Early identification of permanent hearing impairment in children enables appropriate intervention which reduces adverse developmental outcomes. The UK Government has introduced a universal hearing screening programme for neonates. All involved health professionals, including those in Primary Care, need to be aware of the service to enable them to offer appropriate support to their patients. A programme of information dissemination within Primary Care was therefore undertaken. The aim of the current study was to determine the extent to which the information had reached General Practitioners (GPs), the GPs' preferred mode of dissemination and the sources from which GPs accessed informationPostal questionnaire survey of a randomised sample of 1000 GPs in the Phase I pilot sites of the Neonatal Hearing Screening Programme (NHSP).Responses were received from 54.2% of the sample. Just under 50% of those responding had received information, 62.2% of respondents said they would like to receive more information and the preferred methods of dissemination were the written word and web-sites to allow access when needed. Few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject. Many are keen to delegate detail to a third party, usually the health visitor, who has traditionally had responsibility for hearing screening.Dissemination efforts for service developments of relevance to GPs should concentrate on advertising a website address via brief but memorable posted literature and/or articles in relevant journals and magazines. The website should be GP-friendly, and have a dedicated area for GPs including information of specific relevance and downloadable information sheets. Approximately 1.65 per 1000 babies born in the UK have a significant permanent hearing impairment >40 dB HL) detectable at birth . Thus, b0 dB HL dThis study uses the introduction of the Newborn Hearing Screening Programme (NHSP) to illustrate the issues around dissemination to GPs ,7. IntroThe phased implementation of NHSP in the UK began in 2001 in 23 pilot sites and was accompanied by a campaign of information dissemination including distribution of leaflets and posters to all general practices, the development of a dedicated website and appointment of local co-ordinators. The aim of the current study was to determine the extent to which the dissemination information had reached the GPs, the GPs' preferred mode of dissemination and the sources from which GPs accessed information.We designed and piloted a one-page questionnaire see  and usedOf the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website.When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences.To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%).In open comments  it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening.The response rate of more than 50% is acceptable for a survey of GPs. A second mailing might have improved this but its advantage was weighed against the attraction of anonymity. To minimise the number of questions and encourage response, no demographic details were requested. Analysis of any response bias by age of practitioner, size of practice etc was therefore not possible. A response might be more likely from people who knew something of the subject and our estimates for the level of awareness might therefore be high but it is likely that the views expressed concerning resources accessed and preferred channels of dissemination are representative.Although considerable effort had been expended in distributing letters, leaflets and posters to every general practice, less than half the respondents said they had seen it. It is possible that mail had been screened and intercepted by administrative staff in the practice or rejected as "junk mail" by the GP. It is important therefore to ensure such mailed information is identified as relevant. Some health service developments will not be high on the average GP's agenda. In terms of neonatal hearing screening s/he will see only 20 newborns per year on average and approximately one permanently hearing-impaired child in a working lifetime.Nearly two-thirds of respondents said they would like to receive more information even though many complained about "information overload". This is a weakness inherent in surveys of this nature where stated attitudes may reflect socially desirable responses rather than respondents' true attitudes. Further exploration of this issue using qualitative techniques of personal interview would contribute to the general issue of information dissemination to GPs.GPs cannot know everything about everything and do not necessarily need to but they do need to know where they can find the relevant information and to be able to access it easily . Our surWe suggest that any dissemination effort for service developments of relevance to GPs should concentrate on advertising the website address via brief but memorable posted literature and/or articles in relevant journals and magazines. It is also crucial that any website be GP-friendly by having a dedicated area for GPs including information of specific relevance and downloadable information sheets.The author(s) declare that they have no competing interests.PM designed and carried out the survey, performed the analyses and wrote the original report. HF supervised the design and implementation of the study and drafted the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:A survey of awareness of the NHS Newborn Hearing Screening Programme (NHSP) amongst General Practitioners (GPs) QuestionnaireClick here for file


Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.We have elucidated the kinetics of histone methylation during X inactivation using an inducible  In female mammals, one X chromosome is inactivated. Anton Wutz and colleagues find evidence for a very early event that 'marks' the chromosome for future stable inactivation Xist RNA (Xist RNA attaches to chromatin and spreads from its site of transcription in cis over the entire inactive X chromosome (Xi), mediating transcriptional repression. Xist is essential for initiation of silencing, but not for the maintenance of transcriptional repression on the Xi at later stages of cellular differentiation  and lysine 27 (H3-K27) methyltransferase activity , revealing a novel chromosomal memory that is established by Xist expression at an early time point in ES cell differentiation independent of transcriptional repression. Our analysis suggests that this chromosomal memory might have an important role in the transition from the initiation phase to the maintenance phase of X inactivation.We have previously shown that chromosomal silencing can be recapitulated in embryonic stem (ES) cells by expressing romosome , and thiXist RNA . Initiatpression . TherefoXist RNA in the initiation of chromosomal silencing and for analysing the kinetics and relevance of chromosomal modifications. We aimed to delineate a pattern of histone methylation states that define the initial decision for facultative heterochromatin. To achieve this we performed immunofluorescence staining against the various modification states on histone H3 and H4 lysine residues in clone 36 ES cells, in which Xist expression can be induced from a transgene integrated on Chromosome 11 by addition of doxycycline to the culture medium  experiments using antibodies specific for H3K27m3, H4K20m1, H3K4m3, H3K4m2, and H3K9m2 in both undifferentiated and differentiated clone 36 ES cells in the presence or absence of doxycycline . We obseosome 11 . A controsome 11 F and 2L.d sample B. This induction E and 2K.Xist RNA expression in undifferentiated clone 36 ES cells  showing H3K27m3 staining compared to the wild-type Xist RNA , which is cointegrated with the Xist cDNA transgene on Chromosome 11 and can be silenced by transgenic Xist expression. Xist expression was induced either from the beginning or at 24, 48, 72, 96, or 120 h after the onset of differentiation. The ability of Xist to initiate silencing at various time points was monitored by measuring puro expression, and H3K27m3 was analysed in parallel in all cultures at 12 d after differentiation  and a homozygous insertion of the tetracycline-responsive transactivator in the ROSA26 locus . In uninduced cultures and control male fibroblasts no H3K27m3 foci were detected by immunofluorescence in interphase nuclei. However, upon Xist induction 5% (after 48 h of Xist induction) or 15% (after 72 h) of the cells showed focal H3K27m3 staining  and 52% (day 16.5 embryos) of the cells expressed Xist, as determined by RNA fluorescent in situ hybridization (FISH) analysis . We then analysed the kinetics of loss of methylation in differentiated ES cells. Clone 36 ES cells differentiated for 4 d in the presence of doxycycline were differentiated for 24, 48, and 72 more hours in the absence of doxycycline, and H3K27m3 was measured . In cells that had been continuously differentiated in the presence of doxycycline, methylation was detected in 73%–78% of the nuclei. Our data show that efficient methylation at late time points in differentiation did not require continuous Xist expression. Efficient remethylation occurred on a chromosome that had been exposed to Xist in early ES cell differentiation, consistent with the idea that Xist triggers a chromosomal change in early differentiation that is remembered until later time points to enhance H3K27m3 reestablishment. Importantly, the silencing-deficient Xist mutant RNA in J1:XistΔSX-tetOP ES cells gave identical results, showing that this memory is established independent of silencing.To test whether continuous ectively . ParalleXist was turned off until day 8 of differentiation, when doxycycline was added back, and remethylation was assayed by immunofluorescence at day 13 in differentiation . We attribute the different observations in other studies to the various antisera used. We supply peptide blot analysis for our antisera that suggest that the antibodies are highly specific . We suggest that Xist expression and H3K27m3 might be the signal to recruit a chromosomal memory mediating the lock-in of X inactivation . For sections, embryos were sexed (Clone 36 ES cells  and J1:Xeletions  were culeviously . Primaryeviously . Xist exre sexed  and fixe2), centrifuged for 10 min at 1,200 rpm onto Menzel SuperFrost slides  using a Cytospin 3 centrifuge . Staining was performed as described previously , blocked in blocking solution , and incubated with primary antibodies for 3 h. After washing three times for 10 min in TBST  and incubation with secondary antibodies , detection was performed using ECL reagent . Rabbit polyclonal α-Eed , rabbit polyclonal α-Ezh2 , goat polyclonal α-histone H3 , and rabbit polyclonal α-histone H4  were used.For DNA FISH analysis, biotin-labelled STAR*FISH mouse whole chromosome-specific probes  were detected with streptavidin, Alexa Fluor 633 conjugateS-21375 (Molecular Probes). RNA FISH probes were generated by random priming  using Cy3-dCTP (Amersham). Hybridisation and washing were carried out as described previously . SpecimeCells were cross-linked with 1% formaldehyde for 10 min at RT and quenched with 125 mM glycine, and whole-cell extracts were prepared. ChIPs were performed in duplicates as described previously . BrieflyFigure S1Immunodotblot analysis  of the a(611 KB PDF).Click here for additional data file.Figure S2Xist for 3 d using H3K27m1 (A), H3K27m2 (B), H3K27m3 (C), H3K9m1 (D), H3K9m2 (E), H3K9m3 (F), H4K20m1 (G), H4K20m2 (H), H4K20m3 (I), H3K4m3 (J), and H4Ac (K) antisera. Chromosome 11 was identified by a DNA FISH probe  in (J) and (K). Clone 36 ES cells grown in the absence of doxycycline are used as a control for the H4Ac staining without Xist expression (L).Immunofluorescence staining of metaphase spreads of clone 36 ES cells induced to express (4.8 MB TIF).Click here for additional data file.Figure S3Xist expression is shown.