Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns.Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in  Drosophila melanogaster. Our method involves assembling known muscle enhancers, expanding this set with evolutionarily conserved sequences, computationally classifying these enhancers based on their shared sequence features, and scanning the entire Drosophila genome to predict additional related enhancers. Using this approach, we created a map of 5,500 putative muscle enhancers, identified candidate transcription factors to which they bind, observed a strong correlation between mapped enhancers and muscle gene expression, and uncovered extensive heterogeneity among combinations of transcription factor binding sites in validated muscle enhancers, a feature that may contribute to the individual cellular specificities of these regulatory elements. Our strategy can readily be generalized to study transcriptional networks in other organisms and developmental contexts.The development of multicellular organisms requires the formation of a diversity of cell types. Each cell has a unique genetic program that is orchestrated by regulatory sequences called enhancers, comprising multiple short DNA sequences that bind distinct transcription factors. Understanding developmental regulatory networks requires knowledge of the sequence features of functionally related enhancers. We developed an integrated evolutionary and computational approach for deciphering enhancer regulatory codes and applied this method to discover new components of the transcriptional network controlling muscle development in the fruit fly,  Enhancers respond to the combinatorial input of tissue-specific, cell-specific, ubiquitously-expressed and signal-activated transcription factors (TFs) that collectively control gene expression in the appropriate spatial and temporal patterns Complex spatio-temporal gene expression programs guide the progressive determination of pluripotent cells allowing cell fates to become sequentially restricted during embryonic development. These transitions in cell fate are encoded in the genome by de novo motif discovery. In this way, previously described or candidate motifs and/or word profiles can be used to ascertain a training set of enhancers, with the resulting model being used in a genome-wide scan to predict similar enhancers. The enhancer model is validated by testing the activity of these predictions in transgenic reporter assays In recent years, we and others have shown that computational approaches can be used to predict enhancers of a given type with reasonable accuracy when prior knowledge exists of the TFs and their binding sites that contribute to the activity of this enhancer class Drosophila blastoderm embryo The majority of the studies showing the utility of enhancer modeling have focused on regulatory sequences involved in segmentation of the Drosophila larval somatic, visceral and heart muscles from mesodermal progenitors requires the coordinated input of multiple different regulators, including the intrinsic TFs Twist (Twi), Tinman (Tin) and Mef2 Drosophila to vertebrates Drosophila larval somatic muscles are multinucleated myotubes each having unique properties that include their size, shape, orientation, epidermal attachments and innervation The development of the Drosophila mesodermal enhancers having FC activities in order to uncover the motifs that orchestrate gene expression at the level of individual cells, to generate testable hypotheses about the nature of the corresponding FC identity TFs, and to gain insights into the combinations of TFs that contribute to individual FC enhancer specificities. The coordinated input of tissue-specific and signal-activated TFs, combined with the discrete identities of individual FCs, suggests that the regulatory network specifying distinct FC genetic programs is likely to share some common features while differing substantially with respect to others. Furthermore, a series of studies by Erives and colleagues has shown that a family of non-homologous enhancers is characterized by a discrete regulatory signature Here we applied evolutionary and machine learning approaches to model Drosophila species. In vivo testing revealed that these orthologous sequences are functional FC enhancers in spite of having extensive reorganization of their DNA sequences. We show that increasing the training set through the addition of orthologous sequences improves the performance of our enhancer prediction model. By training on this extended set of enhancers, we were able to computationally predict functionally relevant TFBSs and enhancers for the FC gene set. When the resulting classifier was run genome-wide to search for new D. melanogaster FC enhancers, we identified 5,500 high-scoring predictions at a false-positive rate (FPR) of 5%. Moreover, these predicted enhancers were significantly enriched in the noncoding regions associated with known FC genes. While many of the TFBSs learned by the classifier are known to regulate the transcription of muscle FC genes, our classifier predicted additional motifs which have not previously been identified as contributing to FC enhancer activities. Site-directed mutagenesis of five newly discovered motifs in previously characterized FC enhancers demonstrated the critical role played by these TFBSs in supporting full enhancer activity. These validated motifs also suggest plausible candidate TFs acting in the myogenic regulatory network. In one such case—that of the T-box protein encoded by optomotor-blind-related-gene-1 (org-1)—we were able to use loss- and gain-of-function genetic perturbations to establish that this TF functions as a regulator of muscle identity. Furthermore, an analysis of the TFBS compositions of all known FC enhancers revealed an unanticipated complexity in the combinations of TFs that contribute to the unique specificities of individual regulatory elements, a finding that provides a molecular explanation for the well-known diversity of muscle cell identities and their associated gene expression programs.To address this problem, we first compiled a small set of enhancers with activity in FCs. To overcome issues associated with small sample sizes, and to increase the diversity of sequences with similar functions, we extended this set by adding orthologs derived from other in situ hybridization with gene-specific probes; and (4) functional examination of sequence features associated with the computational classification to define novel motifs and TFs regulating myogenesis. An overview of the approach utilized in this study is presented in Here we utilized phylogenetic profiling and machine learning to decipher the motifs and enhancers that underlie the gene expression patterns of individual muscle FCs, which required an array of computational and experimental tools. This study is composed of 4 main components: (1) compiling a training set of FC enhancers from multiple sources including the literature, testing of additional computational predictions from a previous study In situ hybridization of these candidates led to the validation of 180 FC genes  gene is active at different developmental stages and in multiple cell types (including FCs) due to the binding of multiple cell-specific TFs of the same family  reporter and an attB site allowing phiC31 integrase-mediated integration .Of note, in several cases, we observed differences in the organization of TFBSs within enhancer . Neverthxpressed . In thisD. melanogaster counterparts. For example, the ap muscle enhancer is active in a subset of endogenous ap-expressing muscles and was previously shown to depend on the input of Hox TFs D. melanogaster and the D. mojavensis orthologous sequences  gene activates reporter expression in a subset of muscle FCs, pericardial and cardial cells of the heart, and cells of the central nervous system . This gene is expressed in two pericardial cells of the heart and a single dorsal somatic muscle FC D. persimilis sequence is expressed in an identical pattern , though often not identical (Ndg), to the D. melanogaster enhancer. The imprecise activities of some of the orthologous enhancers may reflect the partial level of sequence identity that could affect as yet unidentified binding sites, may result from the extensive shuffling of known binding sites for co-regulatory TFs, or might simply be a reflection of differential gene expression in the orthologous flies In total, 5 out of 5 tested orthologous sequences drove expression in a pattern that is similar  was trained to distinguish between FC enhancers and control sequences based on TFBS occurrences. The ability of the classifier to accurately predict regulatory activity was assessed by a 10-fold cross-validation procedure. The performance of the classifier was evaluated using the AUC, a value ranging from 0.5 (random classification) to 1.0 (perfect classification). The obtained AUC value of 0.89 indicates reliable detection of FC enhancers by the developed classifier see .D. melanogaster putative FC enhancers generally reflects the phylogenetic distances of the species involved in the analysis, with most orthologs in the 50–80% sequence identity range in D. yakuba, D. erecta, D. ananassae, D. pseudoobscura and D. persimillis. However, putative FC enhancer sequences tend to be more deeply conserved than background genomic sequence , and thus, probably functional  of 5%, approximately 5,500 sequences were annotated as putative FC enhancers . Similaronal see . Althougonal see , it was see −10; . The latDrosophila genes have been estimated to be expressed in FCs defective proventriculus (dve) was identified and validated as a novel FC gene using this approach  and ventral transverse 1 (VT1) muscles). This result suggests that additional regulatory elements must account for the complete expression pattern of this FC gene slp1 is also located upstream of the gene and directs reporter activity both to FCs  to each other.To determine if the newly identified motifs are functionally relevant to FC gene expression, we employed site-directed mutagenesis of such putative binding site sequences in otherwise wild-type FC enhancers. We initially concentrated on the potential role of Ets, Myb, POU homeodomain (POUHD) and Fkh binding motifs see . Each ofNdg enhancer reduced or eliminated enhancer activity in subsets of cells which express wild-type Ndg-lacZ (Ndg enhancer (apterous (ap) FC enhancer lead to a complete loss of reporter expression in those FCs in which the wild-type enhancer is active  and optomotor-blind-related-gene-1 (org-1) are the only T-box TFs expressed in muscle FCs org-1 is co-expressed with Slou in the FCs corresponding to muscles LO1 and VT1, and with Lbl in the FC for the segment border muscle (SBM)  .org-1 may directly regulate slou and lbl. To test this hypothesis, we identified potential T-box binding sites in the lbl and slou FC enhancers  for the presence of a total of 11 types of TFBSs that are known to contribute to FC activity. For this purpose, we added 7 motifs from prior studies of FC enhancers to the 4 new motifs discovered here.Having identified and experimentally validated the functions of 4 novel TFBSs that we found to be over-represented in FC enhancers—POUHD, Myb, Fkh and T-box—we were next interested in determining the distribution of all known regulatory motifs in