Little is known about the 'grammar' of interactions between transcription factors and cis-regulatory modules and therefore about the molecular mechanisms that underlie changes in these modules, particularly after gene and genome duplications. We investigated the ar-C midline enhancer of sonic hedgehog (shh) orthologs and paralogs from distantly related vertebrate lineages, from fish to human, including the basal vertebrate Latimeria menadoensis.sonic hedgehog a (shha) paralogs sonic hedgehog b  genes of fishes have a modified ar-C enhancer, which specifies a diverged function at the embryonic midline. We have identified several conserved motifs that are indicative of putative transcription factor binding sites by local alignment of ar-C enhancers of numerous vertebrate sequences. To trace the evolutionary changes among paralog enhancers, phylogenomic reconstruction was carried out and lineage-specific motif changes were identified. The relation between motif composition and observed developmental differences was evaluated through transgenic functional analyses. Altering and exchanging motifs between paralog enhancers resulted in reversal of enhancer specificity in the floor plate and notochord. A model reconstructing enhancer divergence during vertebrate evolution was developed.We demonstrate that the ar-C enhancer function as binary switches that are responsible for specific activity between midline tissues, and that these motifs are adjusted during functional diversification of paralogs. The unraveled motif changes can also account for the complex interpretation of activator and repressor input signals within a single enhancer.Our model suggests that the identified motifs of the  However, divergence in sequence and function of CRMs over large evolutionary distances may hinder the utility of phylogenetic footprinting methodology [Phylogenetic footprinting can predict conserved hodology -5. ThereIt is widely accepted that gene duplication is a major source for the evolution of novel gene function, resulting ultimately in increased organismal complexity and speciation -9. It hacis-acting elements [hox2b, hoxb3a, and hoxb4a enhancers in fish [The diversity of possible mechanisms of subfunctionalization at the level of regulatory elements, however, is still poorly understood because of the lack of thorough comparative molecular evolutionary studies on elements , supportelements ,18. Seve in fish -21.shh genes in various lineages of 'fish', including Latimeria menadoensis. Teleost fish are well suited for analysis of cis-regulatory evolution in vertebrates [Tetraodon nigroviridis), fugu (Takifugu rubripes), zebrafish (Danio rerio), medaka (Oryzias latipes), and stickleback (Gasterosteus aculeatus). Adding them to the many available mammalian and anamniote vertebrate genomes covers a time span of 450 million years of evolution at different levels of genic and genomic divergence. More importantly, gene regulatory elements isolated from fish are suitable for functionality testing by transgenic analysis in well established model species such as zebrafish. Aside from conventional transgenic lines [Here, we report on an investigation into the molecular mechanisms of paralog divergence at the CRM level through the study of the duplicated tebrates ,23. Seveic lines , CRMs caic lines -29. Consic lines -33. Thisic lines -36.hedgehog gene family is well understood [hedgehog gene family [hedgehog gene: sonic hedgehog (shh), indian hedgehog (ihh), and desert hedgehog (dhh). Several lines of evidence indicate that a complete genome duplication occurred early in the evolution of actinopterygian (ray-finned) fishes [shh in the fish lineages resulted in two parlogous genes, namely shha and shhb [ihh [dhh genes as well.The evolutionary history of the derstood , and itsderstood ,39. Compe family ,40 showe) fishes -46, lead) fishes -50. Thusand shhb ,40, as whhb [ihh  and probshha and shhb are both expressed in the midline of the zebrafish embryo [shha is expressed in the floor plate and the notochord, shhb is present only in the floor plate. Etheridge and coworkers [shha is expressed in notochord precursors and shhb is exclusively expressed in the overlying floor plate cells during gastrulation. Later, shha is expressed both in the notochord and floor plate, whereas shhb remains restricted to the floor plate [shhb is very similar to that of shha [shha and shhb are regulated quantitatively by their partially overlapping and tightly controlled level of expression. Thus far, the function only of shha has been studied in genetic mutants [shhb gene. The shhb gene was shown to cooperate with shha in the midline to specify branchiomotor neurons, in somite patterning, but it is also required in the zona limitans intrathalamica and was implicated in eye morphogenesis [The genes h embryo . There aoworkers  have shoor plate . The pro of shha . It is l mutants . Neverthogenesis -60.sonic hedgehog a gene is well characterized, and a substantial amount of data on the functionality of its cis-acting elements exist [shh(a) genes [ar-C directs mainly notochord and weak floor plate expression in zebrafish embryos [cis-regulatory mechanisms involved in regulating shh(a) expression