Current models of obsessive compulsive disorder (OCD) propose abnormalities of cortico-striatal circuits that involve the orbitofrontal cortex, anterior cingulate cortex, thalamus and the striatum. Nevertheless, during the last years, results of morphometric studies were contradictory. Since fully automated whole-brain voxel-based morphometry (VBM) and diffusion tensor imaging (DTI) are used to assess structural changes in OCD patients, increased consistent evidence has been reported that brain abnormalities are not limited exclusively to the “affective” orbitofronto-striatal circuit. Moreover, several studies conducted using a symptom dimensional approach demonstrated that different symptoms are mediated by distinct neural systems.We investigated structural brain abnormalities in 14 carefully selected adult (≥18 years), male and unmedicated patients with OCD - separately for obsession and compulsion scores (Y-BOCS) - compared to 20 healthy controls as reflected according to white matter changes by fractional anisotropy and apparent diffusion coefficient. Moreover, this is the first study in OCD patients, using magnetization transfer imaging (MTI). This method is said to be more sensitive to subtle structural brain changes than conventional volumetric imaging.In our study, we show a positive correlation between MTR and Y-BOCS obsession scores with an increased integrity of tissue structure in the parietal cortex, including myelination and axonal density reflected by the magnetization transfer ratio (MTR) which was used for the first time in our study. Furthermore, Y-BOCS scores for compulsions correlated negatively with ADC-maps in the left nucleus lentiformis and the cingulum.The results support the hypothesis that OCD is a heterogeneous disorder with distinct neural correlates across symptom dimensions and call for a substantial revision of such a model that takes into account the heterogeneity of the disorder. Obsessive-compulsive disorder (OCD) is a disabling anxiety disorder with a lifetime prevalence rate of 2–4 % in the general population. It is characterized by the presence of intrusive and distressing thoughts, images or ideas, termed obsessions and repetitive or ritual behaviours, known as compulsions . HoweverDiffusion tensor imaging (DTI) studies have investigated WM abnormalities in OCD patients compared to healthy controls, showing abnormalities of the fronto-striatal thalamo-cortical loop , 18. TheIt can be assumed that these inconsistencies of GM and WM abnormalities in previous studies can mainly be attributed either to substantial methodological differences or to the inclusion of only small and heterogeneous samples. Since fully automated whole-brain voxel-based morphometry (VBM) and DTI are used to assess structural changes in OCD patients, it has been consistently reported that brain abnormalities are not limited to the “affective” orbitofronto-striatal circuit but also extend to the dorsolateral prefronto-striatal “executive” circuit and additional regions including the parietal and occipital lobes as well as the cerebellum , 23–25. in vivo before it manifests on conventional MR volumetric imaging  / non-MT) × 100. DTI was performed using echoplanar imaging (EPI) . One b = 0 image was obtained. During scanning, all participants were comfortably placed and their heads were fixated within the headcoil with special cushions. All subjects received additional T2-weighted images.Images were acquired on a neuro-optimized 1.5-T GE Signa Horizon LW  using a 3-dimensional T1-weighted spoiled gradient recalled echo (SPGR) sequence generating 124 continguos sagittal slices  and working in an analysis environment . Images were reoriented into oblique axial slices aligned parallel to the anterior-posterior commissural axis with the origin set to the anterior commissure.After calculating the FA and ADC maps, images were pre-processed and analyzed by SPM2 using an approach adopted from VBM. This included an optimized normalisation procedure, together with an automated exclusion of skull and CSF signal values and smoothing (8-mm FWHM). In a first step, all EPI scans were normalized to the EPI-template provided by SPM. The normalized data were then smoothed (8-mm FWHM) and a mean image was created. This provided an EPI site- template appropriate to the population sample and with scanner specific image contrast. In a second step, all EPI images in native space were then normalized to the template derived from step 1. As statistical inference was intended to be only made on the intensities of brain tissue, the individually normalized images were cleaned to remove extracerebral tissue and CSF. This was accomplished by using the segmentation function of SPM2 including the brain extraction step on the normalized images. The white and grey matter partitions were then reunited using the ImCalc function. The resultant image was thresholded by 0.15 to create a binary mask image containing ‘ones’ for voxels with a probability of greater than 15 % to belong to grey ore white matter and ‘zeros’ for voxels outside the brain. The mask image was then multiplied voxel-by-voxel with the corresponding normalized EPI image, thus discarding the majority of extracerebral tissue and CSF and preserving the original voxel intensities. Those cleaned normalized images were then smoothed and a mean image of all subjects – the cleaned EPI template - was created. In a third step, the same cleaning procedure was applied to the EPI images in native space. The resultant cleaned images were then normalized to the cleaned template from step 2 thus preventing any contribution of nonbrain voxels and affording optimal spatial normalization of the cleaned EPI images. The optimized normalisation parameters were reapplied to the coregistered FA images. This resulted in optimally normalized fractional anisotropy images which were then again cleaned from extracerebral tissue and CSF.3. The normalized MTR images were then skull-stripped by applying the corresponding brain mask derived from the optimally normalized PD-weighted images. Analogous to the PD images, this resulted in optimally normalized MTR maps removed from non-brain structures. Images were smoothed to 8 mm using an FWHM Gaussian filter to improve signal-to-noise ratio.The main challenge facing voxel-based MTI analysis involves meeting the requirement for an optimal matching of the brains being compared. Therefore, a complex pre-processing procedure was employed as described before  consistip < 0.001, uncorrected with an extent threshold of 20 voxels. For regions where an effect was hypothesized, namely the fronto-striatal and limbic system (see introduction), a small volume correction (SVC) limited to the volume of that particular region was performed [p < 0.05). Significant voxel maxima were then converted from MNI space to Talairach space. Talairach coordinates were assigned to the anatomical structures by using Talairach Daemon [Processed images of each tissue class were analyzed in the framework of the general linear model. This framework allows the testing, on a voxel-by-voxel basis, of the null hypothesis that the tissue volumes in the two populations (patients and controls) are the same. Group comparison of OCD patients and healthy controls was performed in SPM2 using ANCOVA. Only voxels exceeding an absolute threshold of 15 % were included in the analysis to minimize low signal-to-noise. Correlation with clinical parameters (YBOCS score) were done by using SPM multiple regression. Resulting statistical parametric maps of FA, ADC and MTR were derived at a significance level of erformed . Here, wh Daemon , 41.On visual inspection of the MR images no subject had focal atrophy of any brain region or movement artefacts which may have hindered alignment into standard space or segmentation into gray or white matter. Furthermore, there were no overt involuntary movements, which were monitored during scanning by one of the investigators (AG). Mean (+/− SD) intracranial volume did not significantly differ between patients with OCD and healthy controls.Compared with healthy controls, patients with OCD showed reduced FA in the white matter of the left medial frontal gyrus as well as in the OFC on both sides Fig. , Table 2Compared to normal controls, patients with OCD displayed significant increases of MTR maps in the left middle frontal gyrus and GM of the right inferior frontal gyrus (Brodmann area (BA) 40). No significant decreases in MTR maps were found in OCD patients compared to healthy controls Table .There were no significant correlations between ADC maps and Y-BOCS subscores for obsessions. We found significant negative correlations between the ADC maps and Y-BOCS subscores for compulsions in the left insula BA13), the left nucleus lentiformis and striatum on the same side and WM of the cingulate cortex on the left side Fig. , Table 3, the lefThere were no significant correlations between MTR maps and Y-BOCS subscores for compulsions. Y-BOCS subscores for obsessions correlated positively with the MTR maps in the GM of the left parietal-temporal-occipital association cortex BA 39), WM of the left inferior parietal lobule  of OCD. We therefore performed not only group comparisons between OCD-patients and healthy controls but additionally correlated MTR as well as ADC- and FA-maps with the Y-BOCS subscores for obsessions and compulsions to reassess the hypothesis that symptoms of OCD are mediated by partially distinct neural systems .Three main findings emerged from this study. First, the group comparison between OCD patients and healthy controls demonstrated abnormalities of WM microstructure in multiple sites of the fronto-striatal system. Our patients showed decreased FA-maps in the OFC on both sides and increased diffusivity especially in both sides of the orbitofrontal parts of the brain ) and the left inferior frontal gyrus (BA 47)  and thThe second main finding is a significantly negative correlation between the severity of compulsions (according to Y-BOCS scores) and ADC-maps in the left nucleus lentiformis and the striatum on the same side and WM of the left cingulate cortex. The ADC reflects the degree of apparent water diffusivity: tissues without obstacles such as cerebrospinal fluid have high water diffusivity, whereas those with obstacles such as WM have low diffusivity. ADC-maps, have recently been investigated in psychiatric disorders such as schizophrenia, mood disorders  and OCD In addition, we found a negative correlation between ADC maps and the severity of compulsions in the cingulate cortex. This negative correlation between ADC values and Y-BOCS compulsion scores seems to be contraintuitive at first sight as one would normally expect higher clinical scores to be related to a decrease in structural integrity. Nevertheless, one could assume that increased structure may favor a hypertonic state. Our results are in line with findings from structural and functional neuroimaging studies demonstrating abnormalities in the ACC , 45, butThe third striking finding of this study is a significantly positive correlation between the severity of obsessions and the MTR in the left parietal cortex (BA 39). To our best knowledge, this is the first study using MTI in patients with OCD. We decided to use MTI, since this method is said to be more sensitive to subtle structural brain changes than conventional volumetric imaging . MTI is The following limitations of our study have to be addressed. Firstly, the voxel-by-voxel analysis used was originally intended for the use in large samples and requires smoothing of the images with loss of resolution for small structures. Furthermore, the large number of comparisons required corrections for multiple comparisons. This might explain why we did not find significant MTR differences between OCD patients and healthy controls on a whole brain analysis. Moreover the small size of our group makes the results vulnerable to type I or type II errors, although in recent DTI and MTI studies a similar number of patients was suitable to detect regional differences compared to normal controls , 22. TheThis is the first study using MTI to investigate structural brain abnormalities in OCD patients. In addition we analyzed not only group comparisons between OCD patients and healthy controls but also obsession and compulsion subscores (Y-BOCS) separately using both DTI and MTI in a carefully selected group of adult, unmedicated, male OCD patients without comorbidities and age- and sex-matched normal controls. In our study, we show a positive correlation between MTR and Y-BOCS obsession scores with an increased integrity of tissue structure in the left parietal cortex, including myelination and axonal density reflected by the MTR which was used for the first time in our study. Y-BOCS subscores for compulsions correlated negatively with ADC maps in the left striatum, the left nucleus lentiformis and the left cingulate cortex. Thus, our results support the hypothesis that OCD is characterized by widespread cerebral changes which underlines the heterogeneity of this disorder.


Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism.In many systems, including the social amoeba  Development involves dynamic transcriptional changes. By serial ChIP- and RNA-sequencing, here, the authors show that GtaC, a GATA type transcription factor, exhibits temporally distinctive DNA binding and regulation of gene expression concordant with the development in the social amoeba Dictyostelium discoideum. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is widely used to assay in vivo TF–DNA interactions. This approach provides a genome-wide view of TF-binding preferences, making the discovery of functional cis-regulatory DNA elements possibleDevelopment involves the sequential execution of molecular events achieved by gene-regulatory networks134Dictyostelium development have been well documentedG-box binding factor A)signal transducers and activators of transcription a)serum response factor A)culmination defective A)D. discoideum amoebae initiate a developmental programme and organize into multicellular structures by aggregating in response to 3′-5′-cyclic adenosine monophosphate (cAMP) waves that propagate through the field of cells. After aggregation, cell-type differentiation occurs accompanied by dynamic morphological changes and the developmental process ends in the formation of fruiting bodies consisting of two major cell types—spores and stalks14D. purpureum is an evolutionarily distant dictyostelid species whose developmental morphology is strikingly similar to that of D. discoideum. Both species use cAMP as a chemoattractant and their developmental transcriptomes are remarkably conserved816gtaC, which is abundant in both D. discoideum and D. purpureum during cAMP-mediated aggregation and encodes a GATA-type TFTranscriptional changes accompanying gtaC are defective in aggregation and in morphological and transcriptional progression. Ectopic expression of GtaC fused with green fluorescent protein (GtaC-GFP) in the gtaC-null  strain complements these defects. The expression of a mutated GtaC, in which all four cysteine residues of the zinc-finger domain were substituted with serine residues, does not complement the developmental defects of the KO straingtaC transcript abundance increases twice during development. Once during early aggregation and once starting at late aggregation and continuing through the end of development (dictyExpress (http://dictyexpress.biolab.si/)). This observation is broadly consistent with the early and late phenotypes of the gtaC-mutant strains18gtaC has not been examined yet.GtaC is a key regulator that decodes extracellular cAMP pulses and establishes downstream signalling pathways, ensuring timely development. During early development, GtaC shuttles between the nucleus and cytoplasm in response to, and out-of-phase with, extracellular cAMP pulses. Cells lacking Dictyostelium development is accompanied by sweeping changes in gene expression820gtaC manifest as an early developmental phenotypeDictyostelium developmental gene-regulatory network. In this study, we analyse sequential genomic occupancy data of the wild-type (WT) and cysteine-substituted (CS) forms of GtaC using ChIP-seq to explore the developmental roles of GtaC further. At contemporaneous developmental stages, we also analyse serial transcriptional profiles using RNA sequencing (RNA-seq). By integrating these binding and transcriptome data, we identify GtaC target genes. We find that GtaC targets include developmentally essential TFs and genes involved in cAMP signalling during early development and those relating to cell-type specification at later stages. Our results suggest that GtaC regulates early development by controlling different physiological processes concordant with developmental progression in Dictyostelium.D. discoideum and its nuclear localization is regulated by pulsatile cAMP signals during aggregationgtaC-null mutation, and used the GFP-expressing KO strain as a negative control to exclude potential binding by GFP . Expression of GtaCC-S-GFP in the KO strain does not complement the defects in aggregation and morphological progression  but lower during late aggregation  were closer to each other than to the transcriptomes at early aggregation (5 h) or late aggregation (8 h) stages. Overall, these results suggest that the developmental consequences of carrying various forms of gtaC are accompanied and likely caused by overt transcriptional differences between the strains that express them.To test whether GtaC-binding events were accompanied by gene expression changes, we used our published RNA-seq data of WT, CM and KO strainsC-S–DNA-binding events have no direct role in the regulation of gene expression. We identified 1,188 genes that were differentially expressed between strains with normal  and aberrant  developmental progression and also developmentally regulated in both WT and CM strains. Using hierarchical clustering, we identified five broad clusters of genes that exhibited coordinated changes in their mRNA abundance over time  of the genes in KO and CS strains did not show any significant developmental changes in mRNA abundance, consistent with the lack of transcriptional and morphological progression in these strains 18. It isver time . Genes iver time . This dover time . Genes iC-S binding, we tested the relationship between binding events on the basis of the ChIP-seq data and transcriptional changes in the RNA-seq data at the corresponding time points. We identified genes bound by GtaC and also differentially expressed between CM and KO strains at the two aggregation stages  score to integrate these two parametershttp://dictybase.org/)) and the set of putative TFsTo explore the biological processes regulated by GtaC, we examined gene annotations within the putative direct targets. In a direct comparison with previously defined gene sets, we found the sets of ‘cAMP-responsive'D. discoideum and D. purpureum are two evolutionarily distant dictyostelid species whose aggregation is mediated by cAMP and, their gtaC transcript levels are abundant during the aggregative phases of development816D. purpureum orthologues among GtaC-dependent target genes in D. discoideum. Genes bound by GtaC during the aggregative stages were significantly enriched in the set of orthologous genes. Furthermore, a significant proportion of GtaC targets in the set of D. purpureum orthologues were coordinately expressed during development across the two speciesDictyostelium developmental processes, we performed an unbiased gene ontology (GO) enrichment analysis and identified enriched GO terms at each time point. The identified GO terms were largely associated with the key developmental processes—‘aggregation' at early aggregation and ‘cell differentiation' at late aggregation, for example, as shown in Dictyostelium development.To broaden the association between GtaC targets and In this paper, we showed that GtaC exhibits distinct temporal binding patterns during early development, thus alluding to multiple developmental roles. Such variable binding specificities have been reported in other systems, where they appear to result from various regulatory effectors2830in vitro protein-binding microarray experimentscis-regulatory sequences to modulate gene expression34Here we found that the zinc-finger domain of GtaC was essential for specific binding to the GATA motif. Recent protein-binding microarray experiments revealed that the GtaC DNA-binding domain showed higher-affinity binding to a consensus ‘GATC' sequence, than to ‘GATA' sequencesC-S. While it is tempting to speculate that GtaC binds DNA in different modes, more careful analyses are necessary to characterize the existence and functional consequence of GtaC–DNA interactions. However, regardless of the molecular mechanism, GtaCC-S binding appears to have almost no effect on development because ectopic expression of GtaCC-S in the gtaC-null strain does not significantly modify the developmental or the transcriptional phenotypes.Cysteine substitutions in the GATA zinc-finger domain of GtaC did not affect the ability of the protein to translocate to the nucleus but it seemed to completely inactivate its developmental function25We integrated DNA-binding and transcriptome data from the CM and KO strains to identify putative direct targets of GtaC and found good concordance between the data sets. Despite ectopic expression of GtaC-GFP in CM, remarkable similarities between the CM and WT strains, not merely on the basis of gross morphologies, but also at the more detailed resolution of their transcriptional phenotypes suggest that the artificially constructed CM strain is a faithful representation