enhancers of this class. We reasoned that such a survey might reveal whether TF combinatorics contribute to FC enhancer specificity. Thus, we analyzed all 18 in silico evaluation of candidate transcriptional regulatory models, we demonstrated that Pnt, Twi and Tin alone target a subset of highly Ras-responsive FC genes We had previously constructed and validated a regulatory model of FC enhancer activity which reflected the coordinated input of Tcf, Mad, Pnt, Twi and Tin Using position weight matrices (PWMs) for 3 signal-activated TFs , the ubiquitously expressed Myb, 4 tissue-restricted TFs , DNA sequence pattern analysis, or hybrid methods that combine both of these strategies. ChIP-Seq for p300 using mouse embryonic tissue has proven to be an accurate means for identifying enhancers and their associated activities, with cis-regulatory modules are commonly based on machine learning of arrangements of TFBSs in enhancers that have common functions Drosophila muscle FC enhancers—the size of the training set is limited by the lack of experimentally validated tissue- and cell type-specific enhancers, which results in overfitting of computational models and poor accuracy of predictions. To overcome this problem, and to provide a generalizable approach for increasing the size of the training set, we developed a phylogenetic profiling strategy based on a search for diverged orthologous counterparts of available enhancers from distantly related species. Twenty-four Drosophila orthologs were identified using this approach, which more than doubled the size of the training set. We assessed the ability to accurately distinguish FC enhancers in a cross-validation framework using the extended training set, and determined that the classifier accuracy is 89% as assessed by the AUC approach. We then applied this classifier to scan the entire genome of D. melanogaster for novel FC enhancers, retrieving 5,500 high-scoring predictions at a FPR of 5%. These predictions were significantly associated with genes expressed in FCs, demonstrating that the model was able to capture essential features of FC gene co-regulation. A similar machine learning approach could be applied to a diverse array of datasets, including experimentally-verified regulatory elements from co-expressed targets at either a germ layer, organ, tissue or cellular level from invertebrate and vertebrate databases Computational approaches for predicting et al.Drosophila species to identify over-represented motifs in the regulatory elements of genes expressed in neural progenitor cells, with sequence orthologs used to enrich the training set and to give prominence to conserved motifs. However, our method extends this approach by including suitably diverged orthologous enhancers from other Drosophila species in the dataset used to train the classifier. Our purpose in designing this strategy was two-fold. First, we wanted to enrich for relevant sequence motifs in the training data, allowing for a level of variation that would improve the generalization of the model. Second, we wanted to provide a potentially wider variety of TFBS arrangements that characterize the architecture of authentic FC enhancers. In essence, the addition of orthologous sequences boosts the statistical power of the significance tests, revealing patterns of TFBSs that otherwise could have been neglected.Evolutionary constraint of functional sequences is routinely employed as an effective filter to improve the prediction of regulatory elements D. melanogaster, the overall expression pattern generated was similar to the D. melanogaster counterpart despite extensive evolutionary shuffling of known TFBSs. Similar binding site reorganization has been documented for the enhancers that regulate both the segmentation and mesodermal patterns of eve expression Of note, when 5 of these orthologous sequences were tested in transgenic reporter assays in in vivo functions. Seventy-five percent of the putative enhancers were experimentally validated as having transcriptional activity, demonstrating the effectiveness of our approach to identify regulatory sequences. However, of the sequences showing regulatory functions, only 4 of 9 were active in the mesoderm—including 2 in FCs—and 3 of 9 had nervous system activity. These data suggest that our model has been able to reliably recognize general properties of tissue-specific enhancers without specifically distinguishing an overall muscle FC code, even though numerous individual FC-specific motifs were identified (see below). The former finding is similar to the results of Sinha and colleagues To assess the accuracy of our method, we selected 12 predicted FC enhancers and tested their hunchback gene is also active in the longitudinal visceral mesoderm, and enhancers directing the FC expression of the vestigial, big brain and king-tubby genes are also active in the peripheral nervous system . Given the strong maternal contribution to this gene acj6 transcripts, but this manipulation had no effect on Ndg-GFP reporter activity (data not shown). These findings leave unresolved the identity of the TF that binds to the motif in question. The future characterization of this TF, including exploring the possibility that it is not a POUHD protein, will require searching functional motifs against larger TF databases To extend the components of the myogenic regulatory network beyond these known TFs and motifs, we examined the function of the classifier-defined sequence motifs recognized by POU homeodomain and Myb proteins, transcription factors having no previously known role in Ndg enhancer led to extensive de-repression of the reporter in other mesodermal cells. Myb is a ubiquitously-expressed DNA binding protein which plays a critical role in controlling regulatory decisions during proliferation and differentiation of progenitor cells Myb. In any event, as myogenesis in Drosophila occurs through a series of asymmetric and symmetric cell divisions Inactivating mutations of the Myb binding sites in the Drosophila somatic muscle development, despite widespread functions of this TF class in mesoderm specification and myogenesis in vertebrates Drosophila and vertebrates cis and trans tests of TF function, along with gene co-expression, that Org-1 is a muscle identity TF. In particular, the cis effects of Org-1 were documented in the FC enhancers associated with two known muscle identity TFs, Slou and Lbl, and org-1 expression localizes to the SBM and VT1, muscles in which the lb genes and slou, respectively, are the only previously described determinants of muscle identity Slou function is critical for the proper development of muscles LO1 and VT1 and is further required to repress the lb genes in these cells, suggesting a co-regulatory relationship between slou and lborg-1 acts upstream of slou and lb in this regulatory hierarchy since org-1 expression precedes slou and lb, and the ectopic expression of org-1 causes increased expression of slou and lb  plus 2 tissue-restricted (Twi and Tin) TFs constitute distinct regulatory models for different FC enhancers The marked heterogeneity of FC enhancer architecture uncovered here reflects not only distinct combinations of various TF classes , but also diversity at other biological levels, including the unique identities of the thirty muscle FCs and their differentiated derivatives in each abdominal hemisegment, and the different gene expression patterns exhibited by those particular cells. Thus, TFBS combinatorics provide a plausible molecular explanation for the functional complexity of enhancers having related but non-identical activites at the resolution of individual cells in the context of the developing embryo.de novo motif discovery, classification of enhancer sequence features, empirical testing of candidate enhancers, and cis-trans tests of target gene regulation. We also have established that a small set of training sequences can be expanded with orthologous sequences trans-acting factors regulating a set of co-expressed genes. Finally, we anticipate that an iterative application of this approach, which could include training on datasets of different epigenetic marks associated with active enhancers We have investigated the transcriptional regulatory network specifying individual muscle FCs using an integrated genomics approach that includes identification of orthologous enhancers, Drosophila stocks containing the following transgenes and mutant alleles were used: UAS-org-1 , attP40 and nos-phiC31intNLS lbl-lacZ and Ndg-lacZap-lacZ  6acj6  twi-Gal4in vitro  or PCR-amplified and sequence-verified and then subcloned into the reporter vector pWattB-GFP  or pWattB-nlacZ. The pWattB-nlacZ vector was constructed by cloning the EcoRI-SpeI fragment from a version of pH-pelican in situ hybridization and fluorescent in situ hybridization with tyramide signal amplification  followed standard protocols twi-Gal4 UAS-org-1 were incubated at 25°C. For fluorescent staining, the following antibodies were used: mouse anti-Ladybird early (Lbe) , rabbit anti-Slou , chicken anti-GFP , mouse anti-βgal , rabbit anti-Kirre , rabbit anti-MHC , and guinea pig anti-Eve .Enhancer regions were either synthesized http://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl), two independent gene segments for synthesis of org-1 double-stranded RNA (dsRNA) were selected with lengths of 570 and 473 bp and with less than 20 and 22 bp of identity to any other predicted gene, respectively. These segments of the org-1 gene were PCR-amplified from primary embryonic cDNA using the primers, CGTCCAAAAAGTTCAAGGGA and GCTCGTTCTCATCCAAGGAG (570 bp) and GCTCCAACAGAGCCAGAATC and CCGAACCGTAAAAACTTGGA (473 bp), and transcribed in vitro using the MEGAscript RNAi kit . lbl-GFP, slou-GFP or MHC-tauGFP embryos were dechorionated and injected with negative control (lacZ) or org-1 dsRNA at the syncytial blastoderm stage and allowed to develop to stage 14 or 16 before examination by fluorescence microscopy for assessment of reporter GFP expression. A similar protocol was used to assess the function of acj6 in Ndg-GFP embryos.Embryo RNAi was performed as previously described Drosophila melanogaster FC enhancers range in length from 311 to 2068 bp (average length 1232 bp), in GC-content from 39% to 49% (average GC-content 43%), and in repeat-content from 0% to 7% (average 1.5%). The twenty-four orthologs have similar characteristics, with an average length of 1311 bp, GC-content of 43% and repeat content of 5%. The control set comprised 1000 randomly selected D. melanogaster noncoding genomic sequences with length, GC- and repeat-content matching those of the enhancer set.The sixteen sequences in the training set of Putative TFBSs were identified by searching the sequences with MAST Over-represented TFBSs were determined by comparing the occurrence of the motifs among query sequences and background genomic sequence, and applying Fisher's exact test. We used a P-value threshold of 0.05. When indicated, we adjusted the P-values for multiple testing using the procedure suggested by Benjamini and Hochberg D. melanogaster enhancer sequences with a large number of orthologs, de novo motifs were identified on a restricted set of 34 sequences, including at most two randomly selected orthologs for each D. melanogaster FC enhancer. Features relevant for distinguishing between enhancer sequences and