Early age at infection with hepatitis B virus (HBV) increases the risk of chronic HBV infection. In addition early age at infection may further increase the risk of persistent viral replication beyond its effect on chronicity. The effects of perinatal and early postnatal transmission on the risk of prolonged hepatitis B e antigenaemia in children with chronic HBV infection are not well documented in Africa. We examine these associations using maternal HBV sero-status and the number of HBV-positive older siblings as proxy measures for perinatal and early postnatal transmission, respectively.Hepatitis B e antigen (HBeAg)-positive mothers were identified in six population-based HBV sero-surveys conducted in The Gambia between 1986 and 1990. For every HBeAg-positive mother, a hepatitis B surface antigen (HBsAg)-positive HBeAg-negative mother and HBsAg-negative mother were randomly selected from the population surveyed. These mothers and their family members were tested for HBV sero-markers in a subsequent survey conducted between 1991 and 1993.Thirty-eight HBeAg positive mothers and the same number of HBsAg-positive HBeAg-negative mothers and HBsAg-negative mothers participated in the study. Sixty-nine percent of their children also participated. There was a non-significant positive association between HBeAg prevalence in children and the number of HBeAg-positive older siblings . After adjusting for confounders, having an HBeAg-positive mother was a risk factor for HBeAg positivity in children carrying HBsAg , whilst the number of HBeAg-positive older siblings was not.Maternal HBeAg was associated with positive HBeAg in children with chronic HBV infection. This suggests that interrupting mother-to-infant transmission in sub-Saharan Africa might help reduce the burden of liver disease. A timely dose of HBV vaccine within 24 hours of birth, as recommended by WHO, should be implemented in sub-Saharan Africa. Chronic infection with the hepatitis B virus (HBV) is a cause of hepatocellular carcinoma (HCC) . The risA recent systematic review of observational studies  found a However, the scope of the review was limited since: 1) most of the studies included were from East Asia where mother-to-infant transmission is frequent , and 2) In this study we examined the association between age at HBV infection and the presence of hepatitis B e antigen (HBeAg) in The Gambia, West Africa, using maternal HBV sero-status and number of HBV-positive older siblings as a proxy for perinatal and early postnatal transmission, respectively. The analysis was restricted to study participants with positive hepatitis B surface antigen (HBsAg), because our aim was to identify the effect of age at infection (or mode of transmission) on the risk of hepatitis B e antigenaemia, beyond its effect of increasing the risk of chronic HBV infection .A nation-wide hepatitis B vaccination trial was initiated in The Gambia in 1986 and by 1990 countrywide coverage was achieved . In paraOn the basis of the HBV sero-status determined at these surveys, all HBeAg-positive mothers were invited to participate in “HBeAg Study”. The same numbers of HBsAg-positive HBeAg-negative mothers and HBsAg-negative mothers were randomly selected from the databases used in these surveys. Between 1991 and 1993, these mothers  and their family members were bled for HBV markers to determine the effect of familial HBV sero-markers on presence of HBeAg.After consent was obtained, the index women were bled and interviewed to collect demographic information on dead and living family members. Index women were bled twice, the first bleeding occurred between 1986 and 1990 and the second at the time of the HBeAg Study (1991–1993). We used the serological status recorded in the first survey as a proxy for serological status at birth of their children.Family members of the index woman , were contacted and if they agreed to participate in the study, were bled for HBV serology. All sera were assayed for HBsAg by reverse passive haemagglutination . HBeAg was tested by radioimmunoassay  only when sera were HBsAg-positive. The study was approved by the Gambia Government/MRC  Joint Ethical Committee and the ethics committee at IARC , France.st generation) and index women (2nd generation); and index women (2nd generation) and their children (3rd generation). The effect of the number of older siblings positive for HBsAg or HBeAg was evaluated in index women (2nd generation) and their children (3rd generation).The association between maternal HBV markers  and e antigenaemia was examined in successive generations: mothers of index women (1nd generation). In the children of index women (3rd generation), adjusted odds ratios for the same associations were estimated using generalised estimating equations with an exchangeable correlation structure to account for household clustering . A linear test for trend was used to assess the statistical significance of the number of elder siblings positive for HBeAg and the number positive for HBsAg.Logistic regression was used to estimate adjusted odds ratios for the associations between familial HBV sero-status  and e antigenaemia in index women  were all negative for HBeAg and only one index woman had an HBeAg-positive older sibling. These variables were therefore omitted from multivariable models of e-antigenaemia in index women (2nd generation).A minimally sufficient set of m Figure  by applynd generation) in the database, 38 (71.7%) were traced and agreed to participate in the study. The rate of refusal among HBeAg-negative index women was not recorded. More than 65% of the mothers (1st generation) and children (3rd generation) of index women participated in the study, but participation was less than 35% among older siblings of index women  were HBeAg-positive. The proportion of index women with HBsAg-positive older siblings was similar in HBeAg-positive index women  and HBsAg-positive HBeAg-negative index women . After adjusting for confounding factors, there was no evidence for an association between HBeAg positivity in index women and either maternal HBsAg  or having an HBsAg-positive older sibling .The characteristics of 38 HBsAg-positive HBeAg-positive index women and 38 HBsAg-positive HBeAg-negative index women are presented in Table In total, 96, 102 and 101 children of HBeAg-positive, HBsAg-positive and HBeAg-negative, and HBsAg-negative index women participated, respectively. Of whom, 38 (39.6%), 14 (13.7%) and 6 (5.9%) tested positive for HBsAg, respectively  are presented in Table The associations between familial HBV sero-status and HBeAg positivity in HBsAg-positive children (3rd generation). We found that e antigenaemia in children was positively associated with maternal HBeAg but not with maternal HBsAg. Likewise, the prevalence of e antigenaemia in children was higher if older siblings were HBeAg positive, although this result was not statistically significant. These relationships imply that the early establishment of chronic HBV infection through perinatal transmission from infectious mothers, as well as early postnatal transmission from infectious older siblings is associated with presence of HBeAg. In addition, this is consistent with the observation that positive HBeAg confers greater infectivity than HBsAg positivity alone. For example, the risk of perinatal transmission is 10–17% in HBsAg-positive mothers without HBeAg and 63–67% in HBeAg positive mothers in sSA [We examined the effect of HBV sero-markers in mothers and older siblings on hepatitis B e antigenaemia in children carrying HBsAg  could not be confirmed because all of the mothers of index women (1st generation) tested negative for HBeAg and only one older sibling of index women tested positive for HBeAg. The low prevalence of HBeAg in the mothers and older siblings of index women was expected since HBeAg is lost over time. In The Gambia, 85% of children who established chronic HBV infection during early childhood had lost HBeAg by the second decade of life [The associations of familial HBeAg with e antigenaemia in index women  than in healthy controls  while the prevalence of HBsAg in siblings was similar in cases  and controls  [et al., studied the association between birth order and HCC, and found strong evidence that higher birth order, and hence earlier age at infection, is associated with higher HCC risk (P < 0.005) [Two African case–control studies have investigated the association between age at HBV infection and HCC, ultimate sequelae of chronic HBV infection. Larouze < 0.005) . In the < 0.005) . HoweverThe study has several limitations. Firstly, the sample size was small, and the associations were therefore poorly estimated. Secondly, many family members of the index women did not participate in the HBeAg study, and this might have led to selection bias. Thirdly, important confounding variables might have been omitted, in particular viral genotype. In Taiwan where genotype B (80%) and C (20%) predominate, genotype C was found to be associated with delayed HBeAg seroconversion  and alsoHBV vaccination programmes in Africa and Asia have been successful in preventing postnatal horizontal transmission of HBV, thereby reducing the prevalence of chronic HBV infection ,32. HoweOur study suggests that the risk of prolonged e antigenaemia, an important predictor of HCC , is highHBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; IARC: International agency for research on cancer; MRC: Medical research council; OR: Odds ratio; sSA: Sub-Saharan Africa; WHO: World health organisation.The authors declare that they have no competing interests.MM conducted the surveys, performed laboratory tests and helped to improve the manuscript. YS designed the analysis of the study, performed the statistical analysis and drafted the manuscript. CB supervised the statistical analysis and helped to improve the manuscript. RN supported the conduct of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/532/prepub


Various paper-based mood charting instruments are used in the monitoring of symptoms in bipolar disorder. During recent years an increasing number of electronic self-monitoring tools have been developed. The objectives of this systematic review were 1) to evaluate the validity of electronic self-monitoring tools as a method of evaluating mood compared to clinical rating scales for depression and mania and 2) to investigate the effect of electronic self-monitoring tools on clinically relevant outcomes in bipolar disorder.A systematic review of the scientific literature, reported according to the Preferred Reporting items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was conducted. MEDLINE, Embase, PsycINFO and The Cochrane Library were searched and supplemented by hand search of reference lists. Databases were searched for 1) studies on electronic self-monitoring tools in patients with bipolar disorder reporting on validity of electronically self-reported mood ratings compared to clinical rating scales for depression and mania and 2) randomized controlled trials (RCT) evaluating electronic mood self-monitoring tools in patients with bipolar disorder.A total of 13 published articles were included. Seven articles were RCTs and six were longitudinal studies. Electronic self-monitoring of mood was considered valid compared to clinical rating scales for depression in six out of six studies, and in two out of seven studies compared to clinical rating scales for mania.The included RCTs primarily investigated the effect of heterogeneous electronically delivered interventions; none of the RCTs investigated the sole effect of electronic mood self-monitoring tools. Methodological issues with risk of bias at different levels limited the evidence in the majority of studies.Electronic self-monitoring of mood in depression appears to be a valid measure of mood in contrast to self-monitoring of mood in mania. There are yet few studies on the effect of electronic self-monitoring of mood in bipolar disorder. The evidence of electronic self-monitoring is limited by methodological issues and by a lack of RCTs. Although the idea of electronic self-monitoring of mood seems appealing, studies using rigorous methodology investigating the beneficial as well as possible harmful effects of electronic self-monitoring are needed.The online version of this article (doi:10.1186/s12888-016-0713-0) contains supplementary material, which is available to authorized users. Manyfilling) –13. Durifilling) , includifilling) , a monitHowever, it remains unclear whether the severity of self-monitored mood registered using electronic self-monitoring tools is a valid measure compared to validated clinical rating scales for depression and mania, which are currently used as the golden standard for assessing the severity of depressive and manic symptoms in patients with bipolar disorder. Furthermore, it remains unclear to what extent the use of electronic mood self-monitoring tools affects clinically relevant outcomes, and importantly whether there may in fact, be harmful effects, e.g. self-monitoring of mood symptoms may induce depressive ruminations that may result in increasing severity of depressive symptoms . An undeThe objectives of the present systematic review were thus 1) to evaluate the validity of electronic mood self-monitoring tools compared to validated clinical rating scales for depression and mania and 2) to evaluate the evidence of the effect of electronic mood self-monitoring tools on clinically relevant outcomes in randomized controlled trials (RCT).This is the first systematic review of electronic self-monitoring