Parus major), we tested whether a) these genes are involved in photoperiodic time measurement (PTM) in a wild species, and b) whether predictable species and population differences in expression patterns exist. Using quantitative RT-PCR, we compared gene expression after a single long day in male great tits from Sweden (57°42′N) with that from a German (47°43′N) population. Hypothalamic gene expression key for PTM changed only in the northern population, and occurred earlier after dawn during the single long day than demonstrated in quail; however, gonadotropins (secretion and synthesis) were stimulated in both populations, albeit with different timing. Our data are the first to show acute changes in gene expression in response to photostimulation in any wild species not selected for study of photoperiodism. The pronounced differences in gene expression in response to a single long day between two populations raise exciting new questions about potential environmental selection on photoperiodic cue sensitivity.Measuring day length is critical for timing annual changes in physiology and behavior in many species. Recently, rapid changes in several photoperiodically-controlled genes following exposure to a single long day have been described. Components of this ‘first day release’ model have so far only been tested in highly domesticated species: quail, sheep, goats and rodents. Because artificial selection accompanying domestication acts on genes related to photoperiodicity, we must also study this phenomenon in wild organisms for it to be accepted as universal. In a songbird, the great tit ( Coturnix japonicaSeasonally breeding animals have evolved accurate time-keeping mechanisms to pattern transitions through their annual life cycle. Periods of high energetic demand are timed to coincide with favorable environmental conditions . For mid- to high- latitudes, abundant food resources typically occur in spring when ambient temperatures increase and plant and animal productivity increases by orders of magnitude. Accordingly, for many birds and small mammals with short gestation/incubation times, breeding begins in late winter and early spring during long days (‘long-day breeders’), while in mammals with gestation periods closer to six months, breeding occurs during short days in fall and winter (‘short-day breeders’). Measurement of day length by photoperiodic organisms is critical for timing physiological transitions between life-history stages. Day length is a highly predictable cue that allows individuals to anticipate changing conditions well in advance Parus major). Unlike photoperiodic mammals, birds are thought to detect day length using photoreceptive cells within the brain Here, we provide the first test of this model in a wild avian species, the European great tit  during the light cycle. For quail, the photoinducible phase begins at approximately 12 hours after dawn  and ends approximately 4 hours later Parus major). It is largely sedentary and broadly distributed throughout Europe. Sensitivity of great tit populations to long day lengths  is known to differ with latitude Components of this model appear to be conserved in both mammals and birds, but to date, mainly domesticated species have been used to test the acute changes in gene expression used for PTM, and the changes in photoperiodic ungulates occur over days rather than hours in quail We took advantage of the well-known reproductive ecology and physiology of great tits to test whether a) the proposed ‘first-day release model’ applies to wild vertebrates and b) gene expression in the MBH and circulating concentrations of luteinizing hormone (LH) over one long day differ in a predictable way between birds captured in Sweden (57°42′N) and those in Germany (47°43′N) and kept under controlled conditions. We predicted that changes in the expression of genes involved in PTM should occur later in the Swedish population, matching their longer critical photoperiod and later breeding times, compared to the southern German population. We brought wild great tit males from both populations captured in the fall into common garden conditions and exposed them to a single long day, collecting tissue and blood samples for gene expression and circulating LH concentrations at intervals throughout that day.t(64) = 0.12, p = 0.90), mass  = 0.52, p = 0.61), and had similar fat scores  = 1.08, p = 0.28) at the end of the experiment. Likewise, testis length  = 0.37, p = 0.97) and volume  = 1.23, p = 0.22) were similar between populations.German and Swedish males were similar in body size  until 14 hours after dawn. Although populations had similar expression of Per2 at 18 and 22 hours after dawn, only the Swedish population had significantly lower Per2 expression relative to pre-critical photoperiod levels at 18 hours (p = 0.021) and the German population had slightly higher Per2 expression at 22 hours after dawn  = 5.04, p<0.001), was significantly higher than in the German birds (t(56) = 4.40, p<0.001), and remained somewhat elevated at 12 hours after dawn  = 2.88, p = 0.006), and decreased 11 and 22 hours after dawn (p = 0.03 and 0.01) in the Swedish population, indicating reciprocal switching of DIO2 and DIO3 expression at 11 hours after dawn  = 2.27, p = 0.03) and was significantly greater than the German population (t(56) = 2.38, p = 0.02; In the Swedish population, expression of GnRH mRNA increased 18 hours after dawn from pre-critical photoperiod levels (t(61) = 5.04, p<0.001; (61) = 5.01, p<0.001 ). By 22 hours after dawn both populations had increased FSHβ mRNA expression from pre-critical photoperiod levels (t(61) = 3.53, p = 0.001 and t(61) = 3.99, p<0.001, German and Swedish populations, respectively).In the Swedish population, expression of FSHβ mRNA increased 18 hours after dawn from pre-critical photoperiod levels (t(123) = 3.64, p<0.001) and was higher than in the German population (t(123) = 2.24, p = 0.03; Circulating plasma LH concentration was significantly elevated from pre-bleed concentrations (taken 24 hours before) in the Swedish population , we think this is unlikely. Our study clearly indicates that the DIO2 system is temporally correlated with extremely rapid photoperiodic responses, but that activation of the GnRH neurons, which are located some distance away in the pre-optic area, occurs at a later stage. The combination of the lack of temporal synchronization of DIO2 and GnRH activation along with the physical separation of the DIO2 and GnRH systems implies that photoperiodic up-regulation of GnRH could involve a separate mechanism other than or in addition to the DIO2 system in the mediobasal hypothalamus.Great tits from a Northern European population showed marked changes in gene expression 11 hours after dawn. Expression of DIO2 mRNA increased and DIO3 expression decreased around the presumed critical photoperiod for this species, and the reciprocal switching in expression of these two genes coincided with an increase in LH secretion in the plasma at the same sampling point and a stimulation of GnRH expression six hours later. Our data are the first to show acute changes in gene expression in response to photostimulation in any species not artificially selected for study of photoperiodism. An additional novel component of our experiment is the demonstration of GnRH mRNA activation, which is an important aspect of the first-day release model that has not previously been measured. Further, our data also indicate population differences in the temporal patterns of gene expression under common garden conditions. Interestingly, although reciprocal switching of DIO2/DIO3 occurred only in the Swedish population, The patterns of gene expression in great tit males from the Swedish population are consistent with changes found in quail after the initial rise in LH (at 11L). The local action of T3 production likely has a very rapid action in the MBH and may not involve the GnRH neurons in the pre-optic area (POA) during initial photostimulation. The action of DIO2/T3 could be any one, or all of the following: 1) direct action on the stored gonadotropins in the anterior pituitary, 2) on GnRH terminals in the ME, causing release of GnRH there, or 3) on glial cells, causing retraction and then allowing localized release of GnRH in the fiber terminal fields  of the GnRH system. The combination of a lack of synchronization of DIO2 and GnRH activation along with the physical separation of the DIO2 and GnRH systems implies that photoperiodic up-regulation of GnRH might not involve the rapid activation of the DIO2 system in the geographically distinct mediobasal hypothalamus. This latter finding opens the door to a search for an additional mechanism for photoperiodic timing.This study was performed under the approval of the state of Baden-Wuerttemberg, Germany, Animal Research Commission (protocol no. G-08/63).ad libitum. Birds were allowed to acclimate to the laboratory setting for five weeks.A total of 68 males  were included in the study. Birds from both populations were captured in the fall (Oct/Nov) using passive mist netting and playback of male song to lure birds into mist nets. Males captured in Sweden were held in indoor, light-controlled aviaries 2–3 per cage until transport to Radolfzell, Germany by air on 1 Nov 08. In Germany, all birds were housed indoors in cages with artificial plants inside light-controlled environmental chambers 2–3 per cage. Birds were given food  and water During acclimation, birds were exposed to the same decreasing photoperiods that were similar to the site of capture. For German males, they received 10 hours 13 min of light at the end of October (lights on 0701 h: lights off 1714 h) decreasing to 8 hours 45 min of light by the end of November . Swedish birds received 8 hours 30 min of light at the end of October (lights on 0800 h: lights off 1630 h) decreasing to 7 hours of light by the end of November  to degrade any genomic DNA contamination, 12.5 μg of RNA from each sample was reverse transcribed to cDNA using M-MLV Reverse Transcriptase with oligo dT primers (Invitrogen). We diluted cDNA 1∶50 after testing serially diluted samples for each gene of interest to determine an optimal dilution.P. major sequences (using Primer3 software) for Per2, Cry1, DIO2, DIO3, GnRH, and FSHb , glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein kinase α, and protein kinase φ. Non-template controls were included for each primer pair to check for formation of primer-dimers. These samples always resulted in difference of at least 10 cycles of the Ct values compared to samples containing template. The quantitative real-time polymerase chain reaction (RT-PCR) was performed on samples balancing time points and populations across multiple plates using duplicate 25 μl reactions following manufacturer 's instructions for 2× iQTM SYBER Green Supermix . Specificity of each primer pair was confirmed using a melt curve analysis. The raw fluorescent data were analyzed using the RT-PCR Miner program Partial sequences for all of the genes of interest were first cloned from P. major cDNA see . PrimersPlasma was assayed in 15 μl duplicate samples in a single assay using a micro-modification of the radioimmunoassay originally devised by Follett and colleagues We combined samples from 6 and 10 hours after dawn for each population because both represented ‘pre-threshold’ time points, expression levels were similar, and sample sizes at 10 hours (n = 3 per population) were especially low. These means are shown in graphs as ‘pre-threshold.’ We used student's t-tests to compare average morphology measures between populations. To test whether average mRNA expression values changed over time within populations or differed between populations at each time point, we used planned contrasts Supplementary Materials S1Methods used to partially clone genes of interest in Parus major. These sequences were subsequently used to design primers for qPCR.(DOCX)Click here for additional data file.


