition, we observed strong maternal expression of integrinα5 that could mitigate the zygotic loss of integrinα5 in b926. Thus, the variable penetrance and expressivity of the integrinα5 phenotype could be due to partial activity of mutant Integrinα5 or the presence of maternally supplied Integrinα5. Additionally, other integrins may act redundantly with Integrinα5 in pharyngeal development. A survey of nearly finished genome sequence (http://www.ensembl.org/Danio_rerio/) has uncovered at least 15 additional Integrin α chains, for which no expression or phenotypic data are known in zebrafish. Lastly, integrinα5 is expressed strongly in many tissues, such as the otic placode, for which no overt phenotypes were observed in b926. Future studies, in particular those using animals in which both maternal and zygotic integrinα5 have been eliminated, may reveal new functions of Integrinα5 in zebrafish development.In this work, we isolated and characterized a zebrafish mutant allele −integrinα5 animals to show that early first pouch defects are strongly predictive of later hyoid cartilage defects. Furthermore, transplantation experiments show that wild-type endoderm, but not crest, rescues first pouch and hyoid cartilage development in integrinα5 mutants. We infer that Integrinα5 functions in the pharyngeal endoderm for the formation of the first pouch, and that the first pouch, in turn, interacts with postmigratory neural crest to promote cartilage development in a region of the hyoid arch. A role for the first endodermal pouch in promoting regional hyoid cartilage development is consistent with work in chicken showing that domains of pharyngeal endoderm specify region-specific cartilage shapes . Although more embryological studies of Polypterus need to be done, the evolution of the HM plate in ray-finned fishes such as teleosts appears to have involved a new induction event that led to a new region, pHM, being added to an older region, aHM. We propose that the de novo addition of regions to the skeletal pattern represents another mechanism, in addition to differential growth, of generating skeletal diversity during evolution.Our data show that the first endodermal pouch is required for the development of aHM, yet in mutants that lack all pharyngeal endoderm, such as are lost . Thus, o(Danio rerio) raised at 28.5 °C were staged as previously described  . A morpholino (Genetools, Philomath, Oregon, United States) was designed against the exon13-intron splice site of integrinα5 (itga5-MO) with the following sequence: ATGCTTTCTCACCTGGGTAGCCATT. Embryos were pressure-injected with 5 nl of 2 mg/ml itga5-MO as previously described (escribed .ab fragments (1:20,000, Molecular Probes) and goat anti-rabbit Alexa Fluor 568 antibodies (1:200, Molecular Probes). GFP fluorescence survived the procedure.Alcian Green staining was performed as described . For flaintegrinα5 RNA probes were made from plasmids pINT4150 and pINT4853, constructed by inserting RT-PCR fragments corresponding to nucleotides 254–2,207 and 1,679–2,960 of the integrinα5 cDNA, respectively, into the TA vector . Plasmids were linearized with BamHI, and T7 RNA polymerase was used for probe synthesis. Both probes gave identical expression patterns, and pINT4853 was used for photographs. pea3 (gsc (Probe syntheses and whole-mount in situ hybridizations were performed as previously described . Embryoshs. pea3  and gsc ea3 (gsc  probes wfli1-GFP embryos, 24 hpf, were dechorionated, anesthetized with tricane solution, and bathed in a solution of 5 mg/ml pronase (Sigma) for 1 min to allow passage of the microelectrode through the skin. Agar mounting of embryos on slides was performed as described in The microelectroporation technique was similar to that described by fli1-GFP-expressing hyoid arch was assessed using confocal microscopy. Only embryos with one or two adjacently labeled cells were used in the analysis. Three-dimensional projections were constructed to determine the position of labeled cells in the arch relative to landmarks. All cell distances were made from the mid point of the cell to the landmark. Distance of the labeled cell from the edge of the first pouch was used to determine A-P position. Similarly, distances of labeled cells from the dorsal and lateral edges of the arch were used to determine D-V and mediolateral positions. In electroporations where two adjacent cells were labeled, the positions of each cell were measured and averaged. To control for variation in arch dimensions among individuals, measurements along the three axes were normalized to total axes lengths. At 4 d, embryos were imaged again to determine the fate of labeled cells in hyoid cartilage. The aHM region was defined as the anterior portion of HM that is characteristically lost in integrinα5 mutants. pHM comprises the rest of the HM region. The SY region begins at the point of attachment to pHM. All graphing and statistical analysis were done using JMP (2002, SAS Institute, Cary, North Carolina, United States).Shortly after electroporation, the location of labeled cells relative to the Embryos were manually dechorionated, anesthetized with tricane solution, transferred to 0.2% agarose in embryo media with 10 mM HEPES and tricane, and then mounted onto a drop of 3% methylcellulose on a rectangular coverslip with three superglued #1 square coverslips on each side. A ring of vacuum grease was added around the embryo to make an airtight seal upon addition of the top coverslip. A heated stage kept the embryos at 28.5 °C. Approximately 80-μm Z-stacks at 2-μm intervals were captured every 10 min using a Zeiss LSM 5 Pascal confocal fluorescence microscope. Movies of individual Z-sections were made by manually following cells and concatenating sections; further processing was done with Adobe Premiere. For single time point confocal sections, embryos were mounted without vacuum grease.fli1-GFP host embryos. For the crest transplants, donor fli1-GFP embryos were injected at the one-cell stage with the Alexa Fluor 568 mixture (“Alexa568”). At shield stage (ca. 6 hpf), donor tissue was taken from the animal cap and moved to a position approximately two germ ring widths from the margin and 90° from dorsal in a fli1-GFP host embryo. Confocal images of host embryos were captured at 38 hpf and 4 d, and embryos were subsequently fixed and Alcian-stained to visualize cartilages. For endoderm transplants, only embryos in which donor tissue contributed to the second and at least one other pouch were included in the analysis. For crest transplants, only embryos in which at least half the hyoid arch was composed of donor tissue were included in the analysis.Transplant techniques