(A) The kinetics of H3K27m3 was measured in undifferentiated clone 36 ES cells. The number of cells showing H3K27m3 staining 6, 12, 24, and 48 h after induction of n > 150) showing H3K27m3 staining was analysed in undifferentiated clone 36 ES cells, which expressed Xist for 3 d (lane 1) or were further grown without inducer for 24 h (lane 2) or 48 h (lane 3). This experiment complements data presented in (B) The stability of H3K27m3 was determined in undifferentiated ES cells. The percentage of metaphase chromosome spreads n > 10 showingXist expression. No progressive accumulation over time was observed, indicating that the steady state of H3K27m3 has been reached at 3 d Xist expression. However, a marked increase in methylation is observed in J1:XistΔSX-tetOP ES cells upon differentiation for 2 d (hatched bar).(C) Levels of H3K27m3 were measured in undifferentiated ES cells after 3 and 10 d of Xist for 3 and 10 d (percentage of nuclei showing a staining is given). Analysis of H3K27m3 and H4 acetylation using an antiserum specific for multiply acetylated forms of H4 in clone 36 and J1:XistΔSX-tetOP ES cells that were grown for 4 d in the presence of doxycycline and then shifted to differentiation conditions for 2 d more in the presence of doxycylcine.(D) Combined Xist RNA FISH (red) immunofluorescence analysis of Ezh2 and H4K20m1 in undifferentiated J1:XistΔSX-tetOP cells expressing (E) Male primary mouse fibroblasts (PMEFs) hemizygous for the inducible Xist-tetOP allele and homozygous for the tetracycline-inducible transactivator were induced with doxycycline for 2 d (lane 1) or 3 d (lane 2), and the number of cells showing H3K27m3 staining in interphase was analysed. Control female PMEFs showed a methylation signal in the large majority of cells (lane 3); uninduced male PMEFs were always negative.Xist RNA triggers less pronounced and less dense foci of H3-K27 trimethylation (green) compared to the female wild-type control.(F) Representative indirect immunofluorescence of uninduced (top) and induced (bottom) male Xist-tetOP PMEFs. The inducible Xist expression, H4-K20 monomethylation (green) is observed in interphase Xist-tetOP PMEFs (left). Focal enrichment colocalises with the site or Xist RNA clusters (red) on the X chromosome. Female wild-type PMEFs (right).(G) Upon (3.0 MF TIF).Click here for additional data file.Figure S4Xist cDNA in given (top): repeats A to E are indicated by arrays of triangles, sequences mediating localisation to chromatin are indicated by boxes underneath (degree of hatching represents importance), and the location of the deletion is indicated by a coloured box. RNA localisation was analysed by FISH (lower left), showing that the RNA localises in small clusters in some cells. The ability of the RNA to induce silencing was measured by cell survival of differentiating cultures under induced versus uninduced conditions (lower right). Controls are cells either having a fully functional Xist cDNA transgene (Xist) or a cDNA lacking repeat A that is incompetent to induce silencing (ΔSX). The ΔXSa RNA shows poor silencing activity, presumably as a consequence of its failure to localise well to the chromosome.The XistΔXSa transgene was integrated by Cre-mediated recombination into the Hprt locus on the single X chromosome in T20 ES cells . A schem(1.5 MB TIF).Click here for additional data file.Figure S5PR/SET domain-containing protein 07 (Pr-Set7), indicating SET domain in black (gi:38080595). Below, region tested for histone methyltransferase (HMTase) activity.(A) Schematic presentation of full-length mouse Pr-Set7 (arrow), H4 peptides (arrowhead), and histones used for in vitro reactions with S-adenosyl-[methyl-14C]-L-methionine as methyl donor. Fluorography (right) indicates HMTase activity on the unmodified H4 peptide comprising residues 12–31 of the histone H4 N-terminus. Notably, no further methyl groups could be transferred to the same peptide if it had been synthetically monomethylated at residue H4 lysine 20 (K20m1) before usage in the in vitro reaction. Free histones are not accepted as substrate.(B) Coomassie stain (left) shows purified recombinant GST-tagged GST–Pr-Set7 selective for the unmodified histone H4 peptide (12–31). H4-K20 monomethylation obviously is the terminal state for Pr-Set7, because synthetically mono- (K20m1), di- (K20m2), and trimethylated H4 peptides (K20m3) could not be significantly methylated.(C) Fluorography indicates histone H4 HMTase activity of (948 KB TIF).Click here for additional data file.Protocol S1(22 KB DOC).Click here for additional data file.


To evaluate the association between mood and anxiety disorders and thyroid autoimmunity in a community sample. Methods: A community based sample of 222 subjects was examined. Psychiatric diagnoses were formulated using the International Composite Diagnostic Interview Simplified (CIDIS), according to DSM-IV criteria. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO).16.6% of the overall sample had an anti-TPO value above the normal cut-off. Subjects with at least one diagnosis of anxiety disorders  or mood disorders  were positive for serum anti-TPO more frequently than subjects without mood or anxiety disorders. A statistically significant association with anti-TPO+ was found in Anxiety Disorder Not Otherwise Specified , in Major Depressive Episode  and Depressive Disorder Not Otherwise Specified .The study seems to suggest that individuals in the community with thyroid autoimmunity may be at high risk for mood and anxiety disorders. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. Should our results be confirmed, the findings may be of great interest for future preventive and case finding projects. Autoimmune thyroid disease may be linked to depression  and anxiThe purpose of this investigation was to evaluate the relationship between mood and anxiety disorders and thyroid autoimmunity in a community survey. This research was carried out on the data base of two epidemiological studies aimed at defining the prevalence of psychiatric  and thyrThis paper present the results of the cross psychiatric and endocrinological evaluation from the common areas of the two surveys.The sample was extracted by randomization (1/10) subsequent to stratification according to age and sex, from the records of 2 Sardinian villages. Probands were interviewed face to face in their homes by specifically trained physicians. Two standardized forms were used to acquire information concerning: demographic data, state of health and use of social and health services. Psychiatric diagnosis was made using the Italian Simplified version of the Composite International Diagnostic Interview (CIDIS) . The comAnti-thyroid peroxidase autoantibodies (anti-TPO), considered as the most sensitive and specific marker of thyroid autoimmunity  was deteAll subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). FT4 and FT3 were measured by means of a chromatographic method based on separation of free T4 on Lisophase columns . TSH was measured by a chemiluminescent method  with normal values ranging from 0.3–3.0 μU/ml. Thyroid echography was performed using a "real time" echograph .2 test in 2 × 2 tables. Odds Ratio confidence intervals were calculated through application of the method of Miettinen [The association of anti- TPO+ with the main diagnoses deriving from CIDIS interview was calculated using Odds Ratio. Statistical significance was calculated using the Xiettinen .Multivariate Logistic Regression was performed in order to evaluate the possible influences of gender and age on the association between anti-TPO+ and mood or anxiety disorders. The analysis was carried out considering mood (or anxiety) disorders as dependent variable, and anti-TPO+ (presence vs absence), gender  and age (≤ 44 vs > 44) and their second order interactions as independent variables, by means of backward stepwise procedure; interactions lacking evidence of association (p > 0.20) were eliminated from the models.From a total of 261 subjects identified (age >18 years), 222 (85.1%), 127 females (57.2%), and 95 males (42.7%); over 44 years 127 (57.2%), 79 females  48 males (37.7%), agreed to take part in the study whilst 20  refused to participate and 19 (7.3%) could not be traced. The final sample did not differ respect to the population of origin with reference to the variables applied in stratification.The lifetime prevalence of anxiety disorders in the sample was: Generalized Anxiety Disorder (GAD) 11.3%, Panic Disorder (PD) 2.7%, Anxiety Disorder Not Otherwise Specified (ADNOS) 5.4%, Social Phobia (SP) 5.4%; 18.5% had been diagnosed with at least one of these anxiety disorders. With regard to mood disorders, Major Depressive Episode (MDE) was present in 14.4%, Dysthymic Disorder (DD) in 2.7%, Depressive Disorder Not Otherwise Specified (DDNOS) in 4.0%; 18.9% had at least one of the above mentioned depressive disorders. 