Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time.In order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans.We identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFβ) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFβ pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFβ-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin β1. Furthermore, addition of GHK or TGFβ restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.These results demonstrate that gene-expression changes associated with regional emphysema severity within an individual's lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFβ and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression. Chronic obstructive pulmonary disease (COPD) is a significant public health problem worldwide and the third leading cause of death in the United States . It is cet al. used micro-CT scans to quantify the degree of emphysema in different regions of lungs from patients with severe COPD by measuring the mean linear intercept (Lm), a morphological measurement of alveolar destruction [Several groups have profiled gene expression in patients with and without COPD or between patients with varying levels of airflow obstruction in order to understand differences in gene expression related to COPD -11. Whiltruction . In ordeWe identified a number of genes whose expression is associated with increasing emphysematous destruction and found that pathways enriched among these genes were involved in the immune response and tissue remodeling. Using the Connectivity Map (CMap) , we founn = 6) were removed from patients treated for severe COPD by double lung transplantation at the University of Pennsylvania. Donor lungs (n = 2) for which no suitable recipient was identified were released for research use from the Gift of Life Organ Procurement Organization in Philadelphia. This study was approved by the institutional review boards and conforms to the Helsinki Declaration. Written informed consent for use of these specimens and the relevant clinical and radiological data required for this research were obtained from each patient prior to surgery and from the next of kin of the persons whose donated lung was released for research. Each lung was removed from the thorax, cooled to 1.6°C, and transported to the laboratory where the bronchial stump was cannulated [2O. The specimen was then held at a transpulmonary pressure of 10 cmH2 Owhile frozen by liquid nitrogen vapor (-130°C). The frozen specimen had a multidetector CT scan followed by being cut into 2-cm thick slices in the same plane as the CT scan. Tissue samples were collected using a sharpened steel cylinder (cork bore diameter of 14 mm). One sample from a cluster of four core samples of lung obtained from each site was processed for micro-CT [et al.[Single lungs . The number of intercepts between these lines and tissue was counted. Lm was calculated as the total length of the test lines divided by the number of cross-overs with tissue .High molecular weight (mRNA-containing fraction) RNA was isolated from tissue cores using the miRNeasy Mini Kit . RNA integrity was assessed using an Agilent 2100 Bioanalyzer and RNA purity was assessed using a NanoDrop spectrophotometer. RNA (1 μg) was processed and hybridized onto the Human Exon 1.0 ST array  according to the manufacturer's protocol as previously described . ExpressTwo linear mixed-effects models were used to identify gene expression profiles associated with the degree of regional emphysema severity as measured by Lm:1. 2. ij is the log2 expression value for sample i in patient j for a single gene. Slice is a fixed effect controlling for the position within the lung from which the sample core was obtained. The random term εij represents the random error, which was assumed to be normally distributed, αj represents the random effect for patient, and β0 represents the intercept. Model 2 contains an additional fixed effect term for emphysema severity measured by the natural log of Lm. A gene's expression profile was considered associated with Lm if model 2 fit better than model 1 as determined by a significant P-value from a likelihood ratio test between the two models after applying a false discovery rate (FDR) correction. In the immunohistochemistry experiments, these linear models were also used to examine the relationship between Lm and the volume fraction of tissue with positive staining by substituting volume fraction (Vv) for gene expression as a dependent variable. All statistical analyses were conducted using R statistical software v2.9.2 and the nlme package in Bioconductor v2.4 [Genetor v2.4 .Homo sapiens were used as a reference set. For GSEA, genes were ranked by the t-statistic of the βLm coefficient in the linear mixed-effects model and then analyzed for the enrichment of canonical pathways and Gene Ontology term gene sets obtained from MSigDB v2.5.Functional enrichment analysis was performed using DAVID 2008 or Gene Set Enrichment Analysis (GSEA) v2.0.7 ,18. For Lm coefficient in the linear mixed-effects model. Conversely, sets of genes we identified as significantly positively or negatively associated with Lm were examined within gene lists ranked by the degree of differential expression as determined by re-analyzing previously published COPD-or TGFβ-related microarray studies. See Additional file Using GSEA, sets of genes reported to change with COPD-related phenotypes or with TGFβ treatment in other gene-expression studies were examined in a ranked list of genes ordered from most induced in severe emphysema to most repressed in severe emphysema by the t-statistic of the βet al.[1), 3) FEV1/forced vital capacity (FVC), or 4) between cases versus controls in Bhattacharya et al.[et al.[1 or 8) diffusing capacity of carbon monoxide (DLCO) in Spira et al.[1, 10) FEV1/FVC, 11) DLCO, 12) non-smokers versus GOLD2, or 13) non-smokers versus GOLD3 in Wang et al.[et al.[et al.[et al.[et al.[et al.[in vitro studies of the effects of GHK in primary lung fibroblasts, raw data for GHK-treated and control samples were downloaded from the CMap website and normalized using MAS5.0 with the Affymetrix CDF. Genes were ranked by a paired t-test between treatment and controls of different batches and compared to gene sets of GHK and TGFβ treatment using GSEA.In order to find compounds that reverse gene-expression patterns associated with emphysema severity, we generated separate signatures for each COPD or TGFβ gene-expression dataset examined in this study. Signatures were generated by identifying the 50 genes most up-regulated and the 50 genes most down-regulated with respect to a COPD or TGFβ-related phenotype. Each signature was queried against the CMap using the algorithm described by Lamb et al.. See Addya et al.; genes tl.[et al.; genes tra et al.; genes tl.[et al., 15) Clal.[et al., 16) Renl.[et al., 17) Koil.[et al., and 18)l.[et al.. For comLung tissue of former smokers (defined as quitting smoking for at least one year before surgery) with normal lung function or GOLD stage IV COPD was obtained from patients undergoing surgery for resection for pulmonary carcinoma or lung transplantation. Fibroblast cultures were established from parenchymal lung tissue by an explant technique as previously described . IsolateFibroblast cultures at passage 3 were cultured in eight-well chamber slides  in growth medium , penicillin, and streptavidin from Invitrogen, Burlington, ON, Canada). After reaching 70% confluence, fibroblasts were cultured for 24 h in 1% FBS DMEM and then incubated with either TGFβ1 10ng/ml , GHK 10 nM  or control media  for a further 48 h. After stimulations, chamber slides were fixed with 4% paraformaldehyde for 20 minutes, blocked in 10% goat serum in phosphate-buffered saline (PBS) with 0.1% saponin for 1 h and then stained with integrin-β1 antibody  in 0.1% saponin in PBS for 2 h at room temperature. Following washing in PBS with 0.1% saponin and 0.1% Tween 20, secondary antibody conjugated with goat anti-Mouse IgG Alexa Fluor 488 and Phalloidin conjugated with Alexa Fluor 594 were incubated for 2 h at room temperature. Following final washes, cultures were incubated with DAPI 1 ng/ml and then coverslipped with cytoseal. Confocal images were acquired with a Leica AOBS SP2 laser scanning confocal microscope . The images were overlaid and the contrast enhancements were performed on the images using Volocity software™  as previously described .5 cells/500 µl of 1% FBS DMEM, penicillin, streptavidin in duplicate on the collagen gels and cultured for an additional 24 h at 37°C in 5% CO2. The gels were imaged before and after and the extent of gel contraction measured using Image Pro Software.Fibroblast cultures at passage 3 were cultured in six-well tissue culture plates  in growth medium . After reaching 70% confluence, fibroblasts were cultured for 24 h in 1% FBS DMEM and then incubated with TGFβ1 10 ng/ml , GHK 10 nM  or growth media control for a further 48 h. Prior to the end of the treatment time point, a 12-well tissue culture plate was incubated with 1% bovine serum albumin in DMEM for 2 h. The medium was removed and then 500 µl of 0.4 mg/ml type I collagen  was added and allowed to polymerize for 8 h at 37°C. The treated fibroblasts were then trypsinized and seeded at 2 × 10The collagen gels were fixed with 4% paraformaldehyde for 20 minutes and washed in PBS with 0.1% saponin and 0.1% Tween 20 before being incubated with phalloidin conjugated with Alexa Fluor 594 for 1 h at room temp. Gels were then mounted on to a glass slide using Secure-seal™ imaging spacers  and aqueous mounting media. The gels were then imaged using second harmonic generation microscopy to determine fibrilar collagen as previously described . For eacLm was quantified using micro-CT scans in eight samples taken at regular intervals from apex to base of lungs from six subjects that required transplantation for COPD and two organ donors Figure . Table 1P < 0.05; Additional file Using linear mixed-effect models, the expression levels of 127 genes were significantly associated with Lm and thus associated with regional emphysema severity Figure , and theIn order to demonstrate that the 127 gene signature is related to regional emphysema severity within individuals and not to differences between donors and COPD patients or to differences in levels of emphysema between COPD patients, we repeated the same statistical analysis while only including the five COPD patients with emphysema and standardizing the Lm measurements within each patient core to a mean of zero and a standard deviation of one (Z-score). Using GSEA, the sets of up-and down-regulated genes in the 127-gene signature identified in the previous analysis with all eight patients and unscaled Lm measurements were concordantly enriched among genes differentially expressed when only the five emphysema patients were analyzed with Z-scored Lm measurements, indicating that this gene signature is associated with regional emphysema severity , indicating that B cell abundance increases as emphysema severity increases.In order to investigate whether the up-regulation of components of the B cell receptor signaling pathway is associated with a change in the quantity of B cells in lung tissue, we quantified the Vv of CD79A protein, a marker for B cells, in relation to Lm by immunohistochemistry  and SMAD7 , also showed evidence of modest down-regulation while SMAD1 showed evidence of modest up-regulation . To determine whether the TGFβ pathway might be affected by emphysema pathogenesis, we used seven previously published studies that had examined the effect of TGFβ ligands on gene expression to develop a collection of signatures of TGFβ pathway activation [et al.