controls were identified using linear support vector machine (SVM). We used a standard ten-fold cross-validation procedure to assess the accuracy of the classifier. In each fold of the cross-validation procedure, the de novo motifs were extracted using the training data, only, thereby ensuring that the test data were completely unseen before the predictions were made. The cross-validation procedure should help to prevent overfitting of the classifier.Each nucleotide sequence in the FC enhancer and control sets was represented by the number of putative TFBSs per base pair. Putative TFBSs were identified by searching the sequences for motifs derived from three different sources: (1) 892 TF binding specificities characterized in TRANSFAC, (2) 3 binding sequences for Tin, Twi, and Pnt from the literature We used Support Vector Machines 1, …, xlx1, …, ylyw can be used to decide the relevance of each feature Given a training set of instances D. melanogaster (BDGP Release 5 assembly) with a sliding window of length 1000 base pairs and overlaps of 500 base pairs. The length of the window corresponds approximately to the average length of the sequences in the training data set (1280 base pairs). We scored 137,364 sequences after excluding sequences which overlap annotated coding regions by at least 50%. The cut-off for the genome scan was defined to obtain a false positive rate (FPR) of at most 5% by training and testing 100 classifiers on random partitions of the training data .We scanned the whole-genome of TF annotation for PWMs was obtained from TRANSFAC and the Broad Institute MsigDB database Drosophila melanogaster 62 enhancers active in mesoderm from the REDfly database Drosophila melanogaster enhancer in REDfly. As controls, we randomly sampled for each enhancer (and orthologs) 10 non-coding sequences from the Drosophila melanogaster genome with similar length, GC- and repeat-content.To understand the effects of a sample size on the classifier performances, we first extracted a dataset of Each nucleotide sequence in the enhancer and control sets was represented by the number of putative TFBSs per base pair. Putative TFBSs were identified by searching the sequences with MAST Figure S1in situ hybridization analysis of stage 11 embryos containing RhoGEF3-lacZ (A), FBX011-lacZ (B), cib-lacZ (C) or fra-lacZ (D) transgenes using probes for endogenous RhoGEF3 (A), FBX011 (B), cib (C), and fra (D) transcripts. Panels A′ to D′ show the corresponding signals for lacZ transcripts, and panels A″ to D″ show the merged channels. All enhancers were selected from previously identified candidates Empirical validation of predicted FC enhancers conforming to a previously described regulatory model. Fluorescent (TIF)Click here for additional data file.Figure S2D. melanogaster enhancers active in various mesodermal cell types. The sample size was varied from 10 to 60 by an increment of 5. Each sample was used to train a Support Vector Machine (SVM) classifier. For each sample size, we compared the performance of the classifier trained exclusively with D. melanogaster (“dm3 only”) enhancers with that of a classifier that, in addition, was trained with up to two orthologs for each D. melanogaster enhancer (“dm3 with orthologs”). All classifiers were validated on sets comprising only D. melanogaster enhancers. Control sequences were randomly selected from regions of the D. melanogaster genome with comparable length, GC- and repeat-content. The entire process was repeated a total of 1000 times. (A) Performance of each classifier, measured by its AUC, estimated in a 10-fold cross-validation. Classifiers trained on D. melanogaster enhancers and their orthologous sequences with an AUC significantly greater than that of the corresponding classifier trained exclusively on D. melanogaster enhancers  are marked with a red asterisk. (B) Precision of the estimated calculated based on the Root Mean Square (RMS) error. The RMS describes how well the AUC value estimated in the cross-validation represents the true AUC of the classifier and thus, how good is our assessment of the underlying model; the true AUC of each classifier was computed using the enhancers excluded from the randomly selected sample. (C) Number of enhancers recognized as such in at least 50% of the instances in which they were tested. In the cross-validation process each sequence is used exactly once for validation. Thus, for 100 randomly selected samples and their corresponding cross-validation processes, we counted the number of times each sequence scored positively, compared this number with the number of times each sequence had been included in a random sample, and repeated the complete procedure 10 times to estimate the variance of the results. Classifiers trained on D. melanogaster enhancers and their orthologous sequences consistently recognizing a significantly higher number of sequences as compared with the respective classifiers trained only on D. melanogaster enhancers  are marked with a red asterisk. (D) Concordance of prediction outcome between each pair of 1000 classifiers, for each sample size. In this graph, the line segments represent the 95% confidence intervals surrounding the means. We compared the sequences positively scoring in the 10 folds of the cross-validation experiment between each pair of classifiers. Randomly sampled training and test datasets differ. The likelihood of observing a large overlap between two samples taken from the same (finite) population increases with the size of the samples. Therefore, larger datasets produce larger overlapping outcomes. However, for the same sample size, the overlap between the outcomes of classifiers trained on D. melanogaster enhancers and their orthologous sequences is systematically significantly higher as compared to that of classifiers trained only on D. melanogaster.Variation of the classification performance with increasing sample size. Samples were randomly selected from a dataset of 62 (TIF)Click here for additional data file.Figure S3lbl (A), ap (B), Ndg (C) and eve (D) enhancers were aligned against the orthologous enhancers of D. persimilis (D. per), D. ananassae (D. ana), or D. mojavensis (D. moj). Motif matches to Tcf (black), Mad (blue), Ets (red), Twi (green) and Tin (purple) for these co-regulating TFs of the eve MHE are shown. For the Ndg, lbl, and ap enhancers, motif matches to the co-regulating TFs Ets (red), Twi (green) and Tin (purple) are shown. Motifs are based on known functional binding sites  is shown . For ap, Antennapedia (Antp)-protected functional binding sites are shown for D. melanogasterD. mojavensis (Binding site conservation and evolutionary flux in orthologous FC enhancers. Sequences of the (TIF)Click here for additional data file.Figure S4TFs most relevant to the FC enhancer classification. Presence (red)/absence (gray) of the fifty most relevant TF binding motifs in the set of enhancer sequences used for training. TFs were ranked according to the SVM weights of their respective motifs, which represent their discriminating power. We only considered the highest scoring motif for each TF (median ranks computed across 10 random partitions of the training data varied between 12 and 129). Control TFs were randomly chosen among TFs for which the highest scoring motif had a neutral weight (median ranks computed across 10 random partitions of the training data varied between 437 and 450). TFs and sequences have been clustered using average linkage and Euclidean distance. The phylogenetic tree represents the relations among the sequences in the training data, built on the presence/absence of the motifs for the most relevant TFs. De novo motifs were explicitly excluded from this analysis.(TIF)Click here for additional data file.Figure S5de novo motifs exhibiting the highest power discriminating FC enhancers from background sequence resemble motifs of known transcription factors with roles in mesoderm and FC development. The identity of the transcription factors binding to the de novo motifs was queried using STAMP PRIORITY motifs are representations of known motifs. Many of the (TIF)Click here for additional data file.Figure S6D. melanogaster FC enhancers (dm3) and orthologous and control sequences. Position weight matrices for each of these TFs are also shown.Motifs identified by the classifier that are overrepresented in FC enhancers and their orthologs. Graphs comparing the representation of V$ETS_Q4 (A), V$POU1F1_Q6 (B), V$MYB_Q6 (C) V$TBX5_01 (D) and V$FOX03_01 (E) motifs in (TIF)Click here for additional data file.Figure S7Ndg enhancer in which Ets sites are selectively inactivated (ETS-GFPNdg) is extinguished compared to β-Gal (magenta) driven by WT-lacZNdg. We have previously demonstrated the activity of this enhancer in a subset of FCs, two pericardial and two cardial cells of the heart, the gut musculature and two cells of the central nervous system . (C) Activity of the wild-type ap enhancer in lateral transverse muscles, as revealed by GFP expression driven by the WT-GFPap transgene. (D) Complete loss of ap enhancer activity after Fkh binding sites are inactivated (Fkh-GFPap).The wild-type activities of FC enhancers require input from classifier-defined Ets and Fkh TF binding motifs. (A) GFP (green) expression driven by a version of the (TIF)Click here for additional data file.Figure S8org-1 with Slou and Lbl. Expression of org-1 RNA in stage 11 (A) and stage 13 (B) embryos detected by in situ hybridization. Co-expression of org-1 RNA (purple) with Lbl protein (brown) in the Lbl-expressing SBM FC  and myotube . Co-expression of org-1 RNA (purple) with Slou protein (brown) in the LO1 and VT1 FCs  and myotubes . CVM: circular visceral muscle which expresses org-1 but neither lbl nor slou.Co-expression of (TIF)Click here for additional data file.Figure S9Motif distribution in FC enhancers. (A) The heatmap illustrates the occurrence of 11 motifs that have been shown to be relevant for FC regulation in the 18 sequences that have been positively assayed for FC enhancer activity. Columns and rows are clustered using Ward's method and binary distances. (B) Maximum fraction of the 18 assayed FC enhancer sequences sharing N motifs that have been shown to be relevant for FC regulation, for N in {1, 2, …, 11}.(TIF)Click here for additional data file.Table S1D. melanogaster and orthologous enhancer regions used for training, and the list of FC genes considered in this study.Expression and genomic coordinates of (XLSX)Click here for additional data file.Table S2D. melanogaster mesodermal enhancers. The majority of these enhancers have multiple activities.Genomic coordinates and ontology of REDfly (XLSX)Click here for additional data file.Table S3Classifier predictions and the activity and genomic coordinates of the tested enhancer predictions.(XLSX)Click here for additional data file.Table S4Ndg, lbl, slou and ap FC enhancers.Motifs identified by the classifier and the mapping of TFBSs in the (XLSX)Click here for additional data file.Table S5in situ hybridization analysis of T-box and POUHD family members.Summary of (XLSX)Click here for additional data file.Table S6Mapping of TFBSs in all FC enhancers.(XLSX)Click here for additional data file.Text S1Conservation Profile of Candidate FC Enhancers and TFBS Distribution Among Orthologs of Candidate FC Enhancers.(DOCX)Click here for additional data file.