Mindfulness-based interventions (MBIs) can reduce risk of depressive relapse for people with a history of recurrent depression who are currently well. However, the cognitive, affective and motivational features of depression and anxiety might render MBIs ineffective for people experiencing current symptoms. This paper presents a meta-analysis of randomised controlled trials (RCTs) of MBIs where participants met diagnostic criteria for a current episode of an anxiety or depressive disorder.g effect sizes were calculated using a random effects model. Moderator analyses of primary diagnosis, intervention type and control condition were conducted and publication bias was assessed.Post-intervention between-group Hedges g = −0.59, 95% CI = −0.12 to −1.06). Effects were demonstrated for depressive symptom severity , but not for anxiety symptom severity , for RCTs with an inactive control , but not where there was an active control  and effects were found for MBCT  but not for MBSR .Twelve studies met inclusion criteria (n = 578). There were significant post-intervention between-group benefits of MBIs relative to control conditions on primary symptom severity (Hedges This is the first meta-analysis of RCTs of MBIs where all studies included only participants who were diagnosed with a current episode of a depressive or anxiety disorder. Effects of MBIs on primary symptom severity were found for people with a current depressive disorder and it is recommended that MBIs might be considered as an intervention for this population. Mindfulness refers to a state of consciousness that is characterised by the self-regulation of attention towards present-moment experiences coupled with an accepting, non-judgemental stance towards these experiences There is evidence that MBCT approximately halves the risk of relapse in comparison to standard care for people who are currently well but who have experienced at least three prior episodes of depression In contrast to populations with a history of depression who are not currently distressed, there are at least three reasons to suspect that standard MBIs may not be of benefit to populations currently meeting diagnostic criteria for an episode of a depressive or anxiety disorder. First, MBIs invite participants to bring their full awareness to current experiences. For people with a current episode of an anxiety or depressive disorder their current experiences are likely to include aversive automatic thoughts Second, cognitive processes common in anxiety and depressive disorders may run counter to learning mindfulness Finally, there are motivational and concentration difficulties that may present a challenge for people with a current episode of an anxiety or depressive disorder to engaging in mindfulness practice. Learning to self-regulate attention is seen as one of the cornerstones of MBIs Whilst a number of meta-analyses have explored the effectiveness of MBIs in relation to symptoms of anxiety and depression The current meta-analysis tests the effectiveness of MBIs in randomised controlled trials where all participants were diagnosed with a current episode of an anxiety or depressive disorder. This is operationalized as all participants in a study meeting diagnostic criteria for a current episode of a DSM-IV (or later version) or ICD-10 depressive or anxiety disorder This meta-analysis addressed the important question of whether the conclusions about the effectiveness of MBIs can be extended to people experiencing a current episode or an anxiety or depressive disorder based on findings from RCTs. The primary outcome was symptom severity for the target clinical problem. Secondary outcomes of anxiety and depression symptom severity (irrespective of diagnosis) were used. Moderator analyses looking at primary diagnosis (anxiety or depressive disorder), control condition (inactive or active) and intervention type (MBCT or MBSR) were planned in the event of significant outcome heterogeneity.No published protocol has been published for this meta-analysis. The PRISMA guidelines were adhered to Titles and abstracts from the following databases were searched: MEDLINE, Web of Science, Scopus, ProQuest and PsycINFO for published or unpublished studies from the first available year of publishing until 4 July 2013. Reference sections of identified papers were searched manually. The following search terms were used: [(mindfulness or MBCT or MBSR) combined with (anxi* or depress* or OCD or “obsessive compulsive” or “post-traumatic stress disorder” or PTSD or agoraphobia or “panic disorder” or “acute stress disorder” or “acute stress reaction” or phobi*) combined with (random* or RCT)].www.clinicaltrialsregister.eu, clinicaltrials.gov and www.controlled-trials.com/isrctn) were searched to identify completed clinical trials of MBIs that had not been published. The trial registers were searched with the term ‘mindfulness’ (multiple search terms were not possible) with no restrictions placed on the search. All identified research team members from relevant clinical trials were contacted by email for details of their findings. In the event of failing to respond to email requests a further two emails were sent.In order to conduct a replicable search for unpublished data, three leading clinical trial registers (Inclusion criteria were: (1) designs were Randomized Control Trials; (2) participants aged 18 years or over; (3) mindfulness was a core part of the MBI with mindfulness practice in each therapy session and daily practice encouraged between sessions; (4) studies included a psychometrically reliable and valid outcome measure of depression or anxiety; and (5) data was presented for participants who met full diagnostic criteria for a current episode of a DSM-IV (or later version) or ICD-10 anxiety or depressive disorder. Specifically, participants were required to meet full diagnostic criteria for a DSM-IV depressive disorder  or anxiety disorder  or an ICD-10 depressive episode or depressive disorder or anxiety disorder . Hypochondriasis  was also an inclusion criterion given the central role that anxiety plays in the disorder Exclusion criteria were: (1) participants had marked cognitive impairment (e.g. learning disability or brain injury); (2) participants were currently engaged in substance misuse; (4) MBI was not delivered in a group format; (5) MBI was not delivered in-person (e.g. self-help); and (6) studies not in the English language. Duplicate data was also excluded if the same outcomes were fully or partially reported in another study. When this occurred, the study with the larger sample size was retained.g was used to calculate the effect size for each study:Means, standard deviations (sd) and number of participants for the primary symptom measure and for measures of depression and anxiety were entered into Review Manager version 5.2 ; si = pooled standard deviation; n1i = number of participants in group 1; n2i = number of participants in group 2; sd1i = standard deviation of the post-intervention mean for group 1; sd2i = standard deviation of the post-intervention mean for group 2.SMDg effect size is the between-group difference on the post-intervention mean scores for the chosen outcome measure, divided by the pooled standard deviation  and then multiplied by a figure that adjusts the effect size to take account of small sample sizes. This formula gives larger effect sizes as the difference between the post-intervention means of the two groups increase. By Cohen’s convention a small effect size is 0.2, a medium effect size is 0.5 and a large effect size is 0.8.In essence, Hedges Forest plots of post-intervention between-group effect sizes were produced for each of the three outcome variables. To address publication bias Rosenthal’s Fail-Safe N In order to assess the extent to which effect sizes were significantly different from each other heterogeneity was assessed using chi-square. A significant chi-square value indicates heterogeneity and that the studies cannot be considered to have recruited from the same population. In this case, possible reasons for heterogeneity can be explored. In order to do this moderator analyses were planned to explore effects as a function of control group (active or inactive), presenting problem (anxiety or depressive disorder) and MBI-type (e.g. MBCT or MBSR).The Jadad rating scale After duplicates were removed 657 published articles and 43 unpublished dissertations were identified, 30 records remained after screening abstracts  or an anxiety disorder , generalised anxiety disorder , PTSD , or health anxiety . Two studies included participants with a range of DSM-IV anxiety disorders (n = 146).Mindfulness-based interventions were Mindfulness-Based Cognitive Therapy (MBCT = 6), Mindfulness-Based Stress Reduction (MBSR = 5) and Person-Based Cognitive Therapy (PBCT = 1). MBCT and MBSR are group approaches consisting of eight 2 to 3 hour weekly sessions plus one whole day session. They involve a range of mindfulness practices which range between 3 and 40 minutes in length, with more than one practice per session. Daily mindfulness practice between sessions is encouraged and supported through audio recordings. In addition there is in-session discussion of what was learned during mindfulness practice. PBCT for depression involves twelve 90-minute sessions. There are two mindfulness practices in each session (a 5 minute practice and a 10 minute practice) along with Socratic discussion of what was learned and daily mindfulness practice is encouraged and supported through mindfulness practice audio recordings. Both MBCT and PBCT include elements of cognitive therapy with a greater emphasis placed on cognitive therapy in PBCT than in MBCT.There were five active control conditions  and seven inactive control conditions .There was wide variability in the number of participants dropping out from MBI with attrition ranging from 8 percent to 38 percent (median attrition = 15.5%).g = −0.59, 95% CI = −1.06 to −0.12) that was statistically significant (z(11) = 2.48, p = 0.01). Heterogeneity was significant (2χ(11) = 76.32, p<.001) and so moderator analyses were performed on primary diagnosis (depressive or anxiety disorder), type of control condition (active or inactive) and type of intervention.A random effects model see  showed t2χ(1) = 0.15, p = 0.69). However, analyses within subgroups  = 2.24, p = .03) effects for anxiety disorders were non-significant  = 1.69, p = .09).Moderator analysis showed no significant differences in primary symptom severity between those studies targeting depressive disorders and those studies targeting anxiety disorders (oups see  showed t2χ(1) = 6.60, p = .001). Whilst MBI outperformed inactive control conditions with a large effect size when looking at primary symptom severity  = 3.20, p = .001) MBI was not significantly different