1.1% of the overall sample was affected by hypothyroidism, 16.6% had an anti-TPO value above the normal cut-off (anti-TPO+).Table The Multivariate Logistic Regression showed that gender and age do not interact with anti-TPO either in mood , or in anxiety disorders . Interactions were therefore eliminated from the models.The final Logistic Regression models clearly indicated that gender and age do not influence the risk of one mood or anxiety diagnosis either as independent variables or as confounders Table .The present study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+ in a general population sample which had not been selected from medical or psychiatric health facilities. This association is independent by gender and age. Regarding specific diagnosis, MDE, DDNOS and ADNOS were associated with anti-TPO+.This finding is consistent with several previous clinical studies providing evidence for a significant association of mood disorders or post-partum depression and symptomless autoimmune thyroiditis with or without sub-clinical hypothyroidism . AssociaA sub-clinical dysfunction of axis Thyrotropin Releasing Hormone (TRH) – Thyroid Stimulating Hormone (TSH) with consequent alteration of circadian rhythms of TSH has been hypothesized in some depressive disorders. Indeed, this hypothesis may explain why some forms of mood disorders were associated with anti-TPO+ or thyroid autoimmunity without hypothyroidism, as defined by routine blood tests. A slight reduction in thyroid hormone secretion such as that found in sub-clinical hypothyroidism may affect cognition and mood . At variMoreover, a study carried out in a large community sample found no association between thyroid dysfunction, including hypothyroidism defined by thyroid blood test, and the presence of depression or anxiety symptoms . This suWith regard to the public heath aspects of this research, should these findings be confirmed, they would constitute a most important public health issue, due to the high attributable risk found. The attributable risk is a useful measure to document the burden of risk to a community. Attributable risk depends both on the magnitude of relative risk and on the prevalence of the risk factor in the population . The higThe potential of the study is reduced by the small sample size, particularly regard to psychiatric diagnoses less frequently observed in the general population, such as Panic Disorder; the extension of the findings is therefore rather limited.This study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. If the findings are confirmed, they may prove to be of interest for future projects of case finding: a systematic screening for mood disorders in anti-TPO+ subjects and a systematic evaluation for thyroid diseases and thyroid autoimmunity in subjects with mood disorders may be advisable.None declared.MGC conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. AL, SM and CS participated in the statistical analysis and drafted the manuscript. MCH, SM, MC, BC, LDO participated in its design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Statins also inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases—Rho A, B, and C—as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs). Several cell-surface molecules are substrates for activated α-secretase-type ectodomain shedding, and regulation of shedding typically occurs via activation of protein kinase C or extracellular-signal-regulated protein kinases, or via inactivation of protein phosphatase 1 or 2A. However, the possibility that these enzymes play a role in statin-stimulated shedding has been excluded, leading us to investigate whether the Rho/ROCK1 protein phosphorylation pathway might be involved.Statins are widely used cholesterol-lowering drugs that act by inhibiting HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drugs' ability to activate α-secretase-type shedding of the α-secretase-cleaved soluble Alzheimer amyloid precursor protein ectodomain (sAPPα shedding from a neuroblastoma cell line via a subcellular mechanism apparently located upstream of endocytosis. A farnesyl transferase inhibitor also increased sAPPα shedding, as did a dominant negative form of ROCK1. Most conclusively, a constitutively active ROCK1 molecule inhibited statin-stimulated sAPPα shedding.We found that both atorvastatin and simvastatin stimulated sAPPα from cultured cells, at least in part, by modulation of the isoprenoid pathway and ROCK1.Together, these data suggest that statins exert their effects on shedding of sAPP The cholesterol-lowering statins seem to have a protective effect against Alzheimer disease. In cultured cells, statins affect cleavage of the amyloid precursor protein via the ROCK kinase. This could explain some of their beneficial effects Typical maturation and half-life of mature cellular holoAPP were observed, as was subsequent release of sAPPα [t = 0 versus a control [vehicle treatment] of 150% of the level of immature APP at t = 0; also, at t = 30 min, the relative percent values for drug versus vehicle were 380% and 200%, respectively). Fold increases in released sAPPα in the same experiments were approximately 3- to 4-fold . Secretory maturation toxicity is one possible mechanism for elevated levels of intracellular mature holoAPP and causes retarded conversion of mature holoAPP to sAPPα. This pattern was not observed following statin treatment, excluding maturation toxicity as a mechanism underlying the altered levels of mature cellular holoAPP.A pulse-chase protocol was also used to study post-transcriptional regulation of APP metabolsm by statins (data not shown). This protocol avoids any confound that might arise because of altered APP transcription. Pulse-chase studies were performed using a 10-min pulse with 3Smethioniof sAPPα . In the α. However, the fold effect on reduced intracellular turnover in the endosomal/lysosomal pathway  is apparently insufficient to explain the fold effect on sAPPα generation , indicating a contribution from a downstream site in the processing pathway. When these results are taken together with independent work on regulated shedding of transforming growth factor α (TGFα) [Instead, the pattern that we observed raises the possibility that statins, presumably via isoprenoids , as discussed in the next section, may alter sorting of cellular holoAPP, diverting holoproteins away from terminal degradation in the endosomal/lysosomal pathway and into the constitutive secretory pathway that generates sAPPα (TGFα) ,36, a paα or TGFα [The identification of the regulatory components of the ectodomain shedding machinery have been long-sought in other studies employing phorbol esters to stimulate shedding of sAPP or TGFα ,35,36,37 or TGFα . In our NPXY clathrin-coated vesicle targeting sequence [α shedding, presumably by extending the half-life of co-localized α-secretase and APP on the plasma membrane. In order to exclude the possible contribution of altered endocytosis to statin-stimulated shedding, we evaluated the effect of phenylarsine oxide (PO), an inhibitor of endocytosis, on statin-stimulated shedding . The additivity of statin- and PO-stimulated shedding is consistent with the hypothesis that statins act at or near the plasma membrane, prior to internalization of csAPP. Under all circumstances, stimulated sAPPα shedding was completely blocked using TNFα protease inhibitor 2, a standard α-secretase/metalloproteinase inhibitor . In the same experiment, levels of holoAPP were modestly increased but, again, to an extent insufficient to account for the increase in shed sAPPα  of mevalonic acid did not affect statin-induced sAPPα shedding, but complete inhibition of statin-activated sAPPα shedding was achieved with higher doses of mevalonic acid (100 μM). As predicted, the shedding observed following treatment with FTI-1 was not inhibited by any of the concentrations of mevalonic acid tested . Conversely, DN ROCK1 alone activated shedding of sAPPα. Cellular levels of holoAPP were not affected by transfection (p < 0.05 versus GFP control). In studies aimed at independent confirmation of the involvement of ROCK activation in sAPPα shedding, we treated SweAPP N2a cells with arachidonic acid, an activator of ROCK. As shown in α without altering levels of holoAPP. Based on this series of results, we concluded that both basal and activated sAPPα shedding from cultured cells are controlled by ROCK activity.Since many isoprenoid-mediated Rho effects converge on ROCKs, we next transfected N2a cells with cDNAs encoding either green fluorescent protein (GFP) (control), CA ROCK1, or DN ROCK1 . Simvastα release and blocked statin-activated sAPPα shedding (data not shown). This result was unexpected in light of the effects of DN ROCK1. Given the internally consistent actions of DN ROCK1 and CA ROCK1, as well as the results employing either FTI-1 or arachidonate, we concluded that the Y-27632 result might be due to inhibition by Y-27632 of protein kinases other than ROCK1 [In some experiments, cells were treated with Y-27632 (10 nM to 50 μM), a compound that can inhibit ROCKs. Y-27632 showed no effect on basal sAPPan ROCK1 . The posThe isoprenoid pathway involves lipid modification of various members of the Rho family of small GTPases by the addition of either farnesyl or geranylgeranyl moieties ,42. Isopα in the cerebrospinal fluid and blood. This aspect of APP metabolism bears resemblance to the proteolytic signal transduction pathways involved in processing pro-TGFα [The first evidence that APP might be a substrate for ectodomain shedding was provided by Weidemann et al.  who idenpro-TGFα ,36 and Npro-TGFα . In the pro-TGFα ,29,45. Ipro-TGFα . Ultimatpro-TGFα .α rises from 2 mol shed per 10 mol synthesized to 4 mol shed per mole synthesized. Most of this shedding is catalyzed by the α-secretase pathway, but a trace amount  is cataunt  or by terminating intercellular signaling . A popular model holds that sAPPα may function as a neurotrophic and/or neuroprotective factor, and may promote neurite outgrowth [What is the function of shed sAPPutgrowth . More reutgrowth . Targeteutgrowth , presumautgrowth . Mice wiCao and Sudhof  have recThe potential role of cholesterol in α-secretase-mediated shedding was discovered by Bodovitz and Klein  who usedα following statin application.Maillet et al.  implicatα shedding, i.e., activation of ROCK1 blocks basal and stimulated shedding while ROCK1 inhibition apparently relieves a tonic negative influence exerted on shedding by ROCK1 activity.CA ROCK1 and DN ROCK1 molecules yielded direct and complementary evidence that ROCK1 was indeed a candidate for modulation of statin-activated α-secretase action. Further, we were able to demonstrate that α-secretase activity could be modulated by molecules further upstream in the isoprenoid pathway see . FTI-1, As in PKC- and ERK-activated shedding, the ROCK1 substrate effector molecule or molecules that regulate proteolysis by ADAMs remain to be identified. The cytoplasmic domains of both APP and ADAM-17 have been evaluated as candidates for important targets of protein phosphorylation during the regulated shedding process, but neither “substrate activation” nor “enzyme activation” appears to explain the phenomenon, i.e., phosphorylation of neither APP nor ADAM-17 dramatically increases the efficiency of α-secretion ,62, indiOur data using statins and PO localize the mediator of statin-activated shedding to the plasma membrane, upstream of endocytosis, as appears to be the case for PKC-activated shedding ,63,64,65α-Secretase activation is a potential therapeutic strategy for modifying cerebral amyloidosis in Alzheimer disease . This prPreliminary results from a pilot proof-of-concept using atorvastatin in a human clinical treatment trial are consistent with the proposed beneficial effects of this class of compounds . Since aThe results reported here point to several areas for additional investigation. As described above, the