[Several members of the TGFβ pathway were among the genes that had decreased expression as a function of regional emphysema severity. These genes included tivation -23,30,31To further validate these findings, we cultured human lung fibroblasts with and without TGFβ1 and found that the set of genes most induced by TGFβ1 were enriched among genes that decrease in expression with increasing regional emphysema severity  and the COPD-related expression patterns observed in Bhattacharya et al.[et al.[P < 0.05). In addition, the gene expression effects of GHK are similar to the effects of TGFβ treatment observed by Malizia et al.[P = 0.004).In order to identify compounds that might reverse the gene-expression pattern associated with progression of emphysema, we utilized the CMap , a compeya et al. and Golpia et al. . Resolution of damaged tissue requires mesenchymal cells to attach to collagen fibers through integrin-dependent mechanisms and generate mechanical tension via the actin cytoskeleton to promote tissue contraction and wound size reduction. Using distal lung fibroblasts isolated from former smokers with and without COPD, we found that GHK (10 nM), like TGFβ1 (10 ng/ml), induced alterations in integin-β1 localization  were unable to fully contract collagen I gels compared to fibroblasts obtained from former smokers without COPD . These included integrins involved in collagen attachment, such as ITGB1. The goal of this study was to identify gene expression changes associated with regional emphysema severity in order to elucidate biological processes underlying the progression of emphysema and to identify potential COPD therapeutics. By measuring gene expression from regions of varying emphysema severity within the same lung and by using a morphologic measurement of airspace size (Lm), which reflects the degree of alveolar destruction, we were able to identify gene expression changes associated specifically with the emphysematous component of COPD.Interestingly, there was significant enrichment between genes differentially expressed in COPD or associated with worsening lung function in other datasets and those we found to be associated with regional emphysema severity. Importantly, this similarity supports the notion that regional differences in emphysema severity reflect the processes that occur with general COPD pathogenesis and progression and are not only present in patients with end-stage disease. Overall, these observations suggest a similarity in the gene expression alterations that accompany airflow obstruction, gas exchange abnormalities, and alveolar destruction measured by Lm.A common characteristic in the pathology of COPD is progressive lymphocyte infiltration of the small airways and alveolar walls . In addiet al.[TGFβ1 gene that associate with increased risk for COPD [The TGFβ signaling pathway is involved in a variety of cellular processes, including immune response, extracellular matrix remodeling, angiogenesis, and cell differentiation. This pathway has also been implicated in a variety of diseases such as cancer and fibrosis . It has et al. found thet al., SMAD3 [et al., SMAD6 [et al., and SMAet al.,43 have et al.. In normet al.,45. Furtfor COPD -48. In tin vitro, showing that this computational method is a viable approach for identifying novel therapeutics.As COPD remains a major public health concern due to lack of effective therapeutic strategies, we sought to use computational methods to identify compounds that might modulate molecular processes associated with emphysema pathogenesis. The CMap is a large compendium of microarray experiments that measures the effect of over 1,000 compounds on gene expression in several cell lines . By querUsing the CMap dataset, we identified a relationship between the gene expression changes induced by the tripeptide GHK and those that are repressed with increasing emphysema severity. Intriguingly, we further found that GHK-treatment induced a pattern of gene expression similar to that resulting from TGFβ pathway activation. We replicated both of these findings in human lung fibroblasts, which are the major interstitial cells that maintain tissue structural integrity by sculpting the connective tissue. GHK-Cu is a natural tripeptide that, in human plasma, can be found at a concentration of 200 ng/ml at the age of 20 years but drops to around 80 ng/ml by the age of 60 years . CharactWe further assessed the ability of GHK and TGFβ to induce tissue contraction. As in previous studies , we demoin vitro is associated with the regional disease severity in the tissue from which the fibroblasts are derived.While the number of subjects in this study for genomic analysis was small, the analysis of eight specimens per lung representing different degrees of emphysema from each individual allowed us to detect gene expression changes specifically associated with regional emphysema severity. We further demonstrated that these genes are concordantly differentially expressed in previous cross-sectional studies involving larger numbers of individuals with varying degrees of airflow limitation. These results validate the gene expression differences associated with regional emphysema severity in independent cohorts from different clinical settings and support the hypothesis that the genes whose expression is associated with regional emphysema severity reflect the activity of true disease-associated processes. As demonstrated by our micro-CT data, COPD is a heterogeneous disease within the lung . FurtherThis study has provided insights into molecular processes associated with emphysematous destruction of the lung and revealed mechanisms that contribute to the pathogenesis of COPD. Whole genome gene-expression analysis supports the role of the immune response in regional emphysema and elucidates additional pathways involved in the process of emphysematous destruction. The suggestion that progressive emphysematous destruction is associated with down-regulation of genes involved in or downstream of tissue remodeling and wound repair pathways supports a role for defects in ECM homeostasis and angiogenesis in the emphysematous destruction that occurs with chronic inflammation in COPD. We propose that these processes could be linked through decreased TGFβ pathway activation. These data are supported by our identification of GHK as a compound with the potential to mimic TGFβ pathway activity and induce collagen contraction, an important functional component of wound repair.1: forced expiratory volume in 1 second; FVC: forced vital capacity; GOLD: Global Initiative for Chronic Obstructive Lung Disease; GSEA: Gene Set Enrichment Analysis; Lm: mean linear intercept; PBS: phosphate-buffered saline; TGF: transforming growth factor; Vv: volume fraction.BMP: bone morphogenetic protein; CMap: Connectivity Map; COPD: chronic obstructive pulmonary disease; CT: computed tomography; DLCO: diffusing capacity of carbon monoxide; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FBS: fetal bovine serum; FDR: false discovery rate; FEVBoston University has intellectual property related to the work described in this manuscript.AS, JCH, MEL, DAK, DSP, and WT conceived and designed the experiments. JEM, TLH, DVP, CAB, MS, JVG, GL, YOA, JX, XZ, and SH performed the experiments. JDC, JEZ, TLH, DVP, and CAB analyzed the data. JDC and JCH contributed materials. JDC, MEL, JCH, and AS wrote the manuscript. All authors read and approved the final version for publication.Supplementary methods.Click here for fileStatistical results for gene expression analysis.Click here for fileFunctional categories enriched among genes associated with regional emphysema severity.Click here for fileGene expression relevance network. Dark blue circles are genes that have expression significantly correlated with Lm; light blue circles are all other genes. Edges are indicated by green (positive correlation) or red (negative correlation) lines.Click here for fileRT-PCR validation of 14 genes associated with regional emphysema severity.Click here for fileConfirmation of gene expression changes associated with regional emphysema severity (Lm) within individuals with emphysema using GSEA. Genes associated with unscaled Lm measurements identified using all eight patients in the analysis are concordantly enriched among genes associated with scaled Lm measurements (Z-scored within each patient) using only the five emphysema patients (FDR <0.001). These results demonstrate that the 127 gene signature is related to regional emphysema severity within individuals and not to differences between donors and COPD patients or to differences in levels of emphysema between COPD patients. Orange and blue color bars represent the t-statistics from correlations of gene expression with Lm. The vertical black lines represent the position of genes in the gene set among the ranked gene list. The length of the black lines corresponds to the magnitude of the running enrichment score from GSEA.Click here for fileRelation between gene expression changes associated with regional emphysema severity (Lm) and studies of TGFβ-related gene expression using GSEA. Genes associated with Lm are enriched among the genes that are differentially expressed in response to TGFβ treatment in datasets from (a) Classen et al.[(b) Koinuma et al.[(c) Malizia et al.[(d) Genes most induced by TGFβ in seven studies [en et al., (b) Koima et al., and (c)ia et al.. (d) Gen studies -23,30,31Click here for fileRelation between gene expression changes associated with regional emphysema severity (Lm) and gene expression changes that occur with treatment of GHK or TGFβ in fibroblast cell lines using GSEA. (a) Genes increasing in expression in response to treatment with GHK or TGFβ are enriched among genes that decrease with increasing emphysema severity. (b) Genes differentially expressed with TGFβ treatment or in response to GHK in the Connectivity Map are enriched among genes that change in expression with GHK (0.1 nM) in fibroblast cell lines. (c) Genes that are differentially expressed with TGFβ treatment or that are down-regulated with increasing emphysema severity are enriched among genes that change in expression with GHK (10 nM) in fibroblast cell lines. (d) Genes that are differentially expressed in response to GHK are concordantly enriched among genes that change in expression with TGFβ treatment in fibroblast cell lines. Orange and blue color bars represent the t-statistics from correlations of gene expression with a continuous variable. Red and green color bars represent the t-statistic between treated and untreated samples. The vertical black lines represent the position of genes among the ranked gene list. The length of the black lines corresponds to the magnitude of the running enrichment score from GSEA. Enrichments with an FDR q-value <0.05 were considered significant.Click here for fileLocalization of members of the TGFβ superfamily using immunohistochemistry. Representative images of positive SMAD2 staining (arrows) in the (a) alveolar and (b) small airway wall tissue. (c) Representative image of positive SMAD6 staining in vascular endothelial cells (arrows) and macrophages (arrowheads). (d) Representative image of weak SMAD1 staining in vascular endothelial cells (arrows). Representative images are shown for control IgG staining in the (e) alveolar wall tissue, (f) airway wall tissue, and (g) blood vessels. Scale bar = 200 µm.Click here for fileRelation between gene expression changes associated with regional emphysema severity (Lm) and cross-sectional studies of COPD-related gene expression using GSEA. Genes associated with Lm are enriched among the genes found to associated with the presence of COPD or degree of airflow obstruction in datasets from (a) Golpon et al.