were as described . For endVideo S1fli1-GFP animal from 38 hpf to 86 hpf. At the beginning of the video, the mandibular (1) and hyoid (2) arches are numbered and an arrow denotes the normal position of the first pouch. At the end of the video .Click here for additional data file.Video S2fli1-GFP animal from 38 hpf to 86 hpf. This video is a different depth of the same time-lapse recording as Confocal time-lapse recording shows hyoid cartilage development in a wild-type (7.1 mB MOV).Click here for additional data file.Video S3−; fli1integrinα5-GFP animal from 38 hpf to 86 hpf. At the beginning of the video, the mandibular (1) and hyoid (2) arches are numbered and an arrow denotes the normal position of the first pouch. At the end of the video .Click here for additional data file.http://www.ncbi.nlm.nih.gov) accession number for the zebrafish integrinα5 cDNA is AY550244. Accession numbers for the related Integrinα5 proteins described in Homo sapiens (Genbank P08648), Mus musculus (Genbank P11688), Xenopus laevis (Genbank Q06274), and Fugu rubripes .The Genbank (


Alopecia areata (AA) is a common and chronic skin disease with an unknown etiology. It may significantly affect the patient quality of life. This study was designed to evaluate the illness perception in patients with AA.A questionnaire consisting of 25 questions about causes, timeline, consequences and control of disease were given to 80 patients with AA attending a skin clinic in Tehran, Iran. The impact of age, gender, duration of disease, education, extent of disease and family history of AA were also assessed.Eighty patients  with a mean age of 27.5 years (SD = 9.3) and disease duration of 7.8 years (SD = 7.7) completed the questionnaire. 76.9% of the patients believed that the role of stress was the cause of disease. 17.1 % believed genetic background to be the main cause, this found to be more frequent in patients with positive family history of AA. More than half of patients believed that their illness had major consequences on their lives and 40% of patients believed that their illness would be likely to be permanent rather than temporary, more in patients with longer duration of disease. Only 57.5% of patients considered their treatments to be effective.AA may considerably affect various aspects of patients' lives. The patient knowledge about the causes and course of this disease is limited. Alopecia areata (AA) is manifested as a sudden loss of hairs without any inflammation or scarring. The hair loss might be seen in a circumscribed area or the whole scalp  or whole body  . It is aThe onset of a chronic condition brings with it a range of difficulties that may show considerable variation in their nature and severity as perceived by the patient. In order to make sense of and respond to the difficulties that chronic illness may present, patients construct their own common-sense cognitive model of their condition. Such models are based upon information received from a range of sources including their physician, family, friends, and existing social and cultural notions about health and illness. The resulting system of beliefs can of course be flawed or inaccurate; however there is evidence that it is those beliefs that drive attempts to cope with a condition and issues of compliance with treatment. Patient-held beliefs have important implications for the clinical management of their disease. Studies on patients with psoriasis and acne vulgaris have shown that knowledge of patients about their condition, the course of their disease and current treatments is not appropriate ,7.In this study, we examined the system of beliefs held by AA patients and the factors that might influence such beliefs.The Illness Perception Questionnaire (IPQ)  with a fCause subscale (10 items) measures personal ideas about the cause of AA.Time line (3 items) deals with perceptions about how long the disease will last.Consequences (6 items) are concerned with expected effects and outcomes of the illness.Cure/Control (6 items) details beliefs about recovery from or control of the condition.There were four possible answers for each item in the IPQ to be chosen by patients: I strongly agree, I agree, I do not know, I disagree.Furthermore, some demographic information such as age, sex, family history of AA, and duration and extent of disease , and level of education were obtained from the patients to evaluate their influence on patients' beliefs.Statistical analysis was conducted by means of SPSS statistical software, version 11.0. Because the data were not normally distributed, nonparametric statistics were used. Correlations were processed by Spearman's rank correlation, and differences between means were computed by means of the Mann-Whitney U test. For simplicity of analysis and increasing the power of study, the answers of "I strongly agree" and "I agree" were grouped together and compared with the answers of "I don't know" and "I disagree" which were grouped together as "I do not agree". A p value of less than 0.05 was considered as significant.A total of 80 patients with AA , with a mean age of 27.5 years (SD 9.3, ranged from 13 to 56 years) were recruited to the study. The mean duration of illness was 7.8 years (SD 7.7, ranged from 1 month to 30 years). In 75% of patients, AA was patchy and it was totalis or universalis in 25%. Fifteen percent of patients had a positive family history of AA in their first degree relatives. Physicians were the main source of patients' information about their disease in 66.2% of them.cause item. A total of 76.9% of patients believed that stress was a major factor in onset of their illness and older patients were more likely to believe in this (p < 0.05). Patients who had a belief that their disease was a result of genetic factor were more likely to have a family history of AA and longer duration of the disease (P < 0.05). Younger patients and those with extensive disease (AT/AU) believed that their illness was because of chance or fate (P < 0.05).Table. consequence item.Majority of the patients 58.2%) believed that their illness had a major consequence on their lives, 53.8% of patients also felt that AA had strongly affected their self-esteem, and 50.6% considered AA as a serious condition. These believes were stronger in younger patients, and in patients who had the disease for a long time (p < 0.05). Table % believeHalf of the patients believed whether their disease cleared, it would always come back and forty percent of patients believed that their illness would be likely to be permanent rather than temporary. They were more likely to have a longer duration of disease (p < 0.05). The minority of patients (25.0%) believed that their illness would last a short time.More than 60% of patients believed that their behavior could determine improvement or worsening of their illness table . This beAlopecia areata is a chronic disease which may influence individual or social aspects of patients' lives. The results of our study confirmed this fact as the majority of patients believed that their illness had strongly affected their lives. It also influenced their self-esteem. The results of studies in other chronic diseases with periods of remission and exacerbation have had different results in this respect. For example in a study on acne patients, the disease had affected patients' self-image in nearly all of them, but it had no impact on interpersonal relationships, work, or school activities in majority of patients . On the The present study also investigated cognitive appraisals held by patients about their illness and showed that such beliefs were not associated in any significant manner with the extent of their condition. Fortune et al also did not find an association between the clinical severity of psoriasis and beliefs held by patients about their condition . Thus thThe results of our study also showed that the beliefs about the consequences of having AA were not influenced by the gender of the patients. Thus men are as vulnerable as women in suffering from the consequences of AA.et al. showed that emotional stress is not an important factor in the initiation of AA [et al. did not find a significant role of stress in the onset of AA but stressful life events had an important role in triggering of some episodes of disease [et al. found that AA patients who were depressed, were more likely to mention stress as the cause of their disease [Patients with AA, including 77% of patients in this study, often attribute the onset of their disease to a specific stressful life event. In a study on 178 patients, Van der Steen on of AA . Brajac  disease . On the  disease .In recent studies, psychologic and psychopathologic factors have been analyzed as modulators of neuroendocrinologic, vascular, and immunologic variables; this is far from the initial concept of stress being the causal agent in the illness. In fact, stress may cause its effect by making alterations in immune responses related to neuropeptides, such as the migration of the macrophages, vasodilator or vasoconstrictor responses, phagocytosis, lymphocytic cellular immunity, and expression of some factors of leukocytic adhesion to the microvascular endothelium . In addiThis study also showed that as AA lasts, the patients feel more hopeless about time line and treatment modalities of their disease. The majority of patients had no hope to get rid of their disease. Almost half of patients expected their disease to relapse after it disappeared. Such perpetual stresses are hardly endurable. The psychiatrists' intervention may alleviate patients' stress and improve their quality of life.This study was performed in a private dermatology clinic. The possibility of socioeconomic homogeneity among recruited patients may be biasing the results. So it should be considered that these results may be different in patients with AA in different socioeconomic and cultural backgrounds.There is a need for accessible, accurate, community-based education on the natural history of AA, the effectiveness and expected duration of treatment. The inadequacy of information provided by current sources is evident in ongoing misconceptions on causality and the perceptions of respondents. Incorporating information on this disease may facilitate patient into therapeutic selection, enhance understanding of treatment options and improve patient compliance.The author(s) declare that they have no competing interests.AF participated in the design and conduct of the study and preparation of the manuscript.MRF participated in the conduct of the study and statistical analysis.BG participated in the conduct of the study and statistical analysis.YD participated in the design of the study and preparation of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Imagination and creativity are essential traits that medicine, and medical insurers, must again learn to recognize and reward Galvanized by rising costs, increased calls for greater, accountability, and an Institute of Medicine (Washington, D. C., United States) report suggesting that medical errors may kill nearly 100,000 Americans every year The problem is, most of these reform efforts, while critically important, only capture half the picture. Efficiency isn't everything, and unless we learn to cultivate creativity as avidly as we pursue consistency, future generations of patients may find themselves stuck with the same basic treatments they're receiving today. It will be the same medicine, just served quickly.From its earliest days, medical training was based on an apprenticeship model, in which junior acolytes learned the art from senior practitioners. Even with the evolution of modern medical schools, which offered future physicians a rigorous common training, once doctors entered the real world they essentially did as they pleased. Consequently, there were pronounced differences in approaches to common problems from one clinician to another.There was also little to guarantee that once doctors had hung out their shingle, they were actually competent (and remained competent) to practice their craft. While most physicians remained committed to the general professional standard—do the best that you can for each individual patient— many well-meaning doctors ultimately were not delivering their patients the best care available.More recently, and largely due to the contagious spread of the so-called “business model,” there has been an increased emphasis on the consistency and quality of care. The clear goal is ensuring that all patients truly receive the very best care available, as defined by rigorous scientific studies.This discrepancy between what patients should be receiving and what patients are actually receiving is the major focus of quality reform, and reflects the new recognition that there are truly preferred approaches—pathways—to guide disease management. These pathways are not meant to represent a rigid algorithm reflexively applied to each patient, but are intended as a summary of the best available data, a useful template to guide further medical decisions.The renewed emphasis on quality has also resulted in a newfound appreciation for the role of experience and repetition in patient care. Study after study has shown that the best physician to treat a particular problem is the one who has treated it the most The great paradox