To this end, we utilized the genome sequence of the late blight pathogen Phytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation.Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic  Phytophthora contains several well-known species such as the potato and tomato late blight pathogen Phytophthora infestansPhytophthora sojaePhytophthora ramorumOomycetes are an important class of eukaryotic pathogens that have severe ecological and economic impact cis-regulatory elements), which are bound by diverse transcription factors, and the remodeling of the chromatin structure. Elements in proximity to the transcription start site include the eukaryotic core promoter elements as well as specific regulatory elements. The basic transcriptional activity is determined by the eukaryotic core promoter, which is typically present within 70 nucleotides (nt) surrounding the transcription start site (TSS) and directs the mediator complex, general transcription factors, and the RNA polymerase II (RNA Pol II) into a functional pre-initiation complex Cdc14 and Pks1 contain, next to the Inr- or Inr/FPR- element, specific but distinct elements that are required for correct gene expression cis-regulatory elements in Phytophthora is in sharp contrast to the abundance of the predicted genes encoding the diversity of transcription factors in Phytophthora and related non-pathogenic species  and four in planta stages et al. in planta growth were identified using t-tests between two groups . The tests were independently applied for each day after inoculation and genes were deemed significantly differentially expressed (up- and down-regulated) with a p-value cutoff of 0.05. False discovery rates were assessed by computing q-values  for each comparison. Subsequently, the identified significantly differentially expressed genes were clustered based on their expression profiles, i.e. intensities relative to the average expression intensity in growth media, using Spearman correlation coefficient utilizing the Markov clustering algorithm  P. infestans genes. The correlation coefficient cutoff was determined by the 95 percent quantile, corresponding to value of 0.86. Single, non-clustered genes were discarded before further analysis.We retrieved NimbleGen microarray data of P. infestans) of all predicted proteins in the analyzed Phytophthora spp. using OrthoMCL  P. infestans to 91 percent in P. ramorum. Subsequently, we combined the upstream regions of co-expressed P. infestans genes (clusters with size > = 2) with their orthologs in P. ramorum and P. sojae  and used these to identify conserved DNA motifs. The upstream region per gene was defined as the 1,000 nt upstream of the translation start site ‘ATG’ as annotated by the coding sequence. Upstream sequences without an associated annotated coding gene were discarded. If a coding gene occurred within the 1,000 nt, the upstream region was truncated. For genes located on the negative strand the extracted DNA sequence was converted to its reverse complement. The upstream regions were filtered for the remnants of non-annotated genes by similarity search against the NCBI nr database  were identified using the expectation maximization algorithm implemented in MEME  rd order background Markov model based on the upstream region of all Phytophthora spp. genes .DNA motifs in the upstream regions of different clusters of co-expressed ab initio determined motifs. These scores are transformed to p-values and subsequently to q-values to address false discovery rates due to multiple testing. We applied a q-value cutoff 0.1 to define the genome-wide abundance for each motif. The location of the motifs in the upstream regions is displayed for the first 1,000 nt using bins of the size 50. To account for shorter upstream regions due to coding genes within the first 1,000 nt, the abundance was weighted accordingly. Similarity to known motifs was assessed using Tomtom  Similar motifs were clustered into families based on their pairwise similarity using the Markov clustering algorithm (inflation 2). Combined motif logos were produced using Weblogo 3 Phytophthora spp. . We subsequently calculated the number of cases where both orthologous groups maintained the motif and assessed the significance of the overlap . The values were compared to a set of randomized motifs (the column of each identified motif was shuffled twenty times); the poly-C motif-6 was excluded for this and all subsequent analyses. As expected, the majority of these motifs did not yield any significant hits against the Phytophthora upstream regions. Based on the motifs with significant hits we chose the 95 percent quantile as a conservation cutoff, corresponding to a p-value of 0.04. Applying this cutoff to the set of motif families yield a conserved subset that exceed this score between P. infestans and at least one of the other Phytophthora.To estimate the evolutionary conservation of the identified motifs, we calculated a conservation score that is based on the network-level conservation principle The identified motifs, their genome wide abundance, their conservation score and location  are accessible as ‘Supplementary data 1’.cis-regulatory motifs are DNA elements that modulate the expression of genes upon binding of a transcription factor. They were identified in P. infestans by searching for motifs where their presence within the upstream regions significantly correlates with expression levels of the downstream genes similar to the approach outlined by Bussemaker and colleagues 2-fold change compared to the growth media, were identified by forward variable selection as implemented in R and multiple testing correction was applied to the p-values by computing q-values . Motifs with a q-value <0.01 were deemed significant. For each motif in each condition a ‘time course value’  was calculated: the correlation between the motif score and the expression level at each time point (growth media +2–5 dpi) was transformed into a T-value by multiplying the correlation (r) with the square root of the number of genes (G) (T = r*sqrt(G)) Functional Phytophthora spp. were functionally annotated using BLAST2GO algorithm (default parameters) http://www.broadinstitute.org/). Significance of this overrepresentation was assessed using Fisher exact test .Genes in the analyzed CBF-B, OrthoMCL clustered the human gene solitarily and the orthologs of the Arabidopsis thaliana CBF-B gene were reported.Known transcription factors that bind to common eukaryotic promoter elements were identified by determining orthologs of the human genes (proteins) in oomycetes using OrthoMCL  Phytophthora spp. and four non-pathogenic sister taxa  and in planta growth (four conditions) 2–5 days post inoculation (dpi) of potato plants P. infestans genes using Spearman correlation and a graph based clustering algorithm (MCL) P. sojae and P. ramorum) and filtered the upstream regions for remnants of transposable elements .To predict potential P. infestans genes and their orthologs. Similar motifs, especially common eukaryotic DNA elements, were identified in different clusters of co-expressed genes. Hence, we grouped the total of 80 motifs into 24 distinct motif families , based on the assumption that all motifs within a family represent a binding site for a specific DNA binding protein or complex. To enrich our results for conserved functional DNA motifs, these were filtered by applying an evolutionary conservation filter between P. infestans and at least one of the other Phytophthora yielding 19 conserved DNA motifs for which the genome-wide abundance was determined using FIMO .Within 136 co-expressed clusters, we identified 80 motifs representing putative regulatory DNA elements in the upstream regions of co-expressed in silico approach recovered the previously described oomycete-specific Inr/FPR element (motif-0). In the set of 1,493 P. infestans genes included in the motif search it occurs 652 times. Genome-wide, the Inr/FPR-element is the most abundant motif : It is predicted in 18,138 upstream regions of all annotated genes in the three analyzed Phytophthora spp., and in 6,511 or 37 percent of all P. infestans. It has a distinct localization at a median of 81 nt upstream of the translation start site (TLS)  . In otherthologs , suggestPhytophthora genes Phytophthora spp., we predicted the CCAAT-box in the upstream regions of 8,225 genes, 3,418 of which are from P. infestans. It is primarily localized at 192 nt upstream of the TLS. When the CCAAT-box co-occurs with the Inr/FPR-elements , these motifs are approximately 180 nt apart (80nt for the 25th percentile). Interestingly, we found more occurrences of the CCAAT-box on the negative strand than on the positive strand , consistent with the observation that the CCAAT-box is found in both orientations Phytophthora and in other oomycetes species analyzed  in the biotrophic fungal plant pathogen Uromyces fabae are responsible for the production of mannitol in haustoria We identified a novel DNA motif (motif-1) that does not show any significant similarity to known motifs as determined by a Tomtom search against the JASPAR database . Motif-1nfestans . The invductases . The difductases . MannitoPhytophthora genes, 940 of which are from P. infestans  in 1,388 nfestans . This moPhytophthora genomes, are correlated with the infection-related gene expression levels and are enriched in specific functional categories. Moreover, one of the four positively correlated motifs is the Inr/FPR-element, further emphasizing the adaptation of basic cellular machinery towards pathogenicity . These conserved DNA motifs  are highly abundant in genicity . Hence, Phytophthora species. These ten motifs include the two common promoter elements (Inr/FPR and CCAAT-box) earlier described, three motifs whose presence is correlated with gene expression levels during infection  and five additional candidate motifs , two motifs that are frequently observed as core elements in eukaryotic promoters. However, we found orthologs of the transcription factor II B (TFIIB) as well as the binding factors for the DPE-elements in all omycetes . Hence, ipiO1 gene in which the Inr/FPR-element was initially described Only 37% of all genes have an Inr/FPR-element; a percentage that is lower than reported for the eukaryotic Inr-element present in various eukaryotes such as human and yeast  Phytophthora  they are highly abundant in the analyzed Phytophthora spp., (ii) their presence in the promoter of genes significantly correlate with the gene expression level during infection and (iii) the set of corresponding proteins is enriched for interesting functions. Within the four motifs whose presence significantly correlate with up-regulation during infection, we revealed, next to the Inr/FPR and the putative AT-rich TATA-like element, two novel abundant motifs. This number is slightly lower, most likely due to limitations in the gene expression data, but still comparable to a study in Plasmodium falciparum that identified twelve motifs which are significantly correlated with gene expression levels We identified 17 additional conserved DNA motifs next to the two common eukaryotic promoter