of mood in patients with bipolar disorder.This systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement . MethodsOriginal studies involving IT platforms for electronic self-monitoring of mood used by adult patients with bipolar disorder ≥18 years of age and either reporting on correlations between electronically self-monitored mood and validated clinical rating scales for depression and mania or RCTs assessing the effect of electronic self-monitoring tools as an intervention were eligible for review. The language of the publications was restricted to English. The types of IT platforms for electronic self-monitoring of mood were defined as cell phones, computers, tablets, PDAs, smartphones and online devices. Papers only describing the technical part of the IT platforms, not including patients with bipolar disorder and/or not reporting on correlations between electronic mood self-monitoring and validated clinical rating scales for depression and mania or the effect of electronic self-monitoring as an intervention in RCTs in patients with bipolar disorder were excluded from review. Where multiple articles reported on different validated clinical rating scales deriving from the same study and reported on the same patient sample, all of these articles were included for review.Published studies were identified through searching the electronic databases MEDLINE (January 1950 to July 2015), PsychINFO (1806 to July 2015), Embase (1974 to July 2015) and The Cochrane Library  supplemented by hand search of reference lists of retrieved articles.The literature search was conducted by one researcher (MFJ) and employed the search terms/key words: (telephone or mobile phone or cellular phone or cell phone or smartphone or computer or telecommunications or electronic or electronic device or text message or application or app) and (bipolar disorder or manic depressive psychosis or manic depressive disorder or mania or bipolar affective disorder or manic or bipolar depression).The flow diagram of the study selection process is presented in Fig. The present systematic review did not need ethical approval. Data presented are from individual studies approved by local ethical commitees and consent obtained in individual studies.n = 10) [n = 13) [n = 2) [n = 10) [The results of the literature search and selection of studies are presented in Fig. n = 10) –28 2) no[n = 13) –41, 3) b [n = 2) , 43, and[n = 10) –53. A to[n = 10) , 54–64.Of the 13 included articles seven were RCTs , 60–63 aSeven articles evaluated the validity of different electronic mood self-monitoring tools compared to validated clinical rating scales for depression and mania , 59, 64 n = 179) of the seven articles evaluated the validity of electronic self-monitoring of mood compared to clinical rating scales for depression using the Montgomery Asberg Depression Rating Scale (MADRS) [Six ( (MADRS) , the Ham (MADRS)  or the I (MADRS) . All of The monitoring tools used were PDAs , 64, comn = 64) found a significant correlation between self-monitored mood and the clinical rating scale for mania [n = 18) found a significant positive correlation between self-monitored mood and the clinical rating scale for mania after 6 weeks of mood self-monitoring, but not at baseline [All of the seven articles evaluated the validity of electronic self-monitoring of mood compared to clinical rating scales for mania using the Young Mania Rating Scale (YMRS) . Two artor mania , 64, indbaseline . Regressbaseline , 64, combaseline , 55, 64 baseline , 59, 64,baseline  investigOf the 13 included articles seven were RCTs including a total of 759 patients with bipolar disorder involving a follow-up period ranging from 12 weeks to 12 months , 60–63 (n = 48) reported that the intervention group showed significantly higher compliance to mood self-monitoring compared to the control group. No difference in Clinical Global Impression Severity (CGI-S) score was found between the two groups [n = 40) reported that the intervention group showed significantly higher variability of self-monitored mood compared to the control group. Further, a higher compliance to mood self-monitoring was found in the control group [Two of the RCTs aimed primarily at investigating differences in compliance rates between using an electronic mood self-monitoring tool compared to using paper-and-pencil mood self-monitoring , 58 and o groups . The secFive of the seven included RCTs investigated the effect of different electronically delivered intervention programmes including a self-monitoring tool on symptoms of illness activity in patients with bipolar disorder , 60–63.n = 122) reported on a RCT investigating the effect of an online interactive intervention based on principles of Cognitive Behavioral Therapy and psychoeducation including self-monitoring (‘Living with Bipolar’) (intervention group) compared to treatment-as-usual (TAU) (control group). The study reported that the intervention group showed significant improvement in self-assessed quality of life, recovery, symptoms severity and social functioning compared to the control group [One study (ol group .n = 233) reported on a RCT investigating the effect of a web-based psychoeducational program including self-monitoring (Recovery Road for Bipolar Disorder) (intervention group) compared to a control group directed to web-sites on healthy lifestyles. The study reported that there were no significant differences between the two groups in any of the defined outcome [Another study ( outcome .n = 82) reported on a RCT investigating the effect of smartphone-based personalized self-management strategies based on self-reported mood scores (intervention group) compared to paper-and-pencil mood self-monitoring (control group). The study reported no significant differences in depressive and manic symptoms between the two groups at the end of study [The third study (of study .n = 156) reported on a RCT investigating the effect of an online psychoeducation, mood tracking, discussion boards and cognitive behavioral therapy program (MoodSwings Plus) (intervention group) compared to an online psychoeducation, mood tracking and discussion boards program (MoodSwings) (control group). The study reported that the intervention group showed significant reductions in self-assessed manic symptoms compared to the control group, but no difference was observed in relation to self-assessed depressive symptoms [n = 78) reported on a RCT investigating the effect of smartphone-based self-monitoring including a feedback loop to the clinicians (MONARCA) (intervention group) compared to a placebo smartphone and TAU (control group). The study reported no significant differences between the two groups in any of the defined outcomes. Sub-analyses showed that the intervention group had more depressive symptoms and fewer manic symptoms than the control group during the study period [The fourth study . Whether that would be a threat to the reliability and validity is unknown and should be investigated further.In westerns countries nearly everyone has at least one device that would be able to handle electronic self-monitoring. The use of computers and/or tablets as tools for electronic self-monitoring of mood require some technical skills by the user and can be quite expensive to acquire. However, most people have access to a computer or tablet and know how to interact with simple software systems. Computers and tablets allow for storage and visual presentation of self-monitored data making recognition of symptom patterns possible, thus potentially providing tools for increasing the patients’ illness insight and empowerment. PDAs represent a tool that is possible for the patients to carry with them during the day making continuous real-time electronic self-monitoring in naturalistic settings is possible. However, when using a separate and non-standard electronic device for self-monitoring of illness activity the risk of stigmatization is present . Other ePatients willing to participate in studies using these kinds of novel and technical electronic interventions could represent a more motivated and technically oriented group of patients with higher degree of illness insight and willingness to use the electronic self-monitoring tools in question. As can be seen from some of the included articles, compliance to self-monitoring was highly variable, and patients participating were quite young. Elucidation of possible technical barriers for using an electronic device for self-monitoring among non-technically oriented patient groups, perhaps in patients with higher age than in the included studies, could be of interest. In addition, future studies should provide more information on excluded patients and reasons for declining to participate in studies in order to allow the readers to better assess the level of generalizability of the study results.None of the included studies provided information regarding the economical part of the development and maintenance of the electronic self-monitoring IT platforms, and is likely a relevant factor in the development of future efforts in this area as well as for the clinical implementation.Some limitations to the present review should be mentioned. Telemedicine in general and e-mental health are areas under great expansion and the investigations in this area are published in very diverse forms and places reflecting that this is an research area in the intersection between two areas of research- medical research and IT research. Therefore, conducting a search strategy that is able to capture all relevant scientific literature is a challenge. A search on Google Scholar alone on electronic self-monitoring in bipolar disorder resulted in 595.000 hits. Many commercial websites and smartphone applications  for electronic self-monitoring exist in the App store and the Google Play store. The search strategy for the present review reflects that we aimed at systematically collecting and reviewing published scientific studies in order to get an overview of the validity of electronic mood self-monitoring compared to validated clinical rating scales and the evidence status related to using these kinds of electronic self-monitoring tools.Most of the included studies had a daily and momentary frequency of self-monitoring, but one study used a frequency of self-monitoring on a weekly basis. A recent paper discussed differences in momentary and retrospective trait self-report techniques pointing out that retrospective self-monitoring is influenced by peak moments with greater salience of moments that occur closets in time to the assessment .The interventions of the included RCTs were heterogeneous and often mood self-monitoring was a part of an intervention, which incorporated mood self-monitoring alongside other psychological interventions. In addition, the RCTS employed various monitoring IT platforms and included different clinically relevant outcomes. Thus, comparing not only different mood self-monitoring tools but also different interventions as well as outcomes is a big challenge.It should be noted that the authors did not have access to the various electronic self-monitoring tools reviewed apart from one of the tools for smartphones , 59. FurOne author (MFJ) selected all papers and extracted all data, and one of the co-authors (KM) independently checked the extracted data.It should also be noted that in most of the articles the patients had to provide the hardware for the electronic self-monitoring themselves and no information regarding economical compensation was given. None of the included articles provided information regarding the cost of developing and maintaining the electronic software and the amount of possible technical problems with the electronic self-monitoring systems. These aspects are likely relevant factors that should be evaluated in future studies.It would be interesting to investigate the validity between electronic mood self-monitored and validated clinical rating scales for depression and mania and the effect of electronic self-monitoring as an intervention in a non-technically oriented group of patients with bipolar disorder, and further to elucidate these aspects during full-blown affective episodes.This systematic review identified relatively few studies investigating IT platforms for electronic self-monitoring in adult patients with bipolar disorder. Electronic self-monitoring of mood in depression appears to be a valid measure of mood in contrast to self-monitoring of mood in mania. This calls for other new and more objective real-time electronic methods for monitoring the severity of manic symptoms.The evidence of the effect of electronic self-monitoring tools investigated in RCTs was limited by methodological issues and, by small number of RCTs primarily investigating the effect of different electronically delivered intervention programmes. Crucially, the potential beneficial or harmful effect of electronic self-monitoring tools on clinically relevant researcher blinded outcomes has scarcely been investigated and is unknown. Whether electronic self-monitoring should be considered an instrument or as an intervention or both is unknown and future research should investigate this further.There is a need for further research using rigorous methodology and more RCTs investigating the effect and economic consequences of electronic self-monitoring using different types of IT platforms in patients with bipolar disorder. Furthermore, future RCTs should elucidate possible harmful effects to inform whether the potential benefits are worth the costs and potential risks.