here is that the same reforms that are improving our current care may also be endangering our future health. As medicine has become more standardized and increasingly regulated, it turns out there is much less room for innovation. The spirited pursuit of the unknown—so long a defining quality of medicine—now seems seriously endangered. The new world of rapid throughput and endless documentation provides little time to reflect upon important clinical problems and consider fresh approaches. If anything, thinking about a patient or a question too much is now implicitly discouraged because it slows doctors down; contemplation is bad for productivity.Academic medical centers like our own have played a particularly important role in the history of medical discovery; the hallmark of these institutions is our commitment to thinking and reflecting about the patients we see, patients who are often extremely sick and whose management is exceptionally complex. Unfortunately, many of the measurements now used by insurance companies to assess quality pay little attention—if any at all—to the complexity of a patient's illness, or to the importance of spending time trying to define the underlying malady. Insurance companies' major concern seems to be how fast a patient is “processed,” ideally with as few tests as possible. These measures provide no mechanism for distinguishing between the addled physician who inappropriately orders every test that springs to mind, and the reflective physician who is trying to get to the bottom of a patient's complaint, rather than simply throw a Band-Aid over the symptoms Situated on the front lines, clinicians have a unique opportunity to provide new medical insights and to identify critical, unanswered questions. Classic examples include Archibald Garrod, a British physician whose desire to understand why a patient produced black urine led to the hypothesis that diseases can result from defective metabolic enzymes, and Fuller Albright, a clinical investigator at Harvard whose thoughtful approach to his patients yielded insights that revolutionized the field of endocrinology. More recently, the astute clinical observations of UCLA immunologist Michael Gottlieb resulted in the original description of the Acquired Immune Deficiency Syndrome (AIDS) in 1981 But where are these types of insights going to come from today? It seems difficult to imagine that a medical care environment characterized by staccato-quick patient visits covering an ever-increasing number of compulsory topics will support or encourage such reflection and innovation. Our failure to nourish and sustain inquisitive physicians seems particularly tragic because medicine has traditionally attracted some of our brightest and most imaginative individuals. Even at the height of the dot-com boom, for example, there were still more medical school applicants than there were spaces to train them. But if current trends continue, many of these creative minds will head elsewhere, while those who stay will risk becoming stultified by repetitious routine.www.pasteur.hms.harvard.edu) Several medical schools and a handful of foundations have recognized this emerging problem, and have initiated programs aimed at sparking curiosity in young doctors (our own school's program is called the PASTEUR initiative—see Even as we strive to improve the consistency of care—and striving is clearly a very good idea—we must continue to cultivate novelty and originality, rather than penalize it. Imagination is perhaps the most essential trait that medicine, and medical insurers, must again learn to recognize and reward. Even with the best algorithms and the brightest computers, the future of health care ultimately depends upon the creativity of the hardy men and women still entrusted with its delivery.


While sleep disturbance is widespread in schizophrenia it is less clear whether sleep disturbance is uniquely related to impaired coping and perceived quality of life.We simultaneously assessed sleep quality, symptoms, and coping in 29 persons with schizophrenia or schizoaffective disorder in a post acute phase of illness. Assessment instruments included the Pittsburgh Sleep Quality Index; the Positive and Negative Symptom Scale; the Heinrichs Quality of Life Scale; and the Ways of Coping Scale. Multiple regressions were performed predicting quality of life and coping from sleep quality controlling for age and symptom severity. On a subset of seven subjects non-dominant wrist actigraphy was used as an objective check of their self-reported poor sleep.Analyses revealed that poor sleep quality predicted low quality of life  and reduced preference for employing positive reappraisal when facing a stressor . Actigraphy confirmed poor sleep quality in a subset of subjects. They had shorter sleep duration (p < .0005), shorter average sleep episodes (p < .005) and more episodes of long awakening (p < 0.05) than community norms.The results are consistent with the hypotheses that poor sleep may play a unique role in sustaining poor quality of life and impaired coping in patients with schizophrenia. These associations may hold for community controls as well. Many persons with schizophrenia report chronically disturbed sleep ,2. IndepWhile sleep disturbance appears widespread in schizophrenia and is related to clinical features, less clear is whether it is also related to impaired coping that characterizes the disorder. Is poor sleep quality another factor that uniquely contributes to the difficulties persons with schizophrenia experience coping with stressors, and sustaining relationships and work? There appear to be several reasons to hypothesize that it may. Impaired sleep may make it difficult for persons with schizophrenia to cope with stressors . PersonsTo examine relationships between sleep quality, coping and quality of life, we simultaneously assessed these domains along with symptom level in a group of patients with chronic schizophrenia and schizoffective disorder in a post acute phase of illness. To assess sleep quality we used the Pittsburgh Sleep Quality Index (PSI ), a stanSome doubt the abilities of persons with schizophrenia to report their own sleep patterns accurately. However, a study comparing polysomnography with subjective measures of sleep found that they were highly correlated in a group of patients with chronic schizophrenia . SimilarIn addition, we wanted to know if actigraphy is an effective way of demonstrating sleep problems in the post-acute phase of schizophrenia. To address this question, we used actigraphy to monitor sleep patterns in a subset of this patient population. Wrist actigraphy consists of monitoring