[(b) Spira et al.[(c) Wang et al.[(d) Bhattacharya et al.[(e) Genes previously found to be associated with COPD-related clinical variables [t-test between cases and controls. The vertical black lines represent the position of genes in the gene set among the ranked gene list. The length of the black lines corresponds to the magnitude of the running enrichment score from GSEA. Enrichments with an FDR q-value <0.05 were considered significant.on et al., (b) Spira et al., (c) Wanng et al., and (d)ya et al.. (e) Genariables ,8-10 areClick here for fileExamples of genes associated with Lm and validated by quantitative RT-PCR that were also differentially expressed in other COPD-related gene-expression datasets. Genes such as ACVRL1, SMAD6, CCR7, and CXCL13 were associated with increasing regional emphysema severity and concordantly differentially expressed in other datasets such as Golpon et al.[et al.[l.[et al..Click here for fileSubject demographics for lung fibroblast cultures.Click here for file


Acromegaly is a rare disease most frequently due to a GH secreting pituitary adenoma. Without an appropriate therapy, life of patients with acromegaly can be shortened with ten years. Pituitary surgery is usually the first line therapy for GH secreting pituitary adenomas. A meta-analysis proved that mortality is much lower in operated patients, even uncured, than the entire group of patients and is similar with the general population in patients with GH<1 μg/ L. For the patients with hypersecreting postoperative remnant tumor, those with low chance of surgical cure or with life-threatening comorbidities, medical therapies are available: somatostatin receptor analogues (SRA), dopamine agonists (DA) and GH receptor antagonists. Studies with >30% utilization of SRAs reported a lower mortality ratio than studies with lower percentages of SRA administration. Although therapy with DA has long been used in patients with acromegaly, there are no studies reporting its effect on mortality, but its efficacy is limited by the low remission rate obtained. The use of conventional external radiotherapy, although with good remission rate in time, was linked with increased mortality, mostly due to cerebrovascular diseases. Conclusion. Mortality in acromegaly can be reduced to expected levels from general population by using modern therapies either in monotherapy or by using multimodal approaches in experienced centers. Without an appropriate therapy, life of patients with acromegaly can be shortened with ten years [2]. Before current therapies became available, the standard mortality rate (SMR) for these patients was reported to be of 2-3 [3]. However, in some studies there are meta-analyses reporting mortality similar with the general population in patients with normal GH after successful therapy and even normal insulin-like growth hormone 1 (IGF1) [4]. Using sensitive assays, cut-off level for “safe” GH is still a matter of debate, but recent consensus suggested this could be of 1 ng/ mL [5].Acromegaly is a rare disease most frequently due to a growth hormone (GH) secreting pituitary adenoma. Although usually with an indolent, slow evolution, diagnosed 5 years after first signs of acromegaly [Pituitary surgery6]. Surgery related deaths are very rare nowadays. In experienced centers, the cure rate of surgery in macroadenomas achieved up to 74% cured patients, depending on GH criteria of cure and the technique used [7]. One study reported a better surgical result with an endoscopic approach, versus microscopic neurosurgery in patients with macroadenomas and suprasellar extension [8]. However, the success rate in other centers is much lower [9].Current guidelines suggest pituitary surgery to be the first line therapy for GH secreting pituitary adenomas, especially when experienced neurosurgeons are available and tumor is small and well delimitated [10]. In a meta-analysis, Dekkers reported an overall standard mortality ratio (SMR) of 1.72 in all studied patients (n=16 studies), 1.32 in operated patients by transsphenoidal approach and 1.09 in patients with GH<1 μg/ L, similar with the reference population. The best predictor markers of mortality in operated patients were found to be GH at last follow up, GH three years postoperatively and IGF1 levels at last follow up [11]. An elevated GH level in the first three years post-surgery could be a better predictor factor for mortality than IGF1, probably due to the lack of standardization of IGF1 tests, lack of availability of IGF1 levels in all patients, susceptibility of interferences from binding proteins. In patients with discordant values of GH and IGF1 levels, the authors found that the benefits of reducing GH to <2 μg/ L outweigh the benefits of normalizing IGF1 levels. Behind these data, there are studies reporting better surgery results in patients with acromegaly pre-treated with somatostatin receptor analogues (SRA) [12], while others did not find such differences [13].The impact of pituitary surgery on mortality depends on the ability of the neurosurgeon to remove the tumor, and this procedure is best performed in dedicated neurosurgical centers. Even in experienced centers, the long term follow-up, of up to 10 years, found a lower cure rate of patients initially considered successfully operated , but is usually 30% in unselected patients using strict criteria of GH and IGF1 normalization . Long-term treatment (i.e. 10-15 years) with SRA, improves the biochemical response rate up to 80% of the patients [12]. Also, the dose optimization increases the rate of responders to medical treatment in patients with inadequate biochemical control [17]. Clinically significant tumor shrinkage (>20%) is obtained in 75% of the patients  and also rebound regrowth of pituitary tumor has been found after discontinuing treatment even in patients with inadequate biochemical control [20]. Currently available SRA: Octreotide LAR and Lanreotide Autogel have been reported to be similar in efficacy in both biochemical and tumor control  and slightly better than Lanreotide SR.SRA are usually the first line medical therapy indicated in acromegaly. Biochemical control rate reaches 60% in selected patients [23]. A meta-analysis published in 2013 enrolling almost 1000 patients from 10 studies revealed a significant effect of SRA pretreatment, on outcome only when analyzed the 3 RCT as a group  [24]. In contrast with this, in a randomized controlled trial, Colao did not find any difference in patients submitted to surgery or treated with SRA [25]. Moreover, a recent meta-analysis enrolling 2629 patients from 39 studies found a higher remission rate at the longest follow-up (i.e.>24 months) in patients submitted to surgery compared to medical treatment (67% vs. 45%) [26]. These data supported the current Endocrine Society acromegaly guideline recommending primary medical therapy in patients with severe pharyngeal thickness and sleep apnea, high-output heart failure and suggesting against the routine use of preoperative medical therapy to improve biochemical control after surgery [13]. On the contrary, surgical debulking has been found to improve the rate of response to medical treatment [27].SRA can be administered as primary therapy in patients with stable macroadenomas without chiasm compression, in poor surgical candidates or who refuse surgery. There were studies reporting a better acromegaly outcome in terms of better GH and/ or IGF1 control and tumor volume reduction in patients treated with SRA before surgery. The first randomized, controlled study of preoperative Octreotide treatment of acromegaly (the POTA study) in 2008 concluded that patients with macroadenoma treated 6 months with Octreotide s.c. had a better cure rate at the 3 months post-operatory. However, a non-significant impact of this approach was observed in the same cohort followed at 1 and 5 years after surgery [28]. The profile of safety in the study was similar with other SRA, but hyperglycemia was 35% more frequent in pasireotide treated arm than in Octreotide treated arm. A promising new therapy for patients with acromegaly resistant to commercial available SRA is pasireotide, a multireceptor-targeted somatostatin analog with high affinity for 4 of the 5 somatostatin receptor subtypes (sst), including sst2 and sst5, which are the most prevalent sst on GH-secreting pituitary adenomas. One randomized, controlled trial compared the efficacy of pasireotide LAR and Octreotide LAR in naïve to medical therapy. Pasireotide LAR provided a strong suppression of IGF-1, and patients were 63% more likely to achieve biochemical control with pasireotide LAR than with Octreotide LAR, both in post-surgery and in de novo patients [29]. A meta-analysis by Holdaway et al. demonstrated that studies with >30% utilization of SRAs or >70% of remission rates reported lower SMR ) than studies with lower percentages of SRA administration and remission rates  [4].There are no conclusive data in literature regarding the effect of SRA treatment on mortality in patients with acromegaly. One study compared the effect of surgery and medical therapy on mortality in Italian patients with acromegaly. Surgery resulted in a better survival rate than medically treated patients, but there was a selection bias in this study, patients treated with SRA being more likely to have comorbidities making them more unsuitable for surgery [Dopamine agonist therapy30]. It may exert antiproliferative and pro-apoptotic effects on GH secreting tumors, ACTH secreting tumors, besides known effects on prolactinomas and it has the advantage of oral administration and low cost compared to SRA or GH receptor antagonists. A large multicenter study in Belgium reported IGF1 reduction in 39% of the patients and GH<2 μg/ L in 46% of the patients treated with cabergoline monotherapy. Better results were obtained in patients with mixed GH and PRL secreting pituitary tumors and in those with marginally elevated GH/ IGF1 levels [31]. In another study on patients from the Bulgarian Acromegaly database, control rates on cabergoline therapy were 18.2% and on bromocriptine 16.3%, in patients without prior radiotherapy. Predictors of response to dopamine agonist therapy were active acromegaly duration, radiotherapy and medication dose [32]. There are safety