elements (Data S1). Several of these motifs are candidates for functional active Caenorhabditis elegans, taCATGta motifs are rare footprints of Tc1 transposable elements excision Phytophthora genomes, we do not expect motif-1 to be a transposon footprint. Moreover, a recent analysis of the binding preference of homeodomain DNA-binding domains has identified TACATGTA as the preferred binding site for Irx family transcription factors Drosophila as well as in vertebrates and containing the Homeobox KN domain (PF05920). Interestingly, this domain is also present in several predicted transcription factors in the analyzed Phytophthora (Tab S1A); hence, these might be interesting candidate transcription factors for motif-1 binding.Motif-1 is highly abundant and correlates with up-regulation of genes expression levels during infection. The set of genes containing this motif in their upstream region is enriched for RXLR effector genes as well as genes with catalytic activity such as glycosyl-hydrolases. In P. infestans genes in this set is not differentially expressed during infection. Nevertheless, the high abundance of this motif in the Phytophthora genomes and its enrichment in genes encoding nuclear and DNA binding proteins suggests that motif-3 is a functional binding site for an unknown transcription factor that in turn regulates many other transcription factors.Like motif-1, motif-3 is highly abundant. Notably, it is also present upstream of ∼700 genes that encode proteins with predicted organelle localization such as the nucleus or DNA binding activity. Of these, 119 have both predicted functional annotations and include several transcription factors of the Myb-like family. The majority of In vivo there are many factors that influence the transcription of genes such as the chromatin state, the availability of the binding transcription factors and also the presence of other motifs in the proximity that may act together or antagonistic in a regulatory module. Nevertheless, the combination of different criteria, including significant correlation of motif presence with the gene expression level, allows us to generate a concise list of interesting candidates for pending experimental validation; both of the motif itself as well as of the binding transcription factor.The identification of potential biologically relevant motifs solely by correlating their presence with the gene expression levels is a simplified approach. This is especially apparent in the high variability of expression levels between genes that have one of the correlated motifs in their upstream region . In vivoP. infestans and two closely related Phytophthora species. The identified cis-regulatory elements are promising candidates for further experimental validation and identification of the binding transcription factors. In general, biochemical and genetic approaches such as ChIP-Seq are lagging in oomycetes and pathogenic fungi. However, whole genome transcriptomics and thereby derived gene expression data as well as genomic sequences of close relatives will be available in the close future. In silico methods such as the one outlined in this study are in an exceptional position to take advantage of these data to gradually close the knowledge gap between well-established model organisms and these important and intriguing groups of pathogens.This analysis provides the first systematic insights in the transcriptional regulation of the late blight pathogen Figure S1Positively correlated core promoter motif with gene expression levels. The Inr/FPR-element is positively correlated with the gene expression levels during infection. The motif logo, a graph displaying the T-value per data point and the gene expression of all differentially expressed genes that contributed to the correlation are displayed.(EPS)Click here for additional data file.Table S1Predicted DNA binding domains, orthologs of known transcription factors and analyzed genomes in this study. (A) List of the identified DNA-binding domains in the predicted proteomes of the three analyzed Phytophthora spp. and four non-pathogenic sister taxa and their relative abundances. Domains were either derived from DBD (D) or manually added (M). (B) List of known transcription factors binding eukaryotic core promoter elements and their orthologs in eighteen species including five oomycetes. (C) Names, versions and sources of different eukaryotic proteomes that were used to define the orthologs of known transcription factors as well as the predicted transcription factor repertoire in Phytophthora spp.(XLS)Click here for additional data file.Table S2Tabular overview of the Tomtom results for the analyzed motifs. Tabular overview of all results retrieved by the Tomtom search against the JASPAR motif database. Significant hits  are highlighted in orange.(XLS)Click here for additional data file.Table S3Tabular overview of enriched GO terms. Tabular overview of all significantly enriched GO terms for the set of genes that contain the Inr/FPR element or the CCAAT-box. Redundant GO terms as defined by REVIGO are also indicated.(XLS)Click here for additional data file.Data S1Overview of the identified motif and their occurrence per gene.(ZIP)Click here for additional data file.


Objective: Acceptance-based behavior therapy (ABBT) is a new psychotherapy for generalized anxiety disorder (GAD). The current study intended to compare severity of symptoms, worry and quality of life of GAD female patients between ABBT and control. Methods: This study was a randomized clinical trial. The sample included 18 women with GAD referred to psychiatrists in Isfahan, Iran. Patients were assigned in 2 groups randomly (ABBT and control group without any psychotherapy). Both groups received medication. The intervention in ABBT group was conducted based on Roemer & Orsillo's manual for Acceptance-based Behavior Therapy for GAD. 12 therapeutic sessions administered in Shariati psychiatric clinic of Isfahan. The instruments included the GAD-7 Inventory, Penn State Worry Questionnaire (PSWQ) and Short Form Health Survey -12 revised Version (SF-12V2). The data were analyzed using the Multivariate Analysis of Variance (MANCOVA). Results: Overall, clients receiving ABBT compared to control group reported a significantly decrease in severity of GAD symptoms, and improve in quality of life at post- treatment state. They reported decrease in severity of worry but it was not statistically significant compared to control group. Conclusion: ABBT was effective in alleviating symptoms of GAD.  Generalized anxiety disorder (GAD), is characterized by anxiety, tension, and chronic and persistent worry  and is aIn an effort to improve the efficacy of treatment for GAD, several promising new treatments have recently been developed. One of these treatments is acceptance-based behavior therapy (ABBT) . ABBT aiAn assumption underlying ABBT is that it is not the initial worry, feeling, physical sensation, or image that is problematic, but rather the rigid unwillingness to have these internal experiences. In other words, developing an acceptance of, or a willingness to have these internal experiences should reduce distress and interference associated with the internal experiences, thus reducing the negative reactivity and the cycle of anxiety. From this perspective, the focus of treatment is not on eliminating worry, but rather on decreasing the distress and interference associated with this cognitive activity . Clients receiving ABBT have shown significant improvements in symptoms in both a small open trial  and a waHayes and colleagues  examinedBecause ABBT is a new psychotherapy, we decided to examine its efficacy in an Iranian sample. In addition we compared ABBT to a control group that received only pharmacotherapy. Many of the previous studies directly used advertisements to recruit subjects , 18. By Participants and procedureStatistical population included female patients with GAD referred to some psychiatrist offices in Isfahan, Iran. Inclusion criteria were:  primary The treatment method used in this study was ABBT for GAD. Basic ABBT protocol  was usedMeasuresStructured Clinical Interview for DSMIV Axis I disorders (SCID-I) This is a comprehensive and standardized instrument for assessing major mental disorders in clinical and research atmosphere . SCID-I Structured Clinical Interview for DSMIV Axis II disorders SCID-II SCID-II like SCID-I is a structured diagnostic interview for personality disorder to assess ten personality disorders at the DSMIV Axis II as well as NOS (not otherwise specified) depressive and aggressive disorders . This quGAD-7:This scale is a recent, simple 7-item tool based on DSM-IV criteria that is easy to administer and without undue burden on the patient or the clinician. It has been developed to identify probable cases of GAD and has shown strong properties for screening of likely GAD cases ,26. AlsoPenn State Worry Questionnaire (PSWQ):The PSWQ is a self-report measure was designed by Meyer et al. . This 16Short Form Health Survey-12 revised version (SF-12V2)The Short Form (SF)-12 Health Survey is a shortened version of the Medical Outcomes Study 36-item Short Form Health Survey (SF-36), a generic and popular health-related quality of life assessment instrument for adult populations with eight scale scores and two physical and mental component summary (PCS and MCS) scores. The first version of SF-12 reproduced SF-36 summary scores, but the revised version, SF-12V2, also included the eight scale scores. SF-12V2 was designed by Ware et al . The breSubjects reporting better overall health would have higher scores on SF-12V2 scales than those reporting worse overall health. SF-12V2 has all of eight scale scores of SF-36 but with only 1-2 items in each scale. That’s why in this study we used two physical and mental component summary (PCS and MCS) scores. In this study we administered SF-12V2 in Iranian population. We administered this scale on 30 dormitory students of Tehran University of Medical Sciences. In this sample, the SF-12v2 demonstrated adequate internal consistency (α= .86).  