PTEN gene in the Basal-like SUM149 breast cancer cell line.In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH  was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations .PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival.Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to gene inactivation. These events may contribute to tumor formation through mechanisms not detected using conventional DNA copy number analyses. Copy Number Aberrations (CNA) that can also define genetic events associated with different expression subtypes A hallmark of many human cancers is genomic instability, and cancer itself can be thought of as the result of an altered ploidy As knowledge about breast and other cancers advances, we are finding that there are numerous complex ways by which the genome can be disrupted. While certainly gross chromosomal aberrations and rearrangements have been seen to initiate disease All samples used in this study were collected using IRB-approved protocols and all patients signed informed consent forms and the data were analyzed anonymously.2 ratio data were taken from the UNC Microarray Database (UMD), filtering for a lowess normalized intensity value of 10 or above for each channel, and 70% present data values, and then used for further analyses. Data is available from Gene Expression Omnibus under GSE36889 Sample information including clinical data, subtype, source, GEO Sample ID and overlap with copy number information, can be found in The dataset used here contained both gene expression and high-density array comparative genomic hybridization (HD-aCGH) copy number data from a set of breast tumors from UNC “HD-UNC94” (n = 94). Additionally, the SUM102 and SUM149 breast cancer cell lines 2 ratio data from the HD-UNC94 data set used different gene expression microarray platforms. The dataset was therefore then limited to the probes/genes shared across both platforms. After column standardization of both platforms ), Distance Weighted Discrimination (DWD) 2 ratio data was collapsed (via averaging) from probes to HGNC gene symbols. The PAM50 gene set predictor The Lowess normalized gene expression R/G Loghttps://earray.chem.agilent.com/earray/) software and built on the Human 244 k Custom Oligo platform (GPL15359 Agilent UNC Perou Lab 1X244 k Custom Tiling CGH Array). 230,606 probes cover a total region of 45 Mb, which includes the full genomic sequence of the 128 genes of interest as well as the region 150 kb upstream and downstream of each of these genes (www.genome.unc.edu).The custom HD-aCGH tiling platform was designed using Agilent’s E-array v5.0 online , we utilized the SWITCHdna algorithm In order to identify the regions of genes most commonly affected by micro-aberrations, each gene was divided into four quadrants based upon a proportional splitting of each gene into four equal segments: 5′ End , 5′ Middle (first ½ of gene), 3′ Middle (second ½ of gene), 3′ End . For every micro-aberration instance, the affected quadrants were tallied for each quadrant, and the proportion of affected quadrants out of all possible quadrants was calculated. Similarly, each micro-aberration instance was assessed in terms of whether it encompassed the promoter or 5′ untranslated region (UTR) of each gene, with the promoter region defined as genomic space upstream of the transcription start site., and mapped using MapSplice de novo assembly with the Trinity de novo assembled contigs then compared to the reference using BLAST.mRNA-seq was performed on total RNA isolated from cell lines and tumors using the Qiagen RNeasy Mini Kit (Cat. No. 74104). Library preparation was performed using the TruSeq RNA Sample Kit from Illumina (Cat. No. RS-930-2001) following the low input protocol detailed in the manufacturer’s guidelines. 1×76 bp nucleotide reads were generated using an Illumina GAII sequencer for the SUM102 cell line. For the two tumor samples (UNC990141B and UNC040182B), we sequenced using a 2×50 bp configuration using an Illumina HiSeq2000. In all cases, the read data were aligned to the human HG19 reference genome from the UCSC genome browser The patients in the dataset were rank ordered by total number of SWITCHdna-defined aberrations, and micro-aberrations, and separated in the top 67% and bottom 33%; additional rank order splits were also evaluated, but data not shown. Survival analyses were performed using the Kaplan-Meier test in R In order to test the hypothesis that primary breast cancer genomes contain areas of small-scale copy number gains and losses, termed micro-aberrations, we designed a custom, high-resolution, high-density, comparative genomic hybridization tiling array (HD-aCGH) with an average probe spacing of 200 base pairs, and which was focused on 128 selected genes . We asseTo objectively define a micro-aberration, we established a size definition of the smallest 25% of SWITCHdna identified aberrations in this dataset, which resulted in the size cut off of approximately 64 contiguous probes (∼15 kb). The previously identified PTEN micro-amplification in the SUM149 cell line is able to be identified using these criteria. The genomic landscape of the basal-like subtype exhibited many of these micro-aberrations, as basal-like tumors showed the highest incidence of these events .The value of the high-density tiling array platform is also shown in another example of a RB1 alteration . Each idBeyond simply determining the frequency of micro-aberrations present within each gene or tumor, we wanted to assess whether the presence of micro-aberrations would result in functional consequences. We first assigned a copy number status for each gene and each sample  and then for each gene, determined whether the corresponding gene expression was concordant with the type of genomic aberration observed (i.e. micro-amplifications result in increased expression and/or micro-deletions result in decreased expression of the involved gene). We found similar rates of concordant expression between micro-aberrations and gross aberrations, with 30–40% of the tested genes showing 100% agreement between aberration type and gene expression, meaning that for these genes, every sample that displayed a micro-amplification in the gene also had greater than median expression of the gene and likewise every sample that had a micro-deletion in the gene had less than median expression of the gene. Another 50–75% of the tested genes showed at least 50% concordance between aberration type and gene expression meaning at least half of the samples that displayed a copy number aberration had altered expression of the affected gene in the same direction as the CNA . These fWe performed a closer examination of the micro-amplification of the PTEN gene in the SUM149 cell line, as it is a validated aberration that has now been identified by multiple groups. Using mRNA-seq data, we assessed the expression of the PTEN gene on an exon level to determine the functional consequence of the DNA micro-amplification. For comparison, we also examined the data for the SUM102 cell line, which has no genomic alterations in PTEN (data not shown). The distribution of aligned reads for each exon is shown for each cell line , with thEGFR gene results in many mRNA-seq reads that mapped outside of the exon, and that often contain misalignments , we noted the quadrants that it occupied, and then for each quadrant, determined what proportion of the total possible quadrants were affected by a micro-aberration event . We founThe survival outcomes of patients with varying levels of copy number aberrations were also assessed to determine if an association was present. We identified the number of copy number micro-aberrations per sample using our SWITCHdna criteria and rank-ordered the patients in terms of micro-aberration frequency. Each patient in the HD-UNC 94 dataset was assigned to one of two groups depending on whether they were in the top 67% of microCNA or the bottom 33%. Kaplan-Meier analysis was performed examining overall  and relaMYC and PIK3CA, known to be activated in many cancers tend to show more micro-amplifications; others known to be inactivated in cancer such as RB1 and PTEN display comparatively more micro-deletions. Gene Set Enrichment Analysis was performed using DAVID Lastly, we examined the frequency of micro-aberrations for each of the 128 genes tested . Here, wThe previous discovery of micro-aberrations within genes By utilizing a previously tested segmentation and aberration calling algorithm called SWITCHdna Our data also shows that at least in some cases, these events have functional downstream consequences  and S2. EGFR, and the UNC040182B tumor sample and BCL11A, may cause mRNA mis-processing because the micro-aberration alters the DNA sequence in such a way that splice-site junctions are altered and we are able to observe inter-exon mRNA-seq reads. Using targeted de novo assembly of 990141B mRNA-seq data, we are able to generate contigs that suggest an in-place tandem duplication of a region of EGFR that may be the cause of the micro-amplification detected in the tiling array and the source of expression disruption. When examining data on the UNC040182B tumor sample, we are able to produce a contig that covers only the latter portion of the affected exon, but extends out into intronic space. The micro-deletion located in this region may knock out the initial portion of the exon, resulting in the production of the observed aberrant mRNA  mirror whole genome study findings, and 2) potentially showcase an alternative means of gene disruption that unravels previously unexplained expression data. As one example, RB1 dysfunction has been shown to be associated with the basal-like subtype de novo whole genome assembly Future studies with whole-genome sequencing technology and data are also an additional avenue by which these micro-aberrations can be validated, detected, and further defined. Varying amounts of sequencing depth would, however, be needed depending on the nature of the micro-aberration. Micro-deletions should be comparatively easier to detect as they are due to the absence of DNA, thus reasonably low coverage should be sufficient to call these events. Micro-amplifications would be more complex, especially if one did not know in advance what the nature of the micro-amplification was. Thus, in order to reliably identify all micro-aberrations, one would need sequencing depth capable of performing a RB1 and PTEN were recently described in melanoma cell lines  and SUM102 cell line sample (bottom lane) for the EGFR gene is visualized using IGV. The corresponding tiling array copy number plot for the tumor sample and gene and the associated area of genomic coverage is highlighted in the bottom panel.(PDF)Click here for additional data file.Figure S2mRNA-seq read distribution for BCL11A in the UNC040182B tumor sample. The distribution and alignment of mRNA-seq data for the UNC040182B tumor sample (top lane) and SUM102 cell line sample (bottom lane) for the BCL11A gene is visualized using IGV. The corresponding tiling array copy number plot for the tumor sample and gene and the associated area of genomic coverage is highlighted in the bottom panel.(PDF)Click here for additional data file.Figure S3de novo assembly of targeted EGFR micro-amplification mRNA-seq data in the 990141B tumor sample. A) The two contigs aligned to the region of EGFR micro-amplification are visualized in space using the UCSC genome browser. B) The aligned region of the contigs are displayed in red with the unaligned base pairs at the start and end of the sequence written out. Sites within the aligned region where the unaligned GACCT sequence are observed are highlighted with black boxes.(PDF)Click here for additional data file.Figure S4de novo assembly of targeted BCL11A micro-deletion mRNA-seq data in the UNC040182B tumor sample. The contig aligned to the region of BCL11A micro-deletion is visualized in space using the UCSC genome browser, with the location of the reference exon site displayed. A magnified view of the region is also provided.(PDF)Click here for additional data file.Table S1Clinical data table. Table containing all information on the human breast samples used in this study including outcomes data, microarray and GEO accession number IDs.(XLSX)Click here for additional data file.Table S2Gene list, tiling array coverage, and analysis windows. List of the 128 genes on the tiling array with EntrezGene ID along with their genomic coordinates by NCBI build 36.1 (hg18) of the reference human genome. Coordinates are also provided showing the total coverage on the tiling array for each gene as well as the +/−5 kb analysis window that was used.(XLSX)Click here for additional data file.Table S3Micro-aberration information table. Table containing information on all micro-aberrations identified using the defined parameters described in this manuscript from the tiling array. Information for each listed micro-aberrant segment includes Sample name, Subtype, Affected gene, Start position, Stop position, and Average log-ratio for the segment.(XLSX)Click here for additional data file.