locomotor activity with a motion sensor slightly bigger than a wrist-watch that is worn on the non-dominant wrist. It is a common method used to assess sleep . ActigraAll subjects met the Structured Clinical Interview for DSM IV (SCID ) diagnosSensors (Model # 24.000, Ambulatory Monitoring Inc, Ardsley, NY) slightly bigger than a wristwatch were worn by subjects on their non-dominant wrists for 21 days. Actigraphic logs of the non-dominant arm have a high correlation with gross locomotor activity . ActigraThe PSI is an effective instrument used to measure the quality and patterns of sleep. It differentiates "poor" from "good" sleep by measuring seven subscales: Subjective Sleep Quality, Sleep Latency, Sleep Duration, Habitual Sleep Efficiency, Sleep Disturbances, Use of Sleeping Medication, and Daytime Dysfunction over the last month. The client self-rates each of these seven areas of sleep by answering nine questions. Scoring of answers is based on a zero to three scale, and a score of three reflects the negative extreme on the Likert Scale. A global sum of "5" or greater indicates a "poor" sleeper. The PSI has internal consistency and a reliability coefficient  of 0.83 for its seven components. Numerous studies using the PSI have supported high validity and reliability.The PANSS is a 30 item rating scale completed by clinically-trained research staff at the conclusion of chart review and a semi-structured interview. For the purposes of this study the five PANSS factor analytically derived components are used: Positive, Negative, Cognitive, Excitement and Emotional discomfort .The tool is a 21 item scale completed by clinically trained staff after a semi-structured interview and chart review that assesses quality of life. Items are scored on a 7-point scale with higher ratings representing higher levels of satisfaction. Items tap a range of essential aspects of psychosocial interactions incorporating four subscales: Interpsychic Foundations; Occupational Functions; Commonplace Objects; and Activities and Interpersonal Relationships. For the purposes of this study we used the sum of all items as an index of overall quality of life. Interrater reliability for this instrument has been reported to range between 0.85 and 0.93 .The WCQ is a self-report instrument that asks participants to call to mind a recent stressor and then rate how often they have used 66 different behaviours to cope with that stressor. Scale scores are additively derived from individual items and divided by a total score to provide a relative score. This relative score reflects participants' relative preferences among a set of discrete coping strategies. Relative scores are generally preferable because, among other things, they control for response bias. For the purposes of this study we calculated the relative scores for two subscales: "Escape Avoidance," and "Positive Reappraisal." Escape Avoidance describes wishful thinking and behavioural efforts to actively escape or avoid the problem and includes items such as: "I refused to believe it had happened" and "I wished the situation would go away or be over with." Positive Reappraisal describes efforts to create positive meaning by focusing on personal growth and includes items such as: "I changed or grew as a person in a good way" and "I found new faith." Internal consistency assessed using Cronbach's alpha have been reported to range from 0.61 to 0.79.Following informed consent participants were administered the PSI. PANSS, QLS, and WCQ were completed within the previous month for another study of the effects of personality on function in schizophrenia. Diagnosis was determined using the SCID. Ratings of sleep quality were elicited by a research assistant blind to the results of the PANSS, QLS and WCQ. We fitted a subset of patients with actigraphs that they wore for 21 days.® software. We also repeated the correlations for patients with schizophrenia separate from the patients with schizoaffective disorder.The statistical analyses were Pearson correlations and Mixed Models analyses in SASMean scores ± SD on the tests were: Quality of Life total 51.73 ± 16.16; Pittsburgh Sleep Quality Index 11.6 ± 4.57; Ways of Coping Questionnaire subscales: "Escape Avoidance," 0.138 ± 0.062 and "Positive Reappraisal" 0.125 ± 0.079. There was no correlation between QLS and "Escape Avoidance" scores, but a modest positive correlation between QLS and "Positive Reappraisal" (r = 0.421, p = 0.021).To examine the associations between sleep and quality of life we next performed partial correlations, using the PSI total score to predict QLS total score and QLS subscores with age and positive and negative component scores as covariates. Similarly, we used PSI subscores to predict QLS total score. Results revealed that poor sleep was related to poor quality of life . The PSI accounted for 24% of the variance in QLS. Total PSI did not correlate with any of the QLS subscores. The PSI subscores: "Subjective Sleep Quality", "Sleep Duration", and "Sleep Disturbances" correlated with total QLS at p < 0.05 but none were significant after making the Bonferroni correction for multiple comparisons.To examine the associations between sleep and coping style we next performed partial correlations, using the PSI total score to predict WCQ Escape Avoidance, and WCQ Positive Reappraisal score with age and positive and negative component scores as covariates. Results revealed that poor sleep was related to a reduced preference for Positive Reappraisal . Sleep quality was found to be unrelated to Escape Avoidance. The PSI accounted for over 37 % of the variance in coping.Patients with schizoaffective disorder had worse sleep as measured by the PSI than did patients with schizophrenia . The two groups did not differ in total scores of the QLS or WCQ. The correlations between total PSI and total QLS, and between total PSI and Positive Reappraisal for patients with schizophrenia were significant . The same correlations were not significant for patients with schizoaffective disorder.Seven participants with schizophrenia accepted the activity monitors and were willing to wear them continually for up to 3 weeks. Seven patients completed actigraphic monitoring, and the actigraphic results are in Table The results are consistent with the hypothesis that poor quality sleep that typically characterizes schizophrenia may have a powerful impact on both patients' perception of their quality of life and their ability to cope with stress. Independent of age and symptoms, poorer sleep quality predicted poorer quality of life and greater difficulties appraising stressors in a positive light. This may suggest that among the