Access to effective healthcare is in particular challenging for vulnerable and socially disadvantaged patients. Patients with chronic conditions are over-represented in these lower socioeconomic (LSES) groups. No generic review integrating the evidence on Self-Management support interventions in LSES patients with different chronic conditions exists.To provide an overview of the effectiveness concerning patient-relevant outcomes of Self-Management support interventions and their components in chronically ill patients with a LSES.Six databases were explored from 2000 up to December 2013. 3195 abstracts were screened. 27 studies were included. Data extraction and quality assessment were performed by five pairs of independent reviewers.The main focus was on SMSI in patients with diabetes, in the USA and 2 studies were conducted in Europe. 14 studies explicitly cited an underlying theoretical basis for the SMSI. The most frequently used Self-Management support interventions components were: lifestyle advice (n=25), information provision (n=24), and symptom management (n=22). Problem solving (n=13) and goal setting (n=13) were frequently integrated in the Self-Management support interventions. In 11 studies the Self-Management support interventions was specifically adapted to the needs of LSES patients. No differences in positive outcomes were found when stratified for theory-based Self-Management support interventions or for number of patients included. Very few RCTs on Self-Management support interventions were performed in Europe. Limited evidence exists for the effectiveness of Self-Management support interventions in LSES patients and its linkages with components and strategies of these interventions. Therefore, to improve access to healthcare for all patients, it is important to develop and evaluate effective SMSI for these vulnerable patients.


Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs) and controlled abortions . Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated  and three upregulated  in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans. Ectopic pregnancy (EP) is an early pregnancy complication in which a fertilized ovum implants outside the uterine cavity. Implantation may occur anywhere along the reproductive tract with the most common implantation site being the fallopian tube. The etiology of EP is not completely known, but it is probably due to retention of the embryo caused when the embryo-tubal environment is impaired, thus allowing tubal implantation to occur Recent evidence has conclusively demonstrated that the regulation of numerous key biological processes does not depend only on classical transcriptional mechanisms, and that other regulatory phenomena such as epigenetic mechanisms do have important roles. These epigenetic mechanisms include not only DNA methylation and the post-translational modification of histones, but also small non-coding RNAs, including microRNAs (miRNAs) MiRNAs are expressed in a cell-specific manner Hospital Universitario La Fe, Valencia, Spain (2012/0340). Early embryonic tissue (mostly trophoblast) was collected after obtaining written informed consent from each patient for this research under the principles of the Declaration of Helsinki for medical research involving human subjects.This study was approved by the Institutional Review Board/Independent Ethics Committee of the Twenty-four patients suffering from EP and twenty-nine control patients were recruited for the study. The study design included a first series of eight patients suffering from tubal EP and eight patients scheduled for a voluntary termination of pregnancy (VTOP) for the microarray discovery study. To validate the differentially expressed miRNAs found using microarray technology we used real time PCR on samples from a second series of 15 EP and 21 VTOP patients. The characteristics of the patients included in series one and two are described in see below), frozen, and stored at −80°C until use. A piece of each sample was sent to the Pathology Department , who provided histological confirmation of ectopic pregnancy and the absence of tubal tissue. The fetal dilation and evacuation method or fetal aspiration technique were performed in VTOP women to obtain the embryonic tissue.All the samples obtained from patients included in this study after signed informed consent (EP and VTOP) were properly dated according to the date of the last menstrual period (week of pregnancy). The diagnosis of EP was based on clinical and physical examination, transvaginal ultrasound, and serial quantitative β-hCG levels, and confirmed by laparoscopy in which the tissue was removed. The EP patients did not receive methotrexate treatment, and laparoscopy was performed as follows: ectopic pregnancies selected for this study were unruptured gestations located in the isthmus or the proximal ampulla. The tube containing the ectopic pregnancy was grasped at both sides (approximately 1 cm away from the gestation) and bipolar coagulation applied. Similarly, the adjacent mesentery was also coagulated. Then, salpingectomy was performed employing scissors. Then, a longitudinal antimesenteric incision into the surface of the tube and applied mild pressure with two fingers to extract the gestational sac. Embryonic tissue was carefully separated from obvious blood clots or tubal tissue in the operating room under a stereomicroscope and were immediately placed in TRIzol reagent . The quantity (ng/ml) and purity (260/280 and 260/230 ratio) of the RNAs obtained was measured using a NanoDrop 2000 spectrophotometer (Thermo-Scientific) and the presence, proportion, and quality of miRNAs were assessed with Small RNA LabChips and a BioAnalyzer (Agilent).To compare the pregnancy tissue miRNA profiles between VTOP and EP samples, human miRNA v3.0 8x15K microarrays  that evaluate the expression of 866 human miRNAs were used (See GEO data for a complete list of these miRNAs). Total RNA from each sample was amplified, labeled, and hybridized according to Agilent's instructions The microarray results were validated by real time PCR using miScript reverse transcription and miScript SYBR Green PCR commercial kits  according to the manufacturer's protocols. Specific primer assays for hsa-miR-196b, hsa-miR-223, hsa-mir-30a, and hsa-mir-451 were purchased from Qiagen and real time PCR quantification was carried out in a LightCycler 480 (Roche). To normalize the expression values, a small nuclear RNA with constitutive expression, SNORD96A, was used as a housekeeping control in silico analysis we used three different software programs: the web-based tool ‘DIANA-miRPATH’ together with microRNA.org We performed computational analysis to identify genes and pathways that might be modulated by the altered-expression miRNAs that we identified. For the In vitro cultured trophoblast cell line JEG3 at 50% of confluency was transiently transfected with 50 nM of either miR-196b and miR-223 mimic or scramble miRNA using HiPerfect, following the manufacturer's instructions ; after 24, 48 and 72 h RNA was extracted from the cells. To ensure that the miRNA fraction was recovered we performed RNA extraction using the miRNeasy Kit (Qiagen). The RNA extracted was quantified using a NanoDrop spectrophotometer  and the quality of RNA samples was assessed using a Nano LabChip BioAnalyzer 2100 . Gene expression of predicted targets was performed by Real Time PCR. The experiments were repeated three times.To evaluate the transfection efficiency, levels of miR-196b and miR-223 in JEG3 (compared with the housekeeping gene RNAU6-2) were measured by quantitative PCR with the miScript reverse transcription kit, miScript SYBR Green PCR kit and miScript primer assays according to the manufacturer's recommendations (Qiagen). In both cases miRNAs trasfected were increased between 1000 and 10.000 fold change compared to controls. Furthermore, approximately 95% of the cells transfected with scramble showed up fluorescence for GFP, meaning that the percentage of transfection was close to 100%.p-value.For microarray statistical analysis, MeV software was used. Samples were grouped into cluster hierarchies using Pearson correlation, and then they were subjected to PCA. Following this, a group variable was assigned to each sample and significance analysis of microarrays (SAM) analysis was used to estimate the changes in the expression level (fold change) of the miRNAs between the different samples. A false discovery rate (FDR) correction of less than 5% was used. For qPCR statistical analysis the expression levels of samples were subjected to a Mann-Whitney U test using SPSS v17.0 software. For biological process predictions the right-tailed Fisher's exact test was used to calculate a A total of 866 known miRNAs in first trimester embryonic tissue from eight EP patients were compared with the miRNAs from embryonic tissue derived from eight VTOP patients using miRNA microarrays. All 16 samples used in this microarray study passed extensive RNA and miRNA quality controls see . Among tTo confirm that the results obtained from the array could separate EP and VTOP samples, we first applied a unsupervised PCA including all the miRNAs measured in the array experiment. . Then, ap<0.001) increase in hsa-mir-451 (p<0.05) in EP samples compared to VTOP control samples, which was an even higher increase than in our microarray study. Significant (p<0.05) hsa-mir-196b downregulation was also detected in EP samples compared to controls in this set of samples . The most significant pathways found were the mucin type O-Glycan biosynthesis and the ECM-receptor-interaction pathways. We found three different predicted genes which were altered in the mucin pathway: GALNT7 and GALNT13 (repressed by 196b), and GALNT1 . The action of miR-196 mimic produced no significant changes in expression of the predicted target genes studied48 and 72 hours after transfection . When wein vivo embryonic tissue from tubal EP patients using voluntarily terminated normal gestations as controls. Although we have investigated previously the Lin28/Let-7 system in ectopic and voluntary termination of pregnancy samples To our knowledge this is the first study which has investigated the global differential miRNA repertoire of Of these seven miRNAs, three of them  were validated by real time PCR in a larger sample of EP and control tissues. These miRNAs might alter the downstream gene expression of dozens of genes, and thus investigating their gene targets may help us to elucidate the molecular mechanisms that intervene in ectopic pregnancy.mucin biosynthesis was the most statistically significant. Two miRNAs (hsa-miR196b and hsa-miR223) are able to target three important genes in the mucin biosynthesis pathway: GALNT7, GALNT1, and GALNT13. These two miRNAs were found to be altered in EP tissues compared to controls, meaning that their expression could also be potentially altered in EP embryonic tissue, and therefore they were likely to be implicated in altering mucin expression. In fact, when we checked if GALNT13 gene expression was increased in EP samples, we found that it was upregulated as we had expected. It is noteworthy that not all the genes achieved significant differences in expression between both situations as expected. This is the case of GALNT7, GALNT1 and COL1A2, casting some doubts about the real implication of these pathways in vivo.We are aware that we don't know the exact origin of these mRNA in our samples and could be very interesting to address this issue in future experiments. Ectopic and VTOP samples are complex tissues and the origin of these miRNAs could be important to understand the autocrine/paracrine effects of these miRNAs. Using bioinformatics tools, we analyzed all the predicted gene targets of these three miRNAs, obtaining the main pathways that could be altered