than active control conditions  = 0.13, p = .90). This suggests that the effect of MBIs on primary symptom severity varied as a function of control condition with MBIs being more effective than inactive control conditions but not more effective than active control conditions.2χ(1) = 0.42, p = .52). However, when looking at these subgroups separately there was no significant effect of MBSR on primary symptom severity  = 1.39, p = .16) but there was a significant effect of MBCT with a small to medium effect size  = 3.23, p<.01).Moderator analysis was also conducted on the effect of intervention type on primary symptom severity for studies MBCT and MBSR see . The sing = −0.64, 95% CI = −1.00 to −0.28) that was statistically significant (z(11) = 3.45, p<.001) but effect sizes were heterogeneous (2χ(11) = 46.69, p<.001). Nine studies included a measure of anxiety symptom severity and there was a non-significant post-MBI between-group difference in anxiety symptom severity  = 1.77, p = .08) and heterogeneous effect sizes (2χ(8) = 63.55, p<.001). These results suggest that MBIs had an effect of depressive symptom severity relative to control conditions but did not have a significant effect on anxiety symptom severity.Given the high co-morbidity between anxiety and depression In terms of publication bias, the funnel plot for primary symptom severity see  suggestsr(12) = −.20, p = .54), depressive symptom severity (r(12) = −.14, p = .66) and for anxiety symptom severity (r(8) = .26, p = .53). This shows no significant relationship between study quality and study effect size.Jadad scores for studies ranged from 2 to 5 . The correlation between Jadad ratings and study effect sizes was non-significant for primary symptom severity , the 95% confidence interval for this effect crossed zero (−1.11 to 0.06). Moreover, one of the studies included in this analysis had a low Jadad rating g = −0.17; 95% confidence interval: −0.54 to 0.21). Overall, we suggest that caution should be applied in offering MBIs for populations with anxiety disorders or where anxiety symptom severity is a target. However, the failure to find an effect on anxiety symptom severity could be due to a lack of power in this analysis and further studies are needed before we can draw definitive conclusions about the beneficial effects of MBIs for people with a current anxiety disorder.Although moderator analyses did not show that the effects of MBIs on primary symptom severity varied as a function of primary problem, the effects on anxiety symptom severity were not statistically significant either when just looking at those people with a confirmed anxiety disorder diagnosis or when looking at anxiety outcomes across the full-range of studies. Although the mean effect size for the effect of MBIs on anxiety symptom severity was in the moderate range  for the studies of depressive disorders. Indeed, the effect size was more than twice as large as any other effect size in this particular analysis. The other trials in this analysis were of MBCT and it is of note that PBCT was originally developed for people currently experiencing severe mental health difficulties The single PBCT trial was not included in the moderator analysis however, it produced the largest effect size on depressive symptom severity (Hedges g = 0.03) which suggests that MBIs may be no less effective than group CBT. Whilst future non-inferiority studies are needed to compare MBIs with group CBT, particularly for the full range anxiety disorders, the evidence from the current meta-analysis shows promise for MBI as an alternative to group CBT.Moderator analyses of control condition type showed that whilst effects of MBIs on symptom severity remained for studies using inactive control conditions (such as waiting lists), these effects disappeared when comparing MBIs to active control conditions. Four of the five studies which used an active control condition compared MBIs to group cognitive behaviour therapy (CBT). Two of these studies were for social anxiety disorder A median of 15.5 percent of participants dropped out from the MBI conditions identified in this review. This is similar to the mean drop-out rate of 16.1 percent from a meta-analysis of RCTs of CBT Research exploring the effectiveness of MBIs in populations with current diagnoses of anxiety or depression are in their infancy, and the evidence base is somewhat limited in both quantity and quality. Only 12 studies met our inclusion criteria, and of these the majority of studies (N = 7) compared MBI to inactive control conditions. These studies permit only a weak interpretation of the treatment effects, and do not allow the benefits of mindfulness practice and principles to be separated out from non-specific group therapeutic factors such as universality, altruism and group cohesion g = −0.84 and −0.73 respectively).The methodological quality of several of the included studies was poor, as shown by low Jadad ratings in some cases. Despite this, there were non-significant associations between Jadad ratings and effect sizes which indicates that there was not a bias towards lower quality trials reported larger effect sizes. Whilst seven of the 12 studies reported intention-to-treat data, five studies only reported completer data which potentially introduces some bias in favour of MBI, as it is possible that non-completers would have benefitted less than therapy completers. However this does not appear to be the case here as the mean effect size on primary symptom severity for the studies reporting completer data was similar to the effect size for studies reporting intention-to-treat data  and statistical (Fail-Safe N) methods were used in order to assess for possible publication bias and its potential impact on findings. Whilst the funnel plot indicated a potential for publication bias , the Fail-Safe N analysis suggested that a large number of studies with nil effect would be needed to render the primary analysis non-significant. This allows us to have some confidence in our findings despite the omission of data from unpublished studies.This meta-analysis suggests that people meeting diagnostic criteria for a current episode of a depressive disorder can benefit from MBIs. However, the studies of MBIs for depression were limited to MBCT or PBCT; none of the studies were of MBSR. Therefore our findings only apply to MBCT and PBCT and not to MBSR and we cannot comment on the basis of our analysis on the effects of MBSR on current depression. Our findings suggest that people experiencing a current depressive episode can benefit from MBCT or PBCT despite the negative thoughts and feelings associated with depression, thinking processes that orient attention towards or away from negative content and motivational and attentional problems.The studies targeting depression recruited from primary care and secondary care populations which suggests that MBIs might usefully be offered in both settings to people experiencing a depressive episode. It is of note however that the mindfulness-based intervention used in the PBCT trial Finally, given the paucity of evidence in their favour, we would caution against offering MBIs as a first line intervention for people experiencing a primary anxiety disorder. Not only did we find few studies targeting specific anxiety disorders, but those we did find suggest that MBIs may not be effective at targeting primary symptom severity for people experiencing an anxiety disorder. There are other, well-evidenced interventions for the range of anxiety disorders This is the first published meta-analysis of RCTs that evaluates the effectiveness of MBIs for people experiencing a current episode of a depressive or anxiety disorder. We found significant benefits relative to control conditions for primary symptom severity for people experiencing a current episode of depression following MBIs (namely MBCT or PBCT). Moreover, the analysis indicated that MBIs may produce similar outcomes to group CBT and therefore we suggest that MBCT or PBCT may be offered alongside other evidence-based interventions for people experiencing a current depressive episode in order to increase patient choice. We failed however to find support for MBIs for people experiencing a current episode of an anxiety disorder. This may well be due to the preponderance of small, underpowered studies but until further, adequately powered trials are conducted caution should be applied before offering MBIs as a first line intervention for anxiety disorders.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10−29). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.Oxidative stress is involved in the pathogenesis of airway obstruction in α Chronic obstructive pulmonary disease (COPD) is a complex trait with both genetic and environmental risks factors that is characterized by non-reversible airway obstruction and chronic inflammation. The morphologic manifestations of this disorder include small airway remodeling and emphysema. The predominant environmental risk factor for COPD is cigarette smoking, The genetic component of COPD has been demonstrated by twin studies of disease status 1-antitrypsin and emphysema has been known for several decades 1-antitrypsin is a proteinase inhibitor and acute phase reactant, and its major role is the inhibition of neutrophil elastase. α1-antitrypsin deficiency is caused by alleles of the SERPINA1 gene. Severe deficiency of α1-antitrypsin is most often caused by homozygosity for the Z allele (Glu342Lys) of SERPINA1 and is a risk factor for early-onset emphysema, although the clinical manifestations are highly variable While several novel susceptibility genes for COPD have been identified in recent years, the underlying mechanisms are largely unknown. In contrast, the association between deficiency of αA recent focus of COPD research has been the role of premature aging of the lung and other organs. Emphysema is characterized by reduced cell proliferation Telomeres shorten with each round of cell division and this results in replicative cell senescence. Telomere length is reduced during DNA replication because of the “end replication problem”, i.e., the 5′ end of the lagging strand is unable to be replicated. This loss of telomeric DNA is predicted to be ∼10 base pairs (bp) per cell cycle. However, the observed rate of loss can be higher and in humans has been estimated to be 50–200 bp per division 1-antitrypsin deficiency has not been previously studied. Oxidative stress plays an important role in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients 1-antitrypsin has anti-apoptotic effects 1-antitrypsin deficiency. We investigated telomere length in a group of COPD patients with α1-antitrypsin deficiency and a group of COPD controls. We also determined whether the length of telomeres in peripheral blood DNA is correlated