deficits accompanying schizophrenia, poor sleep is often underappreciated. Impaired sleep may erode the ability of schizophrenic patients to cope with the routine stress associated with work and interpersonal relationships. Furthermore, chronic sleep deprivation may contribute to anergy that impairs attendance and work performance. Since the found associations are independent of symptom level they may be trait variables.These findings are consistent with polysomnographic studies of sleep in schizophrenia which show that many sleep deficits that are not dependent on the acuity of illness -7. The fThe negative relationship between complaints of poor sleep quality and Preference for coping by Positive Reappraisal remained significant when the confounding effects of symptom acuity and age were partialled from the correlation matrix. One interpretation is that chronically disturbed sleep may erode both the ability to find positive meaning and the desire to achieve personal growth. Given the correlative nature of the data analysis, however, it is not possible to infer causality. It may be that poor coping or poor quality of life lead to greater difficulties sleeping or that another unmeasured variable may account for the relationships. We plan future longitudinal studies that may uncover if sleep changes typically precede or follow changes in either coping or quality of life.There are other limitations to this study; most participants were male and in their 40s. Further experiments will be needed which include females and males in earlier phases of illness. The association of disturbed sleep and both quality of life and coping may hold for community controls as well.These findings may have important clinical implications. If poor sleep quality is indeed a critical factor in quality of life and coping impairments in schizophrenia, clinicians will need to focus on and aggressively treat sleep problems. Specific treatments could include training in sleep hygiene with a focus on regular waking and sleep times, avoiding naps, morning bright light, evening melatonin, or other hypnotic agents. Improved sleep may lead to improved ability to cope with stress, and increased energy. These would improve the quality of life and coping in patients with schizophrenia.The author(s) declare that they have no competing interests.JH participated in the design and coordination of the study and performed the statistical analysis and helped to draft the manuscript. PL conceived of the study and participated in its design and helped to draft the manuscript. AM participated in the design of the study and helped to draft the manuscript.The pre-publication history for this paper can be accessed here:


The efficacy of homeopathy is controversial. Homeopathic remedies are made via iterated shaking and dilution, in ethanol or in water, from a starting substance. Remedies of potency 12 C or higher are ultra-dilute (UD), i.e. contain zero molecules of the starting material. Various hypotheses have been advanced to explain how a UD remedy might be different from unprepared solvent. One such hypothesis posits that a remedy contains stable clusters, i.e. localized regions where one or more hydrogen bonds remain fixed on a long time scale. High sensitivity proton nuclear magnetic resonance spectroscopy has not previously been used to look for evidence of differences between UD remedies and controls.Homeopathic remedies made in water were studied via high sensitivity proton nuclear magnetic resonance spectroscopy. A total of 57 remedy samples representing six starting materials and spanning a variety of potencies from 6 C to 10 M were tested along with 46 controls.By presaturating on the water peak, signals could be reliably detected that represented H-containing species at concentrations as low as 5 μM. There were 35 positions where a discrete signal was seen in one or more of the 103 spectra, which should theoretically have been absent from the spectrum of pure water. Of these 35, fifteen were identified as machine-generated artifacts, eight were identified as trace levels of organic contaminants, and twelve were unexplained. Of the unexplained signals, six were seen in just one spectrum each. None of the artifacts or unexplained signals occurred more frequently in remedies than in controls, using a p < .05 cutoff. Some commercially prepared samples were found to contain traces of one or more of these small organic molecules: ethanol, acetate, formate, methanol, and acetone.1H-NMR spectroscopy. The results failed to support a hypothesis that remedies made in water contain long-lived non-dynamic alterations of the H-bonding pattern of the solvent.No discrete signals suggesting a difference between remedies and controls were seen, via high sensitivity  The mechanism of action of homeopathic remedies has baffled practitioners and scientists for two centuries. A widely accepted premise of those doing research in this field is that if remedies are more than placebos, then the process of making remedies by alternating dilution and succussion must alter the solvent, encoding in it a "memory" or "information" that biological systems can detect. Experiments attempting to measure or document solvent alteration through direct study of the physical and chemical properties of remedies have so far failed to yield any independently replicated positive effects.1H-NMR, or simply NMR) is among the techniques that have been used to look for differences between remedies and control samples. The term "NMR" encompasses both solvent mobility studies (results are given as a pair of relaxation times denoted T1 and T2) and analytical studies. In analytical studies, also called spectroscopy, the results are displayed as a graph or spectrum plotting concentration against a variable called chemical shift. There are also more complex applications of NMR such as imaging and two-dimensional NMR that are not relevant to the study of discrete remedy samples. The chemical shift of a proton in a molecule in a sample reflects the (time-averaged) amount of magnetic shielding provided by the electrons making up the covalent or hydrogen bond(s) in which the proton participates, with greater electron density generally correlating with lower chemical shift numbers. Chemical shifts are measured in units of parts per million (ppm) deviation from a reference shift. Recent literature reviews by Baumgärtner [et al.[et al.