in an ectopic pregnancy compared to an intrauterine gestation. Among these pathways, extracellular matrix (ECM) receptor interactions, with hsa-miR-196b and hsa-miR-223 able to interact with two targeted genes ; we also confirmed that integrin A2 (ITGA2) was significantly downregulated in EP samples.Although we saw some disparity in our results, some recent studies have reported deregulation of MUC1 and altered glycosylation in the tubal epithelium in EPs compared to controls Another study addressing tubal pregnancies showed that all trophoblastic cell types express matrix metalloproteinases (MMPs), and their regulators, tissue inhibitors of MMPs (TIMPs) in vitro if the transfection of these miRNAs into trophoblast cell lines could alter the gene expression of these predicted gene targets. Although these miRNAs targets additional genes, we focused on the mucin biosynthesis and the extracellular matrix (ECM) receptor interactions. In vitro over-expression of these miRNAs triggered a clear downregulation of some predicted genes such as ITGA2 in the case of hsa-mi-196b and GALNT1 in the case of hsa-miR-223, but the expression of other predicted genes remains unaltered. This observation means that although some miRNA could directly alter the expression of predicted genes, the alteration of these proposed pathways is not completely demonstrated, requiring further research. We should note that miRNA target prediction software foretells ‘theorical’ gene targets with different ranges of probability, but usually these predictions do not match with real data We finally checked In summary, we have discovered a clear differential pattern of miRNA expression in embryonic tissues derived from normal and eutopic/ectopic pregnancies, and have pinpointed some possible pathways that could be implicated in the processes of implantation and early placentation, although the real implication of these pathways in the ectopic pregnancy is yet to be determined.Figure S1Real time PCR miRNA microarray validation in ectopic pregnancies and controls. The same 8 VTOP and 8 EP embryonic samples that were used in the microarray experiments were used to validate by Real Time PCR the miRNA array. We observed a very significant decrease (p<0.001) in the EP tissue samples compared to VTOP samples for hsa-miR-196b (A) expression, while hsa-miR-223 (B) showed a significant (p<0.05) increase compared to VTOP controls. All data are presented as relative miRNA expression levels. *p-value <0.05; **p-value <0.01; ***p-value <0.001. Box plots represent the first quartile, median and third quartile; error bars show maximum and minimum relative expression levels.(TIF)Click here for additional data file.


The cell membrane is a highly selective barrier. This limits the cellular uptake of molecules including DNA, oligonucleotides, peptides and proteins used as therapeutic agents. Different approaches have been employed to increase the membrane permeability and intracellular delivery of these therapeutic molecules. One such approach is the use of Cell Penetrating Peptides (CPPs). CPPs represent a new and innovative concept, which bypasses the problem of bioavailability of drugs. The success of CPPs lies in their ability to unlock intracellular and even intranuclear targets for the delivery of agents ranging from peptides to antibodies and drug-loaded nanoparticles. This review highlights the development of cell penetrating peptides for cell-specific delivery strategies involving biomolecules that can be triggered spatially and temporally within a cell transport pathway by change in physiological conditions. The review also discusses conjugations of therapeutic agents to CPPs for enhanced intracellular delivery and bioavailability that are at the clinical stage of development. Cell penetrating peptides (CPPs) are short peptide sequences that are able to transport molecules across the cell membrane. The CPPs, also known as protein transduction domains (PTDs) are made up of three to 30 peptide residues . They arDrosophila antennapedia homeodomain was illustrated to be internalized by neuronal cells. This was the origin of the discovery in 1994 of the first PTD or CPP, a 16-merpeptide derived from the third helix of the homeodomain of Antennapedia termed Penetratin (RQIKIYFQNRRMKWKK)  originating from human heparin binding proteins and/or anti-DNA antibodies with both enhanced and safe cell penetration characteristics have been identified. These new peptidic sequences are reported to deliver small and large active molecules inside cells that would otherwise have limited or no bioavailability . It consists of a Morpholino oligo conjugated with the CPP [(RXR)4-XB-CPP]. The goal of this conjugate is to prevent eventual blockage of a transplanted vein after cardiovascular bypass surgery [Avi Biopharma is working on the clinical development of CPPs for the  surgery . Several surgery .Several reviews have elaborated on examples of novel drug delivery systems involving CPPs. All these systems are geared towards targeting a specific cell or tissue. Many of the strategies described have proved successful in rategies . Such stPhosphorodiamidate Morpholino Oligomers (PMOs) are antisense DNA oligonucleotide analogues with a backbone composed of morpholine rings joined by uncharged phosphodiamidate linkages in place of the sugar and anionic phosphodiester linkage of DNA. They are water-soluble, nuclease-resistant, and have an uncharged backbone, which can interact weakly with serum and cellular proteins thereby reducing toxicity. They therefore offer great promise in clinical applications . As PMOsPMO-technology to inhibit viral infections has been exploited as extensively reviewed by Stein  and receIn addition to improving PMO cellular uptake, the CPP moiety has also been reported to intensify PMO antisense activity against Ebola virus (EBOV) 10- to 100-fold in cell-free translation assays. These results are consistent with other findings. The authors proposed that the arginine-rich peptides enhance RNA-PMO binding affinity, thereby increasing specific antisense activity .N terminal to various cell-penetrating peptides, including Transportan, TAT and penetrain, via disulfide bridges. Upon coupling to carrier peptides, the PNA therapeutic agents were rapidly taken up by different types of human cells in culture. When added to culture medium, these anti-HIV-1 PNA–CPP conjugates effectively inhibited HIV-1 replication, tat-dependent trans-activation and viral production by infected cells. No significant decrease was observed with the unconjugated PNA, emphasizing the great promise held by these conjugates as antiviral agents [Due to poor cellular uptake of the uncharged peptide-nucleic acids (PNAs), Pandey and co-workers covalently conjugated PNA at its l agents .Preliminary toxicity, immunological and pharmacokinetic studies in mice for the anti-HIV-1 PNATAR-Penetratin conjugate have just started. Investigating the tissue distribution and clearance of 125I-labeled PNATAR, PNATAR-penetrain and PNATAR-TAT; Ganguly’s group reported the distribution of the conjugates throughout the mouse major internal organs when administered by oral route as well as their slow release and clearance from different organs . The uncThe use of CPPs to deliver siRNAs into cells has received rather less attention especially for antiviral siRNAs due to the fact that siRNAs is less amenable to CPP delivery as a result of charge interactions between the peptide and the siRNA which results in inefficient endosomal escape of the conjugates. However a cell-permeable CPP-siRNA conjugate targeting hepatitis C virus 5’ untranslated region has been formulated . The celIn addition to targeting HIV-1, CPP-mediated protein delivery has also been used to inhibit human papillomavirus type 18 (HPV-18) in cell culture. Delivery of artificial zinc-finger proteins (AZPs) into cultured cells by expressing AZPs in fusion with the 9R-peptide has been reported. These AZP-9R conjugates strongly reduced HPV-18 replication to 3% at 250 nM while non-conjugated AZP showed only 12% reduction. PTD4 conjugates were also tested but proved to be less effective than AZPs fused to 9R .PNA-CPP complexes have also been investigated for virucidal activity microbicides . The stuin vitro uptake studies revealed high cellular uptake values, but no specificity toward any of the cell lines. Their biodistribution in PC-3 tumor-bearing nude mice showed a high transient accumulation in well-perfused organs and a rapid clearance from the blood [in vivo application provided that their cell specificity and pharmacokinetic properties are also considered in design and development of CPP-based drug delivery systems.However CPPs have their limitations as drug delivery carriers. The he blood . Data obet al. conducting a comprehensive search for novel CPPs using an in vitro virus library of peptides consisting of 15 amino acids [Apart from functional group modification of amino acids in the peptide chain, modification of cell penetrating peptides have also been shown to involve substitution of amino acids to achieve variability of the peptide properties such as hydrophobicity or cationic nature. This strategy has been noted to result in increased intracellular internalization by certain CPPs. Kaeko no acids  and repono acids  to have no acids .et al. that in low peptide concentrations, two Trp residues were not enough to initiate effective translocation, but three Trp enhanced cellular uptake. However, when the number of Tryptophan residues increased to four the intracellular translocation activity decreased [Increasing amino acids like Tryptophan which has the tendency to be buried in the cell membrane  improvesecreased . This maFunctional group modification of the amino acids in the peptide usually involves the formation of masking groups or linkages to highly reactive sites. In either case, the peptide bonds formed should be labile and easily broken for regeneration of the initial cell penetrating peptide by simple variation of physiological conditions.Helicity of the peptide chain can be exploited for modification. For example through hydrocarbon stapling the overall cell penetrating peptide’s alpha helicity could further be stabilized. This is derived from the fact that the α-helix, as a major structural motif of proteins, frequently mediates intracellular protein-protein interactions that govern many biological pathways .Fei and co workers reported a modification strategy to decrease the non-specific binding and uptake of a CPP, model amphipathic peptide  blocks, the epsilon-amino groups of the Lysine residues formed acid-labile amide linkages. This was confirmed by analysis for internalization in HeLa cells which revealed that 80.8 ± 2.2% of the amino groups were modified in the CA-MAP conjugate. In the study a MAP conjugate was designed to contain acid-labile modifications to mask the cationic charge, and therefore decreasing the non-specific binding and uptake ,72. AfteOver the years of research focus has been on how to optimize drug delivery systems to increase both cell specificity and drug delivery efficiency. The use of cell penetrating peptides demonstrates the possibility that these drug systems can be improved. Efficient targeting by utilizing multi-functional polymer vehicles with capabilities for endosome disruption and nuclear penetration if appropriately considered can yield cell-penetrating peptides with high cellular specificity. CPP modification presents viable options that such delivery systems can further be improved. This could result in biomolecules that could be triggered spatially and temporally within a cell transport pathway hence truly improving drug therapy. There is therefore a need to develop assays to compare the different existing methods in order to study and optimize modified CPP-mediated delivery.