Coronary-artery-bypass-grafting (CABG) with conventional extracorporeal circulation (CECC) is associated with adverse effects such as systemic inflammatory response leading to a decrease in systemic vascular resistance and hemodynamic instability. Modern "less invasive" procedures have been established recently which potentially avoid negative side effects of CECC. The aim of this study was to compare perioperative outcome following coronary revascularization using either a minimized extracorporeal circuit (Mini-HLM) or off-pump technique (OPCAB).In this prospective ethics-approved trial, 120 patients referred for CABG were randomly assigned either to off-pump coronary artery bypass (OPCAB) or to a Mini-HLM procedure. Patient demographics, preoperative characteristics and extensive postoperative outcome were analyzed for both groups. Hemodynamic data were measured at seven time points perioperatively.Operation-time was longer in the Mini-HLM group  compared to OPCAB  with higher graft numbers in Mini-HLM group . There were no significant differences in perioperative hemodynamic criteria, catecholaminergic support, hospital  and intensive care unit stay , ventilation time , blood loss , transfusion requirements, postoperative atrial fibrillation rate  and neurocognitive disturbance . No deaths and no myocardial infarctions were observed.Coronary revascularisation with Mini-HLM represents a suitable and "less invasive" procedure which achieves all benefits of OPCAB but may allow for less demanding revascularisation than OPCAB in special patients with complex coronary anatomy and can therefore be used both on a routine basis and in all "conversion" cases of OPCAB. Coronary artery bypass grafting (CABG) using conventional cardiopulmonary bypass (CCPB) was first introduced into clinical practice in 1950 -5. AlthoIn this prospective randomized clinical trial, 120 patients referred for isolated elective CABG between December 2008 and July 2011 were enrolled in the study and preoperatively randomized either to OPCAB (n = 44) or Mini-HLM (n = 76) group. The study was performed with the approval of the ethics committee of the medical faculty of the University of Cologne and was in compliance with the Helsinki declaration. For randomization, we initially decided to use the patient chart number with even last digits for the OPCAP- and uneven last digits for the mini-HLM cohort. Unfortunately, this method resulted in an asymmetric distribution of patients rather than a 1:1 ratio; however, in order to avoid a change in the randomization procedure in the ongoing study we continued with this method. After assessment of each coronary angiographic examination by the operating surgeon with special emphasis on the general acceptability all patients were informed about the study. The appropriate written informed consent was obtained from each patient of this clinical trial, and all perioperative data were collected electronically. Included were patients with coronary artery disease and indication for CABG. In order to guarantee the comparability of the two study groups, patients with the following conditions were excluded from the trial: significantly impaired left ventricular pump function (ejection fraction <20%), preoperative catecholaminergic support, hemodynamic instability, mechanical circulatory assistance as well as urgent and emergent cases.The primary end point of our clinical trial was the in-hospital mortality, secondary end points were intra- and postoperative characteristics with the emphasis on invasive hemodynamic assessment using a pulmonary artery catheter (PAC) as described below.All operations were performed by only three senior cardiac surgeons with high experience in OPCAB revascularization in cooperation with experienced anesthesiologists and perfusionists in our department. The surgical access was performed via median sternotomy. The left (LIMA) and - when applicable - right internal mammary artery (RIMA) were harvested together with veins and surrounding tissue as a pedicle graft in both groups. Additional graft material (saphenous vein) was harvested using minimal invasive technique. Body temperature was kept as normal as possible, either by the heat exchange system of the Mini-HLM or by specific warming blankets of different manufacturers in the OPCAB group. No patient was discharged from OR with a temperature below 36.0°C. In all cases of CABG using Mini-HLM several doses of Calafiore warm blood cardioplegia were given in an antegrade fashion through the aortic root after establishment of cardiopulmonary bypass with an arterial cannula in the ascending aorta and a two-stage venous cannula introduced through the right atrium. In OPCAB, a Medtronic Urchin Evo Heart Positioner  was used to allow for effective positioning of the heart and enhanced visualization of the anastomotic sites during beating heart surgery. A Medtronic Octopus System was utilized to enhance visibility at the anastomotic site.Postoperatively, all patients were left intubated and were transferred to the cardiac ICU. Perioperative care of the patients was performed according to standard protocols of our department.The ROCsafe Mini-HLM is a closed loop minimized circulatory circuit that consists of a centrifugal pump head , a microporous polypropylene fiber oxygenator  including a heat exchange system and a 40-μm polyester arterial blood filter . The low prime oxygenator of ROCsafe Mini-HLM facilitates high oxygen transfer (oxygen transfer capacity: 337 mL/min at 5 L/min blood flow) in combination with low priming volume (oxygenator priming volume: 135 mL). Additionally this model provides an air removal function on the venous line allowing an effective air elimination before it enters the centrifugal pump. In case of air in the venous line, the ultrasound-controlled bubble detector leads to a speed reduction of the centrifugal pump, closure of the electronic venous line occluder  and removing of trapped air from the bubble trap  via vacuum suction device. Residual micro air can be eliminated by the oxygenator and arterial filter. This process takes a few seconds before circulation can be resumed. The total component priming volume is approximately 500 ml (Table Measurement of perioperative hemodynamic parameters was routinely performed using Swan-Ganz-CCOmbo PAC (Edwards Lifesciences), which was inserted in Seldinger technique during the induction of anesthesia and removed directly after the last measurement 24 hours postoperatively in the ICU. Systemic vascular resistance (SVR), pulmonary vascular resistance (PVR), cardiac output (CO), cardiac index (CI), pulmonary capillary wedge pressure (PCWP), central-venous pressure (CVP), mixed venous oxygen saturation (svO2), arterial systolic (ASP), diastolic (ADP) and mean pressures (AMP) as well as pulmonary arterial systolic (PASP), diastolic (PADP) and mean pressures (PAMP) were recorded at seven time points perioperatively: skin cut ("OP-0"), post Mini-HLM/OPCAB-stabilizer ("OP-1"), skin closure ("OP-2"), one hour ("ICU-1"), six hours ("ICU-2"), 12 hours ("ICU-3") and 24 hours after surgery ("ICU-4") respectively. These data were collected automatically using clinical pressure monitoring equipment in combination with the Vigilance Monitor without performing bolus thermodilution measurements. SvO2 was monitored by fiberoptic reflectance spectrophotometry available in the PAC model 744HF75 that was used in our study. Additionally this model provides an antimicrobial heparin coating in order to minimize viable microbe count of the catheter surface during placement as well as to guarantee a long-term protection against thrombogenesis .Perioperative clinical characteristics were assessed by evaluating the medical charts of the patients during their hospital stay and after their discharge. The data of the perioperative hemodynamic measurements were directly documented at the appropriate time points.Statistical analysis was performed by SPSS 19.0 . All continuous variables are expressed as mean ± standard deviation (SD). Categorical data are expressed as frequencies and percentages. Statistical analysis of metric and dichotomous parameters was carried out using T test and Chi-square test respectively. A p < 0,05 was considered statistically significant.The preoperative data were comparable between patients of both groups of the study. Table Every single patient in both the Mini-HLM and the OPCAB group received a LIMA-graft to the left anterior descending artery (LAD) as a significant LAD-stenosis/occlusion was present in all operated patients. The usage of bilateral internal mammary artery was comparable between the two groups Table . In the Serial assessment of patient’s hemodynamics did not result in statistically significant differences in most instances between the two groups Figure , a-c. DeCABG is the treatment of choice in patients with three-vessel CAD as recommended by recent Guidelines . A consiTo avoid these adverse effects, alternative approaches such as OPCAB, MIDCAB and interdisciplinary hybrid procedures have been introduced into clinical practice. However, these approaches are sometimes technically demanding and therefore do not