[Proton nuclear magnetic resonance  were obtained from Sigma-Aldrich [2 from exposure to air and ions from exposure to glass, and we considered it to be unavoidable.Chemicals including 100.0% D-Aldrich . DistillSepia and Lachesis), two plant (Ignatia and Lycopodium) and two mineral remedies (Natrum Muriaticum and Argentum Nitricum), all of which occur commonly in clinical homeopathy, were used for the project. Commercial remedies were obtained from Helios Pharmacy [Two animal  and 20 μL of a dilute water solution containing a known concentration of a marker molecule (markers tried were acetone (CH3COCH3) and 1,4-cyclohexanedione (C6H8O2)). The remedy or control sample was then added to fill the tube to the 700 μL mark, followed by gentle tilting and turning to mix. The marker served several purposes: it provided a reference line for zeroing the chemical shift scale, it provided a reference peak for comparing concentrations, and its sharpness and shape gave feedback about the accuracy of the shimming process. By carefully varying the marker concentration we also determined the method's limits of detection. [C6H8O2 was considered an "ideal" marker in that it met all of these criteria: it is available cheaply at high purity; it dissolves in water without altering pH and does not evaporate over time; its 1H-NMR spectrum has a single unsplit line; its unique peak occurs at a distinctive location far from the water peak and is not easily confused with other common peaks (2.77 ppm); and it will not normally occur as a contaminant or from other sources, so one can be sure its concentration is exactly what one intends.]To prepare a sample for analysis, a borosilicate glass NMR tube rated for 500 MHz (Wilmad Lab Glass ) was priProton NMR spectra were obtained at the Department of Chemistry Instrumentation Facility at M.I.T. ("DCIF"). All spectra reported here were obtained on a Varian INOVA 500 tuned to 499.759 MHz and equipped with an inverse probe. Tubes were run with the temperature clamped at either 20°C or 21°C and were not spun. Shimming was done manually for Z1 through Z7 and for first and second order XY magnets, using the lock signal. Presaturation used the standard presat pulse sequence (satdly = 1.5 sec), with the optimal presat frequency being determined to within 0.1 Hz via an array method. Locking, shimming and presat frequency optimization were repeated each time a tube was inserted. Between 128 and 200 transients were collected for each sample. Start-to-finish time for each tube was typically around 35 minutes. Spectrum analyses were done via the standard Varian programs running on an SGI workstation provided by DCIF.Randomization and blinding are among the recommendations in Ref.  for how Nat Mur and Arg Nitr remedies of potency 6 C to 12 C. Because the details of the protocol evolved over the course of the project, it could be argued that this total represents the combination of many different experiments. Realizing this, we made a point toward the end of the project, of applying what we considered to be our best methodology to screen a set of what were arguably the best remedies. Specifically, we screened 18 Helios remedies, namely the 12 C, 30 C and 10 M potencies of the six MT's. The screening protocol consistently used 70 μL of D2O for locking and concentrations of 12.5 μM or less of C6H8O2 as marker. These 18 trials are included in the 57 for the purposes of discussion. Spectra can be conveniently split into those obtained during 2002  6H8O2), a peak was always seen above noise, using the 3σ criterion [As indicated above, when marker concentration was 5 μM of H or greater , but no rationale for the 3.60-ppm artifact was identified. We labeled the consistent artifacts as C1, C2, and C3.Intermittent artifacts were labeled R1 through R12 and are listed in Table 3, Additional File Half of the intermittent artifacts were seen in only one spectrum each. Those that occurred repeatedly demonstrated a "burst" pattern in the sense that they were present for spectra collected on a single day or during an interval of weeks or months, but were not seen at other times. For instance, the artifact R5 occurred only on 4-Jun-03 and was seen in every spectrum obtained that day  or f8.44 ppm ) ions at2O or outside this time interval. We pegged it as coming from a contaminant in the DMSO-d6, which was not present in different bottles of DMSO-d6 that were used before April and after November. Its base was broader by a factor of four than the other singlets seen, and despite its trace size we could often make out that it had a symmetric stepped shape like a ziggurat. A good guess, which fits all of these facts, is that it comes from ethylmethylsulfoxide-d7 (CD3SOCD2CD2H), which could plausibly be introduced during the manufacture of DMSO-d6 (CD3SOCD3). We did not find anywhere listed the chemical shift of ethylmethylsulfoxide, but the ethyl's methyl group of the very similar molecule ethylmethylketone resonates at 1.26 ppm [2O. We gave it the identifier 'X' in Table 3. A DMSO-d6 control run on 1-Aug-02 yielded two peaks in the vicinity of 1.3 ppm, one of which may have been 'X'.One signal, an upgoing singlet at 1.28 ppm, occurred in all but one spectrum run using DMSO-d6 between April and November 2002, and did not occur in spectra run using D6H8O2 protocol, results are given in Table 2, Additional File For the 18 Helios remedies screened with the C3COO- (range 22 – 214 μM, median 55 μM) and HCOO- (range 8 – 75 μM, median 44 μM). None contained detectable lactate, but we sometimes saw acetone or methanol (CH3OH, 3.34 ppm [-6), but the fact that the six runs exhibiting lactate were all done on one day with no non-Washington control means that we cannot rule out an extraneous source for the lactate.The 18 Helios remedies of Table 2, Additional File 3.34 ppm ). Six ofTwelve positions for upgoing singlets could neither be ruled out as artifacts nor assigned definitely to any known small organic contaminant. These are the "unexplained" peaks, labeled U1 through U12 in Table 3, Additional File For each of the unexplained signals and artifacts we made a 3 × 2 matrix of their occurrence vs non-occurrence in remedies, succussed controls, and unsuccussed controls. None of the matrices had a p-value below .05. The occurrence counts and p-values are listed in Table 3, Additional File Ignatia-30 C sample (Line 27 of Table 2). Figure 6H8O2 peak at 2.77 ppm being set to its known value of 100 μM of H. The complex signal between approximately 4.3 and 5.3 ppm is the presaturated water signal. This spectrum contains five contaminants, the three consistent artifacts (artifact at 11.53 not shown), and two intermittent artifacts. Note that the artifacts are biphasic, i.e. have a component below as well as above the baseline, whereas all of the signals due to actual molecules have signals that stay above the baseline.Figure -10 M.Concerning the organic contaminants, acetate, formate and lactate are present on human skin and can be introduced at the trace levels seen here through ordinary handling. Acetate and formate derive respectively from the β- and α-oxidation of long-chain fatty acids by fibroblasts , and lacth century, Hahnemann would have used locally obtained well water or spring water to make remedies, and his water would have been far less pure than our most contaminated sample.  