Both loss of function mutations and the expression of a constitutively active Dia led to cells forming both morphologically abnormal hairs and multiple hairs. The conserved frizzled (fz)/starry night (stan) PCP pathway functions to restrict hair initiation and activation of the cytoskeleton to the distal most part of wing cells. It also ensures the formation of a single hair per cell. Our data suggest that the localized inhibition of Dia activity may be part of this mechanism. We found the expression of constitutively active Dia greatly expands the region for activation of the cytoskeleton and that dia functions antagonistically with multiple wing hairs (mwh), the most downstream member of the fz/stan pathway. Further we established that purified fragments of Dia and Mwh could be co-immunoprecipitated suggesting the genetic interaction could reflect a direct physical interaction.The Drosophila wing is covered by an array of distally pointing hairs that has served as a key model system for studying planar cell polarity (PCP). The adult cuticular hairs are formed in the pupae from cell extensions that contain extensive actin filaments and microtubules. The importance of the actin cytoskeleton for hair growth and morphogenesis is clear from the wide range of phenotypes seen in mutations in well-known actin regulators. Formin proteins promote the formation of long actin filaments of the sort thought to be important for hair growth. We report here that the formin encoding  Genetr of PCP . Furtherr of PCP . A charar of PCP ,2. A secates PCP –8. In th pathway –11 and t pathway –14. Althcofilin)  and flarlr-AIP1)  have beesinged (fascin) [forked (forked) [crinkled (myosin VIIa) [zipper (myosin II) [Rho1, Dcdc-42 and the effector Rho kinase (Drok) also result in short, split and multipled hairs [tsr and flr mutations [slingshot phosphatase that dephosphorylates and activates cofilin also produces hair morphology phenotypes [diaphanous (dia) is known to be important for the morphogenesis of denticles in the embryo [dia to be a key gene. Both loss and gain of function mutations result in dramatic abnormalities in hair morphology. We also established that dia also plays an important role in the morphogenesis of sensory bristles, a another polarized cell type where linear actin filaments are prominent and thought to be important [Mutations in genes that regulate the actin cytoskeleton often result in abnormal hairs. Among the good examples are the bundling proteins (fascin)  and fork(forked) ,19 that in VIIa)  and zipposin II) ,22 whiched hairs –25. Perhutations ,16. Mutaenotypes . Drugs tenotypes . The groenotypes . Forminsenotypes ,30 and he embryo . Denticle embryo . We explmportant ,34.Growing hairs also contain centrally localized microtubules that are likely to be important for hair growth ,27,35. Ifz/stan pathway regulates wing PCP by restricting the activation of the cytoskeleton that drives hair morphogenesis to the distal most part of the cell [multiple wing hairs (mwh) gene is the most downstream member of the fz pathway and hence is a strong candidate for mediating at least part of this restriction [mwh mutations result in most wing cells forming 3 or more hairs with aberrant polarity at abnormal locations along cell periphery [mwh leads to a delay in hair initiation, loss of function mutant cells form extra hairs and ectopic actin filaments and the expression of mwh in cultured cells leads to actin phenotypes [The the cell . The multriction ,36,37. Mformin homology 3 domain (FH3) [diaphanous inhibitory domain (DID), and a dimerization domain (DD) [The sequence of the Mwh protein suggests a possible mechanism for mediating PCP control of the actin cytoskeleton. The amino terminal half shows similarity to the same region in Diaphanous family formins ,37. Thisin (FH3) ,39. The ain (DD) ,40–44. Dain (DD) ,45. In tain (DD) . Howeverfz/stan pathway to restrict the activation of the cytoskeleton to a small distal region. We also carried out genetic studies that show mwh and dia act antagonistically in wing hair development and that purified fragments of these two proteins interact in vitro. Surprisingly this physical interaction was not based on the DD domains that we suspected would mediate the dimerization. These data suggest that one possible mechanism by which the fz/stan pathway regulates wing PCP is by the direct inhibition of Dia by Mwh.We report here that the expression of a constitutively active Dia (CA-Dia) in pupal wing cells greatly increases the area where F-actin accumulation is seen at the start of hair formation. Thus, it appears that the presence of CA-Dia antagonizes the ability of the ptc-Gal4 to knock down levels of the Drosophila formins in the wing [dia, cappuccino, DAAM, formin 3, CG32138 and Formin homology 2 domain containing ortholog (Fhos). Of these only dia showed a substantial wing hair phenotype consistent with a possible role as a downstream target of the fz pathway and/or a key role in hair morphogenesis. A hint of a mutant phenotype was seen for CG32138, but its weakness and weak penetrance lead us to give it a low priority for further study. No phenotype was seen in the knockdowns of the other formins. In a separate set of experiments we also used transgene mediated RNAi knockdowns to determine if Arp2/3 complex proteins, which promote the formation of branched actin networks [p16ARC (CG9881), Arp11 (CG12235), Arc-p34 (CG10954), Arpc3A (CG4560), Arpc3B (CG8936) and Arp14D (CG9901) using UAS-dicer2; ptc-Gal4 all produced a similar set of phenotypes with varying severity , branched hairs (red arrowhead), polyploid cells and decreased alignment of neighboring hairs (red arrows). We also failed to see a phenotype with a knock down of spire, another actin regulator.Dia and other formins are known to promote the polymerization of long actin filaments, which are thought to be important for hair morphogenesis ,46. All the wing . We testnetworks ,50 had aseverity .. The moseverity .. This wdia was polyploid cells  down the center of the wing where ptc-Gal4 drives expression at the highest level  that did not appear to be polyploid (The predominant phenotype seen with the knock down (kd) of st level . Polyplookinesis ,52. Greaokinesis . It is wng cells  but it ing cells  we used the wing . Under fap-Gal4 in these experiments leads to minor folds in the wing presumably due to dia being knocked down in and leading to cell shape changes in dorsal but not ventral cells. The presence of such distortions makes it difficult to be certain that the abnormal polarity phenotype is a primary defect. To get around the distortion problem we needed to examine a tissue whose shape was not distorted in the experiment. As an alternative to the wing we chose to examine hair polarity on the notum of ap-Gal4 pTubGal80ts/+; UAS-dia-RNAi/+ flies  [ap-Gal4, presumably due to the expression of ptc-Gal4 strongly declining in the 12 hours prior to hair initiation. In wings displaying the strongest phenotypes the wing did not fully expand  . Much sty expand , wing cey expand  and the y expand . Many ofy expand  due to ay expand .Gal80ts and a temperature shift. We primarily utilized ptc-Ga4 as the driver because the animals were healthier, their developmental timing was more reproducible and the weaker phenotypes were easier to interpret. Prior to hair initiation affected cells showed much stronger cortical F-actin staining than is seen wild type cells . This suggested that the multiple hair phenotype of CA-Dia is due to cells failing to restrict the location for actin polymerization to a small region around the distal vertex. We also noticed F-actin bundles/filaments in the apical regions of cells apart from the region of hair morphogenesis , only occasional double hair cells were observed  resulted in a large increase in the number of cells producing two hairs   . Once ag; mwh/+)   and putative (Mwh) auto-inhibition sites were removed. The use of a fragment of Mwh was also necessary as we were unable to purify full length Mwh in a soluble form. We tested a His tagged Mwh-GBD-FH3 fragment and a GST tagged Dia-CC-FH1-FH2 fragment. In a His pull down assay on E.coli extracts expressing the two proteins we found the GST tagged CC-FH1-FH2 fragment of Dia was pulled down , lane 6.dia produced a strong hair phenotype. We cannot rule out any of the other formins also functioning in hair morphogenesis as we did not determine the effectiveness of the knock downs and we did not do experiments to rule out redundancy. Indeed the very weak phenotype seen with a knock down of CG32138 could be due to either of these. For the DAAM formin published standard genetic tests argue against a role in wing hair morphogenesis [Growing wing hairs contain actin filaments and a variety of data establishes that these filaments are important for hair morphogenesis –25,27,28ogenesis .dia that implicate Dia in both hair morphogenesis and PCP. Many of the hair morphology phenotypes  seem likely to be related to the abnormal activation of the actin cytoskeleton. Given the extensive literature on the role of Dia in regulating the actin cytoskeleton these phenotypes were not surprising. The involvement in PCP was less expected. The severity of the hair morphology phenotypes complicated but did not eliminate our ability to examine the role of Dia in PCP. Alterations in Dia activity led to the formation of multiple hair cells, hairs of abnormal polarity and swirling hair patterns, all of which are classic phenotypes of PCP mutants [A range of phenotypes were seen with both loss (lof) and gain of function (gof) conditions for  mutants ,2,3.fz PCP pathway normally functions to restrict the activation of the cytoskeleton to a small region around the distal most part of wing cells [mwh gene is the most downstream known component of the pathway and hence is a candidate to mediate such a function. Our observations are consistent with this hypothesis. Notably, the multiple hair cell phenotype associated with CA-Dia expression included the formation of tiny hairs, which is a distinctive phenotype of mwh mutants [mwh mutants but not other PCP mutants [dia and mwh is due to both independently regulating the actin cytoskeleton. This hypothesis needs to be considered given that we did not see a strong co-IP from wing discs.The ng cells . When