of native GtaC-regulatory function. Temporal patterns of GtaC binding in promoters and expression patterns of the associated genes are largely cotemporaneous, suggesting a causative relationship between TF binding and transcription of targets. Among the GtaC targets, we found enrichment in two broad developmental processes during the early and late times. Our results are consistent with GtaC's role in cAMP-mediated processes during early developmentD. discoideum and D. purpureum among GtaC targets. Given the similarities between the two, our data suggest that some of the GtaC-dependent regulatory modules are conserved between these evolutionarily distant speciesacaA and carA, are essential for D. discoideum development, reinforcing the notion that GtaC is a critical regulator of development. The functional importance of GtaC targets is also illustrated by the abundance of putative TFs. A number of these TFs, such as GbfA and CudA, also play important roles in Dictyostelium development10D. discoideum development, the essential functions it regulates and the proposed evolutionary conservation establish GtaC as a significant node in the transcriptional regulatory network that governs Dictyostelium development.Another interesting observation is the over-representation of co-regulated orthologues between csaA gene (contact site A), that we identified as a direct GtaC target, is transcribed periodically, with periods similar to that of cAMP pulsesMany studies that utilize high-throughput technologies, such as ChIP-seq and RNA-seq, provide a snapshot of the cellular population average38183940D. discoideum strain at 22 °C in shaking suspension in HL5 medium with the indicated supplements  for 10 min on iceChIP using e points . We harvDictyostelium reference genomeE-value threshold: 10−3) to search for over-represented motifs on the 600 most enriched GtaC- and GtaCC-S peaks.The sequencing data were mapped to the http://dictybase.org/), weighted by the differential enrichment in GtaC coverage between sample and control ChIP-seq experiments.To associate peaks with genes, we defined the promoter region of a gene as the region 5′ of the ORF, up to the nearest upstream gene, and assigned all peaks within a promoter region to the gene. We calculated the binding scores of each gene using all peaks in its promoter region on the basis of a scoring scheme adapted from refs GSE54866 in the Gene Expression Omnibus. We prepared multiplexed cDNA libraries using these RNA samples as described18We collected RNA samples in duplicates from WT, CM, KO and CS strains . The timDictyostelium reference genomeD=1−SC) and complete linkage as the clustering criterion. The transcriptome analyses yielded data on the steady-state mRNA levels of 12,435 genes, ∼3% of which were not expressed under any of the conditions sampled  analyses using baySeqC-S and differentially expressed between CM and KO and CS and KO strains, respectively, at both E and L, and visualized the overlap as Venn diagrams . To test whether the proportion of overlapping genes identified in our data was different from expected by chance, we randomly sampled the same number of genes from the genome, as observed in our data, at both time points. We counted the number of overlapping genes and obtained an empirical distribution by repeating this procedure a million times. At each time point, we used this distribution to calculate the number of instances that showed a larger overlap than in our data and found that probability of such an event was close to zero. In order to test for regulatory relationships between TF binding in the promoters and mRNA abundance of the downstream genes, we obtained distributions  of GtaC- and GtaCC-S-binding scores on genes categorized as UP, DOWN or NDE at each time point in CM and CS cells. We used a nonparametric statistical test  to compare the binding score distributions of genes that are differentially expressed (UP or DOWN) and those that are not (NDE).We identified the number of genes that were bound by GtaC and GtaCTo identify enriched short DNA sequences among developmental-stage-specific gene sets, we first identified genes showing large changes in gene expression in CM compared with KO cells (absolute log-ratio >2) at each of the three time points. Then, we used MEME-ChIPD. discoideum were obtained from dictyBase (http://dictybase.org/).We ranked the genes on the basis of the absolute values of their DE scores in comparisons between CM and KO strains  at each time point. We also ranked genes on the basis of their GtaC-binding scores such that genes with the high binding scores were ranked first. We computed the RP of genes on the basis of their ranked DE scores and their ranked binding scores at each time pointAccession codes: The relevant data sets have been deposited in the Gene Expression Omnibus database of National Center for Biotechnology Information under the accession number GSE63151.How to cite this article: Santhanam, B. et al. The GATA transcription factor GtaC regulates early developmental gene expression dynamics in Dictyostelium. Nat. Commun. 6:7551 doi: 10.1038/ncomms8551 (2015).Supplementary Figures 1-5 and Supplementary Tables 1-4http://dictybase.org) and second column header is the name of the gene. This data included the normalized and raw read counts for each gene on each strain  at 0, 1, 2, 5 and 8 hr of development. The strain information is provided in Table S1. Each RNA-seq run was performed in two biological replicates. Columns showing the raw read counts have a suffix ‘r' and those with the normalized read counts have a suffix ‘n'. The average on both biological replicates of the normalized data has a suffix ‘mavg'. The time points are indicated in the column headers for each. Thus the column header ‘CS_1hr_n1' refers to the normalized expression in the first biological replicate of genes in the CS strain at 1 hour of development (0 hour is considered to be the beginning of starvation). Similarly, ‘CS_1hr_r1' refers to the raw reads counts of genes in this data set. The average of both biological replicates (i.e ‘CS_1hr_n1' and ‘CS_1hr_n2') is under the column header ‘CSmavg.1hr'. Each baySeq comparison performed with two predefined models  between two strains at the same time point or between time points in each strain produces a total of three outputs: Likelihood (Lik), False Discovery Rate (FDR) and additionally provides directionality of expression difference . Thus the column headers ‘Lik.DE_2hrWTvs.CS', ‘DE_2hrWTvsCS' and ‘FDR.DE_2hrWTvsCS' refer to the Likelihood, direction of differential expression and the false discovery rate, respectively, in a comparison between the 2 hr WT and CS transcriptomes. Similarly, the column headers ‘Lik.DE_CS0hrvs.1hr' and ‘FDR.DE_CS0hrvs.1hr' refer to the likelihood and false discovery rates, respectively, in a comparison between the 0 hr and 1hr CS transcriptomes.Transcriptome data and differential expression comparisons. This file contains the transcriptome data and differential expression comparisons. The first column header ‘ddb_g' is the unique identifier as defined by dictyBase (http://dictybase.org) and column B to names of genes and. Columns C-E show the GtaC rank-product scores  at starvation sensing, early aggregation and late aggregation respectively. Columns F and G show GtaCC-S rank-product scores at early and late aggregation. The differential expression scores  for comparisons between CM and KO at all three times are in columns H-J, respectively and those for comparisons between CS and KO at early and late aggregation in columns K and L, respectively. GtaC binding scores  at all three time points are in columns M-O respectively and GtaCC-S binding scores at the latter two time points in columns P and Q respectively.Set of putative GtaC target genes. This file contains genes bound by GtaC and the set of putative GtaC target genes (multiple data sheets). Column A refers to the unique identifier as defined by dictyBase (Gene ontology (GO) analyses results for GtaC-targets by time point. This files includes gene ontology (GO) analyses results for GtaC-targets by time point (multiple data sheets). Each data sheet contains results on gene sets at starvation sensing (S), early aggregation (E), late aggregation (L), S & E, S & L, E & L and S & E & L, respectively. The unique GO identifier (‘GO.ID'), a short description of the GO term (‘Term'), the number of genes in the genome with that annotation (‘Annotated'), the number of genes in the chosen gene set with the same annotation (‘Significant'), the GO term category (‘GO category') along with all the relevant statistical parameters  are shown for each gene.