issues regarding the long-term treatment with cabergoline: patients with Parkinson disease treated with high dose of cabergoline were reported to have an increased risk for severe valvular regurgitation [33]. There are no data regarding clinically significant valvular regurgitations in patients with acromegaly treated with cabergoline, acromegaly itself raising the risk of valvular disease. Also, one study showed that impulse control disorders are 9.9 times more common in male patients with prolactinomas taking DA than in the control group patients [34].The mostly used DA is cabergoline, which leads to the normalization of GH and IGF1 in 27% of the patients [35].Although this therapy has been long used in patients with acromegaly, there are no studies reporting the effect of monotherapy with DA on mortality. One study compared patients from Bulgaria, with low cure rate of surgery, high usage of radiotherapy and dopamine agonists and usage of SSA only after 2008 with patients from Campania, Italy and life expectancy was significantly shorter in patients from Bulgaria [GH receptor antagonist therapy36]. Its long-term safety  has been studied in 1288 patients from ACROSTUDY cohort: a central MRI reading found a 3.2% increase/ decrease in tumor size from 936 patients with at least two available MRI. 2.5% of the patients had liver enzyme increases greater than 3 times the upper limit of normal. Mortality ratio was 1.2% during the study; the most frequent cause of death was heart failure and cancer related.Pegvisomant is a genetically modified analog of human GH and acts as a competitive GH receptor antagonist. Biochemical control is obtained in 75-92% of the patients from randomized control trials [Combination therapy30]. Also, a weekly dose of 60-80 mg pegvisomant in association with a monthly dose of SSA improve the response rate of both therapies to 95% [37]. Pegvisomant can also be used in combination with cabergoline therapy, combination therapy proving a better acromegaly control than monotherapy with either medication [38].In patients with partial resistance to SSA, cabergoline adding could provide a better control rate, independent of prolactin levels, especially in those with mild elevated GH levels [39]. Clomiphene citrate, a SERM usually used for ovulation induction, was successfully used in men with acromegaly not controlled after surgery, radiotherapy and conventional medical treatment and with testosterone level within or below the third inferior tertile of normality, with an additionally increase of testosterone level [40]. Estrogens, selective estrogen receptor modulators (SERMs), alone or in combination with SSA, have been observed to decrease IGF-1 in women and to improve symptoms of acromegaly [41]. Remission rate of conventional radiotherapy defined as serum basal GH levels <2.5 μg/ L reported is up to 77% after 20 years [42]. Its efficacy is tempered by long time needed until radiotherapy became effective and by concerns about long-term toxicity: second tumor induction, mortality due to cerebrovascular disease in patients already exposed to a high risk of vascular diseases as proved by carotid artery modifications. A study on 462 patients with pituitary adenomas, including 139 patients with acromegaly, found no differences between intracranial tumors incidence and no death events in patients with postoperative radiotherapy as compared to patients submitted to surgery alone [43].Radiotherapy is now considered an adjuvant therapy in uncontrolled patients with acromegaly after pituitary surgery and medical therapy [44]. The new techniques of radiotherapy: intensity modulated RT (IMRT), fractionated stereotactic radiosurgery (FSR), stereotactic radiosurgery (SR), and proton beam techniques have promising results. Rates of hypopituitarism ranged from 28 to 80% in patients with pituitary adenomas, depending on the time from the pituitary irradiation. Hypopituitarism is another factor contributing to the increased mortality rate after radiotherapy, its incidence being more reduced with new focused modalities of radiotherapy [45]. The use of external radiotherapy was linked with increased mortality, with a SMR of 1.58 in the West Midlands Acromegaly Study. Also, in the Finnish Nationwide Survey of Mortality in Acromegaly, treatment with radiotherapy was one of the determinants of mortality, but with borderline statistically significance [Mortality in acromegaly can be reduced to expected levels from general population by using modern therapies either in monotherapy or by using multimodal approaches in experienced centers.Acknowledgement”This work received financial support through the project entitled “CERO – Career profile: Romanian Researcher”, grant number POSDRU/159/1.5/S/135760, cofinanced by the European Social Fund for Sectoral Operational Programme Human Resources Development 2007-2013”.Conflict of interestThe authors declare there were no conflicts of interest.


Protein–protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein–protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins. Protein–protein interactions are fundamental chemical events in living organisms and thus of prime interest to biochemical research in many fields of life science. Although protein X-ray crystallography has delivered an enormous amount of highly refined structural data of large protein aggregates its applicability is limited to high-affinity and stably structured protein complexes. In the recent past, it has yet become clear that also weak protein–protein interactions leading to transiently formed complexes play key roles in diverse biological processes, such as cell signaling, post-translational modifications, general transport phenomena, cellular trafficking, enzyme catalysis, and transcription as well as translational regulation. Additionally, there is growing awareness of the importance of intrinsically disordered proteins (IDPs) in eukaryotic life. Their central role in protein interaction networks has been established. IDPs efficiently sample a vast and heterogeneous conformational space allowing them to interact with multiple and diverse binding partners. It is therefore a great and rewarding challenge for modern structural biology research to access these proteins in the completeness of their native states and to adequately describe their inherent structural plasticity. In this context it is of interest that sparsely populated protein conformational states  are essential for the functionality of all kinds of proteins—ranging from IDPs to tightly structured enzymes.–3 It is KD). For more details and broader coverage of NMR applications, the reader is referred to excellent reviews published over the years.[Evidently disordered systems are not amenable to X-ray crystallography, which is by far the most widespread structural biology technique. Instead these systems require alternative technological strategies. Nuclear magnetic resonance (NMR) spectroscopy offers unique possibilities for this quest. Because NMR is performed in solution there are different sample requirements. As such, it can also be applied to molecular systems displaying substantial degrees of conformational flexibility. Over the years, NMR methodologies were developed to investigate protein complexes. NMR chemical shift changes and saturation techniques are used to locate protein interaction sites. Residual dipolar couplings (RDC), 5 togethe years., 8, 9 Heτ. Conformational transitions between ground and excited state lead to alterations of the precession frequencies  the number of refocusing pulses will alter the observed signal intensity. If the exchange rate k is slower than 1/τ, the precessing magnetization will be completely refocused and signal intensity is unaffected by the exchange process and hence maximized. Yet, if k is in the range of or larger than 1/τ the observed signal intensity will be modified and depend on the frequency (υCPMG) of 180° inversion (refocusing) pulses. In other words, the effective (detected) transverse relaxation rate, R2,eff will be affected by the exchange process and the time interval τ , in the CPMG train  will decrease with increasing υCPMG values . Fitting R2,eff(υCPMG) as a function of υCPMG allows to determine the exchange rate k between the two states. Furthermore, the chemical shift difference between A and B and the populations of these states can be extracted through measurements at different magnetic field strengths.[Biomolecules are characterized by rugged energy surfaces resulting from attractive and repulsive forces of similar strength. The accessible conformational space governs thermodynamics and kinetics of conformational transitions between different substates. In the past, it has become increasingly evident that sparsely populated, high-energy conformational states of a protein can be essential for protein function., 10 A prtrengths. Dependintrengths.υCPMG) must be shorter than the exchange rate k in order to observe enhanced signal attenuation. Thus, by varying υCPMG and observing the consequent effect on the signal intensity one can yield information about k and the details of conformational exchange processes .The CPMG experiment can also be explained from a kinetic point of view by taking into account the deleterious effects of randomly changing precession frequencies on echo formation. The observed NMR signal, that is, a spin echo, will become weaker the more the precision frequencies of the individual spins in an ensemble differ from each other. The time needed for the spins to be randomly distributed in the transverse plane after RF excitation is inversely proportional to the spectrum of precision frequencies (line width). Conformational exchange modulation of precision frequencies due to changes in the local environment of some spins leads to additional line broadening and concomitant signal attenuation. Taking a look at the CPMG pulse train in Figure 15N–1H amide resonances. Implementation of the CPMG train into a two dimensional data acquisition yields residue-dependent R2,eff values. In this case, a residues exchange frequency might be affected by local conformational interconversion processes, for example, flapping of aromatic side chains,[In the realm of protein NMR, one frequently observes the relaxation dispersion of backbone e chains, and by ge chains, Here we Escherichia coli cold-shock protein CspA  experiment is a promising technique to supply information about the sparsely populated RNA-bound state of CspA. Here, we provide 8 Figure  to illus15N-dispersion relaxation profiles were measured in the absence and in the presence of ACB RNA . Thus, CPMG analysis allows for determination of kinetics of a state as lowly populated as 1.6 %, which would be difficult to assess by other techniques. Further, the method used here enables one to obtain information not only on kinetics and population, but also yields the nitrogen chemical shift differences between the free and the bound state of residues observed by CPMG data that correlate with the magnitude of changes in the chemical environment. Thus, in principle one could extract structural information about the bound state of CspA by means of CPMG through the chemical shift difference to the apo-state if a sufficiently high number of residues yield exchange-affected dispersion profiles. The CspA case study presented here demonstrates the power and versatility of the CPMG method in assessing the lowly populated states important in RNA–protein interactions.For CspA residues F12 and F18, A Figure . In the 13C-CMPG RD experiments are the method of choice.