CA mutants ,36,37,66fz/stan pathway was functional but it was not able to block the hyper activation of the cytoskeleton. Indeed, the consequences of CA-Dia expression were more severe than loss of fz/stan pathway function. This could be due to either the over expression of CA-Dia overwhelming the system or to the constitutively active nature of the protein. Of course both factors could be important. It is worth noting that the fz/stan system appears to be finely tuned as both gain and loss of function mutations give rise to PCP phenotypes and it is unable to scale to insure a single distally pointing hair is formed in polyploid cells [In our experiments where CA-Dia was expressed the id cells . Hence iIn vertebrates the DAAM formin has been implicated in PCP  and in mshavenoid is a particularly strong candidate as mutations in this gene lead to the loss of hair formation in some cells and a substantial delay in initiation in others [When we expressed CA-Dia in pupal wing cells we observed increased accumulation of F-actin, consistent with the well-known activity of Dia in other cells types/systems. Prior to hair initiation the increased F-actin was largely restricted to the lateral cell membranes. Thus, the presence of CA-Dia was not sufficient to induce hair formation and other factors must be important in regulating the initiation of hair morphogenesis. The expression of many genes changes dramatically around the time of hair initiation  and one n others .When hair initiation began in cells expressing moderate levels of CA-Dia increased F-actin was primarily seen over the distal apical region of the cell. This region was much larger than normal but still only represented a minority of the cell. Thus, even in the presence of CA-Dia the wing cells still retained significant spatial control over actin polymerization. This seems likely to be due to the PCP mediated localization of factors that regulate the actin cytoskeleton as there was still a distal bias to activation of the cytoskeleton. The Mwh protein could serve this role by inhibiting endogenous Dia and/or CA-Dia proximally leading to a higher level of actin cytoskeleton activation distally where both CA-Dia and endogenous Rho1 activated Dia could stimulate actin polymerization. Mwh could also be acting independently of its interaction with Dia. There is reason to believe that wing PCP involves both negative regulation proximally and positive regulation distally ,69. The dia lof and gof genotypes lead to multiple hair cells. It is an unusual property shared by most of the genes of the fz pathway that both gof and lof genotypes lead to similar mutant phenotypes [dia lof mhc phenotype. The hyper-activation due to the over expression of CA-Dia leads to the activation being spread over a much larger region and this also appears to lead to multiple hairs forming. Proper PCP in the fly wing appears to require the function of an efficient and highly regulated cytoskeleton.It is notable that both enotypes ,73. In tenotypes ,24 and tenotypes  give risenotypes ,73. Thisap>CA-dia flies is also likely to be due to effects on the microtubule cytoskeleton [mwh mutant cells so we think it likely that the mwh-like hair phenotypes are due to effects of CA-Dia on the actin cytoskeleton but specific Dia effector mutations will be required for an unambiguous answer.Diaphanous has primarily been studied with respect to its regulation of the actin cytoskeleton but it is also known to regulate the microtubule cytoskeleton . Indeed skeleton . That DiDrosophila stock center at the Indiana University or were generated in our lab and previously described [UAS-diaΔDad (aka CA-dia) and UAS-FH3FH1FH2 transgenic flies were generous gifts from Mark Peifer at the University of North Carolina. To direct transgene expression, we used the Gal4/UAS system [All flies were raised at 25°C unless otherwise stated. Unless otherwise stated stocks were obtained from the Bloomington escribed . UAS-diaS system . For immAdult wings. Adult wings were dehydrated in 100% ethanol and then mounted in Euparal. To visualize hairs on the notum thoraxes were mounted in Gary’s magic Mountant. Spacers were used to keep the cover slip from crushing the tissue. Mounted samples were examined under the bright field microscope. Images were obtained using a Spot-RT digital camera (Diagnostics Instruments) on a Zeiss Axioskop II microscope. In some cases Metamorph was used to obtain 3D stacks of images. Minimum projections of such stacks were used to increase the effective depth of field and to allow us to visualize 3D structures such as the hairs on the notum.dia and mwh and between CA-dia and mwh.Quantitative analysis of the genetic interaction between  To characterize the genetic interaction between dia and mwh, we scored the number of multiple hair cells in the region distal to the posterior cross vein in 20 wings. The significance of the difference was evaluated using two-sample unequal variance Student t-Test. A similar method was used to quantify genetic interaction between CA-dia and mwh.Image Acquisition and Analysis. We used ImageJ to quantify aspects of both confocal images of pupal wings and bright field images of adult wings. Pupal wings were examined by confocal microscopy using a Zeiss 510 Meta confocal microscope.As evidence for the co-localization of Dia and Mwh in the hair, the grey scale values of the immunostaining signal for GFP-Dia and Mwh were individually determined along a line drawn across the proximal region of 5 different hairs  on both sides. The primers are 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCAAAACATGTACAGCAAGGAAAACCAGCG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGATGCCCTCGTCCTCGTG-3′. The cDNA was cloned into Donor 221 vector (Invitrogen) to generate entry clone which was then cloned into pTWH, one of the Carnegie Gateway Vectors constructed by T. Murphy to generate expression constructs with C-terminal 3HA tag.UAS-GBD-FH3-GFP construct for examining over expression phenotype, the entry clone of GBD-FH3 described above was cloned into pTWG, one of the Carnegie Gateway Vectors constructed by T. Murphy to generate an expression construct with a C-terminal GFP tag.To make UAS-GFP-dia and UAS-CA-dia constructs were generated in Mark Peifer lab at the University of North Carolina.Purified proteins. The 6His-GBD-FH3(Mwh) protein was produced by subcloning DNA encoding aa 60–491 into pET28a and GST-CC-FH1-FH2(Dia) protein was produced by subcloning the region encoding aa 448–1029 into pGEX. They were expressed in E. coli and purified by standard techniques such as affinity chromatography and molecular size exclusion chromatography.Co-immunoprecipitation and Western blotting. 300 wing discs were dissected from third instar larvae for immunoprecipitation assays and an additional 15 wing discs as extract for use as a positive control for Western blotting (WB). Standard immunoprecipitation and WB procedures were performed at 4°C [d at 4°C . SeeBluePolyclonal rabbit anti-GFP antibodies were used for co-immunoprecipitation of GFP-Dia and Mwh. Mouse anti-GFP antibody and rat anti-Mwh antibodies were used in WB to detect GFP-Dia and Mwh respectively. Detection was by chemiluminescence (Amersham) in early experiments and later by infrared fluorescence (Licor)S1 Figptc-Gal4. Aprc3a (A) and Arpc3b (BC) knockdowns are shown as examples. The black arrows point to double hair cells, black arrowheads to swollen bases, red arrows to hairs that have fallen over and are not well aligned with their neighbors, red arrowheads to split hairs and the asterisk to a region where a it appears that a cell did not form a hair.Transgene mediated RNAi was driven by (TIF)Click here for additional data file.S2 FigPolyploid cells at the margin produce oversized  hairs and bristles and in some cases duplicated bristles that lack socket cells. Evidence of cell death is seen by the formation of wing nicks (C).(TIF)Click here for additional data file.S3 Figneur-Gal4 ptub-Gal80ts/UAS-CA-dia flies. The arrows point to bristles that show the stub phenotype. A pair of abnormal scutellar bristles (arrows) from a neur-Gal4 ptub-Gal80ts/dia-RNAi fly. The adult notum of a neur>CA-dia, actin-GFP fly used for in vivo imaging (C). The arrow points to a severely abnormal bristle that was followed in time lapse. The insert shows an image from the in vivo imaging experiment where the relevant bristle was abnormal during bristle growth. A notum with a putative dia5 clone (D) is missing a bristle, contains polyploid cells (arrow) and diploid hair cells (arrowhead). Also note the abnormal hair polarity. Highly abnormal bristles from ap>CA-dia; ptub-Gal80ts/+ notums (arrows) . The severly affected bristles are macrochaetae and the nearby relatively normal bristles are microchaetae.A variety of bristle abnormities are seen in part of a tergite  in (TIF)Click here for additional data file.S4 Figap-Gal4 pTub-Gal80ts/+; UAS-CA-dia/+ pupal wings stained for F-actin. Each image is a maximum projection of 3 optical sections that represent 0.6um along the dorsal/ventral axis. Arrows in A and B point to multiple hairs cells. The arrows in C point to ectopic actin filaments not normally seen in wing cells. The arrows in H point to actin foci in ventral cells found associated with chitin deposition. These are missing from the dorsal cells. The arrows in K point to normal ventral hairs.Starting at the dorsal surface and moving ventrally images of (TIF)Click here for additional data file.S5 Figap>CA-dia (B) arista are shown. The arrow points to the central core which is grossly short and fat in ap>CA-dia. The arrowheads point to laterals which are short and deformed in ap>CA-dia.Oregon R (A) and (TIF)Click here for additional data file.S6 FigThe relative intensity of immunostaining for Dia-GFP (green) and Mwh (red) along the proximal part of a growing hair is shown. The presence of substantial Mwh at the cell periphery prevented us from extending the line of measurement to the full length of the hair.(TIF)Click here for additional data file.S7 FigThe purified proteins were examined by PAGE and western blotting to confirm their purity. The GST-CC-FH1-FH2 protein migrates slightly faster than the 94 kd marker and the his-GBD-FH3 protein migrates at approximately 45kd.(TIF)Click here for additional data file.