Data analysisTo confirm symptomatic differences between the ABBT and control groups, a one-way multivariate analysis of variance (MANOVA) was performed comparing the groups on measures of GAD severity, quality of life and worry. The pretest questionnaire controlled as control variables and we used MANCOVA for analyzing of data.All of the subjects  were femBox's Test of Equality of Covariance Matrices showed that the observed covariance matrices of the dependent variables are equal across groups . Therefore we can use MANCOVA. dependent variables  in pretest entered as covariate variables in MONCOVA model.Since conditions did not differ significantly in educational level, age, marital status and other demographic characteristics, the MANCOVA was done without these variables. Analysis by MANCOVA used pretest scores as covariate variables effects on post-tests.Based on The purpose of the study was to investigate whether a randomized controlled trial of a 12-week group ABBT program, which focused on acceptance of internal experiences and engagement in meaningful activities and bringing mindful awareness to valuable activities, would be an acceptable and effective treatment for patients suffering from GAD. Results from this small randomized controlled trial demonstrate that subjects in the ABBT group experienced a significant decrease in their GAD symptoms and a significant increase in quality of life following a 12-week ABBT. Also, patients while there was a mean increase in worry in pre- to post-intervention state, this difference did not reach statistical significance.A few studies that investigated ABBT for GAD found this therapy could decrease GAD symptoms too. For example Hayes et al  in theirIn general ABBT and mindfulness based therapies are efficient for treatment of anxiety disorders; for example Koszycki et al.  showed gAnother finding in our study showed that quality of life in ABBT group increased significantly compared to control group. Hayes et al.  suggesteIt appears that increasing in quality of life and decreasing in GAD symptoms are related. Clients with GAD often report that choices about how to engage in work, relationships and leisure activities are made based on lessening anxiety rather than maximizing satisfaction and that even when they are engaged in potentially meaningful activities they are often distracted by their worries . Two proFinally, the time spent worrying at the end of therapy did not show significant differences between the two groups. At first the finding that worry frequency did not predict outcome may seem counter intuitive; however, in an acceptance-based model, this finding is consistent with theory. In ABBT, the primary aim is not necessarily to reduce worry, but to change the relationship one has with the worries or other internal experiences, since it appears that attempts to stop worrying paradoxically increase the amount of worry . ABBT inLimitationsThese were small sample size, lack of other psychotherapy groups, and no follow-up about therapeutic outcomes. FZ conceived and designed the evaluation, collected the clinical data, interpreted them and helped to draft the manuscript. AAAF participated in designing the evaluation and performed the statistical analysis, and revised the manuscript. MKAV participated in designing the evaluation; re- analyzed the clinical data and revised the manuscript. , MM, VO and MM participated in referring the GAD patients and primary psychiatric evaluation. All authors read and approved the final manuscript.


There has been considerable interest in high resolution 3D late gadolinium enhancement (LGE) imaging in the atrial fibrillation (AF) population, both pre and post RF ablation. Unlike conventional studies, these are performed with single R-wave gating to reduce the acquisition duration and the effects of gadolinium wash-out. For AF, the heart rate variability results in variable magnetization recovery between sequence repeats which, with a fixed inversion time (TI), causes ghosting and poor nulling of normal tissue. This is exacerbated by missed triggers and the number of poor quality studies is high. An adaptive inversion recovery preparation has previously been demonstrated in a 2D phantom acquisition . The purAn inversion-prepared segmented FLASH sequence was modified so that the inversion time was varied automatically from beat-to-beat based on the time since the last sequence repeat . The gatFigure We have shown that dynamic adaptation of the inversion time for each cardiac cycle is feasible and results in less ghosting and improved nulling of normal myocardium in 2D acquisitions in the AF population. Application to 3D studies should result in improved acceptability rates.Wellcome Trust(grant reference: P32451) and NIHR Cardiovascular Biomedical Research Unit, Royal Brompton Hospital.


Gene expression is controlled by proximal promoters and distal regulatory elements such as enhancers. While the activity of some promoters can be invariant across tissues, enhancers tend to be highly tissue-specific.in vivo using the hydrodynamic tail vein injection assay. Fifty-eight percent of the predictions yielded significant enhancer activity in the mouse liver, whereas a control set of five sequences was completely negative.We compiled sets of tissue-specific promoters based on gene expression profiles of 79 human tissues and cell types. Putative transcription factor binding sites within each set of sequences were used to train a support vector machine classifier capable of distinguishing tissue-specific promoters from control sequences. We obtained reliable classifiers for 92% of the tissues, with an area under the receiver operating characteristic curve between 60%  and 98% (for heart promoters). We next used these classifiers to identify tissue-specific enhancers, scanning distal non-coding sequences in the loci of the 200 most highly and lowly expressed genes. Thirty percent of reliable classifiers produced consistent enhancer predictions, with significantly higher densities in the loci of the most highly expressed compared to lowly expressed genes. Liver enhancer predictions were assessed de novo prediction of enhancers.We conclude that promoters of tissue-specific genes often contain unambiguous tissue-specific signatures that can be learned and used for the  A fundamental question in biology is how cells and tissues differentiate and maintain their identity from essentially the same genome. Wide variation in spatial, temporal and condition-dependent expression patterns of more than 20,000 genes in the human genome  is requicis-regulatory sequences, such as enhancers, are specifically bound by TFs and are central players in the control of transcription in multicellular eukaryotes. The regulation of promoters by distal enhancers involves DNA looping or scanning and/or higher-order conformation changes in chromatin  vector (Promega) using the Gateway system (Life Technologies). Sequence and orientation of the insert were re-verified by Sanger sequencing, and approximately 200 μg of endotoxin-free plasmid DNA was isolated using the EndoFree Plasmid Midi prep (Qiagen).Sequences selected for ApoE liver enhancer  was injected along with 2 μg of pGL4.74[hRluc/TK] vector to correct for injection efficiency, into at least three CD1 mice  using the TransIT EE hydrodynamic gene delivery system  according to the manufacturer’s protocol. Negative (empty pGL4.23[luc2]) and positive  conAUC: Area under the receiver operating characteristic curve; bp: Base pair; CNE: Conserved non-coding element; DHS: DNase I hypersensitive site; NK: Natural killer; PWM: Position-weight matrix; SVM: Support vector machine; TF: Transcription factor; TSS: Transcription start site; UTR: Untranslated region.The authors declare that they have no competing interests.IO conceived the work and supervised the analysis. LT carried out the analysis. RPS and MJK conducted the mouse experiments. NA supervised the experiments. The manuscript was written by LT and IO, with contributions from the other authors. All authors read and approved the final manuscript.Figures S1 to 15 and Supplementary notes.Click here for fileA table listing the motif ranks for the promoter-based models.Click here for fileA table summarizing the performance of the promoter-based models.Click here for fileA summary of results obtained with the hydrodynamic tail vein injection assay.Click here for fileA list of transcription factors known to be relevant for liver function.Click here for file


Hepatic complications are a common side-effect of alcoholism. Without the detoxification capabilities of the liver, alcohol misuse induces changes in gene and protein expression throughout the body. A global proteomics approach was used to identify these protein changes in the brain. We utilised human autopsy tissue from the superior frontal gyrus (SFG) of six cirrhotic alcoholics, six alcoholics without comorbid disease, and six non-alcoholic non-cirrhotic controls. Synaptic proteins were isolated and used in two-dimensional differential in-gel electrophoresis coupled with mass spectrometry. Many expression differences were confined to one or other alcoholic sub-group. Cirrhotic alcoholics showed 99 differences in protein expression levels from controls, of which half also differed from non-comorbid alcoholics. This may reflect differences in disease severity between the sub-groups of alcoholics, or differences in patterns of harmful drinking. Alternatively, the protein profiles may result from differences between cirrhotic and non-comorbid alcoholics in subjects’ responses to alcohol misuse. Ten proteins were identified in at least two spots on the 2D gel; they were involved in basal energy metabolism, synaptic vesicle recycling, and chaperoning. These post-translationally modified isoforms were differentially regulated in cirrhotic alcoholics, indicating a level of epigenetic control not previously observed in this disorder. Excessive consumption of alcohol affects many parts of the body, including the brain and the liver . Liver dThe recent explosion in the application of proteomics to the study of neurodegenerative disease can be attributed to rapid technological advances in methodology. The ‘classical’ proteomics approach consists of two-dimensional electrophoresis (2DE) coupled to mass spectrometry (MS), and is still a powerful tool for comparative protein profiling. It is particularly useful in analyses of post-translationally modified proteins, as this ‘visual’ technique enables each protein and its isoforms to be observed on the 2D gel. Post-translationally modified proteins are highly abundant in the human proteome : in some1.Subjects were categorized according to alcohol intake. Alcoholics were defined by National Health and Medical Research Council/WHO criteria as individuals who had consumed an average of more than 80 g ethanol/day throughout most of their adult life; the alcohol consumption of controls was < 20 g ethanol/day. Cases with multiple drug use or Wernicke-Korsakoff syndrome were excluded. Controls had no history of brain dysfunction, and no significant brain abnormalities were detected by post-mortem examination. Six controls and six alcoholic cases were chosen and matched as closely as possible for age, sex and post-mortem interval. Subject details are shown in Table Samples were collected by qualified pathologists from the Brisbane node of the National Health and Medical Research Council (NHMRC) Brain Bank and the Tissue Resource Centre at the University of Sydney, Australia. Full ethical clearance and informed written consent was obtained from the next of kin.per [Synaptosomes were prepared as per .per [CyDye labelling and 2-dimensional electrophoresis were performed as per . A dye-spost-hoc test. Only proteins with expression differences greater than 1.2-fold and Newman-Keuls P values ≤ 0.05 were analyzed in this study.Gels were scanned on a Typhoon 9400 scanner  at three wavelengths to record the fluorescence of each of the three Cy Dyes in the gel. Images were cropped and rotated in Image Quant , then imported into the TT900 S2S  image alignment module for gel/spot alignment of all gels to each other. Aligned gels were imported into