key substrate or substrates linking cytoplasmic protein phosphorylation to intralumenal or cell-surface protelysis remain to be identified. Nonetheless, α-secretase activation has been validated as a viable therapeutic strategy for modulating cerebral amyloidosis . Identifhttp://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the proteins discussed in this paper are ROCK1 (NP_005397) and APP695, 595–596 KM/NL (NP_958817).The GenBank  is broken down (or cleaved). There are two ways in which APP can be broken down. “Bad cleavage” releases the toxic amyloid-beta, whereas “good cleavage” destroys it. When researchers gave statins to animals over a long period of time, they found that statins could slow down the formation of amyloid plaques. From the animal experiments, it seemed that statins somehow caused more good cleavage to occur.This study examined how statins can affect APP cleavage.They studied cells to see which of the players known to be involved in APP cleavage were affected by statin.Statin's ability to promote “good cleavage” of APP involves a molecular pathway called the Rho/ROCK1 pathway. It seems that when ROCK1 is active, less good cleavage takes place. But in the presence of statins, ROCK1 is less active, shifting the balance toward good cleavage. Consistent with this, when the scientists blocked the Rho/ROCK1 pathway, they saw the good cleavage pattern even without statin.Inhibition of the Rho/ROCK1 pathway could explain some of the beneficial effects of statins against Alzheimer disease. And the pathway itself seems worth more research to see whether it might be a good target for new ways to prevent and treat Alzheimer disease.Statins are likely to influence the risk for Alzheimer disease by several different pathways, and future studies will need to show how important this particular pathway is in the overall picture. Moreover, studies like this one are by necessity done in cells under carefully controlled laboratory conditions and still a long way from the development of safe and effective drugs.http://www.alz.org/Resources/TopicIndex/statins.aspFactsheet on statins from the Alzheimer's Association: http://www.alzheimers.org/index.htmlGeneral information at the Alzheimer's Disease Education and Referral Center at the United States National Institute of Aging: http://www.alz.co.uk/Homepage of Alzheimer's Disease International, an umbrella organization of Alzheimer disease associations around the world:


The first stage in computerised processing of the electrocardiogram is beat detection. This involves identifying all cardiac cycles and locating the position of the beginning and end of each of the identifiable waveform components. The accuracy at which beat detection is performed has significant impact on the overall classification performance, hence efforts are still being made to improve this process.A new beat detection approach is proposed based on the fundamentals of cross correlation and compared with two benchmarking approaches of non-syntactic and cross correlation beat detection. The new approach can be considered to be a multi-component based variant of traditional cross correlation where each of the individual inter-wave components are sought in isolation as opposed to being sought in one complete process. Each of three techniques were compared based on their performance in detecting the P wave, QRS complex and T wave in addition to onset and offset markers for 3000 cardiac cycles.Results indicated that the approach of multi-component based cross correlation exceeded the performance of the two benchmarking techniques by firstly correctly detecting more cardiac cycles and secondly provided the most accurate marker insertion in 7 out of the 8 categories tested.The main benefit of the multi-component based cross correlation algorithm is seen to be firstly its ability to successfully detect cardiac cycles and secondly the accurate insertion of the beat markers based on pre-defined values as opposed to performing individual gradient searches for wave onsets and offsets following fiducial point location. Computerised classification of the electrocardiogram (ECG) is a complex and multi staged process. The overall goal is to determine if the patient is 'normal' and may remain untreated, or whether the patient exhibits any cardiac abnormalities requiring treatment. The classification of the ECG by computerised techniques has been an active area of research for more than 4 decades. A plethora of algorithmic techniques have been applied and developed ,2 all wiWith a multi-stage computerised approach, the overall classification capabilities of the system are highly dependent on the early stages of processing, i.e., accurate detection of each ECG complex in all recorded leads. Hence the necessity of a reliable beat detection algorithm is of paramount importance. Regardless of the approach employed to analyse and classify the ECG signal, all require accurate detection of each QRS complex. Beat detection algorithms are designed with two main objectives. Firstly, the algorithm employed should provide reliable detection of each cardiac cycle. Secondly, the temporal location of the reference points should be described accurately. In general terms detection of each cardiac cycle involves the location of a fiducial point, usually taken as the peak amplitude of the R-wave or of the QRS complex. From this it is then possible to detect markers for the other interwave components (if present) and features of the ECG; QRS onset and offset, P onset and offset and T offset Figure .Two of the most commonly employed approaches to beat detection involve the use of established non-syntactic algorithms -5 or croA non-syntactic approach to beat detection normally involves an algorithm based on two distinct steps; a pre-processor and a decision rule. The function of the pre-processing stage is to enhance the QRS complex and suppress all other components of the recorded signal i.e. P and T waves and noise artefacts. This is achieved through firstly applying a linear filter to extract the required frequencies, followed by a non-linear transformation with the goals of providing a single positive peak for each QRS complex. The output from the non-linear transformation is then processed by a thresholding function to indicate the presence or absence of a QRS complex. Following location of a reference point remaining inter-wave components can be located to the left (P wave) and the right (T wave) of the QRS complex.A number of non-linear transformations exist which can be applied and can occur in a number of different permutations. A commonly adopted approach is conceptualised in Figure The CC function can be used to measure the similarities between two signals . This prN represents the number of samples, j is the lag factor and x1 and x2 are the two signals. To normalise the results based on the number of sample points, the factor 1/N is introduced. When the value of r(12) is maximal, this is considered as the point of maximal similarity between the two waveforms. As the required amount of lagging between the two signals is initially unknown, various degrees of lags within the specified correlation interval must be performed.where It is possible to use CC for the purposes of beat detection, by locating the point of maximal similarity between an ECG signal and a predefined template and hence identifying the temporal location of the QRS complex. It is also necessary for the CC algorithm to store a template or a reference signal of the ECG signal. The origin of the template may be from a variety of sources. It may be of an adaptive nature, whereby a section of the patient's recorded ECG is averaged and stored prior to the analysis. Alternatively, a mathematical model may be used or a collection of ECG recordings from a database used to produce a generic template.et al. [et al. [et al. [Many studies in the past have successfully reported the use of CC as a means of automated beat detection. Abboud et al.  used theet al.  and averet al. . Methods [et al.  were ada [et al.  to facilIt is the aim of the current study to investigate the potential of adapting the general CC based approach as an accurate and reliable means for beat detection. This approach in the past has been shown to be successful and hence is investigated with the aims of further enhancement. Individual templates for P waves, QRS complexes and T waves are generated and the CC approach applied individually to identify each of these components as opposed to a one template CC based approach. Standard approaches have also been developed for benchmarking purposes.The structure of the remainder of the paper is as follows: Section 2 describes the new beat detection method based on the fundamentals of CC. In addition a description of the benchmarking methods developed are also presented. Section 3 describes the structure of the data set and presents the results and discussions for the beat detection algorithms. Final conclusions to the study are presented in Section 4.The aims of the current study were to investigate the possibilities of developing a new approach to beat detection which would offer enhanced accuracy and reliability in comparison to established techniques. Two different approaches, one based on a standard non-syntactic approach and one based on a standard CC approach were developed for the purposes of benchmarking. Developments of both of these algorithms were based on existing published approaches. A new approach, based on a multi-component based CC algorithm was additionally developed. Details of the 3 beat detection approaches are as follows.The first stage of the non-syntactic approach is the inclusion of a bandpass filter, centred at 17 Hz. The purpose of this is to isolate the predominant QRS energy and attenuate the low-frequency characteristics of the P and T waves, any baseline drift present and the higher frequencies associated with electromyographic noise and power line interference .  = 0.1(2 + z-1 - z-3 + 2z-4)     (3)H(z) in Equation 3 behaves in a similar manner to a parabolic smoothing filter and does not amplify any high-frequency noise. The advantage of such a filter is that it goes to zero at half of the sampling frequency. The non-linear transformation is performed by the point-by-point squaring of the signal samples. This derivative approximates an ideal derivative in the dc through to 30 Hz frequency range. This is the necessary frequency range since all higher frequencies are significantly attenuated by the bandpass filter. Finally, the squared waveform is passed through a moving window integrator. A window integrator with the difference equation as presented in Equation 4 was employed.This has been chosen as the function N is the number of samples in the width of the moving window. The integrator sums the area under the squared waveform over an approximate 180 ms interval, advances one sample interval and integrates a new 180 ms window.where To locate the fiducial point, the maximum of the QRS complex is required. Following application of the aforementioned processes to the recorded signal, the maximum amplitude of the resultant signal is sought. An initial threshold value based on 80% of the maximum amplitude located during an initial training period is used to locate potential QRS complexes. Within a 125 ms window, following exceeding this value, the maximum amplitude located is considered to be the fiducial point. A refractory blanking stage of 200 ms is employed prior to repeating the process for the next cardiac cycle. The markers for the onset and offset are inserted following gradient searches to the right and left of the fiducial point within a window of 200 ms on each side. A thresholding technique was exercised, whereby following the location of 6% of the gradient, for a duration of at least 25 ms, to the left and right of the fiducial point the start and end points were identified respectively.Prior to location of the T wave offset, the original signal was low-pass filtered at 12 Hz in an effort to maximise the frequency content of the T wave whilst suppressing the remaining inter-wave components. With knowledge of the end of the QRS-complex and the beginning of the next adjacent QRS-complex a value of the peak of the T wave, i.e. the maximum value within the identified window, was calculated. The T-offset is located based on a gradient descent thresholding search method. The condition of thresholding was set at 25% of the maximum gradient of the T wave, for a duration of at least 25 ms, to the right of its peak.Following filtering and smoothing, the P wave was sought in a window of 300 ms prior to the QRS onset. Similar to the T wave offset location, the maximum (or peak) is located within the defined window prior to the location of the onsets and offsets. The onset is located following detection of 75% of the maximum gradient, for a duration of at least 25 ms, to the left of the peak and the offset is located following detection of 75% of the maximum gradient, for a duration of at least 25 ms, to the right of the peak.k can be defined as in Equation 5:The necessary templates required for the CC were generated during an initial training stage prior to any CC based analysis. During this stage templates required on an individual lead-per-lead basis were generated by averaging cardiac cycles in the initial section of the patient's ECG recording. This requirement for a training stage generally results in CC based approaches being more suitable for ambulatory processing situations, for example Holter recordings, as opposed to analysis of short term rest ECGs. Given that infinite signals are to be analysed, the CC function for a lag where the correlation coefficient is defined as:z can be set to either x or y. The CC approach is only used to detect the fiducial point. The signal is cross correlated with a complete PQRST template. The correlation function calculates a number of correlation coefficients and returns the position of the highest value. Providing this value is greater than a predefined threshold, a QRS is deemed detected. The threshold value can be changed for each signal and template, if required. This ensures adaptability to each individual patient's recording.where The QRS onset and offset as well as the P wave onset and offset and T wave offset are detected with similar rule-based gradient search techniques as described in the non-syntactic approach in the previous section. The main difference being that the search windows in which the gradient searches are performed are dependent upon the distances specified in the template with which the ECG signal is cross correlated.With the proposed multi-component based CC detection method, separate templates for each interwave component are used. Each template consists of an individual component of the complete cardiac cycle, detailing accurate marker positions for the respective inter-wave component onset and offset. In the given approach, 3 templates were employed, one for the P wave, one for the QRS complex and one for the T wave  and standard deviation (SD) of this error were used. The me value is used to determine how close the detector is to the annotated markers, with the SD value providing information relating to the stability of the detection criteria. For the purposes of validation, the database used for testing [To quantify the accuracy of each of the three aforementioned beat detection algorithms in terms of their correct positioning of beat markers, measurements of mean error ( testing  has asso testing .SD (Equation 6) of the me can be generated with the following equation:Average values for the xid is the detected marker position for ECG trace i as identified by the algorithms used and xim is the original stored marker position from the database annotated by experts for ECG trace i.Where Table In terms of the overall accuracy in detecting cardiac cycles, as shown in Table SD of 1.8 ms with the traditional CC approach. The non-syntactic and CC approaches performed to a similar level with the former providing superior results in 5 out of the 8 marker values. The SD values, however, only differ slightly. This can be considered to be the result of both techniques using a similar approach of gradient descent searching to identify onsets and offsets. The difference in the results can be attributed to the different manner in which each technique defines the search window within which the gradient searching is performed.Considering the accuracy with which the three algorithms were able to detect the correct position for the marker insertion as shown in Table The increased accuracy of marker insertion of the multi-component based CC approach can be attributed to a number of factors. The major factor being the avoidance of any gradient searching techniques for the marker positioning as is required by the two benchmarking approaches. As the multi-component based CC approach has pre-defined marker positions in-built as an inherent feature of its design, the ability to detect the fiducial point accurately is the most important process the algorithm initially undertakes. Following this process, markers are inserted based on the values stored within the templates used during the correlation process. Methods used in the two benchmarking techniques of gradient searches are prone to false detection of local gradients and noise still remaining in the signal under examination.Considering the accepted tolerances for the 5 markers as identified in Table Overall the multi-component based CC approach outperformed the two benchmarking techniques in both accuracy of cardiac detection and marker insertions, however, 2 parameters within the algorithm were found to have a significant influence on the algorithm's performance; correlation interval and threshold parameter. Values for these parameters can be established during a training period.Results following the testing process indicated that care must be taken when selecting the appropriate correlation interval for CC based approaches. If this value is too large, interwave components of adjacent waves maybe considered during the correlation process. On the other hand, if the interval is too small, the templates used during the correlation process may not have the ability to discriminate between different desired portions of the underlying signal. Figure In the first instance detection rates are low as the interval is too large, however, when the interval is reduced to 280 ms, the rate of detection of the markers for the algorithm increases significantly. Given that under normal conditions the duration of the QRS complex is in the region of 100 ms  and a seThe threshold parameter can be considered to be the minimum value for a correlation result to be considered as a true wave detection. For example, P waves can be considered to have relatively low energy content. If the threshold value is initially set at too large a value then it will not be possible for the point at which the CC function returns the point of maximum similarity to exceed this and subsequently indicate a waveform detection. Figure The multi-component based CC approach is affected by both the correlation interval and threshold parameters of the algorithm, hence a training/tuning process is required. Each of the other two algorithms suffer from similar inherent algorithmic drawbacks and hence this is not considered to be a disadvantage of the approach provided it is taken into consideration during application of the algorithm.Although, as previously mentioned, the QT database has a large variety of abnormal ECG recordings, two specific examples are highlighted at this point to further compare the performance of the multi-component based CC approach and the non-syntactic based approach. In the first instance a recording where an inverted T wave is present is examined and in the second instance a recording similar to conditions exhibited by First Degree Heart Block is examined.Figure Figure The accurate detection of the interwave components of the ECG can be considered to significantly effect the overall performance of the computerised classification process. Three approaches to beat detection were developed and extensively tested on 3000 cardiac cycles to assess their performance. Non-syntactic beat detection and CC algorithms were used as means of benchmarking algorithms. A new approach of multi-component based CC was proposed and results showed it to out perform the two benchmarking techniques in both accuracy of cardiac cycle detection and marker insertions. The multi-component approach identified beat markers based on 3 individual CC processes addressing the QRS complex, the P wave and the T wave individually. For each of these correlation steps, once a correlation match had been established, markers were inserted for onsets and offsets based on predefined values. It is proposed that the increase in performance of this approach in comparison with the benchmarks can be attributed to the lack of heuristic gradient searching required for marker insertion. In addition, the ability to match a portion of the ECG signal, namely the QRS, during CC as opposed to matching to the entire PQRST reduces the complexity of the overall process and subsequently enhances performance.Overall, the results have shown the benefits of employing a multi-component based CC approach. Further studies are currently underway to investigate the performance of the algorithms under instances of noise conditions and further variants of non-normal ECG recordings and in addition possible improvements to the stage of P wave detection.TL carried out the development of the algorithms and performed the testing procedures. TL, CDN and FJO collaboratively designed the study. TL, CDN and FJO co-authored the paper.