always represent the best available technique in specific patients. Particularly off-pump procedures require sufficient exposition of target vessels which can be very demanding particularly when marginal branches need to be revascularized which may result in severe hemodynamic instability due to cardiac displacement. Furthermore, diffuse CAD with severe calcified target vessels may not always be suitable for revascularization during beating heart surgery. In these instances, the utilization of minimized extracorporeal circuits may offer benefits for these patients as these Mini-HLM systems have been proposed to avoid the potentially harmful effects of CCPB. The basic idea of Mini-HLM is to ensure adequate perfusion by a closed, extremely minimized circuit based on a rotary blood pump and a high-performance membrane oxygenator with elimination of blood-to-air contact by avoiding a venous reservoir, minimizing hemodilution and mechanical blood trauma and significant reduction of contact activation by reduced foreign surfaces . MeanwhiIn the present study we compared OPCAB revascularization with on-pump procedures utilizing a Mini-HLM, and comparable excellent clinical results could be demonstrated in both groups without any significant differences. Particularly mortality, postoperative morbidities and complication rates as well as clinical variables of the perioperative period, e.g., blood loss, transfusion requirements, ventilation- and total-hospital-times were not significantly different between both groups. Nevertheless, this is an extremely important result with high clinical impact as the use of CCPB has been associated with a couple of the above-mentioned negative side effects. Based on these concerns with CCPB, a significant amount of studies deal with the OPCAB procedure as as a well-established and safe minimal invasive procedure for coronary artery revascularization ,22. ThisIn our study the utilization of Mini-HLM, however, resulted in a completely comparable low complication rate compared to the off-pump group. Therefore, broader introduction of minimized circulatory systems into clinical practice has the promising potential to reduce negative side effects of standard CPB systems. Moreover, hemodynamic measurements in the present study did not show any significant differences in the postoperative course between the two groups suggesting that the use of Mini-HLM is safe and did not lead to hemodynamic instability.A significant finding of our study was a higher rate of distal anastomoses in the Mini-HLM group. As an often quoted limitation of OPCAB procedures, complete coronary revascularization might not be achievable in all patients by off-pump techniques owing to the complex anatomy of coronary lesions and the possibility of hemodynamic instability while the beating heart is manipulated. In a study with 2203 patients undergoing off-pump and on-pump procedures, Shroyer et al.  have repHowever, in the available literature there is a substantial uncertainty on the definition and evaluation of the “adequacy” of myocardial revascularization, and numerous inconsistent definitions for “complete (CR)” and “incomplete (IR)” revascularization account for some controversies regarding mid- and long-term event-free survival . GeneralTherefore, due to these difficulties in definition and impact of the “completeness of revascularization” and as each patient of our two cohorts did receive a LIMA bypass to the LAD, it was not the primary goal of this preliminary study to specifically focus on complete or incomplete revascularization rates but rather to evaluate the immediate perioperative clinical outcome including intensive care data and hemodynamic assessment. Nevertheless, anatomically complete revascularization was intended in all Mini-HLM- and OPCAB-cases of our series, and it was guaranteed by surgical expertise that “inappropriate incomplete revascularization” as recently described by Taggart  was avoiOur study has revealed comparable clinical and hemodynamic parameters between the two groups even though the operating time was significantly longer in the Mini-HLM group as compared to the OPCAB group Tables  and 3. TFor the first time internationally, in this investigation perioperative hemodynamic parameters were directly compared in order to appraise the extent of systemic inflammation induced by surgical technique with may lead to potential adverse effects such as deterioration of hemodynamics. The results of this study did not show any significant differences in the perioperative hemodynamics between both groups in spite of total aortic cross-clamping and cardiac arrest in Mini-HLM operations. Furthermore, similar amounts of catecholamines were used in both groups. A possible explanation of our favourable results may be the numerous conceptional advantages of Mini-HLM, resulting in less perioperative systemic inflammation and hemodynamics comparable to off-pump procedures. Our findings which showed that there were no significant differences between the two groups at most time points are also in accordance with clinical observations. The few statistical differences have no clinical significance because the corresponding values remain within the normal range.This preliminary study has a number of certain limitations. First, it was a single center study with a rather small sample size. Therefore caution must be applied, as the findings might not be transferable to larger cohorts. Furthermore, clinical outcome analysis focused initially on short-term peri- and postoperative observations, and no long-term results are provided yet. However, ongoing experience with this continuous ethics-approved study will result in specific intermediate- and long-term evaluations of bypass function and persistent relief or recurrence of ischemic symptoms.Based on the results of this study, coronary revascularization by use of the ROCsafe™ -Mini-HLM system represents a suitable and "less invasive" procedure which achieves all benefits of OPCAB but may allow for less demanding revascularization than OPCAB in special patients with complex coronary anatomy. In these patients the use of Mini-HLM and circulatory arrest might improve the surgical approach to the target vessels resulting in potentially improved quality of anastomoses, while retaining the benefits of off-pump procedures compared to standard HLM such as comparable incidence of atrial fibrillation, ventilation time, postoperative bleeding, transfusion requirements, catecholaminergic support etc. as shown in our study. This promising technique can therefore be used both on a routine basis and especially in all patients who are considered to be no optimal candidates for OPCAB.The authors declare that they have no competing interests.TW: concept of the study, senior cardiac surgeon, extensive revision + final approval of manuscript. AS: Acquisition of data, drafting of manuscript, statistical analysis. PBR: Statistical analysis, revision of manuscript. Y-HC: Concept of the study, revision of manuscript. MZ: Acquisition of data, statistical analysis. TOM: Concept of the study, revision of manuscript. TW: Final approval of manuscript. All authors read and approved the final manuscript.


HIV-1 cure remains elusive despite HAART due to the reservoirs of proviral DNA integrated into the human genome. Efforts to cure HIV-1 therefore need to aim at eliminating proviral DNA from cellular reservoirs. The first epigenetic signal identified in virus infected and uninfected cells has been promoter methylation. Compelling evidence confirms that specific promoter methylation can lead to gene silencing. Previous studies have examined HIV-1-epigenetics mostly in vitro.We determined methylation patterns in HIV-1 proviral genomes from PBMCs obtained from 21 individuals with a spectrum of disease progression. The CpGs in the long terminal repeats (LTRs) of proviral DNA were investigated by bisulfite sequencing in up to 85 genomic variants per individual. This approach facilitates the study of the full range of CpG methylation and sequence variability of HIV-1 proviruses under conditions of natural selection in human populations.In patients with advanced disease, the HIV-1 proviruses remained essentially unmethylated in their LTRs. In one long-term nonprogresssor, the percentage of methylated proviruses varied from 0-77% at different times after infection. More important and unexpected was the detection of three specific LTR-located CpG dinucleotides that had been selectively mutated to TpAs in >20 out of the 32 samples analyzed. Comparison to 11 HIV-1 LTR sequences in the Los Alamos HIV data base demonstrated that mutations in the sites identified by our study occurred more frequently than at other locations, although the mutations were different from TpAs.These specific CpGs, possibly including their abutting sequences, might indicate weak spots in the proviral genomes whose sacrifice by mutation to TpAs could enhance the HIV-1 potential for long-term proviral survival. These data suggest that the sites of the mutated CpGs occurring at conserved sites may serve as potential targets for therapeutic interventions to eliminate integrated proviruses.