Progenesis PG240 (NonLinear Dynamics) then automatically analyzed using the SameSpots module to accurately compare the same protein spot in each gel. Automatic analysis included background subtraction and radiometric normalization. Differences in normalized spot volumes between gels were analyzed in Statistica  using analysis of covariance (ANCOVA) with post-mortem delay and age as covariants. Pairwise differences were assessed with Newman-Keuls Trypsin digest, matrix assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry and data processing were performed as described in .1) and the sample size. The groups were quite well matched on the delay between death and autopsy  but the spread in these values was also quite notable (Table 1). As these factors might influence the amounts of proteins detected on the gels, and also contribute to sample variance, age and PMI were included as covariants in the statistical analysis.The synaptic proteins were isolated from human brain tissue collected at autopsy that had been slowly frozen and stored in isotonic 0.32 M sucrose solution to minimise damage to sub-cellular structures by ice crystals . Both grThe proteomic analysis aimed to identify two sets of proteins representing the effects on the brain of alcohol misuse combined with liver dysfunction. To do this, we obtained synaptosomal proteins from three groups of subjects. The first comparison identified the set of proteins that differed significantly between cirrhotic alcoholics and normal, non-alcoholic controls. These proteins represent the end result of the effects of chronic alcoholism combined with the additional effects of cirrhosis. Ninety-nine proteins fell into this category. Alcoholics who develop cirrhosis of the liver generally have higher lifetime alcohol consumption than non-cirrhotic alcoholics ; hence, 1)). Brain proteins in cirrhotic alcoholics appeared to have been regulated in a different manner from those in non-cirrhotic alcoholics. This differential regulation might be due to genetic, nutritional, or other factors; but the data suggest that consumption alone is not sufficient to explain the disparity.This result was further supported when proteins that were differentially expressed between non-cirrhotic and cirrhotic alcoholics were identified. In this second comparison, 118 proteins were differentially regulated; less than 50% of these were the same as those that differed from controls  after trypsin digestion using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The PMF of each protein was compared against a database containing the theoretical PMFs of all known human proteins. The identification was considered accurate when at least five peptides from the unknown PMF matched a theoretical PMF with a difference in mass of 0.0030% or less. These and other criteria (see Methods) reduced the probability of false identification.2). These multiple protein expression forms (MPEFs) are either the result of alternate splicing, or they are post-translationally modified isoforms of the same transcripts [2); Table 2). Other, undetected MPEFs of these proteins may be present on the gel; however, our aim was to study the proteins that were differentially regulated by liver dysfunction in alcoholics: not all proteins on the gel were subjected to MALDI-TOF MS.Several of the proteins differentially expressed in cirrhotic alcoholics were identified in more than one spot on the 2D gel ), although one protein, L-lactate dehydrogenase H subunit ), was identified in two isoforms of the same pI but slightly different molecular weights. No splice variants of LDH-B are known that could produce two proteins of different sizes. This indicates that these isoforms may be post-translationally modified in a way that does not alter pI. All of these proteins are known to have phosphorylated MPEFs (Table 3), and some are known to be methylated, acetylated, S-nitrosylated or ubiquitin-like-protein conjugated (Table 3). The combination of these and other, yet to be discovered, modifications may be responsible for the different isoforms of these proteins observed on the 2D gel.Most of these proteins were found in ‘strings’ of the same molecular weight but with varying isoelectric points (pI) ), indicative of an alteration in carbohydrate metabolism in brain. This metabolic change may have led to the higher expression of PYG-B in cirrhotic alcoholics ), since this enzyme catalyses the conversion of brain glycogen to glucose. PYG-B expression may be increased as a compensatory response to reduced or impaired glycolysis. LDH-B expression was also higher in cirrhotic alcoholics ), most likely indicating the cytotoxic effects of alcohol on the brain [Many of the identified proteins are involved in basal energy metabolism . In our study, several isoforms of ACO2, ATP-syn β, ATP-syn α  and GAPDH showed lower expression in cirrhotic alcoholics than in controls ; HSP70). Additionally, most isoforms studied showed different regulation in non-comorbid alcoholics ; HSP70; [Ethanol administration induces 70 kDa heat shock proteins (HSP70s) expression , 11 as p; HSP70; ). Oxidat; HSP70; , and thu2)). Dynamin is essential to synaptic signalling [N-ethylmaleimide-sensitive factor (NSF) and dynamin 1 (DYN1) also demonstrated significant variation in the brain’s response to alcoholism in the presence and absence of cirrhosis. These proteins are both related to synaptic vesicle fusion and recycling; the levels of both differed significantly between non-comorbid and cirrhotic alcoholics, particularly DYN1, for which MPEFs were significantly lower in non-comorbid alcoholics than in controls , but notgnalling , 15, andAnalysis of the MPEFs identified in this study showed significant epigenetic regulation of proteins in the superior frontal gyrus of cirrhotic alcoholics. This regulation was disease-state specific, with significant differences between MPEF regulation in non-comorbid and cirrhotic alcoholics. Further analysis of the types of post-translational modifications responsible for MPEF formation, and their regulation in different disease states, will provide insight into the mechanisms causing cerebral damage in comorbid and non-comorbid alcoholics.


Local recurrence of glioblastomas is a major cause of patient mortality after definitive treatment. This review discusses the roles of the chemokine stromal cell-derived factor-1 and its receptor CXC chemokine receptor 4 (CXCR4) in affecting the sensitivity of glioblastomas to irradiation. Blocking these molecules prevents or delays tumour recurrence after irradiation by inhibiting the recruitment of CD11b+ monocytes/macrophages that participate in revascularising the tumour. We review the literature pertaining to the mechanism by which revascularisation occurs following tumour irradiation using experimental models. Areas of interest and debate in the literature include the process by which endothelial cells die after irradiation and the identity/origin of the cells that reconstitute the tumour blood vessels after injury. Understanding the processes that mediate tumour revascularisation will guide the improvement of clinical strategies for preventing recurrence of glioblastoma after irradiation. Despite advances in techniques for administering radiotherapy, local recurrence of glioblastomas (GBMs) typically leads to patient mortality. In this review, we focus on the chemokine stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1/CXCL12) and its receptor CXCR4 in affecting the sensitivity of GBM to irradiation. We begin with an overview of the discovery and characterisation of these molecules, followed by an examination of their role in tumour metastasis and tissue injury. Finally, we discuss the data and controversies surrounding their role in revascularisation and tumour recurrence. Our understanding of the role of SDF-1 and CXCR4 in tumour recurrence post irradiation is largely gained from pre-clinical studies using mouse xenografts of human tumours. The pre-clinical data will be the focus of this review. Clinical studies in this area are limited, but serve as an exciting area for future investigation.The CXC chemokine family has multiple roles in leukocyte migration, immune response, angiogenesis, and tumour metastasis. CXC chemokines have four highly conserved cystine amino-acid residues, with a non-conserved amino acid separating the first two cystine residues. A second structural domain determines their function. The CXC chemokine family contains both angiogenesis-promoting and angiogenesis-inhibiting molecules. Stromal cell-derived factor-1 is regarded as an angiogenesis-promoting chemokine.2+ in response to human SDF-1. The mouse CXCR4 receptor was subsequently found by cloning candidate chemokine receptors and comparing the amino acid sequence with the human cDNA  was not examined. Chemotaxis of CXCR4-expressing murine muscle satellite cells towards SDF-1 has also been demonstrated, suggesting that migration of tissue-specific stem cells may be regulated by SDF-1 , a key proangiogenic molecule in circulating CD11b+ cells. We demonstrated that tumours cannot grow in an irradiated site (given 20 Gy) of an MMP-9 knockout (KO) mouse but can grow in a non-irradiated MMP-9 KO mouse. Tumour growth is restored following irradiation if the bone marrow in the MMP-9 KO mouse is replaced with wild-type bone marrow  or by circulating cells – including endothelial cells (ECs) or endothelial progenitor cells (EPCs), a process known as vasculogenesis (see the section ‘Restoration of tumour blood vessels after irradiation’). In pre-clinical models, several investigators have reported that radiation inhibits local angiogenesis, the generation of new vessels from surrounding vessels  following irradiation lead to accumulation of CXCR4-expressing monocytes/macrophages in the irradiated tumour. This is also suggested by data in the literature showing that the recruitment and retention of proangiogenic hematopoietic cells to sites of ischaemic tissue damage or to tumours is mediated by the interaction of the SDF-1 with CXCR4  or at TCD50 doses (doses that control 50% of the tumours) typical for transplanted tumours (40–100 Gy), and particularly in SCID mice in which all the stromal cells are highly radiosensitive . CD11b+ cells from an irradiated tumour share many of the same properties as ECs and have been shown to interact with newly forming tumour blood vessels  after a median follow-up period of more than 18 months. Although the majority of recurrences were located in the radiation field, in 17 patients GBM recurred outside the radiation field. As recurrences outside the radiation field were observed after a longer follow-up time, different mechanisms may be responsible for in-field and out-of-field recurrences . These strategies have been successfully used in pre-clinical studies. Of these, the use of plerixafor is currently the most promising for further investigations in human clinical trials as a proof of principle, as this drug is already approved for clinical use. Understanding the mechanism by which tumours revascularise after irradiation offers promising therapeutic strategies for treating GBMs.