with that in lung tissue samples in order to test the hypothesis that COPD is a “systemic” disease, and that telomeric shortening in this condition affects both lung and the hematopoietic systems.Several studies have examined telomere length in the context of COPD but there is little consistency in the results 1-antitrypsin deficient patients and 217 COPD control patients (1-antitrypsin deficient patients were selected from the Alpha-1 Foundation (AATF) DNA and Tissue Bank located at the University of Florida (IRB 659-2002). The COPD controls were selected from the Lung Health Study (LHS), a clinical trial sponsored by the National Heart, Lung and Blood Institute 1) to forced vital capacity ≤0.70 and FEV1 between 55–90% of predicted. Selected LHS samples were matched to the α1-antitrypsin deficient samples for age, gender, ethnicity and pack years. An additional 51 patients were selected from the lung tissue biobank at the James Hogg Research Centre (JHRC). For the JHRC samples, both lung tissue and blood samples were obtained from patients admitted to St. Paul's Hospital who underwent lobar or lung resection surgery for localized lung cancer. The lung tissue samples were taken from a site distant from the tumor. All subjects provided written informed consent.We studied 217 αpatients . ApprovaA sample of peripheral blood DNA was used to measure telomere length in the AATF DNA and Tissue Bank samples. Measurement of telomere length in the LHS samples was performed as previously reported GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT; tel 2: TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA; 36B4u: CAGCAAGTGGGAAGGTGTAATCC; and 36B4d: CCCATTCTATCATCAACGGGTACAA. Six qPCR reactions (i.e. triplicates of the telomere and reference gene assays) were performed for each individual in 20 µL reactions including 10 µL QuantiTect SYBR Green PCR Master Mix (QIAGEN), and final primer concentrations of tel 1: 270 nM, tel 2: 900 nM, 36B4u: 300 nM, and 36B4d, 500 nM. Reactions were performed on the ViiA 7 Real-Time PCR Instrument (Life Technologies). Cycling conditions for the measurement of telomere length were as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 sec and an annealing temperature of 54°C for 2 min. Cycling conditions for measurement of the 36B4 reference gene were the same except 35 cycles, with an annealing temperature of 58°C for 1 min were used. Telomere length was calculated as a ratio of telomere to 36B4, using Cawthon's formula Telomere length was measured using a previously published qPCR based protocol 10 transformed to approximate a normal distribution. Student's t-test was used to compare mean telomere length between groups. Multiple linear regression was performed to test for the effect of α1-antitrypsin deficiency on telomere length and lung function, with adjustments for significant confounders including age, gender and pack years. JMP software  was used for all statistical analyses.Telomere length measurements were log1-antitrypsin deficiency and telomere length in peripheral blood cells, telomere length was measured in 217 α1-antitrypsin deficient patients and 217 control patients matched for ethnicity, gender, age and pack years. Mean telomere length was compared between the two groups using Student's t-test. Median telomere length (untransformed) was 2.45 fold longer in peripheral blood DNA from COPD patients with α1-antitrypsin deficiency compared with COPD controls (p = 1×10−29) .To test for association between α1×10−29) . The meaTo test for a correlation between telomere length in the blood and in the lungs, telomere length was measured in lung and blood DNA from 51 patients from the JHRC. There was a significant correlation between telomere length in the blood and telomere length in the lungs  . On aver1-antitrypsin deficient patients and all COPD controls. The effect of telomere length in the blood on FEV1 % predicted was tested. There was no significant association between telomere length in the blood and lung function in either the α1-antitrypsin deficient patients (p = 0.3122), or in the controls with adjustment for age and pack years (p = 0.2503). In addition, lung function data were available for 49 patients from the JHRC with telomere length measurements in the lung. There was no association between telomere length in the lung and FEV1% predicted (p = 0.8057).Lung function data were available for 157/217 α1-antitrypsin deficiency have longer telomere lengths in peripheral leukocytes compared with COPD patients who do not have α1-antitrypsin deficiency. However, there was no significant relationship between telomere length in blood and lung function as measured by FEV1% predicted in α1-antitrypsin deficient patients or in COPD controls. We also found that within subjects, there was a significant relationship of telomere length in peripheral leukocytes with that in lung tissue, although on average the telomere length of peripheral leukocytes was shorter than that in lung tissue.The most important finding of this study was that COPD patients with α1-antitrypsin deficient patients, a group who we hypothesized may be particularly susceptible to accelerated reduction in telomeres and the subsequent cellular senescence.Telomere length has been positively correlated with lung function in some studies in vitroThe role of premature aging in COPD has been shown by studies of explanted lung fibroblasts from emphysema patients that showed reduced proliferation rate 1-antitrypsin deficiency and telomere length. However, the direction of effect was contrary to our hypothesis. Telomere length was longer in patients with α1-antitrypsin deficiency, despite the fact that they are likely exposed to higher levels of oxidative stress than usual COPD patients, as measured by oxidation of nucleic acids 1-antitrypsin deficiency have lower levels of myeloperoxidase (MPO) and neutrophil counts in sputum than non-deficient COPD patients 1-antitrypsin deficient patients In our study we found a relationship between αWe found that there was a significant correlation between telomere length in the blood and telomere length in lung tissue. Many studies of telomere length are performed using DNA from peripheral blood cells, and results are extrapolated to biological processes occurring in other tissues. For example, the majority of studies investigating telomere length in COPD patients have been performed in DNA from blood cells et al.We also demonstrated that the telomere length of peripheral leukocytes was shorter than that in lung tissue. Telomere length is known to vary between different human tissues 1-antitrypsin deficient patients. Another limitation is that all of the α1-antitrypsin deficient patients included in this study were current or ex-smokers. Therefore, it was not possible to test for the effect of α1-antitrypsin deficiency in non-smokers compared with smokers. Finally, our telomere length measurements in the lung were performed using only a small piece of lung tissue, therefore the telomere length measured may not reflect the whole lung.One limitation of our study is that lung function was only measured at one time point; therefore we could not test the effect of telomere length on rate of decline in α1-antitrypsin deficient patients, replicative senescence does not appear to play a significant role in the pathogenesis of COPD. Importantly, for the respiratory community, we found that telomere length of peripheral leukocytes is a good biomarker of telomere length in lung tissue.Our data indicate that in α


Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse. ► Generation of a transgenic mouse line that lacks Bassoon ► Normal basal transmission at mossy fiber to granule cell synapses in Bassoon mutants ► Enhanced synaptic depression within milliseconds during high-frequency transmission ► Halved rate of vesicle reloading at active zones in Bassoon knockout mice Many sensory systems, such as the vestibular , proprioC. elegans and Drosophila), homologs of Bassoon and Piccolo  mice. These glutamatergic synapses appear ideally suited to investigate the mechanisms of vesicle reloading because they show rapid vesicle reloading at a limited number of release sites (We investigated the role of Bassoon by comparing the properties of transmission at cerebellar mossy fiber to granule cell (MF-GC) synaptic connections in control and Bassoon null mutant . Previous studies investigating the function of Bassoon have used a Bassoon mutant , their coefficients of variation , and their kinetic parameters  were not significantly different in control and −/−Bsn mice  was reduced by 14%, corresponding to the previously determined ratio of the stimulus- and rise-aligned quantal responses . Biexponential fits to the recovery revealed time constants of 26 and 36 ms with amplitudes of 62% and 46% for the fast component for control and −/−Bsn, respectively, and a slower component with a time constant of ∼2 s for both control and −/−Bsn  was ∼73% and ∼100%, respectively (data not shown). These estimates are comparable to previous estimates with 100 Hz experiments at MF-GC synapses in rats (−/−Bsn and controls cannot be attributed to postsynaptic mechanisms.To dissect presynaptic and postsynaptic components of the recovery in control and recovery A and 4B.recovery A and 4B. in rats . Further in rats . These dq) during and after high-frequency transmission, the presynaptic component of short-term plasticity could be isolated by assuming that the presynaptic and postsynaptic components multiply to give the overall short-term plasticity of the phasic EPSC amplitude , consistent with the estimates of desensitization from the spontaneous EPSCs. To estimate the number of functional release sites  and the initial probability of vesicular release from each site (rp), the variance of the first EPSC during the train and a fixed q determined independently from the amplitude of the spontaneous EPSCs at that MF-GC connection . These data suggest that neither the rp nor the N were significantly different between Bsn−/− and control. Furthermore, the enhanced synaptic depression in Bsn−/− was not due to enhanced postsynaptic depression in Bsn−/−. Our data therefore suggest that the reloading of vesicles at each release site might be impaired in Bsn−/− mice.To determine the reduction in q during the train (cf. N) with a release probability (rp) and a rate of vesicle reloading (k) from an inexhaustible reserve pool  was lowered to 1.25 mM . An example of such an individual experiment is superimposed with the corresponding predictions of model 3 in Bsn−/− mice matched well to that measured directly from the same MF-GC connections   and the release probabilities (r1p and r2p) were on average not significantly different . These results are consistent with the findings obtained at the calyx of Held, where an increase