Drosophila sperm proteome  led us to hypothesize that this gene family may have experienced such a diversification during insect evolution.The evolutionary diversification of gene families through gene creation (and loss) is a dynamic process believed to be critical to the evolution of functional novelty. Previous identification of a closely related family of eight annotated metalloprotease genes of the M17 Merops family in the To assess putative functional activities of S-LAPs, we (i) demonstrated that all S-LAPs are specifically expressed in the testis, (ii) confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, (iii) determined that they represent a major portion of the total protein in sperm and (iv) identified aminopeptidase enzymatic activity in sperm extracts using LAP-specific substrates. Functionally significant divergence at the canonical M17 active site indicates that the largest phylogenetic group of S-LAPs lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family.Drosophila evolution and copy number heterogeneity in the genomes of related insects. This finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function.Comparative genomic and phylogenetic analyses revealed the dramatic expansion of the S-LAP gene family during  Gene duplication is considered a fundamental process in the generation of genes with novel functions and ultimately the evolution of biological diversity . A wide Drosophila. One example is the strict paternal-effect gene ms(3)K81 (termed "K81"), a gene created by retrotransposition prior to the divergence of the melanogaster subgroup . S-LAP activity was determined by measuring the increase in fluorescence emission at 460 nm (340 nm excitation) using a Luminescence Spectrometer LS50B (Perkin Elmer). The reaction was started by the addition of the sperm extract. To calculate the units of activity a calibration curve was performed with 7-amino-4-methylcoumarin [Sigma] in the range 0-7 nmol.The authors declare that they have no competing interests.TLK and SD conceived the experiments and wrote the paper and ECW performed the PCR experiments. All authors read and approved the final manuscript.Complete S-LAP phylogeny. This file includes a complete S-LAP phylogeny with all Drosophila orthologs.Click here for fileS-LAP protein alignment. This file includes the S-LAP protein alignment denoting the seven characterized functional residues of the active site.Click here for fileS-LAP primers. This file includes a table listing the primers used in S-LAP RT-PCR experiments.Click here for fileName and symbol key. This file includes a table listing the names and symbols of the genes encoding aminopeptidases referred to in this study.Click here for file


Selecting a marginal donor in liver transplantation (LT) remains controversial but is necessary because of the small number of available donors.de novo malignancy. Neither of the siblings has developed an HCV infection.A 46-year-old Japanese woman was a candidate to donate her liver to her brother, who had decompensated liver cirrhosis of unknown origin. Eight years before the donation, she had a mild liver dysfunction that was diagnosed as a hepatitis C virus (HCV) infection (serotype 2). She had received anti-viral therapy with interferon α-2b three times weekly for 24 weeks and had a sustained viral response (SVR). A biopsy of her liver before the donation showed normal findings without any active hepatitis, and her serum was negative for HCV-RNA. Only 67 patients have undergone LT from a cadaveric donor in Japan. The family in this case decided to have living donor LT. A careful selection for the liver graft donation was made; however, since she was the only candidate, we approved her as a living donor. She was discharged nine days after the liver donation. Her liver function recovered immediately. A computed tomography scan showed sufficient liver regeneration one year later. Her brother also had good liver function after LT and had no HCV infection 48 months after surgery and no A patient with SVR status after interferon therapy might be considered a candidate for living donor LT but only if there are no other possibilities of LT for the recipient. A careful follow-up of the donor after donation is needed. The recipient also must have a very close follow-up because it is difficult to predict what might happen to the graft with post-transplant immunosuppression. The number of deceased donor liver transplantations (DDLTs) in Japan is extremely small. There were 67 cases between February 1999 and January 2010, according to the Japan Organ Transplant Network . In thisA 46-year-old Japanese woman donated the right lobe of her liver to her 36-year-old brother, who had decompensated cirrhosis of unknown origin. She experienced mild liver dysfunction  eight years before the donation. Her condition was diagnosed as chronic active HCV infection (serotype 2) on the basis of a liver biopsy and viral study that showed that her level of HCV-RNA was 13 kcopy/mL by real-time polymerase chain reaction analysis. The histological diagnosis showed chronic hepatitis A1/F1 (Figure de novo malignancy.She was doing well and no HCV-RNA had been detected. She maintained an SVR without any complications until she was evaluated as a living donor. The donor evaluation revealed anti-HCV antibody, but her liver function test results were normal and HCV-RNA was negative by polymerase chain reaction analysis. She underwent an ultrasound-guided needle biopsy of her liver, and the pathological findings were normal and there were no findings of active hepatitis Figure . She wasSelecting a living donor in this case might be controversial, although a marginal donor can also sometimes be a candidate. The risks in this case included HCV transmission to the recipient, HCV reactivation in the recipient after LDLT, and donor risk during surgery. A number of studies have reported that the results with recipients of an HCV-infected graft were comparable to those of recipients of an HCV-negative graft -13. The In this case, it was difficult to determine the indications for the donor selection before transplantation. Patients with serotype 2 HCV are more likely to achieve an SVR after interferon therapy than those with serotype 1. Our donor achieved an SVR of HCV. She and her brother were fully informed of the risk of peri-operative complications and the possibility that he would receive an HCV infection from the graft although she had obtained an SVR after anti-viral therapy. In theory, it is unlikely that a recipient would develop a viral infection from a graft that achieved an SVR.We report a case of LDLT from a donor previously treated with interferon for HCV. A patient with SVR status after interferon therapy might be considered a candidate for LDLT only if there are no other possibilities of LT for the recipient. A careful follow-up of the donor after donation is needed. The recipient also must have a very close follow-up because it is difficult to predict what might happen to the graft with post-transplant immunosuppression.DDLT: deceased donor liver transplantation; HBV: hepatitis B virus; HCV: hepatitis C virus; LDLT: living donor liver transplantation; LT: liver transplantation; SVR: sustained viral response.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MH and SE shared responsibility for the management of this patient and were involved in drafting the manuscript or revising it critically for important intellectual content. MT shared responsibility for the management of this patient. All authors read and approved the final manuscript.


Changes in conscious level have been associated with changes in dynamical integration and segregation among distributed brain regions. Recent theoretical developments emphasize changes in directed functional  connectivity as reflected in quantities such as ‘integrated information’ and ‘causal density’. Here we develop and illustrate a rigorous methodology for assessing causal connectivity from electroencephalographic (EEG) signals using Granger causality (GC). Our method addresses the challenges of non-stationarity and bias by dividing data into short segments and applying permutation analysis. We apply the method to EEG data obtained from subjects undergoing propofol-induced anaesthesia, with signals source-localized to the anterior and posterior cingulate cortices. We found significant increases in bidirectional GC in most subjects during loss-of-consciousness, especially in the beta and gamma frequency ranges. Corroborating a previous analysis we also found increases in synchrony in these ranges; importantly, the Granger causality analysis showed higher inter-subject consistency than the synchrony analysis. Finally, we validate our method using simulated data generated from a model for which GC values can be analytically derived. In summary, our findings advance the methodology of Granger causality analysis of EEG data and carry implications for integrated information and causal density theories of consciousness. An important challenge for cognitive neuroscience is to characterize directed functional  connectivity between brain regions, either in the absence of identifiable behaviour or during task performance. In particular, characterizing causal connectivity patterns across different conscious levels  is likely to shed important new light on the neural mechanisms underlying consciousness, and may also provide new clinical methods for assessment of intraoperative anaesthetic depth One powerful approach to identifying causal connectivity from time series data, originally developed in the 1960s by Norbert Wiener and Clive Granger, is ‘Granger causality’ (GC) We illustrate our method by application to high-density, steady-state, source-localized EEG data acquired from subjects during wakeful resting (WR) and when undergoing propofol-induced general anaesthesia . Extending a previous analysis, we focused on time series localized to the anterior and posterior cingulate cortices (ACC and PCC respectively), see The data analysed in this study were obtained from a previous study We re-analyzed a subset of the data comprising 5 min of spontaneous high-density EEG recordings sampled at 1000 Hz from each of 7 subjects during both WR and LOC, with LOC defined as clinical unconsciousness  In this section we rehearse the formalism of Granger causality in the time and frequency domains. Given two wide-sense stationary time series http://tauday.com for several reasons why we adopt Importantly, GC has a spectral decomposition that can be used to restrict inferences about causal influence to particular frequency bands To obtain the ‘band-limited’ GC for a frequency band defined by the range To validate our GC methodology, we simulated data from a multivariate autoregressive model with white noise error terms, a model for which we were able to derive true GC values analytically. The general such system is specified by:GC analysis was conducted on artifact-free epoched time series, following notch filtering and downsampling, reflecting mean EEG activity within two source-localized brain areas, the ACC and the PCC, recorded from subjects during normal wakeful resting (WR) and under propofol sedation (LOC). We next computed, for each segment, the recommended model order , direction, and frequency band . First, for each 2 sec segment we calculated numerical estimates of GC using Eqs. (1), (2), (8) and (10), using the model order Numerical GC values obtained directly from finite data yield biased estimates of the true GC of the underlying process. In our next step we estimated and removed the bias by applying the following permutation procedure. For each subject, condition, direction and frequency band, we selected 1000 random pairs of 2 sec segments from the ACC with (non-corresponding) 2 sec segments from the PCC. We then computed the numerical GC for each pair, Finally, for each subject, condition, direction and frequency band, we obtained an estimate of the mean GC, We repeated the above procedure for the time-domain, with time-domain GC values computed in approximation by taking the mean over frequencies from 0.5–40 Hz (Eq. (10)). We avoided the explicit time-domain GC (3) because that was found to sometimes contain residual spurious contributions from the line noise at 50 Hz et