TLR agonists have demonstrated anti-tumor activity in both animals and humans. Clinically, successful anti-tumor activity with TLR agonists was most often observed when the molecules were applied locally to dermatological cancers. This mode of administration was also associated with fewer and less severe adverse events (AEs) when compared to systemic administration. The efficacy of TLR agonists in human cancer patients has been limited at best, in part reflecting the dose-limiting toxicity associated with systemic administration of this form of therapy. Moreover, recent nonclinical studies indicate that intratumoral administration of TLR agonists helps reverse the immunosuppressive microenvironment that protects large established tumors from immune elimination. In light of these findings, we developed a new class of lipophilic TLR7/8 agonists that are ideal for intratumoral injection. The molecules are retained at the site of injection, thereby supporting the focused induction of anti-tumor immunity. As release into the systemic circulation is low, we postulate that the generation of AE-inducing systemic cytokines is low. Results from studies in mice and rats using 3M-052 confirms that this lipophilic TLR 7/8 agonist is retained at the injection site. Direct activation of innate immune cells with anti-tumor activity was observed, as was a reduction in the immunosuppressive local environment. 3M-052 also induces the accumulation of mononuclear phagocytes concomitant with the induction of histologically established tumor liquifactive necrosis. Moreover, 3M-052 induces antitumor activity when injected into tumors but not when injected distal from tumors. These findings are based on inhibition of tumor growth and increased long-term survival. 3M-052 is under consideration for clinical use in patients with cancers suitable for intratumoral therapy and as being evaluated as a cancer vaccine adjuvant.


Meta-analyses are particularly vulnerable to the effects of publication bias. Despite methodologists’ best efforts to locate all evidence for a given topic the most comprehensive searches are likely to miss unpublished studies and studies that are published in the gray literature only. If the results of the missing studies differ systematically from the published ones, a meta-analysis will be biased with an inaccurate assessment of the intervention’s effects.http://www.open-project.eu) we will conduct a systematic review with the following objectives:As part of the OPEN project .▪ To assess whether the inclusion of unpublished studies or studies published in the gray literature leads to different conclusions in meta-analyses .Inclusion criteria: Methodological research projects of a cohort of meta-analyses which compare the effect of the inclusion or exclusion of unpublished studies or studies published in the gray literature.Literature search: To identify relevant research projects we will conduct electronic searches in Medline, Embase and The Cochrane Library; check reference lists; and contact experts.Outcomes: 1) The extent to which the effect estimate in a meta-analyses changes with the inclusion or exclusion of studies that were not published or published in the gray literature; and 2) the extent to which the inclusion of unpublished studies impacts the meta-analyses’ conclusions.Data collection: Information will be collected on the area of health care; the number of meta-analyses included in the methodological research project; the number of studies included in the meta-analyses; the number of study participants; the number and type of unpublished studies; studies published in the gray literature and published studies; the sources used to retrieve studies that are unpublished, published in the gray literature, or commercially published; and the validity of the methodological research project.Data synthesis: Data synthesis will involve descriptive and statistical summaries of the findings of the included methodological research projects.Results are expected to be publicly available in the middle of 2013. A meta-analyses as part of a systematic review aims to provide a thorough, comprehensive and unbiased account of the literature ,2. HowevHowever, the impact of gray literature and unpublished studies on the conclusions of meta-analyses has not been comprehensively clarified. For example, there is some evidence that suggests that published randomized controlled trials (RCTs) tend to be larger and show an overall greater treatment effect than gray trials . But theIn terms of the above mentioned controversies regarding the inclusion of unpublished studies and studies published in the gray literature on the results of meta-analyses, we will conduct a systematic review with the following objectives:▪ To assess the impact of studies that were not published or only published in the gray literature on pooled effect estimates in meta-analyses (quantitative measure)▪ To assess whether the inclusion of unpublished studies or studies published in the gray literature impacts the conclusions of meta-analyses Overcome failure to Publish nEgative fiNdings) which was developed with the goal of elucidating the scope of non-publication of studies through a series of systematic reviews. In an earlier issue of this journal (‘Systematic Reviews’), our group has already published a protocol for a systematic review which evaluates the extent of non-publication of research studies, which were approved by ethics committees, registered in trial registries or presented as conference abstracts [This systematic review will be part of the OPEN Project (To bstracts .Ovid Medline (1946 to present), Ovid Medline Daily Update, Ovid Medline in process & other non-indexed citations, Ovid Embase (1980 to present), The Cochrane Library (most current issue) and Web of Science. No language restrictions will be applied.To identify the relevant research evidence we will conduct electronic literature searches in the following databases: In addition, the bibliographies of any eligible articles identified will be checked for additional references and citation searches will be carried out for all included references using ISI Web of Knowledge.Ovid Medline has already been designed with the support of a librarian/information specialist. This strategy was translated as appropriate for the other databases (for the full search strategies see Appendix A). In addition, we will contact various experts in the field for further eligible studies.A search strategy for the electronic literature search in A methodological research project will be considered eligible for inclusion in this systematic review if it reviews a cohort of meta-analyses  that:▪ compare pooled effect estimates of meta-analyses of health care interventions according to publication status  or▪ examine whether the inclusion of unpublished studies or gray literature impacts the overall findings or conclusions of a meta-analysesWe will consider ‘published’ articles to be manuscripts that appeared in peer-reviewed journals. Our working definition of gray literature will correspond to the definitions used by the authors of eligible methodological research projects and which also conforms to the definition of ‘gray literature’ described earlier in this protocol (see Background). A meta-analysis is defined as the calculation of a summary estimate of treatment effect by pooling the results of two or more studies.A specifically designed data extraction form will be developed and two reviewers will independently extract all relevant data from eligible methodological research projects. The following information will be collected:1. Characteristics of the methodological research projecta. Baseline data )a. Area of health care/medical specialtya. Number of meta-analyses included2. Characteristics of the meta-analyses included in the methodological research projecta. Type of meta-analyses a. Number of studies included in meta-analyses a. Number of participants included in meta-analyses a. Main purpose of meta-analyses (efficacy versus safety)a. Source used to retrieve unpublished studies, studies published in the gray literature and published studies3. Characteristics of the studies included in the meta-analysesa. Number of unpublished studies, studies published in the gray literature and published studiesa. Number of participant in unpublished studies, in studies published in the gray literature and in published studiesa. Number of statistically significant positive or negative unpublished studies, studies published in the gray literature and published studiesa. Type of unpublished studies , studies published in the gray literature  and published studies a. Year of publication of unpublished studies, studies published in the gray literature and published studiesa. Language and country of unpublished studies, studies published in the gray literature and published studiesa. Funding source of unpublished studies, studies published in the gray literature and published studiesa. Type of data source in which gray, unpublished and published studies were identifieda. Methodological quality  of unpublished studies, studies published in the gray literature and published studies We will systematically consider the validity and generalizability of the identified evidence provided by each of the methodological research projects by evaluating the following aspects:1. Internal validity:a. Role of confounding factors: The results of published studies may differ from those of unpublished studies because of factors other than publication status, such as study design, type of participants, characteristics of the intervention, and methodological quality; in this context, did the researcher of the meta-analyses select comparison groups that were matched ?; if not, were suitable adjustments for potentially confounding factors made?a. Definition of publication status: Are explicit criteria given to categorize or define unpublished studies, studies published in the gray literature and published studies?a. Selection process: Are search criteria given to identify unpublished studies, studies published in the gray literature and published studies?2. External validity :a. Did the researcher of the methodological research project select a broad-ranging sample of meta-analyses that reflect the current literature in the field of interest ?a. How was the sample determined ?a. Did two researchers carry out data extraction independently?a. Did the researchers provide a complete dataset ?