are at least in part conserved between zebrafish and mouse. However, the mouse enhancer, SFPE2 (sonic floor plate enhancer 2), which exhibits sequence similarity with ar-C of zebrafish, is floor plate specific [ar-C enhancer in fish and mouse, we postulated that this enhancer might have been a target of enhancer divergence between shha and shhb paralogs in zebrafish during evolution.The genomic locus of the zebrafish ts exist ,61,62. Ea) genes ,63. Compa) genes ,64,65. T embryos ,62. This embryos , suggestspecific  and exhispecific . This diar-C homolog exists in the shha paralog shhb. Shhb ar-C is diverged in function and became predominantly floor plate specific, similar to what has been found in the mouse ar-C homolog SFPE2. By phylogenetic reconstruction, we were able to predict the motifs that are required for the tissue-specific activity of the paralog enhancers, and we identified the putative transcription factor binding sites that were the likely targets of evolutionary changes underlying the functional divergence of the two ar-C enhancers of the shh paralogs. By engineering and exchanging mutations in both of the enhancers of shha and shhb, followed by transgenic analysis of the mutated enhancers, we were able to recapitulate the predicted evolutionary events and thus provide evidence for the likely mechanism of enhancer evolution after gene duplication.Here, we show that a functional shh loci indicate a high degree of sequence similarity between zebrafish, fugu, chick, mouse, and human  genes (orange peaks), and the degree of their conservation is in accordance with the evolutionary distance between the species compared. In contrast, the zebrafish shhb gene exhibits no obvious conservation with the shha ar-A, ar-B, ar-C, and ar-D CRMs. Apart from Shuffle Lagan, Valis [shhb (data not shown). Taken together, these findings indicate that although orthologous regulatory elements may exist between shhb and shha, they are much less conserved at the DNA sequence level than are shha elements, as detected by the applied alignment programs.Comparisons of multiple vertebrate n Figure . A globan Figure . The CRMn, Valis  has alsoar-C, which is conserved between fish and mouse (SFPE2) and which has been analyzed in considerable detail in both species [ar-C enhancer or its mouse ortholog SFPE2 is detectable across shh(a) loci in various vertebrate species from different lineages that diverged before and after the gene duplication event leading to the evolution of shh paralogs in zebrafish. Because the zebrafish shha ar-C enhancer is located in the second intron of shha and exhibits high sequence similarity to human and mouse counterparts, candidate ar-C containing intronic fragments of several vertebrate species were amplified by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We cloned and sequenced the relevant genomic DNA fragments from several fish species that experienced the genome duplication, such as the cyprinid tench (Tinca tinca), fugu, and medaka [Latimeria menadoensis were used in the analysis. All sarcopterygians diverged from the common ancestor with actinopterygians before the fish-specific genome duplication in the ray-finned fish lineage. A sequence comparison of intron 2 sequences from the available vertebrate model systems revealed a high degree of sequence similarity in all species specifically in the region that spans the ar-C enhancer in zebrafish and the SFPE2 enhancers of mouse  may contain a shhb homolog. Analysis of the available genome sequences of these four species of teleost fish indicated that none of them carries a discernible shhb homolog, suggesting that these lineages . This finding further supports the hypothesis that a shhb gene was originally present after duplication but has been lost secondarily during evolution.The evolutionary functional divergence of paralogous yprinids ) may havshhb and its intron 2 from another cyprinid species, tench, by PCR using degenerate oligonucleotides that were designed in conserved exon sequences. Importantly, the isolation of more than one shhb intron 2 sequences from cyprinids allowed for phylogenetic footprinting of shhb genes and a search for a putative ar-C homolog. We have compared the shha and shhb intron 2 sequences between zebrafish and tench  fragment of nonconserved and a 380 bp conserved sequence. As shown in Figure ar-C enhancer has been discovered in the shhb intron 2, which is consistent with the floor plate specific expression of shhb in zebrafish.The 2.7 kb upstream and proximal promoter sequence of r Figure . Moreoved Figure . The prear-C sequences may indicate conserved motifs, which may reflect conserved transcription factor binding sites [ar-C from large numbers of pre-duplicated and post-duplicated shh orthologs and paralogs. To this end, a CHAOS/DIALIGN [ar-C enhancer of zebrafish  and equshh genes. C1 and C3 are homology blocks, which are present in all shh sequences, including shhb paralogs, in all species analyzed. In contrast, C2 and C4 are homology blocks that are present only in shh(a) genes but absent in shhb genes. Because C2 and C4 are present in pre-duplicated enhancers of sarcopterygians, the lack of C2 and C4 in shhb enhancers is probably