We show that Nestin-positive human mesenchymal stem cells (MSCs) support expansion of a range of biologically and clinically distinct patient-derived ALL samples. Adherent ALL cells showed an increased accumulation in the S phase of the cell cycle and diminished apoptosis when compared with cells in the suspension fraction. Moreover, surface expression of adhesion molecules CD34, CDH2 and CD10 increased several fold. Approximately 20% of the ALL cells were in G0 phase of the cell cycle, suggesting that MSCs may support quiescent ALL cells. Cellular barcoding demonstrated long-term preservation of clonal abundance. Expansion of ALL cells for >3 months compromised neither feeder dependence nor cancer initiating ability as judged by their engraftment potential in immunocompromised mice. Finally, we demonstrate the suitability of this co-culture approach for the investigation of drug combinations with luciferase-expressing primograft ALL cells. Taken together, we have developed a preclinical platform with patient-derived material that will facilitate the development of clinically effective combination therapies for ALL.Lack of suitable  More importantly, cell lines have adapted to suspension culture and grow without niche support. The combination of low complexity and reduced dependence on cell-extrinsic signalling can affect the translation of cell line data to the clinical situation, for example, in relation to clinically relevant mechanisms of drug resistance; 9 thus affecting the ability of cell line models to reflect the original disease.Robust preclinical models for childhood acute lymphoblastic leukaemia (ALL) are essential for dissecting mechanisms that drive malignant growth and survival and to test and develop novel targeted therapies that may improve current therapies with regard to efficacy and toxicity. Cell line models have been widely used in functional in vitro. ALL blasts are highly dependent on their in vivo environment and rapidly undergo apoptosis ex vivo.12 Short-term in vitro assays have been developed for testing drug sensitivity;13 however, their use has not been widely implemented because of the rapid decline of ALL cells in these assays, even without exposure to any anti-leukaemic compounds.Functional studies with primary blasts from children with ALL, however, have been hampered by the difficulty in expanding ALL cells 16 These studies have demonstrated that the murine bone marrow and lymphoid microenvironment is highly conserved between mice and men and able to support malignant human lymphoid cells. Although there is some evidence of clonal selection in the mice,20 clonal complexity and niche dependency are preserved.18 Xenograft mouse models have been used for a wide range of studies including the phenotypic definition and homing of leukaemia propagating cells and for preclinical drug testing.24 However, animal experiments are labour intensive, expensive and time consuming, thus limiting their application.The vast majority of ALL will engraft at low cell numbers and proliferate in highly immunodeficient mice.25 has pioneered novel culture systems providing stroma support for the leukaemia blasts using immortalised mesenchymal stem cells (MSCs). These and similar bone marrow stromal co-cultures have been successfully used to model leukaemia–stroma adhesions and interactions.30 Stroma cells provide crucial survival signals to the leukaemic cells that affect drug resistance, thus mimicking the situation in patients.34 However, despite the ability of allogeneic bone marrow stromal cells to support proliferation of primary leukaemia blasts, acquisition of additional mutations following long-term cell expansion has been observed. This reduces the complexity and feeder dependence of the cultivated blasts, sometimes leading to the outgrowth of cell lines.35The group of Campanain vitro without compromising sample clonal composition and self-renewal capacity. We confirm early-passage primary MSCs to provide superior support than their counterpart feeders in B-ALL expansion. Combining MSC co-culture with lentiviral luciferase transduction of ALL cells generates an experimentally accessible medium–high throughput in vitro system that is suitable for preclinical drug testing in patient-derived primary leukaemic cells.We describe an optimised primary MSC culture system for the long-term propagation of a cytogenetically and clinically distinct panel of primary and primograft ALL blasts 36 For drug treatment, green fluorescent protein-positive ALL cells were seeded onto the 96-well plates at 5000 cells per well followed by incubation with clinically relevant drug concentrations. Dose response was analysed 5 days later by adding luciferin at 30 μg/ml  and measuring bioluminescence using the FLUOstar Omega microplate reader . Combination indices were calculated for drug combinations in constant ratio to each other using software CalcuSyn Version 2.1.37This assay was carried on white frame and clear flat-bottom 96-well plates  seeded with 10 000 MSCs per well. L707 cells were lentivirally transduced with pHR-cppt-SLIEW for stable expression of enhanced green fluorescent protein and firefly luciferase followed by sorting for green fluorescent protein positivity on a FACS Aria III  and expansion on MSCs for 2 weeks.To compare the impact of stromal cell lines and primary human MSCs on B-ALL propagation, we examined ALL cell proliferation on either the murine stromal cell line M2-10B4, human TERT-immortalised MSC or primary human MSCs obtained from hip surgery.Primary MSCs were characterised as NES+ CD73+ CDH2+ CD90++ TEK+ ; absence of haematopoietic cells was confirmed by lack of CD45 and CD19 surface expression . We examex vivo expansion of ALL primograft material on these feeders. Replacement of RPMI-1640 by serum-free expansion medium and 20% fetal calf serum resulted in superior survival of primograft material  that had not been previously passaged through a mouse . WhereasCell cycle analysis showed that co-culture on MSCs caused a twofold increase in ALL cells being in the S phase and a 20-fold reduction in subG1 cell numbers indicating a substantial inhibition of apoptosis . InteresA more detailed examination showed that this effect is limited to ALL cells adhering to the MSC layer. Nonadherent ALL cells showed subG1 and S-phase distributions similar to ALL cells in standard suspension culture , that isMost importantly, adherence to MSC affects the expression of genes likely to be involved in cell–cell communication. Surface expression of CD34 (a cell surface glycoprotein involved in cell adhesion), Cadherin 2  and CD10  was between two- and five-fold higher in adherent than nonadherent ALL cells . Some noin vitro.35 Importantly, we observed a drastic reduction in CD10 levels in suspension cultures as opposed to the ALL cells on MSC that continually retained CD10 expression over a 4-week period  and two primary samples  representing a spectrum of cytogenetically distinct ALLs . All samSelected primograft samples, including L707, could be maintained for more than 80 days in continuous culture, indicating a potentially unrestricted growth potential under these conditions . ALL cel41 This clonal analysis technique is commonly used for the purposes of evaluating clonal dynamics such as clonal complexity, clonal dominance and founder effects.43 We used this cellular barcoding approach as an in vitro tool for clonal tracing so as to evaluate whether such prolonged ex vivo expansion would cause any MSC-related bottleneck. L4951-BCR/ABL cells were lentivirally transduced with a custom-made barcoded vector library.43 Five million cells containing 897 different barcoded clones were seeded onto MSCs. Following 4 weeks of in vitro culture on MSCs, 100% of the barcodes in the baseline sample were retrieved, indicating unaltered clonal complexity on the MSC cultures. In addition, there was no selection of dominant clones (in vitro.Cellular barcoding is a process by which cells are tagged by a unique DNA sequence (the ‘barcode') that upon stable integration confers a heritable mark so as to trace clonal progenies.t clones . Additiot clones . Barcode34 Therefore, we examined whether extended MSC co-culture affects the engraftment potential of primograft cells. We used two primografts (L707 and L826) that had been transduced to stably express luciferase.36 Consequently, engraftment of these primografts can be monitored in vivo using bioluminescent imaging. For the t-positive L707 sample, co-culture on MSC feeder for 8 weeks did not diminish the engraftment potential when compared with uncultured primograft cells  with dexamethasone . At diag36 that allows live monitoring of cell numbers in 96-well plates in response to single and combinations of candidate anti-leukaemic drugs (44 we confirm synergism of dexamethasone/ABT-199 (combination index (CI)=0.32) in our MSC bioluminescence-based drug assay. We repeated the drug-combination experiment with another primary sample (L826-MLL/AF4) and in accordance with reported literature45 confirmed a combination of DOT1L inhibitor EPZ5676 and ABT-199 (CI=0.39) to be synergistic in MLL rearranged leukaemia  .We and others have previously shown BCL2 to be active in t ALL.CI=0.88) . These dex vivo often over prolonged periods of time, without loss of leukaemic self-renewal potential and while maintaining the multitude of founder clones in these cultures.In this paper we show that primary and patient-derived ALL cells can be successfully expanded 49 In particular, in vivo studies have shown that following cytotoxic treatment MSC cooperate with B-ALL cells to create a supportive microenvironment that confers chemoprotection.48 Consistent with these in vivo and previous in vitro studies,50 we confirm that freshly sourced low-passage Nestin-positive MSCs are able to provide superior support for long-term in vitro expansion of primary and patient-derived lymphoid blasts compared with murine M210B4 or human TERT-immortalised MSCs. Through fate mapping we show that hundreds of founder clones establish the leukaemia in these cultures, continue to contribute to the in vitro expanding leukaemia over time and maintain the ability to re-establish the disease in immunodeficient mice.MSCs play an important role in the normal haematopoietic and leukaemic bone marrow niche.51 Adherence to MSCs is an interactive process leading to the upregulation of key molecules on the surface of the leukaemic blasts that are presumed to play a role in cell–cell contacts. This was shown for the cell adhesion molecule CD34 and, more importantly, for cadherin 2 that is involved in establishing tight adhesion junctions between cells. Another molecule upregulated upon adhesion to MSCs but lost in suspension culture was CD10. CD10 is a cell surface molecule with metalloprotease activity that is able to inactivate a range of peptide hormones and as such expected to regulate signals from the microenvironment. Similar to our observation, CD10 has been shown to be induced and to correlate with progression of ALL in vivo while being lost in vitro during suspension culture;40 it has also been shown to correlate with dissemination and progression of melanoma and other types of cancer.52Direct MSC contact is required to support proliferation and viability of B-ALL and cannot be substituted by MSC conditioned medium. A possible mechanism behind this contact-mediated survival may be attributed to the formation of tunnelling nanotubules between the MSCs