One of the most important steps is accurate impression making for fabrication of fixed partial denture.The two different putty-wash techniques that are commonly used are: (1) Putty-wash one-step technique, (2) putty-wash two-step technique.A uniform wash space is needed for an accurate impression. Nissan et al recommended the use of two-step technique for accurate impression making as there is uniform wash space for the light body material to polymerize.The aim of the present study was to compare the accuracy of stone casts obtained from different putty-wash impression techniques using various spacer thickness.The critical factor that influences the accuracy of putty-wash impression techniques is the controlled wash bulk which is absent in one-step putty-wash impression technique and with polyethylene spacer was used.How to cite this article: Chugh A, Arora A, Singh VP. Accuracy of Different Putty-Wash Impression Techniques with Various Spacer Thickness. Int J Clin Pediatr Dent 2012;5(1):33-38. One of the most important steps is accurate impression making for fabrication of fixed partial denture. The two different putty-wash techniques that are commonly used are: (1) putty-wash one-step technique, (2) putty-wash two- step technique. A uniform wash space is needed for an accurate impression. Nissan et al recommended the use of two-step technique for accurate impression making as there is uniform wash space for the light body material to polymerize..Putty acts as a tray for wash material. Light body being less viscous has good flow to record the fine details resulting in an accurate impression. An accurate impression produces the stone casts with minimal dimensional change in regard to the vertical and horizontal dimension between the prepared abutments. Clinical success of fixed prosthodontic procedure is dependent upon the dimensional accuracy of elastomeric impression material and impression proceduresTo compare the accuracy of various impression techniques made with putty-wash impression material.To determine the effect of wash space on the accuracy of impressions made with different techniques.Clinical recommendations based on study and observation.In the present study, putty-wash impression techniques with different spacer thickness of 1 and 2 mm and polyethylene spacer has been used. The two putty-wash impression techniques that have been compared for dimensional accuracy are one-step and two-step procedures.Master model, containing three complete crown fixed partial denture abutment preparations.Six metal copings, three each of 1 and 2 mm thickness.Polyethylene separating sheets.Perforated metal tray.Addition silicone impression material.  (easy putty and light-bodied polyvinyl siloxane).Tray adhesive .Die stone .Debubblizer (Dentofill).Vaccum mixerAutomatic mixing syringe and dispensing gun (Heraeus Kulzer)VibratorRubber bowlMixing spatulaBase formerStopwatchCoordinate measurement machine  .A metal master model, containing three complete crown fixed partial denture abutment preparations, was fabricated for making the measurements. The abutments were prepared with occlusal taper of 6° and two perpendicular cross grooves on the occlusal surface as reference points for taking measurements.The impressions were categorized into four groups as follows:Group I: One-step technique in which putty and wash impression materials were used simultaneously and the casts obtained from them were categorized as group I casts  and mean of master model (mmm) divided by mean of master model multiplied by 100 was expressed as percentage deviation from master model for each impression technique of each measurement location:Percentage of deviation = (msm – mmm)/mmm × 100All the measurements obtained for all four groups were tabulated and statistically analyzed .The aim of the present study was to compare the accuracy of stone casts obtained from different putty/wash impression techniques using various spacer thickness  to 4.The critical factor that influences the accuracy of putty- wash impression techniques is the controlled wash bulk which is absent in one-step putty-wash impression technique and with polyethylene spacer was used.The above results showed that when stone casts and master model were compared, the vertical distance (intra- abutment) of the stone dies decreased, whereas horizontal distance (interabutment) increased.In the present study, the controlled wash space is essential for accuracy of putty-wash impressions. The controlled wash space was provided by uniform spacer thickness of 1 and 2 mm. The uncontrolled wash bulk was seen in one-step impression technique and two-step impression technique with polyethylene spacer.The results of present study do not agree with Hung et al and Idris et al. Hung et al and Idris et al investigated the importance of impression techniques and reported that impression accuracy is not technique dependent.Based on the observation of the present study, two-step putty-wash technique with 1 and 2 mm spacer thickness is more acceptable and viable alternative to obtain accurate impressions. The clinical implication of the present study is that to achieve a controlled wash bulk, temporary crowns can be used to create the desired wash space in the putty impression. Further investigation is needed to determine the exact amount and technique of achieving wash space that is essential for accuracy in using two-step putty/wash impression techniques in conjunction with polyvinyl siloxane impression materials. Study can also be undertaken for dimensional accuracies in long-span bridges.A two-step technique with uniform and controlled wash space is recommended for the fabrication of stone dies which will result in precise fitting of the restoration.The two-step putty-wash technique with 1/2 mm spacer thickness produced casts within accepted clinical range. The one-step and two-step with polyethylene spacer produced the most uneven dimensional changes.The clinical implication of this study will be to use temporary crowns to create controlled wash space.


Military Medical Research. This review provided a valuable overview of this major scientific advance. It has been four years since the first publication of the CRISPR-Cas9 breakthrough . The use of this technique has expanded into various scientific areas and is being developed into a systematic technical platform that may contribute to many bioengineering fields involving DNA sequence editing.In May 2015, Professor Xiao Yang authored a review on the development of CRISPR-Cas9 techniques in the journal of  Dear editor,Military Medical Research [In May 2015, Professor Xiao Yang authored a review on the development of CRISPR-Cas9 technique in the journal of The advantages of the CRISPR-Cas9 technology include its economy, high efficiency, precise targeting and flexible technical extension compared with traditional DNA sequence modifying measures such as transcription activator-like effector nuclease  effector nucleases) . The pubThe CRISPR-Cas9 technology does have limitations associated with targeting ability. Several off-target mutations have been detected by genome-sequencing due to its high specificity. This prohibits its potential use in correcting disease-associated mutations . The rapWhile we are joyfully celebrating the progress brought to science by the development of CRISPR-Cas9, we must use caution in applying this technology.


Centrocercus urophasianus) are sensitive to energy development and likely serve as an effective umbrella species for other sagebrush-steppe obligate wildlife. We assessed the response of birds within an energy development area before and after the implementation of mitigation action. Additionally, we quantified changes in habitat distribution and abundance in pre- and post-mitigation landscapes. Sage-grouse avoidance of energy development at large spatial scales is well documented. We limited our research to directly within an energy development field in order to assess the influence of mitigation in close proximity to energy infrastructure. We used nest-location data (n = 488) within an energy development field to develop habitat selection models using logistic regression on data from 4 years of research prior to mitigation and for 4 years following the implementation of extensive mitigation efforts . The post-mitigation habitat selection models indicated less avoidance of wells (well density β = 0.18 ± 0.08) than the pre-mitigation models (well density β = -0.09 ± 0.11). However, birds still avoided areas of high well density and nests were not found in areas with greater than 4 wells per km2 and the majority of nests (63%) were located in areas with ≤ 1 well per km2. Several other model coefficients differed between the two time periods and indicated stronger selection for sagebrush  and less avoidance of rugged terrain . Mitigation efforts implemented may be responsible for the measurable improvement in sage-grouse nesting habitats within the development area. However, we cannot reject alternative hypotheses concerning the influence of population density and intraspecific competition. Additionally, we were unable to assess the actual fitness consequences of mitigation or the source-sink dynamics of the habitats. We compared the pre-mitigation and post-mitigation models predicted as maps with habitats ranked from low to high relative probability of use . We found more improvement in habitat rank between the two time periods around mitigated wells compared to non-mitigated wells. Informed mitigation within energy development fields could help improve habitats within the field. We recommend that any mitigation effort include well-informed plans to monitor the effectiveness of the implemented mitigation actions that assess both habitat use and relevant fitness parameters.Growing global energy demands ensure the continued growth of energy development. Energy development in wildlife areas can significantly impact wildlife populations. Efforts to mitigate development impacts to wildlife are on-going, but the effectiveness of such efforts is seldom monitored or assessed. Greater sage-grouse ( Thus, globally, 87% of terrestrial lands are unprotected and candidates for potential development. Intelligently designed mitigation is the best option to support wildlife population persistence in areas were development will occur.Human-wildlife interactions are one of the primary issues in wildlife ecology and management. Human impacts to the environment are experienced globally  and estiOn-site mitigation involves a hierarchy of avoid, minimize, and restore ,6. BiodiOn-site mitigation suggestions within development areas typically involve techniques aimed at reducing human activity and disturbance. For example, within energy development areas in sagebrush steppe habitats, human use can result in both direct habitat loss and functional habitat loss as animals may avoid habitat in close proximity to infrastructure . HabitatCentrocercus urophasianus; hereafter sage-grouse) are an excellent species to assess the influence of on-site mitigation for a number of reasons. First, sage-grouse are a year-round resident species across sagebrush-steppe habitats in North America, and co-occur with many prime energy development areas. Second, sage-grouse responses to energy development are well documented and the species