(quantitative measurement). If possible, we will calculate a ratio of risks or odds ratios between the results of unpublished studies and studies published in the gray literature and the results of published studies and estimate the percentage change (pooled risk ratio from unpublished studies and gray literature divided by pooled risk ratio from published studies). A weighted pooled overall estimate will be calculated taking into account number of studies, participants and events.1. The extent to which the effect estimate in a meta-analyses changes with the inclusion or exclusion of unpublished studies and gray literature 2. The impact of the inclusion of unpublished studies or studies published in the gray literature on conclusions of meta-analyses . The impact will be estimated by calculating the proportion of meta-analyses which show a change in their conclusions according to publication status of the included studies; categorization will be as follows:a. Change from negatively significant to positively significanta. Change from inconclusive to positively significanta. Change from positively significant to inconclusivea. Change from negatively significant to inconclusivea. Change from inconclusive to negatively significanta. Change from positively significant to negatively significanta. Change from not clinical relevant to clinical relevanta. Change from not clinical relevant to inconclusivea. Change from clinical relevant to inconclusivea. Change from clinical relevant to not clinical relevanta. Change from inconclusive to clinical relevanta. Change from inconclusive to not clinical relevantSignificance and clinical relevance will be defined according to the definitions provided in the methodological research project.The anticipated unit of analyses is the meta-analyses included in the methodological research project.2-test and calculation of the I2 value [Heterogeneity for pooled outcome measures will be assessed by standard methods including ChiI2 value .et al. will be used to test for asymmetry [Funnel plots will be used to assess the association between point estimates of log odds ratio  and a measure of precision if more than ten methodological research projects provide necessary information. Funnel plots will be visually assessed and appropriate formal statistical tests following recommendations formulated by Sterne symmetry . In the Data synthesis will involve a combination of descriptive and statistical summaries of the impact of the inclusion or exclusion of unpublished studies and gray literature on the results of meta-analyses .The decision on whether or not to combine the results of the included methodological research projects will depend on the assessment of heterogeneity. Where methodological research projects will be judged to be sufficiently homogenous in their design a meta-analyses of these research projects will be carried out. The estimated ratios of unpublished and published in the gray literature only versus published treatment effects generated from each methodological evaluation will then be used to summarize the overall difference in risk ratios between unpublished and published in the gray literature only and published studies. The 95% confidence interval for the combined effect will be estimated using a random effects model.The following subgroup analyses are planned:1. On the level of the methodological research projecta. Number of meta-analyses included in the methodological research projecta. Number of participants included in the methodological research project2. On the level of the meta-analysesa. Number of studies (unpublished studies versus studies published in the gray literature versus both)a. Number of participants included in studies (unpublished studies versus studies published in the gray literature versus both)a. Design of studies (unpublished studies versus studies published in the gray literature versus both)a. Source of database: gray literature  published in an easily accessible database versus unpublished studies for which immense efforts are required to be identified a. Type of research work a. Area of health careNo sensitivity analyses are planned. However, should the instance arise where a methodological research project with doubtful eligibility is identified , sensitivity analyses may be undertaken. Possible sensitivity analyses may be based on the following:▪ Methodological quality/validity of the methodological research project; only methodological research projects with low risk of bias will be considered. A methodological research project will be of high risk of bias when the external validity and/or the validity of the included meta-analyses are doubtful.This systematic review seeks to comprehensively synthesize the growing body of research that is related to the impact of including unpublished studies and studies published in the gray literature in meta-analyses. By considering multiple characteristics and potential confounders related to unpublished studies and studies published in the gray literature, we hope to identify sufficient evidence to conclude whether (or to what extent) inclusion of unpublished studies and studies published in the gray literature has an impact on the pooled effect estimates and the conclusions from a meta-analyses. The findings, including risk factors for unpublished studies and studies published in the gray literature, will have important implications for researchers conducting meta-analyses since they need to be informed about the impact and extent of (not) including unpublished and gray studies in meta-analyses. In addition, this systematic review in combination with the results of other systematic reviews that are part of the OPEN Project will serve to raise awareness about the impact of publication bias and the complexity of this issue. These reviews will also serve as a foundation for a recommendations workshop which will enable key members of the biomedical research community  to develop future policies and guidelines to lessen the frequency of non-publication and related biases.We acknowledge that more than half of all systematic reviews do not involve meta-analysis in their analyses. Despite the fact that our main outcomes focus on the impact of unpublished and gray studies on pooled effect estimates in meta-analyses, our findings will also be valuable for systematic reviews. It is obvious, if we find a statistical difference in the pooled effect estimates in meta-analyses, it is also likely that gray and unpublished literature impacts descriptive results of systematic reviews. Beside effect estimates, we will also evaluate differences in the methodological quality and study characteristics  between unpublished, gray and published studies. These results will also be valuable for systematic reviews to appraise the potential impact of publication bias.st systematic review [Line 10 to 14 and 16 to 35 are not shown because they are part of the search strategy for our 1c review  and, the1. exp Publishing/sn2. *publishing/3. publication bias/4. selection bias/5. exp manuscripts as topic/6.  adj6 (unpublish* or un-publish* or unreport* or un-report* or nonpublish* or non-publish* or nonpublicat* or non-publicat* or (publication? adj3 rate?) or "not publish*")).ti,ab.7. ) or (full? adj2 (report* or publish* or publicat* or manuscript? or paper? or article?)) or (subsequent* adj2 (report* or article? or paper? or publi* or manuscript?)) or (sub-sequent* adj2 (report? or article? or paper? or publi* or manuscript?)) or (complete* adj2 (report* or article? or paper? or publish* or publicat* or manuscript?))).ti,ab.8. (bias* adj3 (publish* or publicat*)).ti,ab.9. or/1-815. exp animals/ not humans/36. meta-analysis as topic/37. Guidelines as Topic/ or Practice Guidelines as Topic/38. exp Clinical Trials as Topic/39 meta-analysis.pt.40. (guideline or practice guideline).pt.41. .ti,ab.42. (systematic* adj3 (review* or overview*)).ti,ab.43. exp Technology Assessment, Biomedical/44. .ti,ab.45. or/36-4446. 9 and 4547. 46 not 1548. ((implication? or impact? or influenc* or effect? or differen*) adj6 (publication bias* or unpublish* or un-publish* or unreport* or un-report* or nonpublish* or non-publish* or nonpublicat* or non-publicat* or "not publish*")).ti,ab.49. ((implication? or impact? or influenc* or effect? or differen*) adj6 ) or (full? adj2 (report* or publish* or publicat* or manuscript? or paper? or article?)) or (subsequent* adj2 (report* or article? or paper? or publi* or manuscript?)) or (sub-sequent* adj2 (report? or article? or paper? or publi* or manuscript?)) or (complete* adj2 (report* or article? or paper? or publish* or publicat* or manuscript?)))).ti,ab.50. ((unpublish* or un-publish* or unreport* or un-report* or nonpublish* or non-publish* or nonpublicat* or non-publicat* or "not publish*") adj6 publish*).ti,ab.51. ) or (full? and (report* or publish* or publicat* or manuscript? or paper? or article?)) or (subsequent* and (report* or article? or paper? or publi* or manuscript?)) or (sub-sequent* and (report? or article? or paper? or publi* or manuscript?)) or (complete* and (report* or article? or paper? or publish* or publicat* or manuscript?))).ti.52. (unpublish* or un-publish* or unreport* or un-report* or nonpublish* or non-publish* or nonpublicat* or non-publicat* or "not publish*" or bias*).ti.53. or/48-5254. 47 and 5355. 47 and (3 or 4)56. 47 and (36 or 37)57. 56 and (6 or 7)58. *meta-analysis as topic/59. *Guidelines as Topic/ or *Practice Guidelines as Topic/60. 47 and (58 or 59)61. (6 or 7) and (3 or 8)62. 54 or 55 or 57 or 60 or 6163. (unpublish* or un-publish* or unreport* or un-report* or nonpublish* or non-publish* or nonpublicat* or non-publicat* or "not publish*").ti,ab.64. 7 or 6365. 62 and 6466. 36 or 3767. 3 and 6668. 65 or 6769. 68 not 1570. remove duplicates from 69Table We declare that all authors and contributing members have no competing interests.JM is the lead researcher of this project. JM and CS, along with EvE and SP, developed the methodologies of the systematic review protocol and led the writing of the protocol. EM designed the search strategy. DB, MB and GS contributed significantly to the writing and revision of the protocol. All authors critically revised the protocol and read and approved the final version.