and B-ALL cells.Interestingly, our cell cycle analyses show that a high number of ALL blasts go into cell cycle arrest (G0) upon direct contact with MSCs. This could be seen as a limitation of this assay as not all ALL samples will continue to proliferate and expand long term; however, it provides a tool to study the mechanisms that regulate quiescence/dormancy of leukemic cells. In summary, the ALL/MSC co-culture system provides an experimentally accessible model to study and dissect key components of the leukaemic niche, including how the niche facilitates chemoprotection.in vitro response in the ALL/MSC co-culture assay correlates with clinical and in vivo responses, as shown here for steroid and targeted drug-combination sensitivities. Testing novel agents on primary and primografted cells on MSC co-cultures will increase the predictive value for clinical responses as compared with historic suspension culture assays. To further improve the assay system compared with previously published MSC co-culture models50 and to create an up-scalable tool for high-throughput screening, we introduced bioluminescence-labelled ALL cells allowing live monitoring of cell numbers in 96-well plates in response to combinations of candidate anti-leukaemic drugs.Drug response validation experiments show that in vivo microenvironment while significantly reducing expensive in vivo experiments that are currently regarded as gold standard assays for preclinical drug development. Most importantly, as we show that the MSCs preserve the initial multitude of leukaemic founder clones, this assay will allow complex functional RNAi/CRISPR  screens to study synthetic lethality, thereby facilitating design of novel and clinically effective combination therapies for ALL.Although not all ALL samples grow long term on MSCs, using bioluminescent-labelled patient-derived ALL cells after expansion through immunodeficient mice (primografts), this assay will significantly simplify preclinical drug testing by using a clinically relevant cell source and by improved modelling of the


Recent studies have been shown that functional connectivity of cerebral areas is not a static phenomenon, but exhibits spontaneous fluctuations over time. There is evidence that fluctuating connectivity is an intrinsic phenomenon of brain dynamics that persists during anesthesia. Lately, point process analysis applied on functional data has revealed that much of the information regarding brain connectivity is contained in a fraction of critical time points of a resting state dataset. In the present study we want to extend this methodology for the investigation of resting state fMRI spatial pattern changes during propofol-induced modulation of consciousness, with the aim of extracting new insights on brain networks consciousness-dependent fluctuations.Resting-state fMRI volumes on 18 healthy subjects were acquired in four clinical states during propofol injection: wakefulness, sedation, unconsciousness, and recovery. The dataset was reduced to a spatio-temporal point process by selecting time points in the Posterior Cingulate Cortex (PCC) at which the signal is higher than a given threshold . Spatial clustering on the PCC time frames extracted was then performed (number of clusters = 8), to obtain 8 different PCC co-activation patterns (CAPs) for each level of consciousness.The current analysis shows that the core of the PCC-CAPs throughout consciousness modulation seems to be preserved. Nonetheless, this methodology enables to differentiate region-specific propofol-induced reductions in PCC-CAPs, some of them already present in the functional connectivity literature , some others new .In conclusion, our results indicate that the employed methodology can help in improving and refining the characterization of local functional changes in the brain associated to propofol-induced modulation of consciousness. Functional magnetic resonance imaging (fMRI) technique has been widely used in the investigation of brain connectivity patterns at rest In particular, in altered states of consciousness it's important to see which functional connections remain unaltered and which ones are modified or disrupted Additionally, recent studies have been shown that functional connectivity of cerebral areas is not a static phenomenon, but exhibits spontaneous fluctuations over time Lately, a new approach in exploring functional brain connectivity, using point process analysis, has been proposed by Tagliazucchi et al. In the present study we want to extend and apply this methodology for the investigation of fMRI resting state spatial pattern changes during propofol-induced modulation of consciousness, with the central aim of extracting new information regarding brain networks consciousness-dependent fluctuations.The study was approved by the Ethics Committee of the Medical School of the University of Liège . The subjects provided written informed consent to participate in the study.The present work is a reanalysis of previous published data 3, and matrix size = 64×64×32).The subject was asked to strongly squeeze the hand of the investigator. She/he was considered fully awake or to have recovered consciousness if the response to verbal command (squeeze my hand) was clear and strong (Ramsay 2), in sedation if the response to verbal command was clear but slow (Ramsay 3), and in unconsciousness if there was no response to verbal command (Ramsay 5–6). For each consciousness level assessment, Ramsay scale verbal commands were repeated twice. Functional MRI acquisitions consisted of resting-state functional MRI volumes repeated in four clinical states: normal wakefulness (Ramsay 2), sedation (Ramsay 3), unconsciousness (Ramsay 5), and recovery of consciousness (Ramsay 2). The typical scan duration was half an hour in each condition. The number of scans per session  was matched in each subject to obtain a similar number of scans in all four clinical states. Functional images were acquired on a 3 Tesla Siemens Allegra scanner .The fMRI data were preprocessed using Statistical Parametric Software (SPM8), performing the typical preprocessing steps of functional connectivity analysis. These steps included motion correction, spatial smoothing (FWHM = 8 mm), temporal filtering with a bandpass filter (0.005 to 0.1 Hz), and the removal of linear and quadratic temporal trends. In addition, the brain-averaged signal, the time series of regions of interest in the white matter and cerebrospinal fluid, and six affine motion parameters were regressed out from the dataset. The fMRI data of each subject was first spatially coregistered to high-resolution anatomical images and then to the 152-brain Montreal Neurological Institute (MNI) space. It has recently been shown that even after standard motion correction, residual head movements can still inflate connectivity measures 3 cube centered at the posterior cingulate cortex , which includes wakefulness resting-state functional magnetic resonance imaging (fMRI) collected at multiple sites (247 subjects), as used by Liu and colleagues In order to achieve a spatio-temporal mapping of correlated activity we clustered all the time frames which were significantly co-activated with PCC, in the same way as described in www.nitrc.org/projects/fcon_1000/) were then kept fixed, and spatial clustering on the PCC time frames extracted from our propofol dataset, for each condition , was then performed around these centroids, averaging the within-cluster spatial maps to obtain 8 different PCC-CAPs for each level of consciousness. The clustering with centroid fixed allowed us to compare PCC-CAPs between states , and to follow thus the fluctuation of each PCC-CAP over the course of consciousness modulation.The eight PCC-CAPs centroids obtained from the clustering of the 1000 Functional Connectome Project dataset  with the four clinical conditions as factors: normal wakefulness, sedation, unconsciousness, and recovery of consciousness. The error covariance was not assumed to be independent between regressors, and a correction for non-sphericity was applied. We used one-sided T contrasts, as implemented in Statistical Parametric Mapping software, to test for significant effects in all our analyses. After model estimation, a first T contrast searched for areas co-activated with the PCC during normal wakefulness, sedation, unconsciousness and recovery. Afterwards, in a second analysis a linear one-tailed T contrast was computed for each CAP, searching for a linear relationship between PCC co-activation patterns and the level of consciousness of the subjects across the four conditions . We studied PCC-CAPs in our 18 subjects fMRI resting state dataset, for all the 4 different states of consciousness acquired, i.e. normal wakefulness, sedation, unconsciousness, recovery  during anesthesia has previously been studied in monkeys Similarly, the PCC-related primary motor disconnection is pointed out here (CAP 5 and 8) on fMRI resting state data. These results are in line with previous transcranial magnetic stimulation (TMS) and electroencephalographic (EEG) studies, that indicate intracortical inhibition of central motor circuitry during incremental suppression by a potentiator of GABA agonist (propofol) in a dose-dependent manner A limitation to this approach is the choice of the seed, that needs to be based on a strong priori hypothesis: whole brain connectivity analysis can improve research in this direction. Finally, since it has recently been shown that EEG directional connectivity shows characteristic changes during propofol-induced unconsciousness In conclusion, our result show that functional changes in the brain associated to propofol-induced modulation of consciousness can be efficiently revealed by tracking the patterns of co-activation in the Posterior Cingulate Cortex, an area with a central role in the dynamical connectivity at rest. This methodology, based on point process analysis, can help in refining the characterization of local functional disconnections following the partial or total loss of consciousness.Figure S1CAPs from BOLD and deconvolved BOLD signal. To avoid the possibility that the PCC-coactivation patterns were only due to different hemodynamic response functions in the different areas of the brain, we applied the approach proposed in Wu et al. (TIF)Click here for additional data file.Figure S2CAPs from negative BOLD peaks. CAPs obtained using positive threshold crossing (left) and negative threshold crossing (right), during wake resting state . After the clustering, the specificity of the spatial patterns obtained using positive peaks in BOLD is not reproducible using negative peaks; positive BOLD peaks allow to reconstruct a richer variety of patterns.(TIF)Click here for additional data file.Figure S3Sliding window correlation vs CAPs. PCC co-activation patterns in wakefulness (left) compared to the 8 patterns obtained after spatial clustering of the N PCC-correlation maps computed using sliding window correlation, with window size varying from 5 time points  to 20 time points (i.e. 50 s window). Note that the region-specific patterns obtained with point process on the BOLD peaks are not recovered by using sliding window correlation. This approach seems to add more refined information in the spatial differentiation of functional networks.(TIF)Click here for additional data file.Figure S4Motion correction in CAPs. Example of the procedure discussed in (TIF)Click here for additional data file.