in temperature resulted in a similar rp and vesicle pool size but accelerated vesicle recruitment . In this line, the gene trapping of Bassoon was achieved by insertion of gene trapping vector VICTR 48 into Introns 1–2. Homozygous Bassoon mutant mice are here referred to as −/−Bsn and their wild-type littermates as Bsn+/+ or controls. Bassoon mutant mice lacking the central part of the protein encoded by Exons 4 and 5 of the Bassoon gene (ΔEx4/5Bsn). The mice exhibit a mixed genetic background of C57BL/6J and 129/SvEmsJ strains, which is controlled by using sustained C57BL-backcrossed and 129 inbred mice to breed the heterozygous parents. The mice, bred at the Leibniz Institute for Neurobiology in Magdeburg, Germany, were between 20 and 28 days of age at the time of the experiments. Experiments were performed in an interleaved manner with the corresponding controls.Mutant mice carrying a gene-trapped allele of the oon gene  were useWestern blotting and immunohistochemical staining were performed similarly to that described previously . Rabbit ΔEx4/5Bsn mutants or 37°C ± 0.5°C  in experiments with −/−Bsn mutants. See Acute cerebellar brain slices were prepared similarly to those described previously . The recΔEx4/5Bsn mutant  as the peak EPSC amplitude within a window of 1–2 ms after the stimulation artifact measured relative to a baseline (tonic component) of 0.1–0.5 ms preceding the pulse . For eac5 mutant B as obse5 mutant , which cq during 300 Hz trains with EPSC fluctuation analysis in a similar manner to that formerly reported .The nonparametric Mann-Whitney-U rank sum test was used for statistical analysis unless otherwise noted. The data are reported either as mean ± SEM or as box plots with the middle line, box boundaries, and whiskers indicating the median, the 25%, 75%, 10%, and 90% quantiles, respectively. Analyses were carried out with Igor Pro 6.1 and SigmaPlot . n indicates the sample number and p denotes the significance of a Mann-Whitney-U test (


P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly ( Zn is thought to be involved in Alzheimer's disease (AD) development and progression, however there are many questions that remain unanswered. It is evident that the transcription of amyloid precursor protein (APP) is regulated by the Zn-dependent transcription factors Sp1 and NF-κB It has been suggested that ZnT6, located in the Golgi apparatus, mediates accumulation of Zn therefore enabling Zn to promote Aβ formation by binding to APP and inhibiting α-secretase ZnT3 localises predominantly to the membranes of Zn-rich synaptic vesicles in the hippocampus Manganese (Mn) is another trace element that is essential in the body. It is an important co-factor for several enzymes in the brain, including Mn-superoxide dismutase and glutathione synthetase. It is also needed for adequate neurotransmitter synthesis and metabolism Further understanding of the ZnT family and their interactions in the brain may elucidate a role for ZnTs and Zn and/or Mn in the progression and pathology of AD. To date ZnT10 has not been studied in this context. To determine if dys-regulated expression of ZnT10 could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression.All human tissue was obtained from the Newcastle Brain Tissue Resource at Newcastle University which is a Human Tissue Authority licensed Research Tissue Bank following ethical approval by the National Research Ethics Service. All donations were obtained with fully informed consent following an NRES approved protocol.All mouse experiments were carried out in accordance with the local animal ethics committee requirements  and National Health and Medical Research Council standards of animal care .n = 16; female n = 10, male n = 6) and age-matched controls  and APP/PS1 mice (n = 11). APP/PS1 mice are a mouse model of AD containing two transgenes inserted at a single locus that develop beta-amyloid deposits in the brains by 6 to 7 months of age (http://jaxmice.jax.org/strain/005864.html). Brain samples were removed and snap frozen before being stored in RNA Later at −80°C.Frontal cortex RNA samples from human Alzheimer's disease pathology cases  - 1)*100 and were required to fall within the range of 80–115%. Values for ZnT10 were expressed relative to the value for the reference gene  measured in the same cDNA sample.Total RNA was extracted from brain tissue using TRIzol® reagent. Concentration of RNA was determined by measuring the absorbance on a Nano drop ND-1000 (NanoDrop®). Total RNA preparations were analysed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Samples were stored at −80°C. RNA was DNAse treated and synthesis of RNA into cDNA was carried out using M-MLV RT (Promega) according to manufacturer's instructions in 25 µL reactions containing 0.625 mM oligo dT primers and 1 µg RNA. Primers specific to human and mouse ZnT10, GAPDH and 18S rRNA (reference genes) were designed over intron/exon boundaries and are as listed in t test, taking into account homogeneity of variance. Braak scores and age were compared using non-parametric testing and the Mann Whitney U test. Correlations were carried out using Pearson's correlations and, where appropriate, Spearman's Rho correlation. Statistical analysis was carried out using Microsoft SPSS.Experiments were performed in triplicate and each experiment was repeated at least twice. Results are expressed as a ratio of ZnT10 RNA: reference RNA, normalised to control samples. Data were tested for normality using the Shapiro-Wilk test. For normally distributed data differences between means were considered significant at p<0.05 using a two tailed Student's To investigate if there is any association between levels of ZnT10 expression in relevant regions of the brain and AD, we measured ZnT10 mRNA in frontal cortex of AD patients and controls and in frontal cortex of 12 month old female wild-type and APP/PS1 mice.Demographic data for the human subjects studied are shown in Mean RNA Integrity numbers (RIN) were 5.32 (±1.88 S.D) with a range (from 2.5–7.3). These values indicate that, as expected RNA degradation had occurred in some of the post mortem brain tissue samples. For this reason all amplification products for analysis were less than 250 bp. Amplicons of this length have been shown to be independent of RNA quality P<0.01) in AD patients (median = V) compared with control subjects (median = II). Compared with age-matched controls, we observed a significant decrease in ZnT10 mRNA in frontal cortex of AD patients (p = 0.173) in the AD tissue. An indication that ZnT10 levels increase with age was observed, with a trend evident for controls (p = 0.088) and a near significant correlation in AD cases (p = 0.066). When separated on the basis of sex, brain tissue from female AD patients revealed ZnT10 mRNA levels that correlated significantly with age (p = 0.020). Correlation results are shown in There was no significant difference in age or post mortem delay (PMD) between control and AD subjects. Braak staging scores were significantly elevated (patients . GAPDH aP<0.01 by Mann-Whitney U). Despite a 45% decrease in ZnT10 expression observed in the male AD cases compared with the controls, this did not reach statistical significance. As sample numbers were relatively small, larger numbers are required to draw robust conclusions from this finding. Also, there was no significant difference in ZnT10 mRNA levels between male and female control subjects (p = 0.66) or between male and female AD cases (p = 0.67). The observed down regulation of a transporter of the ZnT family in AD is not without precedence. Znt3 mRNA was found to be reduced by up to 60% in all four cortical regions tested  in AD cases compared with controls. Interestingly there was no significant differences in ZnT3 expression in the cerebellum We observed a significant decrease in ZnT10 mRNA in samples of frontal cortex from AD patients compared with age-matched controls, giving the first indication that ZnT10 regulation is affected in AD. Furthermore, there was some suggestion of a sex-specific effect; the lower levels of ZnT10 observed in female AD cases compared with controls was statistically significant , but not the cerebellum p = 173) in the AD tissue. Intriguingly, however, there is an indication that ZnT10 levels increase with age, with a trend evident for controls (p = 0.088) and a near significant correlation in AD cases (p = 0.066). When separated on the basis of sex, brain tissue from female AD patients revealed ZnT10 mRNA levels that correlated significantly with age (p = 0.020). Details of the implications of age-related effects on ZnT expression have not been established, however it is known that there is a decline in Zn status with age . As in AD, these mice have Aβ deposits and exhibit deficits in spatial learning In concordance with our findings in human subjects, we observed lower levels of Znt10 mRNA in frontal cortex from female APP/PS1 transgenic mice. APP/PS1 mice are a mouse model of AD containing two transgenes inserted at a single locus. The first is the APP sequence that is modified to encode the human Swedish mutations K595N/M596L. Secondly, PS1 corresponds to the human presenilin 1 gene. In this case there is a deletion of exon 9, therefore known as the DeltaE9 mutation  and pre-clinical AD , as well as in AD SLC30A10 gene in patients with the neurological conditions dystonia and Parkinsonism that lead to hypermanganesemia in brain regions Δpmr1Xenopus laevis oocyte system) the precise specificity of this transporter remains unknown, but the results of the current study should nonetheless be interpreted with cognisance of the likelihood that ZnT10 transports manganese. Given the known link between neurological conditions and chronic exposure to Mn, it is possibile that the dys-homeostasis of ZnT10 observed in the AD brain is related to changes in levels of Mn rather than Zn., Mechanisms of Mn-induced neurological changes remain to be elucidated, although oxidative stress, protein aggregation or altered homeostatic conditions of other divalent metals that share similar transport mechanisms have been suggested Recent observations identifying frame-shift mutations in the Overall we uncover, for the first time, changes in ZnT10 mRNA levels in a vulnerable region of AD brain, which is paralleled in the AD transgenic mouse brain. Given the subcellular localisation and predicted function of ZnT10 as both a Mn and Zn transporter it is possible that tdysregulation of these metals could contribute to increased Aβ deposition and senile plaque formation and ultimately disease progression.Table S1Individual data for each subject including individual actual and normalised ratios for each RT-qPCR. – indicates PCR failed quality checks and therefore sample not used in final analysis.(DOCX)Click here for additional data file.