[13C/15N-labeling patterns impair the application of certain NMR experiments to address dynamic features, like the CPMG RD experiment. A uniform 13C/15N-labeling pattern introduces one-bond scalar and dipolar 13C–13C couplings resulting in resolution and sensitivity problems especially for larger RNA comprising more than 30 nucleotides (nt) but also to artifacts in the CPMG RD data sets impairing a reliable analysis.[13C/15N-modified ribonucleotide triphosphates (rNTPs) and T7 RNA polymerase, several approaches were proposed.[19F-based isotope labeling protocol can be very efficiently applied to RNA as all four 2′-fluorine modified nucleotide phosphoramidites are commercially available and can be incorporated into the target RNA without modifying the standard protocol  and a vast chemical shift range about 100 times larger than protons. It represents a bioorthogonal reporter spin for nucleic acids and was efficiently used in RNA folding and RNA ligand binding NMR assays.[19F-CPMG relaxation dispersion experiments (data not shown). We also focused on the chemical synthesis of site-specifically 13C-modified pyrimidine phosphoramidites  residing in the 5′-UTR of the E. coli cspA messenger RNA (mRNA) were replaced by the 6-13C-modified counterparts. We then used 13C-CPMG relaxation dispersion experiments to address the micro- to millisecond dynamics of this very RNA. Preliminary results are shown .[As a third example for the application of CPMG techniques to transient protein interaction studies, we outline an application to the reversible protein dimerization of the Myc-associated factor X (MAX). MAX read, 35 °C). The stro, 35 °C). Fitting , 35 °C). This is , 35 °C).ωN of the bound (dimeric) state of MAX is known, while the chemical shift of the monomeric form can be directly observed in 15N–1H HSQC spectra (since the monomeric form of MAX is the dominating species under these conditions). The differences between monomeric and dimeric chemical shifts should coincide with individual δω’s obtained from a fit of CPMG data. As indicated in Figure δωfit=8.0 ppm and ωN,Dimer−ωN,Monomer=7.8 ppm).[The reliability of the CPMG results can be further tested by comparison of the chemical shifts extracted from the CPMG data with literature data for the dimeric state of MAX. From earlier publications, for example, the 7.8 ppm). Because The hallmark of intrinsically disordered proteins (IDPs) is their vast and heterogeneous conformational ensemble comprising both extended conformations and simultaneously compact reasonably stable structures displaying significant resistance against denaturing agents (like urea). The ruggIn order to perform DEER, one has to modify a protein with two labels that both carry an unpaired electron, hence, the term spin label. The freeP(R) can be obtained by well-established procedures such as the Thikonov regularization.[Δeff, for data interpretation of IDPs. Details of this novel approach were explained elsewhere.[Δeff denotes the signal decay of the time trace, V(t), at a given point of time, teff. Δeff can be defined as: Δeff=1−V(teff)/V(t=0). For large IDPs, we suggest to simply choose the longest experimentally achievable DEER evolution time for teff. Δeff is an approximate measure of average interspin distance for broad P(R)s. Yet, the reader should be aware that Δeff does not linearly depend on the population-weighted average distance in the measured ensemble but is a complex function of several spectroscopic parameters.[Δeff decreases with increasing interspin distance R for broad distance distributions. With Δeff as tool in hand, we probed structural preferences of Osteopontin (OPN), a cytokine involved in metastasis of several kinds of cancer.[In case of folded proteins, after removal of background contributions, the spin label distance distribution rization., 43 Yet,rization. Thus, welsewhere. In shortrameters. To a firf cancer. Typical Δeff with increasing urea concentration is representative for unfolding and expansion of a doubly spin-labeled protein of interest. In Figure Δeff values are shown as a function of urea concentration for a selected double mutant comprising a central segment of the cytokine OPN. The time traces appear as exponential decays because of the aforementioned convergence of cosine functions (one for each conformation in the OPN ensemble). With increasing urea concentration Δeff decreases indicating an expansion of the IDP. As published earlier,[Δeff with increasing urea concentration. This gives rise to a steep slope of the Δeff function and can be regarded as a denaturation profile of an unstably folded protein segments of potentially random-coil-like character. Already for low urea concentrations such segments show significant conformational expansion  in accordance with the idea of very low stability of transient or residual structural elements in IDPs. For other mutants, one might observe an approximately linear decrease of Δeff with urea concentration, indicating that the OPN segment framed by these mutants is on average conformationally more stable than the C-terminal segment. Nevertheless, it is still largely unstructured, random coil- or (pre)molten globule-like. Strikingly, however, for a mutant of OPN with two terminal labels, we observe a sigmoidal development of the Δeff-derived denaturation profiles with urea concentration . This observation of a cooperatively folded conformation is surprising since distance distributions between two labeled residues of OPN are generally quite broad, as deducible from prior studies concerning OPNs conformational space[A sigmoidal denaturation profile is indicative for stably and cooperatively folded tertiary structures of OPN, since for low urea concentrations of up to 0.75 nal space and as rnal space This dedΔeff approach be utilized to elucidate structural preferences of IDPs complementary to NMR data, but also ligand interaction can be observed in high detail. We analyzed the interaction of OPN with heparin,[The applicability of this novel EPR-NMR approach to the observation of transient complex formation of IDPs was also recently demonstrated. Not only can the  heparin, a highly heparin, In a bio heparin, Addition heparin, and our Δeff indicating longer distances between the labeling sites of the six double mutants and an expansion of the protein upon heparin binding. This is shown in a previous paper.[15N NMR relaxation rates and heteronuclear 15N–1H NOEs (hetNOE) of bound and free forms. NMR relaxation reports on motions ranging from picosecond to low-nanosecond timescales. The three measurable NMR relaxation parameters  dynamics of the N−H bond. Figure 15N–1H hetNOE) increase in the region located around residue 120. The central region around residue 150, however, shows increased 15N transverse relaxation rates R2 due to decreased conformational flexibility in the binding cleft. Closer inspection of the charge distribution in OPNs primary sequence reveals that the differential mobility changes upon heparin binding are linked to the electrostatic pattern found in OPN (increased mobility in negatively charged regions and decreased mobility/rigidification in positively charged patches).Upon binding to heparin, OPN largely remains disordered although its structural ensemble is updated. For several doubly spin-labeled mutants of OPN, heparin binding leads to a clear decrease in us paper. This infus paper. When intus paper. Hence, aThrough combination of NMR and EPR a clear picture arises of the OPN–heparin binding event. As the highly negatively charged heparin contacts OPNs positively charged core region, the structural and dynamic properties of the OPN ensemble get drastically altered. Along with an increase in dynamics along most of the proteins backbone, an average increase in distances is observed. This unfolding-upon-binding event is depicted in Figure H and ΔS values of −16.3 kCal mol−1 and −35 cal mol K−1, respectively, can be obtained for the heparin binding event of OPN assuming an average molecular weight of 17.5 kDa for heparin. These quite large ΔH and ΔS values nearly cancel each other at 293 K in the Gibbs energy (ΔG=ΔH−TΔS). Thus, although there is local rigidification in the heparin binding cleft (region around residue 155) the resulting conformational entropy penalty is reduced by a compensatory increase in conformational flexibility of the negatively charged region 90–120. The local  unfolding and expansion of the OPN core segment thereby significantly contributes to the overall thermodynamic equilibrium balanced between counteracting contributions like solvation enthalpy, rotational and translational degrees of freedom and conformational entropy.[From isothermal titration calorimetry (ITC) measurements, Δ entropy., 49Three examples for weak and reversible protein interactions are presented in combination with magnetic resonance based experimental techniques to access these interactions. Relaxation dispersion  experiments allow for determination of sparsely populated states in CspA–RNA complexation and MAX–MAX homodimerization. Since both kinetic data about complex formation (association and dissociation rates) as well as structural details of lowly populated (high-energy) conformational states are accessible by this technique, unique information about protein interactions becomes amenable. Further, the combination of paramagnetic relaxation enhancement (PRE) NMR and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) gives rise to the unprecedented and unexpected observation of cooperatively folded conformational states contained in the conformational ensemble of Osteopontin (OPN) and its changes in the weak OPN–heparin interaction. Our findings suggest that more elaborate conceptual approaches are required for an adequate description of intrinsically disordered proteins and their conformational ensembles.Proteins are characterized by significant structural plasticity and can undergo large structural rearrangements of the time-averaged conformational ensemble. It is therefore evident that classical structural biology approaches are only starting points for a comprehensive analysis of protein function. For intrinsically disordered proteins (IDPs) flat energy landscapes allow for rapid exchange between different conformational isomers (substates). These often only sparsely populated conformational states, yet, lead to the formation of protein complexes that are essential for biological function. Detailed knowledge of the transiently formed intermediates along the reaction trajectory will be highly valuable. Further developments of new techniques but also the amendment of existing ones  will be necessary to provide a more complete picture of how proteins form biologically active molecular aggregates and perform the myriad of essential tasks and how this information can be exploited to manipulate their activities based on knowledge of the underlying chemical mechanisms.