While high purity solvents are necessary for scientific validity and reproducibility, there is no a priori reason to think they matter for remedies' effectiveness. Indeed, one of the hypotheses on the mechanism of homeopathy is that the "information" is in the geometry of the solvation shells of the low levels of impurities that are present in the solvent, or in some structuring of how solvent impurities group together or interact with each other.Despite the implication that commercial remedies may be "impure", we consider the levels of detected impurities to be extremely low. Large doses of methanol are hepatotoxic, but the trace amounts here represent no threat to health. Our findings certainly support the widely accepted view that homeopathic remedies are safe. We also believe that the contaminants found should be unlikely to interfere with remedies' clinical effectiveness. The median value of 310 μM corresponds to less than one part ethanol in 50,000 parts water. Working in the early 19Six of the unexplained signals occurred in just one spectrum each and could easily be artifacts that happened to be in phase with the rest of the spectrum. There is little to be gained by trying to explain these one-time events, five of which were from unsuccussed controls. The signals U1 to U3, being broad and upfield, could be consistent with complex aliphatic mixtures such as finger grease on the outside of the NMR tubes . We have2O runs starting in November 2002, which was when a fresh bottle of D2O was brought into use, and it was not seen in the DMSO-d6 runs from the same period. This pattern suggests it was a contaminant in the D2O, even though it disappeared when the same D2O bottle was used in April 2003. There was also one sporadic occurrence in April 2002 (a DMSO-d6 spectrum). A likely assignment is quinone (p-benzoquinone, C6H4O2), whose spectrum consists of one singlet at 6.80 ppm [2O bottle may have simply evaporated during four months.Signal U8 at 6.80 ppm occurred in 7 of 8 D6.80 ppm . QuinoneIn Table 3, Additional File We found no discrete signals that occurred significantly more frequently in remedies than in controls. Does this mean that NMR spectroscopy cannot show differences between remedies made in water and controls? Earlier NMR studies focused not on additional signals but on peak positions and shape. For samples in water there is only one peak, the water peak at 4.8 ppm, along with the expected peaks for marker and/or DMSO-d5. We did not examine the shape of the water signal. Presaturation completely destroys any information that may have been present in the water signal shape, and the shape of the water signal after presaturation is sensitive to so many variables that it would be hard to track down the effect, if any, of solvent alteration. Still, this is an avenue that could conceivably be pursued. Similarly, we indicated that any information in the range of 4.4 to 5.2 ppm was essentially lost. It might be possible to examine this range closely, perhaps by subtracting off a smoothed spectrum, to look for signals between 4.4 and 5.2. We have not attempted this. We were primarily looking for separate peaks that could be from long-lived stable clusters, and did not find any. For this reason we couch our results carefully by saying merely that "no discrete signals suggesting a difference were seen."2O. Nor do we believe that the addition of the marker could have interfered with the detection of structuring. Consider that Helios's remedies, reputedly among the best anywhere, contained comparable or higher concentrations of organic contaminants to start with, than we added. If one wants to maintain the position that homeopathy works , and that Helios's and Washington's remedies work, then the fact that their remedies come with traces of small organic molecules tells us that such traces cannot be something that interferes with homeopathy's mechanism. Thus it should not "hurt" a remedy to add a tiny bit of organic marker.The absence of any evidence of stable H-bonds above the 5 μM or 10 μM detection limit, in 57 remedy samples, certainly casts doubt on the hypothesis that remedies in water might contain regions of long-lived non-dynamic H-bond structuring. Is this hypothesis salvageable? We do not believe so. We made a point of screening low, medium and high potency remedies, as well as remedies from six common MT's. While it could be argued that even 10% DMSO could disrupt H-bonding and thereby denature (i.e. destroy) H-bond structure , this caab initio simulations [Could there be signals from fixed H-bonds but we didn't see them because they fell in the inaccessible 4.4- to 5.2-ppm region? The chemical shift of an H in a fixed O-H - - O setup depends on the O - - O separation and the details of the geometry, but studies of ice , organiculations ,40 all sWe have ruled out, or more precisely we have rendered highly improbable, only this one hypothesis on the nature of the "active ingredient" of homeopathy. Although a positive finding would certainly have been interesting, our negative findings should not be taken as evidence against clinical homeopathy. In particular, the possibility of dynamic alterations of solvent structuring remains open, but this hypothesis will need to be studied by methods that take a much faster "snapshot" of what is going on in samples. Finally, there are also several non-cluster-based hypotheses that have been proposed to explain homeopathy. These include isotopic patterning, coherence, and chaos-based explanations . These e1H-NMR spectroscopy method to look for discrete signals that could provide evidence of pockets of fixed H-bonding in water-based homeopathic remedies. No such evidence was found. The method did reveal the presence of some small common organic molecules, at levels deemed too low to be problematic. We hope that we have made a contribution to homeopathic research, both by answering a particular question, and by setting a standard for quality hypothesis-driven research that others will be inspired to follow.We used a high sensitivity The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:Table 1: Results of runs during 2002, Table 2: Results of Helios remedies screening, Table 3: List of peak positionsClick here for file