Animated movements of simple geometric shapes can readily be interpreted as depicting social events in which animate agents are engaged in intentional activity. However, the brain regions associated with such intention have not been clearly elucidated. In this study, intentional bias was manipulated using shape and pattern animations while measuring associated brain activity using event-related functional magnetic resonance imaging (fMRI). Twenty-five higher-intention involved and twenty-five lower-intention involved animations were presented to participants. Behavioral results showed that the degree of agency attribution of the mental state increased as intentional involvement increased. fMRI results revealed that the posterior superior temporal sulcus (STS), inferior temporal gyrus (ITG), inferior frontal gyrus (IFG), premotor, temporal pole, supramarginal gyrus, and superior parietal lobule (SPL) were activated while participants viewed the high-intention animations. In contrast, occipital, lingual, and middle frontal gyri were activated while the participants viewed the low-intention animations. These findings suggest that as agent attribution increases, the visual brain changes its functional role to the intentional brain and becomes a flexible network for processing information about social interaction. Recent evidence from cognitive social neuroscience has accelerated our understanding of intricate social brain functions, including processes involving the perception of others and their apparent behavior. However, relatively little research has been conducted to evaluate agency and its role in intentional bias. Moreover, there is limited evidence regarding how the intentional brain can be differentiated from the visual brain. For example, some configural cues such as contingent movement of geometrical patterns trigger an agency or animacy detectors in the brain that can partially explain intentional agents such as other people's minds.We hypothesize that the specific intentional brain function of estimating others' mental states based on agency attribution is an extended version of the visual brain. This extension involves recruiting higher brain regions found in the temporo-parietal cortices like the superior temporal sulcus (STS) Current social neuroscience studies suggest that the superior temporal sulcus (STS) and medial prefrontal cortex (MPFC) are likely essential components of the social brain region involved in intentional tasks. In order to examine this issue, we developed simple animations that manipulated intentional bias (higher- and lower-intention involved animations) by representing geometrical shapes as opposed to complex verbal or visual tasks.In their seminal research, Heider and Simmel (1944) In his theory of interpersonal relations, Heider proposed that individuals perceive and create explanations for the behavior of other's, a process he called “attribution” In mentalization studies in which the ability to estimate another's mind is required, the observer must infer and model the intentions of another person. In this type of paradigm, the observer models the behavior of the other person prospectively by using attributions that are represented as animated dots or cartoons. For example, Baron-Cohen et al. (1994) In another study, Schultz et al. presented short animations to participants in which two moving disks appeared to be either interacting or moving independently from each other Perception of animacy also influences interactive behavior In a study using PET, Castelli, Happe and Frith presented participants with a silent, computer-generated animation involving two simple geometric shapes  that resembled Heider and Simmel patterns The primary goal of the current work was to evaluate the degree to which intentional bias could result in greater STS activation and less MPFC activation. Similar animations were used such that objects always stayed within the same local region. However, animations differed in terms of their movements. Specifically, some animations were designed to give a graded impression of either intentional-oriented interactions or mechanical-oriented movements Twelve healthy, right-handed participants  and fifteen separate participants  were recruited for the fMRI experiment and preliminary rating study, respectively. All had normal or corrected-to-normal vision, and were screened for the presence of current or past neurological and psychiatric disorder.The experiment was conducted in accordance with the guidelines of the ethical committees of the Brain Activity Imaging Center  and of Kyoto University. All individuals voluntarily participated in the study and provided their written, informed consent prior to study participation.The animations used in the study was modeled on that of Heider and Simmel The upper panel of The lower panel of In the preliminary behavioral study, 15 participants rated each animation based on an intentionality score. The intentional score was rated using a Likert-type scale of 1 to 7 . Next we selected 25 “high” and 25 “low” intentionality animations. Observers were asked to rate intentionality between the blue object and the other objects based on their mutual actions.Pairs of high- and low-intention animations were created. Their paths of motion are shown in p<.001] and stimulus number . We also found significant interaction between intentionality and stimulus number . Multiple comparisons using Turkey's HSD revealed significant differences between high- and low-rated scores. Thus, we confirmed that the higher-rated group was significantly more sensitive to intention than the lower-rated group. T-tests comparisons between the number of objects (2 to 3 objects) found no differences in terms of intentionality. Based on these preliminary findings, we adopted all the stimulus objects tested for later experiments.A two-way repeated-measures ANOVA (intention×animation number) revealed a significant main effect for intentionality [F = 768.9, No participants who participated in the preliminary study participated in the fMRI study. In an fMRI session, an animation was presented one second after a beep tone and an evaluation screen appeared which asked the participant to rate the level of intentionality from one (high) to four (low). Participants made ratings by choosing from two sets of four buttons (one set for each hand). One trial took 17 s, resulting in a total of length of 14 min 30 s for each session. For the first session, fifty moving patterns were presented in random order to participants in a counter-balanced manner. Twenty-five patterns were presented to the high group and to the low group, respectively. In the second session, up-down reversed patterns from the first session were presented. The presentation of normal and up-down reversed patterns was counter-balanced for each participant. In the preliminary study, we confirmed that participants could easily decide a response after reading the agent's intention 3 s after presentation. Therefore, the fMRI scan began 3 s after the animation presentation.Animations were back-projected onto a screen viewed through an angled mirror. The size of each animation was 11.5°×11.5°. In one session, participants observed 50 animations presented in random order. The length of each trial was 17 seconds.Whole brain images were acquired on a 1.5-T whole-body magnetic resonance imaging scanner . Head motion was minimized with a forehead strap. Functional MRI was performed with a gradient echo-planer imaging . After the collection of functional images, T1-weighted images (154 slices with no gap) using a conventional spin echo pulse sequence  were collected for anatomical co-registration with the functional images.After image reconstruction, functional images were analyzed using SPM2 . Six initial images were discarded from the analysis to eliminate the non-equilibrium effects of magnetization. All functional images were corrected for between-slice timing differences in image acquisition and realigned to correct for head movement, which was less than 1 mm within runs. The functional images were normalized and spatially smoothed with an isotropic Gaussian filter . Low-frequency noise was removed by high-pass filtering (time constant = 128 s). We conducted the analysis using an event-related design. An onset of an event according to the data analysis occurred three seconds after an animation started based on the results of the preliminary study.t-contrast images were then entered into second-level analysis using a random effects model for all participants. The levels of statistical significance for these analyses were set to p<0.001 (uncorrected).Data were modeled by convolving the vector of expected neural activity with the canonical hemodynamic response function (HRF) included in SPM2 and modulated by ratings of intentionality . Single-participant Two contrasts were specified per single-participant analysis: 1) Low versus High and 2) High versus Low. Low-intention involves activations under participant's button press 1 (highest) and 2 (higher) and high-intention involves that of button press 3 (lowest) and 4 (lower). As shown in In this study, we sought to investigate the differential contributions of the areas involved in visual and intentional cognitive processes. Participants conducted tasks that required them to make social interpretations by looking at moving objects that were presented as low- or high-intentionally biased animations. By varying the stimuli, we varied the extent to which intentional cognitive processing was required, which facilitated the analysis of intentional and perceptual influences on various brain regions.Based upon event-related fMRI data, our results revealed activation of several visual areas including the calcarine sulcus/cuneus and the lingual gyrus , which is near the fusiform gyru when the visual brain operated in a mechanical low-intention-involved context. The middle frontal gyrus is thought to maintain visual attention to groups of moving objects As shown in In an earlier PET study, Castelli et al. The STS has been hypothesized to be closely connected to the perception of biological motion. Studies using transcranial magnetic stimulation Overall, we assumed that activation in the premotor cortex invoked a mirror system when a human acts and when the person observes the same action performed by anothe With close interconnections to the STS, the IFG and the temporal pole provide internally-represented self and other's mental states. Rather than the MPFC per se, it is the ventral side of the IFG, close to the orbitofrontal PFC and temporal pole, along with temporo-parietal-junction areas including the posterior STS and supramarginal gyrus In their examination of the neural correlates of mentalization, Vogeley et al. (2001) To summarize, we investigated how the visual brain transitions to the social brain using event-related fMRI in the present study. Animations consisted of moving patterns evoking various mental states of attribution based on intentions. Among 25 pairs of animations, each participant rated the higher- and lower-intention animation according to their attribution of agency . Results showed that activations of the posterior STS, ITG, IFG, premotor, temporal pole, supramarginal gyrus, and SPL occurred under high-intention–involved animations, whereas occipital, lingual, and middle frontal gyri were activated under lower-intention-involved animations.Findings of the present study suggest that as intentional stance increased, the portion of the social brain involving the representation of an agent's intentional actions became more activated. Thus, developing the capacity to model another's mind could be an evolutionary innovation in the human social brain that developed from the perceptual brain. Previous studies have implicated regions activated by higher intention in self-monitoring in the perception of biological motion and in the attribution of mental states, and regions activated by lower-intention in simple perceptual processing. In the present study, we report how the visual brain shifts to the social brain in an agency attribution experiment. We suggest that as agent attribution increases, the visual brain changes to the intention-assuming social brain and therefore possesses a flexible network for processing information about social interactions based on agency attribution.Movie S1An example movie under high-intention condition.(AVI)Click here for additional data file.Movie S2An example movie under low-intention condition.(AVI)Click here for additional data file.Movie S3An example movie under intermediate high-intention condition.(AVI)Click here for additional data file.Movie S4An example movie under intermediate low-intention condition.(AVI)Click here for additional data file.