alTo confirm validity of application of linear regression models of order increase in GC between the ACC and the PCC during LOC, particularly in the beta and gamma bands.raph see  and 5. TTo validate the GC analysis of the EEG data, we applied the same procedure to the simulation model described in the section ‘Simulation model’, for which analytical GC values could be computed. The model we simulated had the following non-zero regression matricesundirected measure of functional connectivity. Synchrony measures have also been widely applied in studies of both conscious content and conscious level , reconfirming the group-average analysis in For each subject and condition we divided the synchrony data into 10 sec non-overlapping windows. For each window we calculated the proportion of time-points with above-threshold  across all windows, in both the ACC  and the increased dynamical connectivity between the ACC and PCC during LOC at the group level. Changes in spectral power were also consistent across subjects, with power in most frequency bands generally increasing during LOC.We have presented a method for applying GC analysis to steady state EEG data, that (i) accommodates non-stationarity by dividing the data into short approximately-stationary segments, and (ii) systematically removes bias by permutation analysis. Our method is generally applicable in neuroimaging contexts that generate continuous time series data at sampling rates reflecting neural interactions . We demonstrated the efficacy of our method via a rigorous set of simulations for which GC could be solved analytically. We illustrated its value by application to source-localized steady-state high-density EEG data obtained from healthy human subjects undergoing propofol-induced anaesthesia, examining changes in bidirectional GC between the ACC and PCC as subjects transitioned from wakeful resting (WR) to loss-of-consciousness (LOC). We found an increase in bidirectional GC during LOC that was most pronounced in the beta (12–25 Hz) and gamma (25–40 Hz) frequency bands and which was observed consistently across subjects. A comparison with a phase synchrony analysis  and parameter estimation (better for long segments); partition the data into non-overlapping segments of the chosen length, removing the linear trend and mean from each segment. Exclude segments containing artifacts.For each segment, estimate the model order , omitting any frequencies contaminated by line-noise removal.To estimate the bias in GC values for a particular connection and frequency, compute the mean numerical GC at this frequency between Subtract the estimated bias from each raw GC value to obtain an approximately unbiased estimate.Assess significance using a Wilcoxon rank sum test on the distribution of approximately unbiased GC estimates across segments.et altime-varying GC over short time-scales and not on accurate estimation of GC for steady-state data. To our knowledge, the issue of bias has not been examined until now, possibly because most previous studies have been concerned with inference on the statistical significance of individual GC values and not on comparing distributions of GC values across conditions, as we do here. Importantly, we have been able to demonstrate the efficacy of our method with respect to debiasing via our novel analytically solvable model of spectral bidirectional GC.Elements of the above method have been proposed previously. Analysis of short time-windows was advocated by Hesse The context of spectral bidirectional GC between two variables is deliberately simple. The method is however readily extensible to more complicated situations including conditional GC  connectivity, whether directed or undirected. On the face of it, these views contrast with our results which showed increased GC and phase synchrony during LOC, as well as increased power. However, increased functional connectivity could be consistent with overall reduction of causal density and/or information integration under at least two scenarios. First, high values of some measures  could reflect pathologically increased integration at the expense of differentiation. This view aligns with the increased cortical-subcortical synchrony observed during LOC associated with generalized epilepsy We have described a methodological pipeline for GC analysis of steady-state EEG signals, accommodating nonstationarity, eliminating bias, and validated against an analytically solvable model. This pipeline represents a contribution towards the general problem of identifying directed functional connectivity in brain networks


P = 0.02) and systolic blood pressure (P = 0.002) in baseline variables were associated with an elevated CIMTAR for the entire population. Among those not taking lipid-lowering therapy at baseline, only systolic blood pressure remained significant (P = 0.0002). Correlations between low-density lipoprotein (LDL) cholesterol level and maximum carotid wall thickness/CIMTAR were weak for the entire population . Measure of maximum carotid wall thickness may select patients earlier for treatment than traditional risk factors. The addition of CIMTAR to risk algorithms may permit a single-point assignation of subsequent vascular risk that is more efficacious than traditional risk factors.Maximum carotid artery wall thickness was utilized in a primary prevention population and compared with baseline risk factors. Carotid wall thickness was measured between the blood–intima and media–adventitia interfaces by B-mode ultrasonography using software calipers at points of protrusion. Long-axis measures were confirmed by short-axis assessment. The maximum carotid wall thickness for each subject was divided by age in years to yield an annual accretion rate . The entire study population was then divided by median CIMTAR to investigate the association with baseline variables used in standard risk assessments with the bifurcated groups. Traditional risk factors such as age, diabetes, smoking, hyperlipidemia, and obesity were not associated with greater than median CIMTAR. Only male gender ( As atherogenesis is a time-dependent process, preclinical measures of vascular changes may identify subjects at highest risk of cardiovascular disease (CVD). Carotid intima–media thickness (CIMT) is a validated surrogate marker of future risk as increasing carotid wall thickness is reflective of the increasing burden of atherosclerosis  bilaterally . Long- and short-axis views were utilized to identify protrusions of the arterial wall into the lumen. Software cursors were manually placed at the blood–endothelial interface and at the media–adventitia junction yielding the measured carotid wall thickness on long-axis views of these protrusions. Short-axis examination was utilized to insure accuracy of each long-axis measurement point  were also estimated. P-values <0.05, two-tailed, were considered statistically significant, with no adjustment for multiple testing.Subjects were classified based on the median CIMTAR for the entire sample. Group comparisons for the entire sample and for the subset not taking lipid-lowering therapy at baseline were made by P = 0.02, 0.002, respectively). Among those not taking lipid-lowering therapy, only systolic blood pressure remains significant (P = 0.0002). There was a statistically significant association between maximum wall thickness and baseline LDL for the entire population (P = 0.002), but the association is a negative one with a weak correlation . Scatter plots of CIMTAR and baseline LDL for the entire population were only weakly associated .The baseline characteristics of the 393 subjects are delineated in One advantage of risk attribution by a single measure at any point in time such as CIMTAR would be to select subjects earlier in life without having to wait for the time-based increase in wall thickness to pass a threshold beyond normal. Such a single measure might also select subjects at risk prior to the development of risk factors. The similar values of CIMTAR from trials of differing patient populations  suggest a linear accretion of wall thickness independent of baseline LDL-C levels and support the validity of the concept of a snapshot determination of risk. The larger mean CIMTAR in our population may be a result of the selection of the maximum CIMT for each subject and increased baseline risk in the referral population. A CIMTAR of ≤0.016 mm/year may prove to be a useful cutpoint for populations such as ours.At every level of baseline LDL-C, half of the population was variably distributed above the median CIMTAR, while the other half was densely grouped below. Between these two groups, traditional risk factors did not account for their separation, and baseline LDL was not associated with elevated maximum wall thickness or CIMTAR. The increase in carotid wall thickness was not determined by the concentration gradient of LDL between serum and the subendothelial space. In previous studies , similarThe single baseline variable associated with an elevated CIMTAR for both the overall population and those without lipid-lowering therapy at baseline one was systolic blood pressure. In meta-analyses of hypertensive trials, elevations in systolic blood pressure were associated with an increase in risk of vascular outcomes with a 40% increment for every 10-mmHg increase . There aLimitations of our study include the operator dependence of ultrasound measures. Although automated means of maximum wall thickness measure may help reduce operator error, we used short-axis examination to confirm the longitudinal measurements. The addition of morphologic measures of the content of the carotid wall such as grayscale median (GSM) might enhance the accuracy of risk stratification . Serial Earlier identification of vascular risk by a single imaging measure such as CIMTAR may enable earlier treatment and expanded benefit from a longer duration of care. Enhanced communication of such risk may increase adherence to risk reduction programs, which is critical for long-term or lifetime treatment strategies. There is abundant need for more efficient treatments of larger patient populations to reduce vascular outcomes such as acute coronary syndrome, stroke, and sudden death.


HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the ‘Vpu sensitivity sequence’ in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex. Unlike most gammaretrovirus Env proteins, GaLV Env cannot pseudotype HIV-1 particles The CTDs of gammaretroviral Env proteins are predicted to be alpha helical and likely form coiled-coil trimers The Friend murine leukemia virus (F-MLV) Env protein is a trimer + HIV-CMV-GFP was created by replacing the fragment between BamHI and SalI sites in HIV-CMV-GFP with the equivalent BamHI-SalI fragment encoding Vpu from the plasmid ΔR8.2.The ecotropic F-MLV Env (isolate 57) expression construct was kindly provided by Walther Mothes . The F-MLV/GaLV Env Δ8 chimeric construct containing an 8- amino acid truncated version of the GaLV Env CTD was constructed using oligonucleotide linkers coding for the cytoplasmic tail of GaLV The 293FT cell line was obtained from Invitrogen. The cell line expressing the ecotropic F-MLV Env receptor, 293T mCAT-1- HIV-1 pseudotyped with a pair of complemented Envs to the percentage of cells infected with Vpu- HIV-1 pseudotyped with F-MLV Env in the same experiment. Vpu sensitivity for each complementation pair is defined as the ratio of infectivity in the absence of Vpu to the infectivity in the presence of Vpu.Infectivity assays using HIV-CMV-GFP and its derivative were performed by transfection of 293FT cells with 500 ng of the HIV proviral DNA and 500 ng total of the Env and 4 µl of 1 mg/ml polyethylenimine (PEI) in six well plates Within the Env CTD, F-MLV and GaLV Env sequences are highly similar . In the Infectivity assays with HIV-CMV-GFP were performed with pairs of BD and FD Env constructs to confirm that the proteins are able to complement each other as previously described To understand the Vpu sensitivity of the different Env trimers, pairwise transfections with complementation pairs were performed in the presence or absence of Vpu as previously described . Vpu senThe hydrophobic amino acids in the ‘vpu-sensitivity sequence’ may form a coiled-coil interface in the predicted alpha helix