Sleeping Beauty (SB) transposon system, provide a model that recapitulates the spontaneous development and progression of human disease. This approach has been widely used to model a variety of cancers in mice. Comprehensive mutation profiles are generated for individual tumors through amplification of transposon insertion sites followed by high-throughput sequencing. Subsequent statistical analyses identify common insertion sites (CISs), which are predicted to be functionally involved in tumorigenesis. Current methods utilized for SB insertion site analysis have some significant limitations. For one, they do not account for transposon footprints – a class of mutation generated following transposon remobilization. Existing methods also discard quantitative sequence data due to uncertainty regarding the extent to which it accurately reflects mutation abundance within a heterogeneous tumor. Additionally, computational analyses generally assume that all potential insertion sites have an equal probability of being detected under non-selective conditions, an assumption without sufficient relevant data. The goal of our study was to address these potential confounding factors in order to enhance functional interpretation of insertion site data from tumors.Animal models of cancer are useful to generate complementary datasets for comparison to human tumor data. Insertional mutagenesis screens, such as those utilizing the We describe here a novel method to detect footprints generated by transposon remobilization, which revealed minimal evidence of positive selection in tumors. We also present extensive characterization data demonstrating an ability to reproducibly assign semi-quantitative information to individual insertion sites within a tumor sample. Finally, we identify apparent biases for detection of inserted transposons in several genomic regions that may lead to the identification of false positive CISs.The information we provide can be used to refine analyses of data from insertional mutagenesis screens, improving functional interpretation of results and facilitating the identification of genes important in cancer development and progression.The online version of this article (doi:10.1186/1471-2164-15-1150) contains supplementary material, which is available to authorized users. Animal models of cancer represent a complementary approach to direct analysis of patient tumors, allowing a level of experimental control not possible with human studies. The ability to generate large cohorts under precisely controlled conditions facilitates the interpretation of immensely complex datasets obtained from human samples. Currently, one of the greatest challenges to the development of successful cancer therapies is distinguishing so-called “driver” genetic aberrations that functionally contribute to tumors from background “passenger” events that are propagated during tumor development due to their co-occurrence with drivers. Insertional mutagenesis screens in animal models are particularly suited to addressing this issue, as they can provide large collections of tumors with tagged mutations that are considerably less complex at the molecular level than human cancer.Sleeping Beauty (SB) transposon system has proven useful for identifying drivers of tumorigenesis in a wide variety of tissue types  . Additional factors, however, such as PCR amplification bias and differential mapping efficiency could potentially impact coverage, complicating quantitative interpretation of insertion site sequencing data. Assessment of the influence such factors have on sequence coverage is therefore critical to enhancing the ability to infer relative clonality information for individual mutations within a tumor.Typical high-throughput sequencing analyses of genomic DNA from tumors induced by transposon-mediated mutagenesis detect thousands of unique insertion sites in each tumor , 7. The e.g. based on GC content or repetitive nature; see details in Additional file We conducted a standard curve experiment to determine the correlation between sequencing depth of a particular transposon insertion site within a tumor and its actual abundance relative to other sites. A set of 10 plasmids was engineered, each of which contained two artificial templates for amplification by ligation-mediated (LM)-PCR that consisted of either the left or right SB transposon ITR element adjacent to a specific genomic locus identified through prior mutagenesis screening . As shown in Figure Following mapping, the number of sequence reads assigned to each transposon insertion site was normalized to reflect its proportion relative to the most abundant site  or T2/Onc3 (TG12740 and TG12775) transposons. Transposon integration sites were identified in genomic DNA from liver, spleen, skin, and lung tissue by LM-PCR followed by Illumina sequencing. Three independent sections per tissue were analyzed for two males and two females of each strain , and a total of 33,350,101 sequence reads were mapped to 599,938 unique genomic loci. In order to estimate the relative degree of clonal expansion occurring in tissues during the two week transposition period, we compared the percentage of all mapped sequence reads assigned to the top five most frequently identified sites between a set of T2/Onc3 transposon-induced liver tumors [To generate such a dataset, transposition was activated ubiquitously in eight-week-old mice and allowed to persist for two weeks prior to tissue collection. Inducible, ubiquitous activation was achieved through tamoxifen injection into offspring resulting from a cross between the homozygous 2 strain  and an Si.e. TA dinucleotides). After Bonferroni correction for multiple hypothesis testing, this analysis identified 794 distinct windows with significantly more insertion sites than predicted . While this type of mutation could have functional effects regardless of where it occurs in the genome, the greatest impact would be expected within coding exons, due to the generation of frameshift mutations. Measuring the degree of transposon remobilization and selection for maintenance of footprints in tumors is important to ensure that functional contributions from these events are not missed when interpreting data from SB insertional mutagenesis screens. To date, no such characterization has been reported.The relative simplicity of genetic profiling in animal models of cancer induced by insertional mutagenesis is a major advantage when attempting to elucidate molecular mechanisms of carcinogenesis. Because the insertional mutagen’s sequence is known, techniques that amplify adjacent genomic regions for sequencing can readily be implemented. It is important to note, however, that additional types of genetic aberrations are certainly present at some level in tumors induced by this mechanism. Of particular interest for cancer models induced by We have described a novel high-throughput sequencing approach to identify SB transposition footprints. Using this technique, we detected ~100-200 candidate footprints per sample in a set of three SB-induced T-cell leukemias  cells reported some influence of transcriptional activity, regulatory function, and chromatin topology on target site selection [in vivo and in the absence of selection, conditions chosen to match techniques used for tumorigenesis screens. In this experimental context, patterns of detection bias do not appear to be influenced by tissue type or concatemer location, as evidenced by the presence of insertions from nearly all strains and tissues in all 16 non-local windows of preferential detection that could not be explained by mapping artifacts. Additionally, our study is the most comprehensive to date, analyzing five times more sites than the most extensive study conducted previously [Current methods to analyze insertion site data from SB mutagenesis screens typically assume that all TA dinucleotides in the genome outside of the local chromosome have an equal chance of being detected as insertion sites in the absence of selective pressure. This assumption is based on studies that have characterized global patterns of SB transposition and found no major biases , 11–15. election . There ieviously . These qin vivo has previously been utilized. For this approach, integration sites are profiled in DNA from tail clips of weanling mice [An alternative method to generate SB transposon insertion data from unselected tissue ing mice –26 or hiing mice , and anying mice . By usining mice .Consistent with our assumption of a lack of significant clonal expansion of cell subpopulations over a two-week transposition period, the transposon integration profiles for all of the normal tissue samples were strikingly different from those typically obtained from tumor samples. In tumors, the majority of mapped sequence reads are derived from a relatively small number of insertions that have increased in prevalence relative to other insertions due to clonal expansion of the cells harboring them, and many of these clonally-expanded sites are recurrently identified in multiple independent tumors. In contrast, sequence reads derived from insertion sites identified in normal tissues following two weeks of transposition were more evenly distributed, indicating a lack of significant selection for cells harboring any given insertion. Additionally, we did not observe enrichment for insertions within transcription units in normal samples, whereas tumors display such enrichment due to the clonal expansion of cells harboring driver mutations.i.e. it may have a biological basis). Alternatively, it could be caused by technical biases in amplification, sequencing, or mapping. Any of these factors could contribute to enhanced detection of specific insertion sites, and it is likely that each of them does to some extent. In our dataset, mapping artifacts were responsible for the majority of preferentially detected windows. Removal of sequence reads that could not be definitively mapped prior to downstream analysis eliminated the majority of initially identified significant windows, leaving only 16 that were not attributed to this source of bias.In total, our initial analysis identified 116 non-local genomic regions detected to have a significantly higher frequency of insertion than predicted based on a random transposition pattern. In some cases this may result from an increased prevalence for SB transposons to integrate within these regions relative to others  of genes overlapped by the regions we identified have previously been reported as CISs in SB screens Table , accordi/59 of geSfi1 , which has been identified by 12 separate