Removal of aggregates was performed by using the Aggregate Specific Reagent 1 (ASR1) compound which has been previously described to specifically bind misfolded species. Our results show that pre-treatment with ASR1-coupled magnetic beads reduces the in vivo misfolding inducing capability of an AD brain extract. These findings shed light respect to the active principle responsible for the prion-like spreading of Alzheimer's amyloid pathology and open the possibility of using seeds-capturing reagents as a promising target for AD treatment.Recent studies in animal models of Alzheimer's disease (AD) show that amyloid-beta (Aβ) misfolding can be transmissible; however, the mechanisms by which this process occurs have not been fully explored. The goal of this study was to analyze whether depletion of aggregates from an AD brain suppresses its  Misfolding and aggregation of amyloid-β (Aβ) is one of the central pathological features of Alzheimer's disease (AD) Recent experiments have shown that Aβ and other disease-associated proteins can transmit misfolding in a similar way as infectious prions propagate in Transmissible Spongiform Encephalopaties (TSEs) Prion-like propagation not only represents a risk of transmission among individuals, but may also explain the spreading of misfolded particles among different cells and tissues In this study, we tested the ASR1 compound as a putative agent to bind and remove misfolded species from an AD brain sample and its effect on the acceleration of cerebral accumulation of amyloid deposits in a transgenic mouse model of AD. Our results show that ASR1-depletion of Aβ aggregates is able to abolish the seeding capability of an AD brain, supporting the future use of this compound for the diagnosis and treatment of AD and other PMDs.Swe/PSEN1ΔE9 transgenic mice ΔE9). As a result, these animals develop amyloid plaques starting at 4–5 months old ad libitum. All animal manipulations were carried out in accordance to NIH regulations and approved by the Animal Welfare Committee of the University of Texas Medical School at Houston. Surgical procedures were performed under isofluorane anesthesia and all efforts were made in order to minimize suffering. 5–8 animals per group were used for this study.APPin vivo and in vitro analyses. The same process was conducted for a different aliquot of the same brain homogenate using magnetic beads lacking the ASR1 peptoid. 30 days-old anesthetized APPSwe/PSEN1ΔE9 mice were intra-cerebrally injected with 10 µL of treated or untreated brain homogenates into the right hippocampus using the following coordinates: anterioposterior (AP): −1.8 mm; mediolateral (ML): −1.8 mm; dorsoventral (DV): −1.8 mm. Challenged mice and untreated controls were sacrificed by CO2 inhalation at 6 months old. Brains were removed and cut in half by the sagittal plane. The right hemisphere was post-fixed into fixative solution  and embedded in paraffin, while the left hippocampus was dissected out, snap-frozen in liquid nitrogen, and stored at −80°C until use. Written consent was obtained to use human derived samples. Handling of human samples was conducted as directed by the Institutional Review Board and Biological Safety Office of The University of Texas Medical School at Houston.Frozen and paraffin fixed temporal cortex specimens from AD  and non-demented  individuals were supplied by the National Disease Research Interchange . Age difference between control and AD samples were due to the lack of available age-matched controls not displaying any amyloid pathology in the brain. It is well-known that cognitively normal people over the age of 60 years old show scattered presence of Aβ aggregates 2O2/10% methanol in PBS for 20 min. Sections were treated with formic acid 85% for 5 minutes for antigen retrieval. After rinsing, the sections were incubated overnight at room temperature with the 4G8 anti-Aβ monoclonal antibody . Slices were later incubated for 1 hour in HRP-linked sheep anti-mouse antibody . Peroxidase reaction was visualized using DAB Kit (Vector) following the manufacturer's instructions. Finally, sections were dehydrated in graded ethanol, cleared in xylene, and cover slipped with DPX mounting medium . For Thioflavin-S staining, tissue slices were incubated in ThS  solution (0.025% in 50% ethanol) for 5–10 min after deparaffinization. Sections were dehydrated in graded ethanol, cleared in xylene, and cover slipped with DPX mounting medium. Staining of human AD sample was performed using a similar procedure. Anti-hyperphosphorylated tau immunostaining was performed using the AT8 antibody  at a 1∶100 dilution. After incubation with HRP-linked sheep anti-mouse antibody, human samples were developed with DAB and counterstained with hematoxylin.10-µm-thick serial sections  from all animal groups  were processed in parallel for immunostaining. Sections were deparaffinazed and the endogenous peroxidase activity was blocked with 3% HSections were examined under a bright field/epifluorescent DMI6000B microscope  and image analysis quantifications were performed using the ImagePro software . Burden was defined as the area of the brain stained with anti-Aβ antibodies or ThS per total area analyzed. Burden quantification was performed by an investigator blind to the experimental groups. Lateromedial sagittal slides from all animal groups (n = 5–8 mice per group) were quantified in every tenth sections, in a total of 5 sections.42 and Aβ40 were measured using Human Aβ ELISA Kits . Samples were read on an ELISA plate reader  at 450 nm.Mice hippocampi were homogenized at 10% w/v in PBS plus proteinase inhibitors. Resulting extracts were fractioned by using a previously described serial extraction protocol For Coomasie staining, 20 µL of PBS, SDS and formic acid fractions (diluted as previously described) were separated by SDS-PAGE using 4–12% gels . Gels were cleared with water and stained in Coomasie solution  for ∼2–3 h with gentle shaking at room temperature. Later, gels were washed in washing buffer  overnight at 4°C. Next day, gels were washed again for ∼2–3 h at room temperature and pictures were taken. Bicinchoninic acid (BCA) assay was performed in the same samples used for Coomasie staining as recommended by the manufacturer .Data were expressed as means ± standard error (SEM). After confirming normal distribution with Skewness and Kurtosis statistic test, one way analysis of variance (ANOVA) followed by a post-hoc Tukey's multiple comparisons test was used to analyze differences among groups. Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc). Statistical differences for all tests were considered significant at the p<0.05 level.40 . Beads lacking the ASR1 compound were used to control the specificity and efficiency of this molecule. The presence of disease-associated Aβ species was assessed by anti-Aβ ELISA using the same procedure explained above  and 1C. in vivo for their seeding capability in AD transgenic animals. Resulting samples were intra-cerebrally inoculated into 1 month old APPSwe/PSEN1ΔE9 mice, a transgenic line described to show Aβ aggregates in hippocampus and cortex as early as 4–5 months of age Swe/PSEN1ΔE9 mice showed an inflexion point between 6–7 months. The time of animal sacrifice was chosen to allow the longest incubation period in which animals will have minimal accumulation of endogenous Aβ aggregates. In our experience, this enables to most clearly differentiate the effect of exogenous seeding from endogenous accumulation of amyloid deposits. We suspect that results obtained at longer incubation periods could be difficult to interpret due to the exponential appearance of amyloid deposits after 7 months of age. Untreated 6 months old mice showed few deposits at this specific age . Interestingly, the ASR1 effects were stronger over Aβ42, the molecule considered as the most deleterious in this pathology in vivo seeding capability of the aggregate-depleted AD samples in APPSwe/PSEN1ΔE9 mice. By using three independent analysis , we observed that the ASR1-treated AD brain homogenate significantly reduced its ability to induce Aβ aggregation in the hippocampus of challenged animals. Conversely, injection of untreated brain extracts and homogenates treated with control-beads resulted in a highly significant induction of Aβ deposition mostly in the corpus callosum and hippocampal fimbriae. Our results indicate that the ASR1 compound can specifically bind biologically active Aβ seeds and inhibit the seeding of these structures in the brain. Since animals were not observed for longer periods of time, we cannot rule out that the reduction of amyloid deposition observed by treating AD-inoculum with ASR1 may not be so evident at later time points. However, reduction of inducible pathology was clear in this specific experimental setup. These findings open the possibility of using seeds-capturing reagents as a promising target for treatment in AD and several other PMDs. Future experiments should address whether this molecule is suitable for in vivo administration or would be restricted to in vitro pre-treatment of samples designated for human use  to deplete them from misfolded aggregated proteins. In order to use ASR-1 for in vivo administration, it is necessary to elucidate the efficacy, pharmacokinetics, biodistribution and toxic properties of the molecule. It is also unclear whether ASR1-bound amyloids would be inactivated or stabilized in terms of their seeding effects in an in vivo context. This molecule could also be potentially used for basic research by helping in the analysis of common structural motifs present in amyloid assemblies.Taking advantage of the ASR1 ability to bind and capture misfolded aggregates, we studied the effect of incubating an AD brain extract with this molecule on the seeding of amyloid deposition in a mouse model of Aβ amyloidosis. Our results confirmed the efficacy of magnetic beads bound to ASR1 to remove disease-associated Aβ structures  Pictures show the aggregation profile of Aβ and hyper-phosphorylated tau in the temporal cortex of an AD patient. Open and solid arrows show mature and diffuse plaques, respectively. ThS stained image depicts extracellular and intracellular aggregates, the latter associated to neurofibrillary tangles , as shown in the AT8-stained brain slice. The scale bar corresponds to 100 µm. The quantity of soluble/insoluble Aβ40 (B) and Aβ42 (C) was measured by ELISA as described in the text. The values shown are expressed as means ± SEM.(TIF)Click here for additional data file.