due to a secondary loss of these elements after the fish-specific gene duplication. The two sets of putative binding sites  may thus be targets for transcription factors that regulate the differential enhancer activities of shh(a) (predominantly notochord expression) and shhb (predominant floor plate expression). In conclusion, we identified a set of putative targets of mutations that may contribute to the divergence of ar-C enhancer functions after gene duplication.A pattern of conserved motifs is detected in the form of homology blocks extending to 20 to 30 bp. These conserved motifs exhibit distinct distribution characteristics, which reflect phylogenic as well as paralogy and orthology relationships between shha and shhb genes. Furthermore, we carried out exchange of homology blocks between shha and shhb ar-C enhancers to test whether evolutionary changes after gene duplication can be modeled in a transgenic zebrafish system.To test the functional significance of the two sets of homology blocks, we conducted a systematic mutation analysis of the C1 to C4 conserved homology blocks in both shha ar-C enhancer activity. Replacement of C1 with random sequence results in total loss of ar-C enhancer function, indicating that this binding site is critical for shha ar-C activity  results in strong expression of GFP in the floor plate , indicating that the changes observed with the C4 insertion are due to the specific sequence of C4. These results together strongly suggest that the function of C4 is to repress floor plate activation by the shha ar-C enhancer. Together, these findings are consistent with a model in which loss of the C4 motif in the evolution of the shhb ar-C has contributed to its floor plate specific activity.We next asked whether a Figire . We thend Figure , but inty Figure . The effr Figure . In a cocis-regulatory elements of genes. Recent years have provided evidence in support of this hypothesis [shh genes during vertebrate phylogeny. By constructing multiple alignments, we were able to predict which motifs within the ar-C enhancer represent regulatory input. Through specific mutations and exchanges of motifs, we mimicked probable evolutionary events in transgenic analysis and identified the lineage-specific modifications that lead to discernible changes in tissue-specific enhancer activity in embryo development.It has long been suggested ,73 that pothesis -13,74-76pothesis ,77,78. Ishhb genes we have identified a conserved ar-C homolog enhancer in two species of cyprinids. The results of our transgenic analysis indicate that the ar-C sequences in intron 2, together with the promoter activity of shhb [shh(a) enhancers retained significant sequence similarity with their orthologs, the whole of the shhb gene and its ar-C enhancer is grossly changed from that of shha paralogs. This paralog-specific change happened despite the fact that shhb had equal time and chance to diverge as did shha after duplication from an ancestral sonic hedgehog gene. This result is in accordance with observations indicating selective pressure on the CRMs of paralogs in invertebrates [Using phylogenetic footprinting of intron 2 of  of shhb , contribtebrates  as well tebrates ,20,80,81tebrates .ar-C enhancers. Our findings are most consistent with a model in which these motifs are individual or multimeric transcription factor binding sites. Mutation and transgenic analysis verified the functional relevance of these motifs in driving expression in the midline, and therefore the most parsimonious explanation for the conservation of these sequence elements is that they represent functional binding sites for developmental regulatory transcription factors.Use of a local sequence alignment approach of representative species of major vertebrate lineages allowed us to predict functionally relevant motifs within the ar-C enhancer is composed of motifs with different regulatory capacities . shhb and shha enhancers, we were able to reconstruct the probable evolutionary events leading to divergence of the ar-C enhancer function. For example, insertion of the floor plate repressor C4 element into shhb resulted in enhancer activity reminiscent of shha ar-C, in which the C4 site had been identified. These findings indicate that the very changes that resulted in the divergence of the enhancer function have been identified.The use of large numbers of species spanning long evolutionary distances allowed us to generate a phylogenetic reconstruction of enhancer divergence before and after gene duplication Figure . By genear-C enhancer of shha be repressed in the floor plate while the shha gene is well known to be active in this tissue? The level of the Hedgehog morphogen signal emanating from the embryonic midline is critical for correct patterning of the ventral neural tube [shhb) achieve this by controlled activation of shh in the notochord and floor plate as a result of a combination of several synergistic enhancers [shh paralog (shhb) also contributes to Shh production in the floor plate. At least in zebrafish, controlled levels of the floor plate expressed shhb are required, together with the notochord and floor plate derived shha, for normal patterning of branchiomotor