SB screens. Based on our findings, along with the previous identification of this region as underrepresented in the reference genome [Sfi1 was also identified as a potential false-positive CIS through the analysis of unselected SB insertion sites in mES cells [in vivo SB mutagenesis screens, our results are particularly relevant to the functional interpretation of insertion site data from such screens.One particularly striking example of a potential false-positive CIS is e genome , its recES cells . None ofin vivo by methods currently used to identify SB insertion sites in tumors. By shedding light on this potential source of bias, our results reveal an important factor to consider when interpreting insertion site data. Overall, the methods and data that we have presented will facilitate improved identification of cancer genes by insertional mutagenesis strategies, as well as the enhanced interpretation of data generated using this approach.The results that we have described represent significant advancements to the field of cancer gene discovery utilizing insertional mutagenesis models. Our characterization of footprints induced by SB transposon remobilization is the first large-scale analysis of this phenomenon’s prevalence in tumors. We report that remobilization is a relatively rare event in SB-induced tumors and that it is unlikely to result in mutations being missed by traditional screening methods. Through the use of a standard curve experiment, we found that individual insertion sites within heterogeneous tumors can reliably be divided into groups based on a semi-quantitative assignment of clonality. This information will serve to deepen our understanding of the dynamics of mutation acquisition and selection in developing tumors. Finally, we report several genomic regions that are preferentially detected under non-selective conditions CD4-Cre; ROSA26-LsL-SBase; T2/Onc2 mice used for experiments to detect transposon remobilization were generated previously by crossing CD4-Cre transgenic mice (Taconic Farms model #4196) to RosaSBaseLsL;TG6113 double transgenic mice [ROSA26-CreERT2 mice were purchased from The Jackson Laboratory (Strain B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J; Stock #008463). SB mouse strains carrying the Cre-inducible ROSA26-LsL-SBase allele along with one of four distinct transposon concatemers  were generated by the Dupuy laboratory, as previously described [Triple transgenic nic mice . ROSA26-escribed .ROSA26-LsL-SBase allele was allowed to proceed for two weeks, at which point mice were euthanized and tissues were collected.All animal experiments were performed using procedures approved and monitored by the Institutional Animal Care and Use Committee at the University of Iowa. At eight weeks of age, two male and two female mice of each SB strain  were injected intraperitoneally with a single dose of 2 mg tamoxifen (Sigma-Aldrich product # T5648) dissolved in corn oil. Resulting ubiquitous transposon mobilization due to activation of the HpyCH4III (New England Biolabs), followed by heat-inactivation at 80° for 20 minutes. Unique footprinting adaptor oligos (HpyLinker + and HpyLinker-) were annealed by heating to 95° for 5 minutes and cooling slowly to room temperature, leaving a single nucleotide A or T overhang on one end. Annealed adaptors were ligated to digested tumor DNA overnight at 16° using T4 DNA ligase (New England Biolabs). Samples were purified using the MinElute 96 UF PCR purification kit (Qiagen) and resuspended in 20 μl of water prior to a two-stage PCR amplification protocol. Primary PCR was conducted using 3 μl DNA as template with a forward primer complementary to the blunt adaptor region (Linker Primer) and a barcoded reverse primer complementary to the HpyCH4III adaptor region (HpyLinker Primer), each included at a final concentration of 200 nM. Platinum Taq DNA polymerase (Life Technologies) was used for amplification with 30 cycles of: 94° for 30 sec, 57° for 30 sec, and 72° for 60 sec. Primary PCR products were diluted 1:50 in water and incubated at room temperature for 30 min. Secondary PCR was conducted using 8 μl of diluted products as template with a nested primer complementary to the blunt adaptor region (Linker-A2 Primer) and the same barcoded reverse primer, each included at a final concentration of 200nM. Cycling conditions were the same as for primary PCR, except that 20 cycles were performed instead of 30. A portion of each reaction was analyzed by electrophoresis on a 1.5% agarose gel to assess library quality. The remaining secondary PCR products were purified using the MinElute 96 UF PCR purification kit (Qiagen). Approximately 25 ng of each sample was pooled and submitted for sequencing on the Illumina Hi-Seq platform. Oligonucleotide sequences used for footprint detection are listed in Additional file Tumor DNA was isolated, acoustically sheared, end-repaired, and ligated to a blunt adaptor  as previously described . After bbona fide footprint. This ensured that the candidate site contained an intact footprint sequence (i.e. 5’-TACWGTA-3’).Sequences were trimmed and mapped using the IAS pipeline as previously described . The fooHpyCH4III (New England Biolabs), and separated by electrophoresis on 1.5% agarose gels.For validation, ten primer pairs . PCR products were sequenced for verification. Chimeric transposon/genome junctions were cloned into pT2/Onc2 vectors  to simulTransposon insertion sites in mouse tissues were sequenced using the Illumina Hi-Seq platform as previously described . Brieflyi.e. sites present in the test set but absent in the true-positive set) was determined based on this comparison.The false-discovery rate was estimated by generating three independent LM-PCR profiles for six different tumor samples. For each tumor, we treated each single insertion profile as a “test set” of common insertion events found in the remaining two replicate samples (defined as the “true-positive set”). Next, the percentage of false-positive sites (The Poisson Regression Insertion Model (PRIM) was used to calculate the expected insertion rate for non-overlapping 20 kilobase windows along the length of each chromosome in the mouse reference genome . The PRIFirst, we identified the minimum read length required to unambiguously map sequences surrounding each TA dinucleotide in the mouse reference genome (GRCm38/mm10). To do this, mock sequence reads were generated on both DNA strands starting at a length of 10 bp and increasing in 5 bp increments to a maximum length of 65 bp. Fragments were mapped using Bowtie2 . Using these data, the minimum read length required for accurate and unique mapping was determined on both strands for each TA in the reference genome. For this purpose, mapping was characterized as accurate and unique when a mock read mapped to the originating locus with a score >10 and without mapping to any other genomic site having two or more mismatches. We modified our insertion site analysis pipeline such that any experimental sequence read not meeting the minimum required length for accurate and unique mapping was discarded. The remainder of the pipeline functions as previously described .Additional file 1: Figure S1-S5: (PDF 3 MB)Additional file 2: Table S1: Putative SB transposon footprints identified by Illumina sequencing. (XLSX 66 KB)Additional file 3: Table S2: Characteristics of transposon standard plasmid constructs. (XLSX 53 KB)Additional file 4: Table S3: Genomic windows with significant enrichment for detected transposon insertions in unselected tissues. (XLSX 102 KB)Supplemental Methods. Genotyping protocols. (DOCX 110 KB)Additional file 5:Additional file 6: Table S4: Oligonucleotide sequences used for SB footprint sequencing library preparation. (XLSX 26 KB)Additional file 7: Table S5: Oligonucleotide sequences used for validation of candidate footprints. (XLSX 48 KB)Supplemental data. Processed data sets obtained by mapping using both the IAS and the map-corrected IAS method (IASmc). (ZIP 5 MB)Additional file 8:


The absence of dilatation of the right heart on subxiphoid ultrasound argued against the diagnosis of PE, but not conclusively so. In the context of the circulatory collapse induced by cardiac arrest, this aspect was relegated in terms of importance. The second dose of rtPA utilised in this case resulted in return of spontaneous circulation (ROSC) and did not result in haemorrhage or an adverse effect. More than 70% of cardiac arrest cases are caused by acute myocardial infarction (AMI) or pulmonary embolism (PE). Although thrombolytic therapy is a recognised therapy for both AMI and PE, its indiscriminate use is not routinely recommended during cardiopulmonary resuscitation (CPR). We present a case describing the successful use of double dose thrombolysis during cardiac arrest caused by pulmonary embolism. Notwithstanding the relative lack of high-level evidence, this case suggests a scenario in which recombinant tissue Plasminogen Activator (rtPA) may be beneficial in cardiac arrest. In addition to the strong clinical suspicion of pulmonary embolism as the causative agent of the patient's cardiac arrest, the extremely low end-tidal CO Acute pulmonary embolism is a common disease with well-recognised morbidity and mortality –3. It caIn this regard, contemporary guidelines  suggest High level evidence in respect of thrombolysis of PE during cardiac arrest is lacking. While there are case reports and case series that describe successful resuscitation following administration of systemic thrombolytic therapy during cardiac arrest (from suspected PE) –16, therThe most frequently used emergency thrombolysis dosing regimen for PE in cardiac arrest remains the prototypical 2003 British Thoracic Society regime of alteplase 50 mg intravenous (IV) bolus . It is uA 39 year-old gentleman presented to the Emergency Department with a two-day history of pleuritic chest pain, lethargy and associated symptoms of progressively increasing shortness of breath . This occurred on a background of more long-standing non-specific lethargy. There was a history of a recent long-haul flight from Nigeria to Ireland one week previously. At presentation, he exhibited a tachycardia of 116 beats per minute, blood pressure of 131/94 mmHg, and a respiratory rate of 22 breaths per minute.2 of 7.5 kPa, PaCO2 of 3.8 kPa, pH 7.47, and an oxygen saturation of 89%. A D-dimer assay performed at triage was significantly elevated at 10.5 mg/L. Given the working diagnosis of probable pulmonary embolus (high-risk pretest probability), therapeutic low-molecular-weight heparin (Enoxaparin 120 mg subcutaneously) was