experiences several negative impacts within energy development areas [Greater sage-grouse  is located in Northeastern Wyoming and has been the site of extensive Coal Bed Natural Gas (CBNG) development since 2000. Research on the sage-grouse population in the region began in 2003. The research documented the impacts of oil and gas development in the region on sage-grouse from 2003 to 2007 ,13 and pProcyon lotor), red fox (Vulpes vulpes) and striped skunk (Mephitis mephitis). It was impossible to isolate the effect of each unique mitigation action because the actions were implemented simultaneously within the development area. One of our objectives was to assess the cumulative effects of the mitigation actions on the abundance and distribution of important sage-grouse habitat within the energy development field. Essentially, we asked the question: did the mitigation efforts implemented produce measurable improvement in sage-grouse nesting habitats within the development area?Sage-grouse are prey to multiple avian predators  and power lines and tall structures can provide perches for these predators. Additionally, roads and surface disturbance can result in both structural and functional habitat loss for sage-grouse . MitigatWe addressed these questions using a before—after experimental design. We developed nesting habitat selection models for sage-grouse within the development area from 2004 to 2007. This was the pre-mitigation time frame. Therefore, the sage-grouse response to habitat structure characterized by the pre-mitigation habitat selection models represented sage-grouse responses to the pre-mitigated energy landscape. We also developed separate nesting habitat selection models for sage-grouse within the development area from 2008 to 2011. These nests represented the post-mitigation time frame. The sage-grouse response to habitat structure was characterized by the post-mitigation habitat selection models and represented sage-grouse responses to the post-mitigation energy landscape. Mitigation activities were targeted to reduce the impacts of energy infrastructure, including roads. Therefore, we predicted that successful mitigation would result in less avoidance of energy infrastructure and roads. Thus, application of pre- and post-mitigation nesting models to the same landscape should result in a greater abundance of high-quality nesting habitat and the post-mitigation models should predict higher quality habitats in closer proximity to energy infrastructure.2 of which 65% was private, 29% was public land administered by the U.S. Bureau of Land Management (BLM), and 6% was Wyoming state land. Seventy four percent of the study area, including most of the private surface, contained federally owned mineral rights that were under the jurisdiction of the BLM. Cattle and sheep ranching were the primary agricultural uses and energy development, in the form of CBNG, was the primary energy extraction activity occurring in the study area. At the end of the study (2011) approximately 2548 CBNG wells were established in the study area. The study area was within the Great Plains Sage-Grouse Management Zone [Our study area was in the Powder River Basin and primarily in Johnson County with the northern portion extending slightly into Sheridan County, Wyoming, USA . Plains silver sagebrush (Artemisia cana cana) was present but at much lower abundance and was largely limited to drainage corridors. The understory was composed of native and nonnative grasses and forbs including, western wheatgrass (Pascopyrum smithii), thickspike wheatgrass (Elymus lanceolatus), prairie junegrass (Koeleria macrantha), Sandberg bluegrass (Poa secunda), blue grama (Bouteloua gracilis), Japanese brome (Bromus japonicus), cheatgrass (B. tectorum), scarlet globemallow , field sagewort (Artemisia campestris), curlycup gumweed (Grindelia squarrosa), milkvetch , and sweetclover . Conifers, primarily mountain juniper (Juniperus scopulorum), were not abundant in the study area and when present were concentrated along draws.The climate in the study area was semi-arid and characterized by long cold winters and short summers. Monthly average temperatures ranged from 21.6°C in the summer to—5.8°C in the winter. Annual precipitation averaged 33 cm to 43 cm and average annual snowfall ranged from 84 cm to 170 cm. The majority of the study area was shrub-steppe habitat dominated by Wyoming big sagebrush  2008, 2009, 2010, and 2011 and late summer (September) 2009 and 2010. Females were captured using a rocket-net  and a COWe located sage-grouse on the ground using hand-held receivers and 3-element Yagi antennas. We used ground telemetry to monitor radio-marked females through the nesting period (May–June). Nests were confirmed by two consecutive visits that identified the radio-marked grouse using the same shrub or by visually observing the female on a nest with binoculars. We monitored the status of the nests every 2–6 d until the conclusion of the nesting effort. We monitored the nests from a distance of ≥20 m using binoculars or by triangulating to the nest location using radio telemetry in order minimize stress to the female . We retrhttp://www.spatialecology.com/gme). We calculated summary statistics for mean sagebrush cover, mean bare ground cover, mean topographic wetness index (TWI) [http://services.arcgisonline.com/ArcGIS/rest/services/World_Imagery/MapServer). World imagery allowed us to visually confirm sagebrush stands, individual conifer and deciduous trees, and CBNG well structures. GIS data layers were created and processed at a 30-m cell resolution unless stated otherwise.Geographic information system (GIS) data were processed with ArcGIS 10.1 and 10.2 , and Geospatial Modeling Environment  (http://ex (TWI) , vector 2 as in other studies of sage-grouse habitat selection [We developed spatial predictor variables at scales known to be important predictors of sage-grouse habitat selection . We wantelection ,32,33. Eelection ,24,32–342) and 0.564 km radii (1 km²). The finer scale (0.335 km radii) was based on the median distance between consecutive year nests of individual sage-grouse in our post-treatment sample (2008–2011). Moreover, earlier research in the Powder River Basin, Wyoming demonstrated that a similar scale (0.35 km radii) was predictive of nest-site selection [Modeling distribution or occurrence of wildlife can be highly sensitive to scale . Therefoelection . The lare 2008–20. Moreoveelection  that fouArtemisia spp.; [Sage-grouse consistently demonstrate strong selection for sagebrush cover across life-stages and regions ,13,36,37ia spp.; ; Table 1http://www.landfire.gov/, assessed 02 August 2013).We included the predictor variable conifer (primarily mountain juniper) as previous research in the PRB demonstrated influences of conifer stands on nest site selection . ConiferOil and natural gas wells and associated development can affect sage-grouse habitat use ,14,34,39We classified all wells within the study area as either mitigated or unmitigated. We determined whether a well had been mitigated by identifying the producer as APC and within the APC set separating out wells that were drilled after the 2007 field season, when mitigation was implemented. We confirmed the accuracy of mitigated and unmitigated wells with the as-built POD maps.Road development in sagebrush habitat results in direct habitat loss and fragmentation ,41 and hWe compiled topographic wetness index , and VRMIt is important to identify biologically relevant areas that are available for selection by individuals when developing habitat selection models . The seli.e., pre- and post-mitigation) to select the best fit spatial scale, and to assess whether a quadratic term was appropriate for certain variables. We calculated selection ratios as the observed count/expected count across 10 quantiles and plotted these ratios across the range of the variable. We assessed the value of including a quadratic term to the variable if inspection of the selection-ratio graph suggested a quadratic relationship, or it made sense biologically ,. We used Akaike Information Criteria (AIC) to compare models with the same descriptive variable to that of a null logistic regression model. The variable was excluded from all subsequent analyses if the variable was not more predictive than the null model. We selected the best fit term for each variable and then calculated the correlation among variables using pairwise Pearson’s correlation coefficients. Highly correlated variables (r ≥ |0.60|) were not included in the same model. We used the resulting variables to fit global multivariate models to develop pre- and post-mitigation models which we used to generate spatial predictions.We modeled sage-grouse habitat use with a use-availability design in an RSF  paradigmWe evaluated models by partitioning the data using k-fold cross validation with five folds for each model . We iterWe developed two global nesting models using the data and methods outlined above. The first model was developed using only pre-mitigation nesting locations to assess the impact of mitigation on abundance and distribution of nesting habitat. This model characterized the distribution and abundance of nesting habitat within the study area prior to mitigation. Model coefficients thus represented estimated relationships in the pre-mitigated landscape. This model characterized sage-grouse response to the landscape  prior to mitigation. The second global model was developed using only post-mitigation nest data and hence represented the distribution and abundance of nesting habitat within the study area after mitigation efforts had been implemented. The post-mitigation model coefficient estimates thus represent the relationship among nesting habitat use and the predictor variables post-mitigation and characterize sage-grouse response to landscape features post-mitigation. The pre-mitigation model and the post-mitigation model applied to the same landscape should produce different distribution and abundance of nesting habitat. All model characteristics were kept similar in the development of the two models to allow direct comparison. For example, the same predictor variables at the same scales were used to develop each model and the study site boundaries and available habitat were the same between the two models.We visualized the model results for key habitat covariates through estimation of the marginal effects for our global models for each time period. We used the margins command in STATA ver. 12.0. We calculated predictive margins across the range of each variable in our datasets. The margins predictions were developed from non-standardized model covariates to facilitate comparison and for ease of interpretation. This allowed us to assess the changes in the probability of use over the range of a covariate.2. Therefore, these areas were assigned null values in our final maps. We summarized the change in value in proximity of all mitigated and unmitigated well locations.We applied the pre-mitigation and post-mitigation models to the current landscape within the study area. We overlaid the equal-area-binned models and calculated the change in classification for each pixel by subtracting the bin value of each pixel in the post-mitigation model from the bin value of each pixel in the pre-mitigation model. For