Vitis vinifera (common grape or European grape) have been widely used in Ayurveda to treat a variety of common and stress related disorders. In the present investigation, the seed extract of V. vinifera was evaluated for antistress activity in normal and stress induced rats. Furthermore, the extract was studied for nootropic activity in rats and in-vitro antioxidant potential to correlate its antistress activity.The aerial parts of V. vinifera extract. Urinary vanillylmandelic acid (VMA) and ascorbic acid were selected as non-invasive biomarkers to assess the antistress activity. The 24 h urinary excretion of vanillylmandelic acid (VMA) and ascorbic acid were determined by spectrophotometric methods in all groups under normal and stressed conditions. The nootropic activity of the extract as determined from acquisition, retention and retrieval in rats was studied by conditioned avoidance response using Cook's pole climbing apparatus. The in vitro antioxidant activity was determined based on the ability of V. vinifera to scavenge hydroxyl radicals.For the evaluation of antistress activity, groups of rats (n = 6) were subjected to forced swim stress one hour after daily treatment of V. vinifera at doses of 100, 200 and 300 mg/kg body weight one hour prior to induction of stress inhibited the stress induced urinary biochemical changes in a dose dependent manner. However, no change in the urinary excretion of VMA and ascorbic acid was observed in normal animals at all the doses studied. The cognition, as determined by the acquisition, retention and recovery in rats was observed to be dose dependent. The extract also produced significant inhibition of hydroxyl radicals in comparison to ascorbic acid in a dose dependent manner.Daily administration of V. vinifera seed extract and substantiate the traditional claims for the usage of grape fruits and seeds in stress induced disorders.The present study provides scientific support for the antistress (adaptogenic), antioxidant and nootropic activities of  Ocimum sanctum [Withania somnifera [Eleuthrococcus senticosus and Panax ginseng [Stress can be described as the sum total of all the reactions of the body, which disturb the normal physiological condition and result in a state of threatened homeostasis. Stress is an internationally recognized phenomenon fortified by advancement of industrialization in a demanding civilization. Thus, every individual is likely to face stressful situations in day-to-day life. Stress represents a reaction of the body to a stimulus that tends to alter its normal physiological equilibrium or homeostasis and has been defined as a nonspecific response of the body to any demand imposed on it . Since t sanctum , Withaniomnifera  opened a ginseng .Vitis vinifera (Linn.) (Family: Vitaceae) also called as common grape or wine grape or European grape is one of the fruit crops most widely grown throughout the world [V. vinifera have been widely used to treat a variety of common and stress related disorders [V. vinifera are reported to have antimicrobial and free radical scavenging properties [Vitis vinifera was evaluated in-vivo, in normal and stress induced rats following a biochemical approach. The antioxidant potential of the extract was evaluated in-vitro to support the antistress activity. The plant extract was further evaluated for nootropic activity using conditioned avoidance response in rats.he world . In the isorders . The comisorders ,10, reduisorders . In addioperties . In the Vitis vinifera were collected from the Chittor district of Andhra Pradesh, India in the month of October and the seeds were separated from the pulp and shade dried. The dried powdered seed material of Vitis vinifera (5 kg) was extracted with boiling water (25 L) for 45 minutes and the filtrate was evaporated under vacuum below 70°C in a vacuum drier to give a final yield of 50 gm.The riped fruits of Vanillylmandelic acid (VMA) and scopolamine butylbromide (SBB) were purchased from Sigma-Aldrich, St. Louis, USA, while ascorbic acid was purchased from Loba Chemie, Mumbai. All other reagents used were of analytical grade.ad libitum. Animals were kept for seven days in laboratory for habituation.All animal experiments were performed in accordance with our Institutional Animal Ethics Committee and by the animal regulatory body of the government (Regd. No. 516/01/A/CPCSEA). Albino rats of either sex obtained from Ghosh Enterprises, Kolkata were used in the study. They were housed six per cage at a temperature of 22 ± 2°C with 12 h light/ dark cycle under controlled environment. Rats were fed with standard pellet diet , and water N hydrochloric acid for the determination of vanillylmandellic acid (VMA) spectrophotometrically at 360 nm [V. vinifera extract to the same groups of animals after having recovered completely to normal condition. Groups II, III and IV were administered orally with V. vinifera (suspended in 2% gum acacia) at daily doses of 100, 200 and 300 mg/kg body weight respectively for seven consecutive days while group I serving as control. The 24 h urine samples were collected and the levels of both VMA and ascorbic acid were determined. The final phase of the experiment consisted of administration of V. vinifera extract to the same groups of animals after a recovery period of one week. Groups II, III and IV were administered orally with V. vinifera at doses of 100, 200 and 300 mg/kg body weight respectively, one hour prior to the daily induction of stress for seven consecutive days while group I serving as control. The 24 h urine samples were collected and analyzed for VMA and ascorbic acid for seven consecutive days to study the influence of the extract on the stress induced biochemical changes.Rats of either sex weighing between 120–150 gm were divided into four groups  each containing six animals. The 24 h urine sample from each group was collected into two different beakers, one containing 5 ml of 10% oxalic acid for the spectrophotometric determination of ascorbic acid at 550 nm  and the t 360 nm . The expV. vinifera was evaluated by using the conditioned avoidance response (CAR) in rats as described by Cook and Weidley [V. vinifera (suspended in 2% gum acacia) while animals in group I were served as control. After 60 minutes, all the animals were subjected to a training schedule individually by placing inside the perspex chamber of the apparatus. After an accustomed period of five minutes to the chamber, a buzzer was given followed by a shock through the grid floor. The rat had to jump on the pole to avoid foot shock. Jumping on the pole functionally terminates the shock and this was classified as an escape while such jumping prior to the onset of the shock was considered as avoidance. The session was terminated after completion of 60 trials with an interval of 20–30 seconds given for each trial. This procedure was repeated at 24 h intervals until all groups reach 95 to 99% avoidance. After attaining complete training of a particular group, the animals were treated with a single dose of scopolamine butyl bromide , thirty minutes before the next day dosing. The training schedule was continued further with the daily doses of the extract and vehicle until they returned to normal level from scopolamine induced amnesia.The Nootropic activity of  Weidley . Rats weV. vinifera was determined based on its ability to scavenge the hydroxyl radicals [3+-ascorbate-EDTA-H2O2 system. The hydroxyl radicals attacks deoxyribose and eventually results in the formation of thiobarbituric acid reacting substances (TBARS). The reaction mixture containing deoxyribose (2.8 mM), ferric chloride (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM) phosphate buffer  and various quantities of the extracts in a final volume of 1 mL was incubated for 1 h at 37°C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al., 1979 and the percentage free radical inhibition was calculated from control.The antioxidant activity of radicals . Hydroxyt-test. In all the cases, p < 0.05 was considered statistically significant.The results are expressed as means ± standard error of means. Statistical analysis was done using Student's paired V. vinifera to the animals under normal condition produced no change in the excretion of VMA and ascorbic acid compared to normal basal levels indicating that V. vinifera did not alter excretion of VMA and ascorbic acid in normal condition. Daily administration of V. vinifera one hour prior to the induction of stress inhibited the increase in VMA and decrease in ascorbic acid excretion which was manifested during stress alone. The inhibition was found to be significant at all dose levels in a dose dependent manner.The urinary data of VMA and ascorbic acid observed in various phases of the experiment are shown in Fig. CAR of rats administered with the extract of Vitis vinifera or vehicle increased gradually to 95% over seven to ten days. The percent avoidance was always higher in the extract treated groups compared to vehicle treated control group. The acquisition (time to achieve 95% CAR) for the extract treated groups was quicker and found to be dose dependent. Animals receiving 300 mg/kg body weight of the extract have taken seven days whereas, groups treated with 200 and 100 mg/ kg doses of the extract required eight and nine days respectively to reach the point of acquisition  responsible for observed activities of the seed extract.In conclusion, the present study provides scientific support for the antistress (adaptogenic), antioxidant and nootropic activities of VMA: Vanillylmandelic acidCAR: Conditioned avoidance responseSBB: Scopolamine butylbromideEDTA: Ethylene diamine tetra acetic acid2O2: Hydrogen peroxideHThe author(s) declare that they have no competing interests.SS conceived the study, made substantial contributions in data analysis, data interpretation, writing of the manuscript and in coordination of the experiments. SN made substantial contributions in conceptualization of statistical analyses, drafting the final manuscript and designing the illustrations. RK and SK helped to conceptualize the work. KMB made significant contribution in designing the studies, conducting the experiments, interpretation of the data, and drafting the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Current approaches for representing pathways are limited in their capacity to model molecular interactions in their spatial and temporal context. Moreover, the critical knowledge of cause-effect relationships among signaling events is not reflected by most conventional approaches for manipulating pathways.Substantial amounts of data on cell signaling, metabolic, gene regulatory and other biological pathways have been accumulated in literature and electronic databases. Conventionally, this information is stored in the form of pathway diagrams and can be characterized as highly "compartmental" (We have applied a semantic network (SN) approach to develop and implement a model for cell signaling pathways. The semantic model has mapped biological concepts to a set of semantic