administered prior to emergent Computed Tomographic Pulmonary Angiography (CTPA).An electrocardiogram revealed a sinus tachycardia with symmetrical T wave inversion in praecordial lead V3. Arterial blood gas analysis showed a PaO2 was not detected initially. Subxiphoid ultrasound—performed during brief interruption of chest compressions—did not reveal a dilated right side of heart. Despite this, given the overall clinical picture at this juncture, a presumptive diagnosis of massive or saddle pulmonary embolus was made.Two hours later, while awaiting emergent CTPA, the patient collapsed and was found to be in cardiac arrest. Cardiopulmonary resuscitation was promptly initiated for pulseless electrical activity (PEA). Intubation with a cuffed oroendotracheal tube (COETT) was achieved without interruption of chest compressions. Despite primary confirmation of COETT placement, end-tidal CO2, no cardiac output was detected after twenty minutes. A decision was taken to administer a second bolus of rtPA  50 mg. Ten minutes subsequent to this and following on-going advanced life support, return of spontaneous circulation (ROSC) was achieved with an initial non-invasive blood pressure of 144/50 mmHg.Along with conventional ACLS adrenaline therapy, rtPA  50 mg was promptly administered. Despite continuing high quality chest compressions and a gradual rise in quantitative end-tidal COStandard post-ROSC resuscitation care was instituted and this gentleman was admitted to the intensive care unit. A CTPA demonstrated multiple bilateral pulmonary emboli. He was continued on Enoxaparin and bridged to Warfarin once critical care stability was achieved. No major (or minor) bleeding was observed during this gentleman's hospital stay. The patient ultimately recovered to hospital discharge with a Glasgow Outcome Score of 4, secondary to watershed cerebellar infarcts.A number of trials and guidelines address the issue of thrombolysis during a massive pulmonary embolism and sub-massive pulmonary embolism. There are few specific guidelines which directly address the issue of thrombolysis during cardiac arrest in those with (suspected) massive pulmonary embolism , that is “instituted on clinical grounds alone if cardiac arrest is imminent.” The American Heart Association seems to recommend a two-hour infusion of 100 mg of alteplase in those with haemodynamic compromise (though they do not explicitly address the issue of cardiac arrest) [The British Thoracic Society (BTS) recommends a bolus of 50 mg alteplase for massive PE  and stat arrest) . The 201 arrest)  recommen arrest) , though P = 0.06) as well as survival to discharge .There are numerous case reports and case series describing survival post thrombolysis in cardiac arrest caused by PE. Er et al.  retrospeOnce thrombolysis is initiated for a suspected PE in cardiac arrest, guidelines suggest that CPR should be continued for at least 60–90 minutes , 32–36.Domino or double dose thrombolysis also appears in the literature. In the sentinel study by Böttiger et al. , 90 patiP < 0.001). The issue of risks and benefits in terms of haemorrhage remains a major consideration and while the principle of primum non nocere is more difficult to weigh in those in cardiac arrest, it should be remembered that the bleeding risks remain significant. Evidence would suggest that thrombolysis does not seem to be unduly associated with catastrophic haemorrhage in this critically ill patient group [There is general consensus that thrombolysis should be considered in cardiac arrest where pulmonary embolism is strongly suspected. The exact dosage and timing of fibrinolysis remain to be clarified , 37, 41,nt group , 42, 43;nt group  and 3.6%nt group  in conte2 (ETCO2) is seen both in cardiac arrest [2 are seen in recovery from both entities and have prognostic value in those in cardiac arrest in predicting the likelihood of ROSC [Low end-tidal COc arrest –48 and ic arrest –55. Incr of ROSC . The unrCommon echocardiographic findings in massive pulmonary embolism include that of an enlarged right ventricle  which maIn conclusion, this particular case describes a clinical scenario in which double dose thrombolysis was successfully used. A similar strategy might be contemplated in the future by emergency physicians dealing with cardiac arrest caused by massive pulmonary embolism.


Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues.Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the  The function of an individual protein, therefore, is often determined by the identity and range of the proteins to which it binds Intrinsically disordered proteins and protein regions are present in more than one third of protein complexes and are enriched in proteins with multiple partners In contrast to the complementary interface formed by two folded proteins, a subset of disordered regions remain highly dynamic even when bound, either initially through an induced-fit binding mechanism Drosophila melanogaster Hox transcription factor. Ubx is composed of both structured and disordered regions , a  regions  [6], . Alternative splicing, combined with protein partner availability may also impact how Ubx selects DNA binding sites. Ubx binds three different categories of DNA sequences, defined by the protein interactions in which Ubx engages: i) multiple, closely spaced Hox binding sites that permit cooperative Ubx binding, ii) single or multiple Hox binding sites interspersed with binding sites for other transcription factors, or iii) Hox-Exd heterodimer binding sites . The parFinally, whereas the α-α superhelix partners bound specific disordered regions and Ubx isoforms better than others, the DNA/RNA binding 3-helical bundle fold partners tended to bind all three disordered regions equally well and bound UbxIa as well as UbxIVa. The reduced sequence specificity of DNA/RNA binding 3-helical proteins may reflect the fact that all of the disordered regions in Ubx evolved to interact with the Ubx homeodomain to regulate DNA binding in vivo function in vivo. As the Ubx sequence evolves, a specific motif for binding that partner may emerge, and with time eventually become an obligatory binding site. Examples of proteins at each of these stages may be found among the Ubx partner proteins. Most of the partners appear to recognize the disordered regions without any clear sequence or motif preferences, representing a relatively early stage in the evolution of partner binding. However, a 14-3-3ε interaction motif ubx mRNA splicing, allowing tissue-specific control of Ubx's affinity for 14-3-3ε. In our model, the enhancement, but not obligatory reliance, of partner binding by a recognition motif represents an intermediate stage of partner evolution. Finally, Exd/Pbx is an ancient Hox protein partner required for many basic Hox functions. Although the disordered regions in Ubx influence Exd binding, Exd interactions are primarily dependent on specific motifs in the Ubx sequence in vivoThe sequence of intrinsically disordered regions evolves more rapidly than for structured regions via phosphorylation and mRNA splicing provide opportunities for tissue-specific regulation of Ubx-partner interactions.We have demonstrated that the intrinsically disordered regions in Ubx select interacting partner proteins based, in part, on the topology of the protein partner. Furthermore, partner topology determines the affinity of binding to Ubx spliceoforms. The ability of multiple disordered regions in Ubx to bind numerous partners creates a variety of mechanisms for regulating partner binding, including competition or cooperation, preferences of alternatively spliced Ubx isoforms for specific protein – and thus DNA – interactions, and synergistic partner and DNA binding. The overlap of partner binding regions with functional or regulatory domains may provide an additional mechanism for partners to impact molecular functions such as transcription activation and DNA binding. Alteration of the Ubx disordered regions Figure S1Maps of a large-scale Drosophila melanogaster yeast two-hybrid data  (A) All fold•fold interactions with a confidence score of at least 0.5 are shown. Intrafold interactions are depicted as loops which connect back to the originating node. (B) Mapping only fold•fold interactions with a confidence score of at least 0.5 and containing at least 3 protein•protein interactions significantly simplifies the depiction. The total number of protein interactions (for between 3 and 12 interactions) in one fold•fold connection is reflected in the weight of the lines. Connections with 12 or more interactions have the same line weight. Key folds discussed in the text are labeled on both maps.(TIF)Click here for additional data file.Figure S2Probability distribution curves for Drosophila protein interactions from a large-scale yeast two-hybrid experiment parsed by fold. Data were fit to a truncated scale-free model. The scatter observed at high k is often observed in scale-free systems (TIF)Click here for additional data file.Figure S3The distribution of the fold to interaction ratio (F/I) for (A) all single domain proteins and (B) all single domain proteins with more than one partner. Proteins with a high ratio do not select protein partners by fold, whereas interactions with proteins with a low ratio have strong fold preferences. Ubx has an F/I ratio of 0.61, indicating a strong ability to select partners by fold.(TIF)Click here for additional data file.Figure S4AkUbx, a Ubx orthologue with only one intrinsically disordered region, cannot bind Drosophila Ubx partners. Sequence alignment between Akanthokara kaputensis Ubx (AkUbx) and Drosophila melanogaster Ubx showing the locations of disordered residues (red boxes) and the three disordered regions (blue labels).(TIF)Click here for additional data file.Figure S5Ubx variants expression level does not correspond with partner interaction strength. (A) Quantitative Western blotting result for Ubx variants protein expression in yeast (Strain:EGY48 transformed with p8op-LacZ reporter plasmid). (B) Weak correlation between yeast two-hybrid result and Ubx variants protein expression without outliers (R2 = 0.1403). Inset plot shows the influence of the two outliers (UbxIbN103 Pro4 Δ292–389 mCherry and UbxIb Δ292–389 Pro4 mCherry) on the correlation between yeast two-hybrid result and the Ubx variants protein expression.(TIF)Click here for additional data file.Table S1Ubx partners with non-selected folds. A fold with only one partner was classified as a non-selected fold. Folds for Ubx binding partners were classified according to SCOP.(DOCX)Click here for additional data file.