example, if a pixel were classified as a 3 in the post-mitigation model and a 1 in the pre-mitigation model, it was assigned a value of 2. Thus, the range of potential pixel values on the overlaid change maps ranged from -3 to 3. We did not predict to cells with well densities higher than those in our models when we applied our models to the landscape. We did not want to predict beyond the extent of our data . Sage-gr2 of nests was 11.5% ± 1.6  scale, with the exception of roads and VRM  were located in areas with ≤ 1 well per km2. Selection for sagebrush also differed between the two time periods with stronger selection  in the post-mitigation period. This indicates that nests were consistently located in areas with higher sage-brush cover. There are several possible explanations for this pattern. First, the quantification of availability can affect coefficient estimates. For example, in areas where sagebrush is less abundant (as estimated by the available locations) birds tend to show stronger selection for sagebrush [Mitigation efforts seemed to produce measurable improvement in sage-grouse nesting habitats within the boundaries of a development area based on the strength of model coefficients and the observed changes in the distribution of preferred nesting habitats. Sage-grouse response to certain habitat covariates changed between the pre and post time periods. The model coefficients demonstrated less avoidance of wells in the post-mitigation models than pre-mitigation. This suggests that mitigation efforts within the study resulted in less avoidance of wells overall. However, sage-grouse still avoided areas of high density wells. No nests were found in areas with greater than 4 wells per kmagebrush . The sagagebrush  and if dPopulation density can also lead to competition and competitive exclusion from the highest quality habitats . Sage-grwithin energy development fields. Additionally, the roads and well pads within our study extent are developed in some of the best habitat. However, within mitigated development, one of the mitigation efforts focused on avoiding the highest quality sagebrush habitats when siting infrastructure. Energy development can have negative impacts on sage-grouse and birds tend to avoid high density energy developments [The extent of a study area—the population of interest—can strongly influence conclusions regarding the functioning of the system. The location of study boundaries influences our understanding of species ecology and can have profound management implications. We intentionally limited our study extent. We wanted to examine sage-grouse habitat selection within an energy development field to assess the localized impacts of mitigation efforts. Our results represent habitat selection behavior lopments –11,13,52Not all wells within the study area were the subject of mitigation efforts. Our post-mitigation models contained 17% mitigated wells in 2011. It is possible the limited number of mitigated wells may have masked the potential positive impacts of oil and gas mitigation. This suggests that even limited mitigation can have positive influence on the habitat use of sage-grouse. It is likely that mitigation of the additional 83% of well pads would have lead to even greater changes in the habitat selection behavior of sage-grouse between the pre and post mitigated landscapes.This study focused on sage-grouse, but multiple other species can be negatively affected by energy development –55. The Our results suggest that mitigation can affect the habitat selection behavior of sage-grouse within an energy development field. However, habitat is linked with a species fitness components that drive population dynamics and additional work must assess the actual fitness consequences of mitigation. We documented shifts in habitat use, but were unable to address the fitness consequences of these shifts or the source-sink dynamics of the habitat. Future studies that consider the source-sink dynamics of habitat use and potential changes in response to mitigation would be beneficial. It is possible that areas within energy development could be partially mitigated for the use of habitat, but the fitness landscape may not be mitigated, thus potentially resulting in a habitat sink. Previous work has shown that nest and brood , and yea


In vivo and in vitro data support that cardiac fibrosis may be consequent to macrophage Toll-like receptor (TLR) signaling following ligation of the ssRNA complexed to the Ro protein. TLR signaling and fibrotic endpoints may be abrogated by chloroquine (CQ), which inhibits endosomal acidification. This in vitro observation was initially "translated" to patients by evaluating the use of hydroxychloroquine (HCQ) in an extensive case-control retrospective chart review of anti-Ro/La Ab exposed fetuses of mothers with SLE enrolled in three databases. This approach was followed by another study that addressed whether HCQ use reduces the expected recurrence rate of CHB. The collective results were highly encouraging.One of the strongest clinical associations with autoantibodies (Ab) directed to components of the SSA/Ro-SSB/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring. Fetal disease is independent of maternal disease and often anti-Ro Ab are first sought only because CHB has been identified. The first-time risk of 2% is 10-fold higher in women who have had a previous CHB child. Tissue injury in the fetus is presumed to be dependent on the FcγR-mediated transplacental passage of maternal IgG Ab. Despite attempts of large multicenter studies to forestall disease by careful monitoring, irreversible block and extensive myocardial injury have been documented within 7 days of a normal rhythm and PR interval. Based on these studies we initiated an open-label prospective study design using Simon's two-step approach. HCQ at 400 mg is initiated by 10 weeks following conception. Serial echocardiograms  and evaluation of maternal and cord blood biomarkers  are part of the protocol to address maternal compliance, pathobiology and efficacy. The first stage has been completed with 19 subjects and only one recurrence of CHB.Over the next 4 years, 35 subjects will be enrolled. Ultimately, HCQ will be considered efficacious for the prevention of CHB if fewer than six cases occur among a total of 54 subjects evaluated. A positive result will probably change the management of all anti-Ro-positive women who have had a previous child with CHB and illustrate the importance of translational science. Perhaps most relevant to impact, a potential prevention would justify screening of all pregnant women for anti-Ro antibodies, particularly relevant since mothers of affected children are frequently asymptomatic, a point critical to early pregnancy counseling.


The health of doctors who work in primary care is threatened by workforce and workload issues. There is a need to find and appraise ways in which to protect their mental health, including how to achieve the broader, positive outcome of well-being. Our primary outcome was to evaluate systematically the research evidence regarding the effectiveness of interventions designed to improve General Practitioner (GP) well-being across two continua; psychopathology  and ‘languishing to flourishing’ . In addition we explored the extent to which developments in well-being research may be integrated within existing approaches to design an intervention that will promote mental health and prevent mental illness among these doctors.Medline, Embase, Cinahl, PsychINFO, Cochrane Register of Trials and Web of Science were searched from inception to January 2015 for studies where General Practitioners and synonyms were the primary participants. Eligible interventions included mental ill-health prevention strategies (e.g. promotion of early help-seeking) and mental health promotion programmes . A control group was the minimum design requirement for study inclusion and primary outcomes had to be assessed by validated measures of well-being or mental ill-health. Titles and abstracts were assessed independently by two reviewers with 99 % agreement and full papers were appraised critically using validated tools.Only four studies  from 5392 titles met inclusion criteria. The studies reported statistically significant improvement in self-reported mental ill-health. Two interventions used cognitive-behavioural techniques, one was mindfulness-based and one fed-back GHQ scores and self-help information.There is an urgent need for high quality, controlled studies in GP well-being. Research on improving GP well-being is limited by focusing mainly on stressors and not giving systematic attention to the development of positive mental health.The online version of this article (doi:10.1186/s12875-016-0431-1) contains supplementary material, which is available to authorized users. How this fits in: This review has identified a research gap in terms of mental health promotion and disease prevention interventions aimed at GPs especially those that focus on improving the positive or ‘flourishing’ concept of well-being.Within healthcare systems the degree to which they are based on a primary care model relates positively to the delivery of efficient, cost-effective and high quality care . HoweverA more pathogenic work environment appears to be developing for a population already known to be at risk of mental ill-health including burnout, depression and addiction –14. GiveThere is a paucity of evidence on mental ill-health prevention in GPs and reviews of occupational well-being interventions have reported few studies in those working in primary care , 16. A bOver the past decade consensus has been emerging from leading well-being researchers as to what constitutes optimum mental health. Former advocates of either a hedonic (pleasure seeking/happiness) perspective or a eudaimonic (meaning/functioning) perspective now recognize the requirement to incorporate elements from both to capture the construct of optimum well-being or ‘flourishing’ –24. ThisThere remains debate about constituent elements within flourishing. Our detailed discussion of this and the operational definition of well-being developed for the purpose of this review concluded that ‘it is a multidimensional construct that comprises the core dimensions of (i) positive affect, (ii) personal relationships and social engagement and (iii) a life view that is meaningful and optimistic’ .This is the first systematic review of studies of interventions across and within the two continua of ‘mental illness to absence of mental illness’ and ‘languishing to flourishing’. This comprehensive model of well-being is best suited to our combined mental health promotion and mental illness prevention approach. The review aims to evaluate the research evidence regarding the effectiveness of interventions designed to improve GP well-being with either a mental ill health focus or a positive mental health focus or both. This comprehensive approach facilitates exploration of the extent to which research developments and reviews in positive psychology and organizational studies may be integrated within existing health to illness approaches to promote ‘flourishing’ among GPs.The review followed the methodology specified in our PROSPERO-registered protocol and conforms to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines .A specialist subject librarian assisted in the development of a search strategy designed to identify internationally recognised terminology in peer-reviewed journals. Full details of this strategy are available in the published protocol .A scoping review informed the selection of databases. Six databases were searched from inception until January 2015: Cochrane Register of Trials, MEDLINE, EMBASE, PsycINFO, CINAHL and Web of Science. Only studies published in English language peer-reviewed journals were eligible. This decision was made at the protocol stage due to concerns about potential heterogeneity in constructs across languages following operationalisation of the term ‘well-being’. (In practice the paucity of evidence identified did not merit such stringent parameters).As we aimed to evaluate research evidence for the effectiveness of GP well-being interventions a control group was the minimum design requirement for a study to be included and only studies  in which GPs were the primary participants were eligible.In recognition of various perspectives on well-being eligible interventions included ‘distal’ or ‘proximal’ approaches to well-being improvement. Distal-level interventions  comprised, for example, strategies that promoted protective factors including the development and application of personal strengths and psychological capacities. Examples of proximal interventions  included efforts designed to promote early help-seeking behaviours, raise mental health awareness and address stigmatisation. In addition to an operational definition of well-being the protocol provided a process to resolve potential disputes in this context regarding the eligibility of interventions.Primary outcome measures included validated tools that measured either mental illness such as the General Health Questionnaire (GHQ)  or positStudies designed to improve patient management by increasing GP knowledge and clinical skills enhancements were ineligible as were studies of interventions (such as rehabilitation and return-to-work programs) that were delivered at a tertiary level to GPs recovering from mental ill-health. Consensus between reviewers  on eligibility across all criteria was attained without arbitration from the third (MD).In a two stage study selection process titles and abstracts were assessed independently by two reviewers (MM and LM). The study selection pilot identified 99 % agreement. The third reviewer (MD) provided additional quality control by screening 10 % of the titles during Stage 1 of the selection process. In Stage 2 full texts of studies appearing to meet inclusion criteria were independently assessed by two reviewers (MM and LM) to ascertain eligibility. Reasons for excluding studies were recorded and tabulated  following detailed descriptive tabulation. The eligible studies were not considered to be of sufficiently good quality and fit to conduct a meta-analysis.Following de-duplication 5392 studies were screened. Of the thirty-three studies that were assessed at Stage 2 twenty-nine were excluded. Main reasons for exclusion at this stage included lack of intervention and uncontrolled study design. Four studies met the methodological and design criteria for inclusion.   compriseTwo studies used the GHQ-12  as the pInterventions in both Gardiner et al. studies had a cognitive-behavioural basis. They were delivered in different formats. Cognitive-behavioural stress-management techniques were taught in 15-h over 5 weeks in Gardiner et al. (2004) while in Gardiner et al. (2013) GPs were group coached in cognitive-behavioural techniques addressing issues such a coping skills and time management during a 9-h ‘retreat’ with 5-6 weeks individual follow-up via e-mail. In contrast to that approach Holt and Del Mar used baseline GHQ results to develop a mailed brief, individually-tailored guide that interpreted GHQ scores to increase awareness about mental health risk among GP intervention ‘cases’ and provide them with self-help advice. Awareness also underpinned Asuero et al’s mindfulness based education programme. This was modelled on an earlier uncontrolled study set in primary care that reported evidence of decreased burnout and mood disturbance using mindfulness-based stress reduction principles . Asuero The interventions focussed on self-awareness and amelioration of stress response consistent with a mental illness prevention approach. There were not any studies identified that appeared to be designed to promote positive mental health, flourishing. All studies reported statistically significant short-term improvement in psychological distress. Risk of bias was rated high in 4 categories for both Gardiner studies and in 2 categories for the remaining studies. Table Our review aimed to evaluate systematically the research evidence regarding the effectiveness of interventions designed to improve GP well-being with either a mental ill health focus or a positive mental health focus or both and to explore the nature and extent to which research developments in positive mental health may be integrated within existing ‘illness to health’ approaches to promote ‘flourishing’ among GPs. It identified a paucity of evidence across the mental ill-health continuum and no studies specifically designed to effect change within the positive mental health continuum. The focus was mainly on mental illness prevention rather than mental health promotion. All studies were assessed as high risk of bias using the Cochrane Risk of Bias tool. The Quality Assessment Tool for Quantitative Studies (recommended for use in non-randomised intervention studies ) deemed The findings reported in the four included studies suggest that cognitive-behavioural-based and mindfulness–based programmes delivered in a group format may reduce GP distress at least in the short-term. Increasing awareness generally and with specific regard to thoughts, beliefs, self-care, personal health and setting boundaries appeared to improve GP mental health. Potential mechanisms include the support afforded by professional peer-groups; cognitive-behavioural techniques that address emotional distress by modifying ‘maladaptive’ thoughts and thought patterns  and stren = 21) and organizational changes (n = 20). The review found low-moderate quality evidence for both physical relaxation (e.g. massage) and mental relaxation (e.g. mindfulness) - stress levels were reduced by 23 % compared to controls. Although intervention heterogeneity precluded precise identification of potentially active ingredients, these results suggest that approaches that address cognitions and relaxation techniques merit further study.The development of potentially effective well-being interventions for GPs currently requires exploration of evidence within other occupational groups. A recent Cochrane review of occupational stress interventions for healthcare workers  found that cognitive-behavioural training (approximately one third of the 58 studies) had relatively poor impact reducing stress by only 13 % compared to no intervention over periods from one month to two years . Only 5 Further evidence of the potential benefit of a mindfulness-based approach was identified in an additional study which met all our inclusion criteria except the English language restriction as it was reported in Spanish. It was included in a meta-analysis which found that cognitive, behavioural and mindfulness-based interventions significantly reduce stress in doctors . This RCElucidating ‘what works’ to improve doctor well-being is difficult due to the paucity of studies. Comparisons between, for example, medical students and experienced clinicians, physicians and nurses, and primary- and secondary-care doctors provide only limited insights due to pre-existing significant differences. Arbitrary categorisation of intervention type, relatively small sample sizes and simple study designs makes it difficult to achieve clarity and certainty regarding essential active ingredients and mechanisms of effect across various intervention approaches. Whilst there is scope within e.g. mindfulness approaches for mental health promotion the emphasis in healthcare professional well-being interventions appears to be on mental illness prevention (psychopathology continuum). There is negligible evidence within this population for interventions designed to empower and enable optimum mental health through the development of personal resources thereby promoting flourishing.The creation of empowering work environments through organisational-change interventions has received even less research attention than person-directed interventions . Organizational approaches to mental health promotion include enhancing the flexibility of working hours , 46, impThe aforementioned Cochrane review identified 21 study arms examining the effect of organizational change in preventing occupational stress in healthcare workers. These included changing working conditions, provision of support and mentoring, communication skills training and improving work schedules. Shorter or interrupted work schedules were found to decrease stress levels however no clear benefit of other interventions was identified. They concluded that organizational interventions should be more focussed on addressing specific stressors .Empirical evidence for organizational approaches is limited and often includes individual-based approaches. In a review of ‘burnout prevention’ interventions for various occupational groups, only 2/25 interventions were organizational in nature and focus . CognitiResilience enhancement is common to both organizational and person-directed interventions. Furthermore it can be integral to both mental health promotion and mental illness prevention programmes. Definition of resilience and specification of what constitutes resilience training remain topics of considerable debate –55. A reResearch on improving GP well-being is limited by focusing mainly on stressors and not giving systematic attention to aspects of well-being such as positive affect, engagement and optimism. The application of interventions to promote flourishing - so-called positive psychology interventions (PPIs) - in occupational groups is under-researched.The (only) systematic review of PPIs in organizations found that 13/15 had positive effects across 29 measures of well-being including positive emotions, optimism, resilience and life satisfaction   The onlyShifting from the deficit approach that underpins stress response amelioration towards a more proactive mental health promotion approach that empowers and enhances work and personal resources may prove to be more effective in and appropriate to our population of interest , 66.This review is limited by an English language restriction applied at protocol stage to address potential heterogeneity in well-being terminology and constructs. However, it was conducted using robust methodology and identified a substantial research gap. It is the first review to evaluate an extensive body of research pertinent to the optimisation of well-being in GPs.The evidence base in this area is limited. There is a clear need for pragmatic randomised controlled trials using validated assessments of the positive construct of well-being to identify strategies that will help safe-guard the mental health of doctors working in primary care.