Various injectable bulking agents have been used for the treatment of faecal incontinence (FI). However, encouraging early results are not maintained over time. This study aimed to assess short- and medium-term results of a new bulking agent for the treatment of FI.The Gatekeeper™ prosthesis comprises a thin solid polyacrylonitrile cylinder that becomes thicker, shorter and softer within 24 h after implantation. Fourteen patients with FI underwent treatment with Gatekeeper™ under local anaesthesia. Four prostheses were implanted in the intersphincteric space in each patient, under endoanal ultrasound guidance. Number of episodes of major FI, Cleveland Clinic FI score (CCFIS), Vaizey score, anorectal manometry, endoanal ultrasonography (EUS), health status and quality of life  were assessed before and after treatment.P = 0·002). CCFIS improved significantly from 12·7(3·3) to 4·1(3·0), 3·9(2·6) and 5·1(3·0) respectively (P < 0·001), and Vaizey score from 15·4(3·3) to 7·1(3·9), 4·7(3·0) and 6·9(5·0) respectively (P = 0·010). Soiling and ability to postpone defaecation improved significantly, and patients reported significant improvement in health status and quality of life. At follow-up, manometric parameters had not changed and EUS did not demonstrate any prosthesis dislocation.Mean(s.d.) follow-up was 33·5(12·4) months. There were no complications. There was a significant decrease in major FI episodes from 7·1(7·4) per week at baseline to 1·4(4·0), 1·0(3·2) and 0·4(0·6) per week respectively at 1-month, 3-month and last follow-up (The Gatekeeper™ anal implant seemed safe, reliable and effective. Initial clinical improvement was maintained over time, and follow-up data were encouraging. Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. Faecal incontinence (FI) is a common problem that can present with a wide variety of symptoms ranging from involuntary but recognized passage of gas, liquid or solid stool to unrecognized anal leakage of mucus, fluid or stool. Depending on the degree of symptoms, FI can be a highly distressing and socially incapacitating problem. The wide variety of aetiologies and difficulty in accurately defining the cause of the problem make treatment difficult. Appropriate treatment relies on accurate diagnosis and careful patient selection. Various injectable anal bulking agents have been used to treat FIThis aim of this study was assess the short- and medium-term results of a new bulking agent used for the treatment of FI.This study, carried out at the Department of Surgical Sciences, Catholic University, Rome, was approved by the institutional ethics committee.Gatekeeper™ prostheses  were used as anal bulking agent. These are thin solid cylinders  of HYEXPAN™ (polyacrylonitrile), a hydrophilic material that, within 24 h of implantation in contact with human tissue, changes shape and volume, becoming thicker (diameter 7 mm), shorter (length 17 mm) and of softer consistency (Patients with at least a 6-month history of episodes of FI (soiling or incontinence to liquid and/or solid stools) occurring at least once a week that had failed to improve with conservative measures were invited to participate. Patients with isolated incontinence to gas, risk of significant postoperative complications, including uncontrolled diabetes, anal sepsis, inflammatory bowel diseases with anorectal involvement or any colorectal cancer undergoing active treatment, were excluded. Patients with an isolated external anal sphincter (EAS) defect demonstrated on endoanal ultrasonography (EUS) were also excluded. Patient selection was based on data collected from the history, physical examination, continence diary recorded for 14 days , Cleveland Clinic FI score (CCFIS)The procedure was performed as a day case, under local anaesthesia using a posterior perineal block with the patient placed in the lithotomy position. Four 2-mm skin incisions were made at 3, 6, 9 and 12 o'clock positions in the perianal area 2 cm from the anal verge. With an Eisenhammer retractor inserted in the anal canal, a dedicated introducer formed by an introducer guide and an external sheath .Continuous data were presented as mean(s.d.), and compared using the Wilcoxon test. Fisher's exact test was used for analysis of categorical data. Fourteen consecutive patients  with a mean age of 63·5(17·0) (range 28–83) years were enrolled from May 2005 to January 2008. Postevacuation soiling was reported to occur always in nine patients, sometimes in two, rarely in two and never in one patient. The mean number of major incontinence episodes was 7·1(7·4) per week overall, 6·2(6·0) per week to liquids and 0·9(1·4) per week to solids. The mean CCFIS was 12·7(3·3) (range 7–18); only three patients had a CCFIS below 10. The mean Vaizey score was 15·4(3·3) (range 11–21). Patients could postpone defaecation for a mean of 6·1(4·9) min. EUS demonstrated no sphincter injury in eight patients, an isolated internal anal sphincter (IAS) defect in four, and a combined IAS and EAS defect in two patients (the latter patients both had an episiotomy during childbirth). Baseline anal manometric data are reported in All procedures were carried out successfully as a day case. Mean duration of operation was 35(7) min, including local anaesthesia, surgical procedure and control EUS. In all patients, the procedure proved to be easy and safe, and EUS confirmed accurate prosthesis placement. There were no intraoperative complications.There was no postoperative morbidity. None of the patients experienced any degree of local or systemic sepsis, fever or pain. There was no evidence of any acute or chronic inflammatory response around the implanted prostheses. Neither prosthesis dislodgement nor mucosal/skin alteration  was noted. Patients experienced no anal discomfort either at rest or during defaecation.Overall median follow-up was 33·5(12·4) (range 5–48) months. One week after Gatekeeper™ implantation, clinical assessment by digital examination and EUS showed the absence of acute inflammation at the prosthesis sites. All but one patient reported a significant improvement and regarded the treatment as successful. In one patient with an IAS defect secondary to lateral internal sphincterotomy the number of episodes of FI remained relevant ; 5 months after Gatekeeper™ implantation this patient underwent successful sacral nerve stimulation and was excluded from further analysis.P = 0·002). Findings were similar when episodes of incontinence to liquids or solids were analysed separately (P = 0·028) (P < 0·031).The mean total number of episodes of major FI decreased significantly immediately after surgery and the improvement was maintained over time, with a change from 7·1(7·4) per week before operation to 1·4(4·0), 1·0(3·2) and 0·4(0·6) per week at 1-month, 3-month and last follow-up respectively (P < 0·001) (P = 0·010)  (P = 0·001), coping and behaviour (P < 0·001), depression and self-perception (P < 0·001) and embarrassment (P = 0·001) . In 2008, however, the commercial licence for GORD was discontinued and enrolment for the present study was interrupted. The patients already enrolled were followed up according to the study protocol. In the meantime another company acquired the production line and recently obtained the EC mark of approval specifically for the use of Gatekeeper™ in FI, making this new bulking agent available on the market again.Gatekeeper™ prostheses are made of a unique material (HYEXPAN™) that is solid at the time of delivery, but slowly absorbs water and expands once implanted. Within 48 h the prosthesis has reached its final size and shape. At this stage the consistency of the material has changed from hard to soft, giving the implant a pliable texture that makes it compliant to external pressures without losing its original shape. For these reasons it was decided to place the implants in the intersphincteric space, in the belief that this would achieve a more effective distribution of the bulking effect than would be achieved with submucosal positioning, and thus exploiting the physical characteristic of the implant most effectively. The intersphincteric location should also minimize the potential risk of erosion, ulceration, fistulation of the anal canal and possible displacement of the prosthesis. This is particularly important in view of the solid state of the prostheses at the time of implantation. In this series there were no complications related to implantation of the prostheses, and ultrasonographic surveillance for a mean of almost 3 years confirmed that none of the implants had become displaced. The ultrasound results also showed that the size of all prostheses remained virtually unchanged over time, thus confirming the durability of the Gatekeeper™. The present cohort included not only patients with an intact IAS but also those with an IAS tear, or both IAS and EAS defects; patients with isolated EAS defects, however, were excluded. The prostheses were placed in the same position in all patients, irrespective of the location of the sphincter lesion.Skin incisions were made about 2 cm away from the anal verge to minimize the risk of wound contamination during bowel movements. The non-linear tunnelling through the soft subcutaneous tissues to reach the intersphincteric plane from the skin incision should also avoid possible prosthesis extrusion along the track. Prosthesis placement was performed under EUS guidance, to control the procedure step by step and ensure correct positioning of the prostheses. The operator could easily reach the intersphincteric space and decide on the exact position for each prosthesis. Moreover, the introducer could be followed by direct vision and digital palpation, and visualized by EUS. Therefore, lesions in the rectoanal mucosal/submucosal layer could be avoided. The contribution of EUS during Gatekeeper™ placement