neurons and the somites [shh genes emerging from the floor plate and notochord may thus result in one of the paralog floor plate enhancers being subjected to selection pressure. For example, to counter the overproduction of Hedgehog levels, the reduction in transcription can occur by blocking the activity of one of the synergistically active enhancers (in this case ar-C). It is important to note, however, that the shh(a) ar-C enhancers are not exclusively expressed in the notochord, and retained a weaker but still noticeable capacity to activate expression in the floor plate. Thus, the output of Shh levels in zebrafish appears to be a subject of quantitative regulation of paralog enhancer activities. Alternatively, it is feasible that there are time points when the two paralog genes are not overlapping in expression and the complementing specificities of shhb and shha ar-C enhancers reflect the non-overlapping production of Hedgehog proteins in the two midline tissues [An open question remains, however; why should the ral tube . Animalsnhancers ,63. In z somites -58. The  tissues .ar-C enhancer activity specific to one of two midline tissues after gene duplication. By selective removal of repressor and activator binding sites, subfunctionalization of the ar-C enhancer to floor plate or notochord can thus occur  paralleled by modifications either on the cis or on the trans level to restrict activity of the less diverged sister paralog enhancer (shha). The prediction from this model is that fish species without shhb gene (acantopterygii) may have floor plate active ar-C enhancer. Interestingly, the floor plate repressor elements (C2/C4) of shha ar-C of acanthopterygians  are present but diverged from all other shh(a) homologs  he mouse  and can he mouse   [ar-C sequences that are functionally relevant in zebrafish, and this divergence may explain, at least in part, the different results obtained with mouse and fish enhancers.The combination of both negative and positive regulatory sites within a single enhancer indicates the integration of activating and repressing signals to modulate the resulting transcriptional activity. This could be achieved through multiple of mouse ,94, frogof mouse , and fisof mouse . Interesof mouse . This bie (HR-c)  only parshh ar-C enhancers provided novel insights into the functional components of enhancer divergence in an important developmental regulator gene. In particular, our findings demonstrate that phylogenetic reconstruction using large number of vertebrate species can identify a series of lineage specific motifs that were the probable targets of evolutionary change and represent individual regulatory input acting in concert on a developmentally regulated gene enhancer. These findings reinforce the importance of the phylogenomic and functional analysis of duplicated cis-regulatory elements in deciphering the cis-regulatory code of developmental gene regulation.In conclusion, the observed changes in the duplicated shha and shhb intron 2 fragments were isolated by using degenerative oligonucleotides, designed based on conserved amino acid blocks in the second and third exons of shh(a) and shhb genes from several vertebrate species. The PCR products were directly cloned into pCRII-TOPO vector , and the clone containing the right insert was identified by sequencing.The tench Latimeria intron 2 was isolated by screening of genomic bacterial artificial chromosome (BAC) library from Latimeria menadoensis [shh locus, was shotgun sequenced and relevant genomic regions were secondarily amplified by gene specific primers. The correct PCR product was identified by sequencing. The mouse and chick intron 2 were directly amplified from genomic DNA with specific oligonucleotides.The adoensis   into 0.8shha:gfp plasmid. The plasmids 0.8shha:gfp:t-shha-I2 and 0.8shha:gfp:shhb-I2 were made by reamplifying the respective intron 2 fragments from pCRII-TOPO:t-shha-I2 and pCRII-TOPO:t-shhb-I2, and subcloning them in 0.8shha:gfp using NotI/KpnI restriction sites. The 0.8shha:gfp:l-shh-I2 was constructed by reamplifying the intron 2 part from the correct PCR fragment isolated from the BAC clone and cloning it into 0.8shha:gfp (NotI/KpnI). The 0.8shha:gfp:c-shh-I2 and 0.8shha:gfp:m-shh-I2 palsmids were made by direct cloning of the respective intron 2 fragments, amplified from genomic DNA, into 0.8shha:gfp (NotI/KpnI). The 0.8shha:gfp:z-shhb-non-cons and 0.8shha:gfp:z-shhb-arC were made by cloning the PCR-amplified nonconserved 5' part of z-shhb I2 (1032 bp) and the 380 bp 3' part containing the conserved region (ar-C) into 0.8shha:gfp (NotI/KpnI). All plasmids  containing z-shha-ar-C or z-shhb ar-C carrying mutations in one of the conserved motifs (C1 to C4) were created by replacing the respective wild-type sequence of each conserved block with random sequence using a PCR-based approach. The same method was used to introduce the C2 and C4 from z-shha ar-C or random sequence into z-shhb ar-C . The PCR products were cloned into 0.8shha:gfp (NotI/KpnI) and verified by sequencing.The  plasmid   and ha:gfp:C , 2.4shhashha ar-C [shhb ar-C sequences can be found in GenBank under the following accession numbers: AL929206  for the shha