agents and relationships, and characterized cell signaling events and their participants in the hierarchical and spatial context. In particular, the available information on the behaviors and interactions of the PI3K enzyme family has been integrated into the SN environment and a cell signaling network in human macrophages has been constructed. A SN-application has been developed to manipulate the locations and the states of molecules and to observe their actions under different biological scenarios. The approach allowed qualitative simulation of cell signaling events involving PI3Ks and identified pathways of molecular interactions that led to known cellular responses as well as other potential responses during bacterial invasions in macrophages.We concluded from our results that the semantic network is an effective method to model cell signaling pathways. The semantic model allows proper representation and integration of information on biological structures and their interactions at different levels. The reconstruction of the cell signaling network in the macrophage allowed detailed investigation of connections among various essential molecules and reflected the cause-effect relationships among signaling events. The simulation demonstrated the dynamics of the semantic network, where a change of states on a molecule can alter its function and potentially cause a chain-reaction effect in the system. Interactions among genes, gene products and small molecules regulate all cellular processes involving cell survival, cell proliferation, and cell differentiation among others. Such interactions are organized into complex lattice structures conventionally divided into cell signaling, metabolic and gene regulatory networks in a cell . In receConventionally, this information is highly compartmental: various individual signaling pathways are mostly stored in separated and non-linked diagrams. Traditional pathway diagrams, where molecules are represented as nodes and their interactions are depicted as lines and arrows have significant limitations as they lack spatial and temporal context . MoreoveRecently, many models have been proposed for representing, storing and retrieving interactions among various biological entities. BIND  and IntARecently an artificial intelligence approach known as semantic networks (SN) have gained the attention of the biological community as a potentially powerful tool for organizing and integrating large amounts of biological information . For insA semantic network is a method to represent information or knowledge by nodes and edges in a graphic form, where a node represents a concept and an edge represents a relationship . A semanPreviously an application development environment known as Visual Knowledge (VK) has been created, and VK is capable of different formalizations and implementations of semantic networks for various knowledge domains . Visual Mycobacterium tuberculosis [M. tuberculosis and the formation of a phagosome within the macrophage. These processes are mediated by the class I phosphoinositide 3-kinase (PI3K) – one of the most well-characterized enzymes to date [M. tuberculosis can inhibit the activity of the class III PI3K, arresting phagosome maturation process, and ensuring the survival of M. tuberculosis inside the macrophage [It is the current knowledge that many pathogenic bacteria are capable of entering and surviving within mammalian macrophages by modulating the host signaling pathways . One welrculosis . Activat to date . The cla to date . The p11 to date . Activat to date . The for to date . Howevercrophage ,32.In addition to their role in phagocytosis, PI3Ks are essential proteins that regulate cell survival, cell growth, cell cycle and other cellular processes . AlthougIn the paper, the word "model" refers to a set of rules in two different but related contexts. In the context of the semantic network, the model refers to a set of rules that specify how a biological concept is mapped to one or multiple semantic agents/relationships. In the context of cell signaling pathways, the model is a set of rules that specify what, how, and when molecules interact with each other. The model has been formalized and implemented, using BioCAD software system, and it is presented in the following sections.Within the semantic network, all biological structures are modeled by semantic agents that are members in one of the 6 different prototypes. Table To create an adequate semantic model, we have postulated that structures of different levels in the cellular hierarchy can interact with one another. One example of such interactions is the movement of a molecule from one subcellular compartment (e.g. cytosol) to another (e.g. plasma membrane). This is referred to as a translocation event, and it is demonstrated on the left panel of Figure Table Interactions that occur by non-covalent or covalent forces have also been modeled as two distinct "event" prototypes as shown in Table All hypothetical spatial changes occurring in the three-dimensional structure of a given macromolecule have been modeled within the SN as switches in the corresponding conformational states, and the changes do not lead to the creation of new semantic agents. Domains or sites for every protein encoded into the SN model have been assigned to either "Functional" or "Non-functional" conformational states. The "Functional" state represents that a domain/site is currently in a conformation that enables a certain interaction. On the other hand, a "Non-functional" state implies a domain/site is in a conformation that prevents an interaction. To illustrate this construct we have graphed the semantic agents and their relationships created within the developed SN. It should be noted that within the SN, all semantic agents are visualized as icons, and their relationships are depicted as connecting arrows. In addition, all agents are related by pairs of reciprocal relationships, and for simplicity, only one direction of each pair of the relationships was visualized. The left panel of Figure In addition to the conformational states, a protein domain or site has been assigned one of the two binding states: "Bound" or "Not-bound". A "Bound" state implies that this domain/site currently associates with a domain/site of another molecule through a non-covalent interaction. On the other hand, a "Not Bound" state indicates such an association does not exist. Since ligand bindings can affect the conformation of a macromolecule through allosteric regulations, two types of such regulations have been implemented within the SN. A positive allosteric regulation event has been assigned to the scenario when a "Bound" binding state of a domain/site causes the conformational state of another domain/site to switch to "Functional". The right panel of Figure A more complex allosteric regulation event can be specified for mapping the binding states of multiple domains/sites (the condition or the input) to the conformational states of multiple domains/sites (the response or the output). Hence, a domain is switched to "functional" only if a certain combination of ligand bindings has occurred. The utilization of the states on domains/sites and allosteric regulation events in the SN has enabled us to express the cause-effect relationships among various signaling events explicitly.In the developed semantic model, any molecular complex formed due to non-covalent interaction has been treated as a transient state of these two molecules, and a complex was not represented by a new semantic agent. Instead, the existence of a protein complex is inferred from the non-covalent interaction event. Thus, Figure Conventionally, there is often some inconsistency between representing chemical modifications of small molecules in metabolic pathways and modifications of proteins in signaling pathways. In the developed model, two molecules that interacted by covalent forces have resulted the creation of distinct semantic agents within the SN. This rule has been implemented consistently for both macromolecules such as proteins and small molecules such as ATP. As one example, Figure In the semantic model, a molecule can participate in different sets of interactions in different locations. The translocation events define all possible localizations of molecules, and therefore, an interaction can only occur if the participating molecules can be present in the same location at the same time. Alternatively, an interaction  can directly associate with a subcellular compartment, and this interaction is only available to molecules in that location.In addition, all qualitative cellular responses such as cell survival and phagosome formation have been implemented within the SN under a distinct "event" prototype. They have been implemented in a way that the formation or the activation of certain signaling molecules such as PIP3 can trigger the occurrence of these cellular response events in a simulation.As it has been mentioned previously, the behavior of any semantic agent can be clearly defined by its relationships or connections to other agents. Thus, the formalization of the five types of events, which are translocations, non-covalent interactions, covalent interactions, allosteric regulations and cellular responses, has enabled us to model the behaviors of molecules depicted in conventional pathways and to reconstruct a cell signaling network of the human macrophage.The molecular composition of human macrophages and information of known intracellular interactions have been extracted from various research articles ,32,34-47A pathway diagram in the literature or an electronic database, in principle, represents some scenario of what may happen in a call if every depicted molecule is expressed in the correct location, at the correct time and with the correct states. Hence, the aggregation of multiple pathway diagrams describes some, if not all, possible molecular events that can potentially occur in a cell under the right conditions. To utilize such information and build a cell signaling network, we have decomposed conventional pathways into individual pieces of information such as subcellular localization of a protein, a pairwise protein binding, a chemical reaction or a cellular response. Then, using the sets of semantic rules described in the model, we have represented and integrated each piece of those information in the form of semantic agents and their relationships. Table The defined semantic agents have been connected in the semantic network and can be visualized at different levels. Figure Such detailed semantic reconstruction of the cell signaling network has allowed thorough investigation of biochemical relationships between essential proteins. One such example is presented on Figure Another example of successful SN reconstruction is the relationship between CD14 macrophage receptor and the class I PI3K; such a relationship was previously suspected but not clear . By incoIn the implemented semantic model, the "possible" behaviors of a molecule are defined through its relationships to