A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers. Tissues obtained from surgeries or diagnostic procedures are most commonly preserved in formalin-fixed paraffin-embedded (FFPE) tissue blocks. These tissues are fixed in formalin and processed as paraffin-embedded tissue blocks. The embedding process preserves the cellular morphology and allows tissues to be stored at room temperature, causing FFPE fixation to be used by many tissue banks and biorepositories It is well documented that malignant transformation and cancer progression result in fundamental changes in the glycosylation patterns of cell surface and secreted glycoproteins One potential approach to assess glycan changes in tissues is matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). This technique has been used to directly profile multiple protein In this report, we describe the application of MALDI-IMS glycan imaging to various formalin-fixed tissues. Formalin-fixed mouse kidney tissues were used to optimize antigen retrieval, PNGaseF digestion and glycan detection conditions for MALDI-IMS. This was followed by N-glycan analysis of clinical FFPE tissue blocks from prostate and pancreatic cancers, as well as a commercial tissue microarray of hepatocellular carcinoma (HCC). Glycan identity was confirmed by on-tissue collision-induced dissociation (CID) and off-tissue permethylation analysis. An optimized MALDI-IMS workflow is presented that allows routine simultaneous analysis of 30 or more glycans per FFPE tissue, including TMA formats. The approach is amenable to any FFPE tissue, and represents an additional molecular correlate assay for use with the TMA format. Furthermore, depending on the construction of the TMA and targeted tumor type, the approach has the potential to identify novel glycan biomarker panels for cancer detection and prognosis. To our knowledge, this represents the first instance of using MALDI-IMS to profile N-glycans in FFPE tissue blocks or TMAs.Flavobacterium meningosepticum was expressed and purified as previously described The glycan standard NA2 was obtained from ProZyme . Trifluoroacetic acid, sodium hydroxide, dimethyl sulfoxide, iodomethane and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Sigma-Aldrich . HPLC grade methanol, ethanol, acetonitrile, xylene and water were obtained from Fisher Scientific . ITO slides were purchased from Bruker Daltonics  and Tissue Tack microscope slides were purchased from Polysciences, Inc . Citraconic anhydride for antigen retrieval was from Thermo Scientific . Recombinant Peptide N-Glycosidase F (PNGaseF) from All human tissues used were de-identified and determined to be not human research classifications by the respective Institutional Review Boards at MUSC and Van Andel. Mouse kidneys were excised from euthanized C57BL/6 mice and immediately placed in 10% formalin prior to processing for routine histology and paraffin embedding. Mice were housed in an Institutional Animal Care and Use Committee-approved small animal facility at MUSC, and tissues obtained were harvested as part of approved projects unrelated to glycan tissue imaging. A liver TMA was purchased from BioChain consisting of 16 cases of liver cancer in duplicates, and one adjacent non-tumor tissue for each case. Tissues were from 14 male and two female patients with an average age of 47.5 with a range of 33 to 68 years old, with additional information provided in Tissue and TMA blocks were sectioned at 5 µm and mounted on positively charged glass slides measuring 25×75 mm, compatible with the Bruker slide adaptor plate. The slides were heated at 60°C for 1 hr. After cooling, tissue sections were deparaffinized by washing twice in xylene (3 minutes each). Tissue sections were then rehydrated by submerging the slide twice in 100% ethanol (1 minute each), once in 95% ethanol (one minute), once in 70% ethanol (one minute), and twice in water (3 minutes each). Following the wash, the slide was transferred to a coplin jar containing the citraconic anhydride buffer for antigen retrieval and the jar was placed in a vegetable steamer for 25 minutes. Citraconic anhydride (Thermo) buffer was prepared by adding 25 µL citraconic anhydride in 50 mL water, and adjusted to pH 3 with HCl. After allowing the buffer to cool, the buffer was exchanged with water five times by pouring out ½ of the buffer and replacing with water, prior to replacing completely with water on the last time. The slide was then desiccated prior to enzymatic digestion. Tris buffer pH 9–10 was also effective, but citraconic anhydride buffer was used for all experiments in this study.www.functionalglycomics.org). Glycan structures were generated by Glycoworkbench An ImagePrep spray station  was used to coat the slide with a 0.2 ml aqueous solution of PNGaseF  as previously described www.functionalglycomics.org).PNGaseF sprayed mouse kidney tissue slides were incubated for 2 hr at 37°C; 50 µL water was applied on top of the tissue and incubated for 20 minutes to extract the released native N-glycans. The water was removed from the tissue, and then concentrated under vacuum by centrifugation. Permethylation was performed as described Glycan standards were spotted on a stainless steel MALDI plate using CHCA matrix and desiccated to yield a homogenous layer. Tissues were prepared as previously described for MALDI imaging of FFPE tissues. 10 spectra of 1000 laser shots with a laser frequency of 1000 Hz were averaged for each spectra provided. The collision energy varied between 60–70V.Mass spectra from TMA tissue Regions of Interest (ROIs) representing each tissue core were exported directly from FlexImaging and analyzed using an in-house workflow. The peak lists were first deconvoluted followed by calculating the mean peak intensity of points in each ROI, resulting in a monoisotopic peak list corresponding to signal intensity in each region. Comparison of tumor versus non-tumor was accomplished with a Wilcoxon rank sum test. Individual peaks were also evaluated to discriminate between tumor and non-tumor using receiver operator characteristic curves.www.functionalglycomics.org), and previous MALDI-IMS glycan imaging data from our laboratory for fresh/frozen tissue analysis Mouse kidney tissues were fixed in formalin and used as an initial model system to develop MALDI-IMS glycan imaging workflows for FFPE tissues. These tissues were chosen due to the availability of reference glycan structures and spectra  TMA (BioChain) consisting of samples from 16 individual patients, with two tumor tissue cores and one non-tumor tissue core per patient . AdditioMultiple N-linked glycans can be directly profiled from FFPE tissue blocks and TMAs while maintaining intact architecture. The basic methodology, which mirrors that of MALDI-IMS analysis of peptides in FFPE tissues and TMAs In general, the peak intensities of PNGaseF-released glycans in the FFPE tissues seems to be more intense than that obtained with fresh/frozen tissue sections. This may be a result of the more extensive heating and washing steps required in the deparaffinization and rehydration steps. It is this increased detection sensitivity that facilitated CID fragmentation of N-glycans directly from the tissue  & S6. UnOne drawback to using FFPE tissues is residual polymer from the paraffin block adjacent to the tissue. Detection of this polymer is more predominant in the lower mass range of the imaging runs, and can overlap with potential glycan masses, complicating detection and further statistical analysis. This polymer can be observed in the average spectra of the mouse kidney tissue after PNGase application  from m/zIn relation to potential cancer diagnostic applications, the most significant aspect to developing a method to image N-glycans on FFPE tissue blocks could be the ability to use TMAs for high-throughput glycan-based experiments. Not only does the method increase the number of tumor samples that can be analyzed in one experiment, but it could also be used to compare the glycans detected in a TMA core versus the larger source FFPE tissue. N-glycan MALDI-IMS of the HCC TMA  is proviCurrently, MALDI-IMS provides a new approach to effectively visualize and evaluate N-glycan localization in tissue sections. It does not solve the known limitations of MALDI analysis of underivatized glycans like loss of sialic acids, nor does it provide anomeric linkage information for N-glycan structure. Established tandem mass spectrometry methods of glycan extraction, modification and fragmentation are more capable of providing this structural information. Combining the glycan tissue maps generated by MALDI-IMS to target regions of interest for further tandem mass spectrometry analysis of glycans could be a new synergistic approach to more effectively identify tumor-associated glycans and glycoproteins in situ. Use of other glycosidases like sialidase, as we have previously reported Figure S1Panel of Mouse Kidney N-Glycans. Ions detected in the kidney with enzyme application were compared to the control tissue. Ions that were only observed in the tissue following PNGaseF application were compared to the glycans found in the mouse kidney database on the Consortium for Functional Glycomics. The panel provides the glycan species, the projected mass for the sodium adduct, and our observed mass for the sodium adduct.(TIF)Click here for additional data file.Figure S2Permethylation of Mouse Kidney N-Glycans. Mouse kidney N-glycans were extracted from the imaging slide after PNGaseF application and digestion. Glycans were dried down and underwent permethylation as previously described. The permethylated m/z values were then compared to the permethylation data from the Consortium for Functional Glycomics mouse kidney database .(TIF)Click here for additional data file.Figure S3Panel of Mouse Kidney N-Glycans Linked to Known Glycan Database.(TIF)Click here for additional data file.Figure S4Individual N-Glycans from Prostate Cancer FFPE Tissue. The orange ovals highlight the areas of heterogeneous tumor for high mannose glycans.(TIF)Click here for additional data file.Figure S5CID of N-Glycans from Human Pancreas Tissue I.(TIF)Click here for additional data file.Figure S6CID of N-Glycans from Human Pancreas Tissue II.(TIF)Click here for additional data file.Figure S7Images From Ions Corresponding to N-Glycans. The Ions identified in (TIF)Click here for additional data file.Table S1Patient Data Summary for Hepatocellular Carcinoma TMA from Biochain.(TIF)Click here for additional data file.