was fundamental to guiding placement of the prosthesis. However, the authors do not believe that it is necessarily mandatory; the procedure could be performed safely under digital guidance by an experienced clinician. Four prostheses were always implanted; this choice was arbitrary but seemed effective. The prostheses were placed at 3, 6, 9 and 12 o'clock positions for convenience but it is likely that, provided the implants are inserted correctly and distributed equally around the anal canal, the actual position may not influence the outcome. Whether the number of prostheses implanted influences the outcome is not clear at this stage.In recent literature reviews, Vaizey and KammThe major problem with other anal bulking agents used so far is their reduced efficacy with time, probably due to a variable combination of degradation and/or diffusion through the tissue adjacent to the injection site or, sometimes, far from that site . The present data suggest that the Gatekeeper™ overcomes all of these potential problems. In this preliminary experience, implantation of the Gatekeeper™ in the anal canal was safe, without any morbidity, either during or after surgery. The entire procedure was painless and well tolerated. Wounds healed in all patients and no discomfort was noted either at rest or on defaecation. Adverse events, sometimes severe, have been described for some other bulking agents used in urinary incontinence or FI, including pulmonary embolism, fatal stroke, periurethral mass and suspected disease transmissionOf utmost importance was the significant improvement in episodes of major FI and both CCFIS and Vaizey score. Ability to postpone defaecation for longer and postevacuation soiling were both significantly improved in the majority of patients. Of note was the improvement in both SF-36® and FIQL scores in this series, suggesting regained health and quality of life in patients treated with Gatekeeper™. Furthermore, findings observed in the short and medium term appeared substantially unchanged at the final evaluation. This is important as some of the other bulking agents demonstrated very poor long-term results and have had no further clinical use despite preliminary reports suggesting good clinical effectiveness (polytetrafluoroethylene, autologous fatThis preliminary study demonstrated that the Gatekeeper™ anal implant was a safe, reliable and effective treatment for FI, with results maintained over time. These results call for larger series and longer follow-up better to establish the role of the Gatekeeper™ in the management of FI.


Radiation damage propagation was examined in starch granules by synchrotron radiation micro- and nano-diffraction techniques from cryo- to room temperatures. Careful dose limitation allowed raster-diffraction experiments with 500 nm step resolution to be performed. Canna edulis and Phajus grandifolius starch granules. A total loss of crystallinity of granules immersed in water was found at a dose of ∼1.3 photons nm−3. The temperature dependence of radiation damage suggests that primary radiation damage prevails up to about 120 K while secondary radiation damage becomes effective at higher temperatures. Primary radiation damage remains confined to the beam track at 100 K. Propagation of radiation damage beyond the beam track at room temperature is assumed to be due to reactive species generated principally by water radiolysis induced by photoelectrons. By careful dose selection during data collection, raster scans with 500 nm step-resolution could be performed for granules immersed in water.X-ray radiation damage propagation is explored for hydrated starch granules in order to reduce the step resolution in raster-microdiffraction experiments to the nanometre range. Radiation damage was induced by synchrotron radiation microbeams of 5, 1 and 0.3 µm size with ∼0.1 nm wavelength in B-type potato,  The ultimately accessible length scale in a raster-diffraction experiment is limited by the beam-size-defined step-resolution. A nanometre step-resolution allows in principle local inhomogeneities to be resolved in real space on length scales which are accessible in reciprocal space to high Q-resolution scattering techniques. In practice, a ∼200 nm step-resolution has been demonstrated for radiation-hard poly fibres a, 1c               c               b               et al., 2004Experiments were performed on whole granules and sections from the central part of potato starch granules. Granule sections allow the propagation of radiation damage to be studied without the influence of the granule shell structure , reflecting the lateral chain–chain correlation of the hydrated fraction  is assumed for starch granules in saturated water vapour or immersed in water. Complete amorphization  corresponds therefore to the total disappearance of the 100-reflection.Starch is a semicrystalline carbohydrate biopolymer composed of a shell structure containing amorphous and semicrystalline growth rings  structural loss. This is a factor of about four times smaller than the value of ∼5 photons nm−3 determined previously (see supplementary information) hilum).The azimuthal width of the 100 reflection observed in a transmission microdiffraction experiment is sensitive to the granule shell structure as the polysaccharide chains are oriented normal to the shell surface . This result suggests that the primary radiation damage remains confined to the beam track at cryotemperatures. We tentatively assume that primary radiation damage owing to atomic ionization results in a cleavage of the gluco-pyranose ring followed by a further decomposition into fragments including gaseous products. Such decomposition reactions are known from γ-irradiation of cellulose and starch  image of the irradiated section reveals holes at the position of the individual raster-points Phajus grandifolius granule which had been irradiated for 30 s by a 0.3 µm beam of about 4 × 1010 photons s−1 flux. The analogue composite pattern with the integrated 100-reflection intensity is shown in Fig. 5(b)et al., 2009i.e. 6.5 s overall for each consecutive line-scan. The composite pattern shows that the structural loss extends rather symmetrically around the irradiated point with a width of about 7 µm. We have fitted Gaussian functions to the normalized intensity variation through the irradiated centre along a horizontal and vertical line  dependence of azimuthal width but the limited amount of data points would also allow an exponential dependence . A similar structural loss was observed for potato starch granules which had been immersed in water  or the proposed energy-recovery linac sources (Bilderback et al., 2010et al., 2009There are several possible strategies for raster-scan data collection on starch granules at RT. A frequently used strategy is to maintain the dose at each raster point close to amorphization, but to keep the distance between raster points larger than the photoelectron travel range, in order to avoid radiation damage spill-over. Alternatively, as shown in this study, one can collect data at a fraction of the local amorphization dose so that radiation damage spill-over does not significantly reduce reflection intensities at neighbouring raster points. This strategy is valuable for recording the strongest reflections with a nanometre step-resolution. Indeed, a 0.1 s pattern for a flux of 4 × 1010.1107/S0909049510028335/ms5024sup1.pdf            Supplementary material file. DOI:


Chondrocytes are regularly exposed to load-induced stimuli and have the capability to sense and respond to applied mechanical stress. However, the mechanisms involved in chondrocyte mechanotransduction are not clearly understood. The purpose of this study was to explore the effects of cyclic equibiaxial mechanical stretch on the expression of α-BK and TRPV4 channels.Ca and TRPV4 channel proteins. Mechanical stretch increased the expression of BKCa channels by 1.8 fold but TRPV4 expression was not affected.Freshly isolated equine articular chondrocytes were subjected to mechanical stress (8% elongation at frequency of 0.5 Hz for 8 h). Western blotting was used to investigate the expression of BKCa channel may be the result of direct membrane stretch or elevated intracellular Ca2+.Upregulation of BK Chondrocytes are the only resident cells within the extracellular matrix (ECM) of articular cartilage  were dissected from metacarpophalangeal joints of healthy mature horses euthanized for unrelated clinical reasons. Articular chondrocytes were isolated as previously described  as we have described previously  at 2 × 10P ≤ 0.05 considered statistically significant.Statistical analysis: Average data are presented as a mean ± S.E.M, Student’s t-test or ANOVA was used to determine the statistical significance, followed by Bonferroni's test for multiple comparisons. Cyclic stretching significantly increased the expression of the α-BK channel by 1.8 fold) but in contrast the expression of the TRPV4 channel was not effected by mechanical stretch . An alternative hypothesis for BK channel upregulation requires the elevation of intracellular Ca2+ concentration (Ca2+-dependent) that could occur through intracellular Ca2+ release  and/or members of TRP channel family. The level of TRPV4 protein was not significantly effected following exposure to mechanical stress which is interesting as it has been previously shown that the overall activity of this channel is directly related to its expression level (Veys et al., In load bearing joints chondrocytes are exposed to mechanical forces that lead to membrane deformation, which in turn, may significantly alter cartilage matrix production Urban, . ChondroThe results of this study suggest that in equine articular chondrocytes the α-BK but not the TRPV4 channel is upregulated by cyclic equibiaxial membrane stretch.