other agents , and all instances of that prototypical molecule will inherit the same behaviors. However, the action of a molecule at any given time is affected by factors including its current states and its current location with respect to other molecules in the system. Hence, we have built an application that enabled us to produce instances of molecules in various locations and to observe the "current" action of a molecule qualitatively under different biological scenarios. We refer such scenario-play as simulation in this paper.The application or the SN-simulator allows the molecules to move among various locations, to interact with each other and to create events when the conditions are met. In addition, every instance of a molecule has been represented as an individual agent while every instance of a molecular interaction has also been implemented as an individual event agent. Thus, the simulator provides a traceable "trajectory" of all the events that have happened on every molecule during a simulation.As illustrated in Figure Because an Fcγ receptor was the only interacting molecule (for the IgG) present at plasma membrane in the simulation, it has bound to the IgG by a non-covalent interaction event, as illustrated in Figure M. tuberculosis was initially present in the plasma membrane, it would inhibit the class III PI3K and thus arrest the phagosome maturation response in the macrophage. Table M. tuberculosis has caused several known cellular responses as well as additional responses such as cell survival of the macrophage, cell cycle entry, increase of protein synthesis and increase of intracellular glucose level in the simulation. We suspect that some of these responses have not yet be appreciated in previous studies of bacterial invasions. Further experiments can be formalized to test the simulation results.Figure In this study, the SN-simulator has enabled us to "play" different scenarios and observed their effects in the macrophage. It allowed us to investigate how changes on one molecule caused changes of another molecule in the cell signaling network during bacterial invasions.In the present work we have developed a semantic model to represent the properties and the behaviors of molecules and their interactions in the context of cell signaling pathways. The proposed model offers some additional features, compared to other existing pathway models. Those features are essential for characterizing the complex behaviors of biological entities, and they include:The semantic model has specified the hierarchical relationships among the different biological structures, from cells to compartments, molecules and domains/sites. The hierarchy between subcellular compartments and molecules has allowed us to specify the spatial organization of molecules, model the translocation events and represent the effects of locations on the different interactions among molecules.The hierarchy between molecules and their domains/sites has enabled us to explicitly model the relationship between forms and functions for proteins. Through the allosteric regulation events, proteins have been modeled and implemented as integrating and logical devices in the semantic network, and their conformational states (outputs) are switched by the combination of non-covalent ligand bindings or covalent modifications (inputs).Through the prototyping system in the semantic network, any rule or interaction specified on a prototypical molecule automatically define the properties and behaviors of all its instances. As demonstrated by the simulator, the semantic network provided a direct communication from the interaction model to an application where the actions of molecules can be observed under different scenarios. Therefore, the semantic network is dynamic as a change of states on a molecule can alter its function and potentially cause a chain-reaction effect in the system.In addition, the current semantic model is different from the previous models in BioCAD. An essential difference is the representation of functional labels or roles on proteins. The meanings of functional descriptions or association words such as "enzyme", "activator/activates" or "inhibitor/inhibits", which are often used to characterize the behaviors of proteins in most pathway models, have been represented explicitly through events and relationships in the developed semantic network. For example, a protein acts as an "enzyme" if 1) the protein participates in a "covalent interaction event", 2) the presence of a "functional" catalytic domain on the protein is required for the occurrence of the event, and 3) the protein itself is not modified after the event. Similarly, a protein A "activates" a protein B if a non-covalent binding event from protein A turns on the "functional" state of a domain/site on protein B. Hence, the model has reduced the need for labels, which are often confusing or misleading on conventional pathway representation.The use of non-covalent and covalent events has enabled us to model protein-protein interactions and chemical modifications on molecules including proteins and metabolites. The next challenge is to model the complex interactions that govern gene regulations. The current construction of non-covalent interaction events can model the binding of an individual transcription factor to a particular site of a gene, and the covalent interaction event can represent the transcription process that leads to the production of an mRNA, and the translation process that produces a protein. However, a successful transcription in a eukaryotic cell requires the formation of a protein complex that involves more than one hundred subunits, and the complex may be assembled in various orders . We antiThe semantic network representation can be exploited for performing analysis of cell signaling pathways. The examples of Fcγ receptor, CR and the class I PI3K demonstrated that connections can be queried and analyzed among different biological entities. The semantic model is also compatible with other pathway models. Therefore, the number of biological entities and interactions in the semantic network can be greatly increased as pathway data from existing databases is integrated. Previous study has shown the value of combining gene expression profiles with protein-protein interaction networks for identifying active subnetworks . SimilarIn this study, the proposed semantic model has been applied to cell signaling pathways in the macrophage as a case study. The model is not limited to those pathways. The hierarchical classification of the biological structures and the events can model other cell signaling pathways for different cells and organisms. An interactive website is currently under development. We anticipate that through the web, researchers can utilize the semantic network approach for creating pathways in cells of their interest and for analyzing any existing pathways including the PI3K pathways of the human macrophage presented in the paper.In this study, we have developed a simple simulator to demonstrate the dynamics of the semantic network and to observe the actions of molecules qualitatively. In order to perform a realistic cellular simulation in the future, three components need to be improved. First, quantitative factors should be integrated into the model. For example binding affinity, directly associated with non-covalent events, will affect the probability and the duration of the binding of molecules. Reaction kinetics, associated with covalent events, will determine the rate of production.Second, the two parameters, the population of molecules and their localization, which influence the simulation outcome, could be initialized and supported by experimental data. For instance, gene expression data from microarrays supports the relative abundance of transcripts, and protein expression data supports the relative abundance of proteins. Computer algorithms such as PSORT  can assiThird, the proximity of molecules has been represented by subcellular compartments in the simulation. This approximation can be improved in two different ways. First, a compartment can be further divided into smaller sub-locations. Increasing the number of locations and reducing the size of each location will improve the accuracy of the simulation. Second, the occurrence of non-covalent events in the simulation has allowed us to identify molecular complexes and their members effectively. Hence, the proximity can be approximated through molecular complexes, such that molecules in a complex have higher probability to interact with members of the same complex.The simulator has demonstrated that a biological pathway can emerge from the creation of semantic agents and their relationships in the SN, and such a pathway represents a series of consecutive events resulting from the activation of a single molecule. It is anticipated that further development will improve our ability to track and visualize different instances of molecules participated in multiple pathways. Hence, the occurrence of a cellular response event can be triggered by the accumulation of certain molecular species with particular states.M. tuberculosis in the macrophage were investigated by the simulator. As a result, the simulation identified pathways of molecular interactions that led to known cellular responses as well as other potential responses during bacterial invasions.We concluded from our results that the semantic network is an effective method to model cell signaling pathways. Utilizing the semantic agents and the relationships in the model, information on biological structures and their interactions at different levels has been properly represented and integrated in the hierarchical and spatial context. The reconstruction of the cell signaling network in the macrophage has allowed qualitative investigation of connections among various essential molecules and reflected the cause-effect relationships among the events. The simulation demonstrated the dynamics of the semantic network, where actions of molecules are affected by their current states and locations, and the history of events can be traced and analyzed. In addition, changes caused by the invading Visual Knowledge (VK) is an application development environment, and its implementation has been influenced by the theory of semantic networks as well as other approaches including set theory, frame system, object-oriented modeling theory and systems based on networks of active software agents . DiffereBioCAD, a Visual Knowledge-based development environment, is developed by Upstream Biosciences, Inc. and customized to model biological systems . The BioThe semantic model was developed jointly by all authors and implemented by MH, JLB and CS. MH implemented the simulation, collected and analyzed data, constructed pathways in the macrophage and drafted the manuscript. JLB, CS, AC developed general concepts, provided scientific support, participated in the manuscript writing and coordinated the study. All authors read and approved the final version of the manuscript.