Contamination by bacterial or fungal organisms reduces the effectiveness of mycobacterial culture for diagnosis of pulmonary tuberculosis (TB). We evaluated the effect of an anti-microbial and an anti-fungal oral rinse prior to expectoration on culture-contamination rates.We enrolled a consecutive random sample of adults with cough for ≥2 weeks and suspected TB admitted to Mulago Hospital  between October 2008 and June 2009. We randomly assigned patients to oral rinse (60 seconds with chlorhexidine followed by 60 seconds with nystatin) vs. no oral rinse prior to initial sputum collection. Uganda National Tuberculosis Reference Laboratory technicians blinded to the method of sputum collection  processed all sputum specimens for smear microscopy (direct Ziehl-Neelsen) and mycobacterial culture (Lowenstein-Jensen media).Of 220 patients enrolled, 177 (80%) were HIV-seropositive . Baseline characteristics were similar between patients in the oral-rinse (N = 110) and no oral-rinse (N = 110) groups. The proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group . Oral rinse significantly reduced the proportion of contaminated cultures among HIV-infected patients  but not HIV-uninfected  patients. However, the proportion of smear-positive specimens  and culture-positive specimens  were lower in the oral-rinse compared to the no oral-rinse group, although the differences were not statistically significant.Oral rinse prior to sputum expectoration is a promising strategy to reduce mycobacterial culture contamination in areas with high HIV prevalence, if strategies can be devised to reduce the adverse impact of oral rinse on smear- and culture-positivity. Sputum mycobacterial culture services are increasingly offered in low-income countries for the evaluation of patients suspected of pulmonary tuberculosis (TB). This service is in accordance with the latest World Health Organization (WHO) recommendations designed to improve the diagnosis of the more than two million annual smear-negative TB cases and more than 0.5 million multi-drug resistant TB cases However, contamination of mycobacterial culture media resulting from overgrowth by oro-pharyngeal bacteria and/or fungi is of significant concern. Culture contamination reduces the proportion of interpretable results and diminishes the diagnostic value of culture systems Candida species are common causes of culture contamination To reduce the risk of sputum culture contamination, WHO recommends strict adherence to standardized laboratory protocols with regard to sputum collection, transportation, and processing The Makerere University Faculty of Medicine Research and Ethics Committee, the Mulago Hospital Institutional Review Board, the University of California, San Francisco Committee on Human Research, and the Uganda National Council for Science and Technology approved the study protocol. All study participants provided written informed consent.We screened consecutive adult patients admitted to Mulago Hospital  with cough ≥2 weeks but <6 months and with suspected pulmonary TB for study eligibility. We excluded patients who were receiving TB treatment or had a history of TB treatment in the previous two years. Patients unable to expectorate sputum and those individuals whose smear and culture results were unavailable were excluded from the analysis.Medical officers obtained demographic and clinical information from enrolled participants using a standardized questionnaire. We tested for Human Immunodeficiency virus (HIV) infection in all patients without a confirmed diagnosis of HIV, and measured CD4+ T-lymphocyte counts in those whom we found to be HIV seropositive. On day 1, prior to sputum expectoration, patients either performed a 60-second oral rinse with chlorhexidine  followed in immediate succession by another 60-second rinse with nystatin  or no rinse at all. Study participants were allocated to either group using a list of randomly generated study ID numbers. Under supervision, all patients provided two sputum specimens for Acid Fast Bacilli (AFB) Ziehl-Neelsen (ZN) stain smear microscopy and culture. Laboratory technicians provided standardized instructions on proper sputum expectoration NTRL technicians blinded to sputum collection procedures  evaluated sputum specimens for acid-fast bacilli (AFB) using direct light microscopy (Ziehl-Neelsen staining) and Lowenstein-Jensen (LJ) mycobacterial culture. They decontaminated specimens using the NALC-NaOH  method, concentrated them by centrifugation, and then inoculated the concentrated pellet onto LJ media. NTRL staff checked LJ cultures at least weekly for positive results and considered cultures to be negative if no growth was observed after eight weeks. They assessed the presence of culture contamination using the following criteria: 1) any change in colour or consistency of culture media, 2) development of any liquid or water film over the culture media, and/or 3) presence of non-mycobacterial colonies in culture media .We calculated our sample size using an expected reduction of the contamination rate from 18%  to a target of 5% using the chlorhexidine-nystatin rinse. Using an alpha of 0.05 and a power of 0.80, we determined that we needed 109 patients in each group. We compared proportions of contaminated cultures, of positive smears, and of positive cultures in each intervention arm using the chi-squared test or the Fischer’s exact test where appropriate. We carried out sub-analyses stratifying results by HIV status. We defined significance in reference to the probability of a two-tailed, type-I error  less than 0.05. We used STATA 10  for analyses.Of 245 eligible patients enrolled, 20 (8%) were excluded because they failed to expectorate and 5 (2%) were subsequently excluded because their culture results were missing . Thus, wThe proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group  −18% to −3%, p = 0.005) . Oral riThe proportion of patients with positive Ziehl-Neelsen smears was lower in the oral-rinse group compared to the no oral-rinse group, although this difference was not statistically significant  . SimilarTrends for culture positivity were similar to those observed for smear positivity. The proportion of patients with positive cultures  was lower in the oral-rinse group, and this difference was smaller when contaminated cultures were considered to be negative .Culture contamination represents a major threat to the sensitivity of mycobacterial culture, more so in high TB-HIV burden settings, and yet few studies have investigated strategies to lower its occurrence. Recently, one study in HIV-uninfected individuals showed that rinsing with chlorhexidine reduced MGIT culture contamination rates An unexpected finding of the study was that chlorhexidine-nystatin oral rinsing showed a trend towards reducing the overall proportion of positive sputum smears and cultures, as well as smear sensitivity. This finding does not have a clear explanation. The reduction in Ziehl-Neelsen smear sensitivity and proportion of positive smears was especially surprising because acid-fast bacilli should still be identified, even when they are non-viable. The reduction in the proportion of positive cultures on the other hand could be explained by a possible but undocumented anti-mycobacterial effect of the chlorhexidine and nystatin rinse. Perhaps some of the rinse is retained within the mouth, such that these agents are expectorated with the sputum specimen in high enough concentrations to inhibit mycobacterial growth. However, whereas chlorhexidine oral rinse has been used and shown to have antibacterial properties against a wide range of oro-microbial bacteria, evidence has shown that mycobacteria are highly resistant to it This study has some limitations. First, although we have strong evidence of the negative impact of oral rinse on smear and culture positivity, we did not have a sufficient sample size to estimate precisely its effects on smear and culture sensitivity, more so for the HIV-uninfected population. A larger study is needed to explore this unexpected finding. Second, we only evaluated the combined effect of chlorhexidine and nystatin and are unable to distinguish between the individual contributions of these agents on both culture contamination and on smear- and culture-positivity. It is possible that rinsing with a single solution may be as effective as rinsing with both solutions. Finally, we evaluated the effect of oral rinse on contamination of LJ cultures because MGIT cultures for which strategies to reduce contamination are even more important Culture is an important tool for evaluating TB suspects, with special utility for identifying smear-negative TB cases and for testing second-line drug-susceptibility to guide therapy in patients with multi-drug-resistant TB. However, contamination is a major barrier to its efficacy. The use of an antibacterial-antifungal oral rinse prior to sputum expectoration to reduce mycobacterial culture contamination is a promising strategy worth considering, especially in areas with a high HIV prevalence. Further studies are needed to characterize the nature of contaminants in this population and to evaluate the potential adverse impact of each oral rinse in varying concentrations on smear- and culture-positivity.