ar-C and BX510360  for the shhb ar-C. The GenBank accession numbers for tench shha and shhb and Latimeria shh intron 2 sequences are as follows: EF593170, EF593171, and EF593172. For more detailed information about the sequences, which have been mutated and introduced in shha and shhb ar-Cs, see Table 2.7shhb:gfp was constructed by replacing the 2.4shha promoter fragment  from 2.4shha:gfp with the PCR-amplified 2.7 kb shhb promoter fragment (upstream from the translation start site). The plasmid 2.7shhb:gfp:z-shhb-I1 and 2.7shhb:gfp:z-shhb-I2 were made by subcloning the shhb I1 and I2 from 2.4shha:gfp:shhb-I1 and 2.4shha:gfp:shhb-I2  into 2.7shhb:gfp (NotI/KpnI). For sequence information on the oligonucleotides that were used, see Table The zebrafish hha ar-C  and shhbshh(a) and shhb are normally not expressed.All microinjection experiments were performed with injection solution containing circular plasmid at a concentration of 10 to 15 ng/μl, supplemented with 0.1% phenol red. The solution was injected trough the chorion into the cytoplasm of zygotes. The GFP expression was analyzed on 24-hour-old embryos using Leica MZ FLIII fluorescent stereomicroscope . The level of expression was quantified by counting the number of GFP-positive cells in notochord and floor plate, as well as the number of ectopic GFP-positive cells in tissues where hh loci  Transgenic lines and transient transgenic embryos generated with reporter constructs containing the 2.4 kbzebrafish shha promoter and zebrafish ar-C enhancer (B), tench shha intron 2 (C), Latimeria shh intron 2 (D) and zebrafish shhb intron 2. The numbers on the right side of the images of the stable transgenic lines indicate the number of the transgenic lines showing the expression pattern/total number of stable lines generated. ar, activation region; fp, floor plate; I, intron; l, Latimeria; nt, notochord; pr, promoter; t, tench; z, zebrafish.(A) Stable transgenic line (left) and transient transgenic embryos generated with gfp reporter construct, containing the 2.4 kb  zebrafish Click here for fileshha locus alongside medaka chromosome 20 (A), and zebrafish chromosome 2, containing the shhb locus alongside medaka chromosome 17 (B).Shown are Ensembl views of zebrafish chromosome 7, containing the Click here for filear-C enhancer homolog sequences from several vertebrate species, performed with two alignment-algorithms, CHAOS-DIALIGN (A) and MUSCLE (B), reveals specific changes in the conserved putative transcription factor binding sites 2 and 4 (C2 and C4) of acanthopterygian fishes, which lack a sonic hedgehog b gene , as compared with ostaryophisian fishes, which have sonic hedgehog b . The C2 and C4 sites are marked with blue frames, and the differences in the C2 and C4 sequences in the acanthopterygian fishes are highlighted in yellow and orange, respectively.Multiple sequence alignment of Click here for file


Hyperglycemia is common in critically ill patients and is associated with increased morbidity and mortality. Strict glycemic control improves outcomes in some adult populations and may have similar effects in children. While glycemic control has become standard care in adults, little is known regarding hyperglycemia management strategies used by pediatric critical care practitioners. We sought to assess both the beliefs and practice habits regarding glycemic control in pediatric intensive care units (ICUs) in the United States (US).We surveyed 30 US pediatric ICUs from January to May 2009. Surveys were conducted by phone between the investigators and participating centers and consisted of a 22-point questionnaire devised to assess physician perceptions and center-specific management strategies regarding glycemic control.adults should be treated, while 3/30 believe all critically ill children should be treated. Twenty-nine of 30 believe some subsets of adults with hyperglycemia should be treated, while 20/30 believe some subsets of children should receive glycemic control. A total of 70%, 73%, 80%, 27%, and 40% of centers believe hyperglycemia adversely affects outcomes in cardiac, trauma, traumatic brain injury, general medical, and general surgical pediatric patients, respectively. However, only six centers use a standard, uniform approach to treat hyperglycemia at their institution. Sixty percent of centers believe hypoglycemia is more dangerous than hyperglycemia. Seventy percent listed fear of management-induced hypoglycemia as a barrier to glycemic control at their center.ICUs included a cross section of centers throughout the US. Fourteen out of 30 centers believe all critically ill hyperglycemic Considerable disparity exists between physician beliefs and actual practice habits regarding glycemic control among pediatric practitioners, with few centers reporting the use of any consistent standard approach to screening and management. Physicians wishing to practice glycemic control in their critically ill pediatric patients may want to consider adopting center-wide uniform approaches to improve safety and efficacy of treatment. Hyperglycemia in critically ill patients occurs frequently, is associated with increased morbidity and mortality, and studies in adults suggest that tight glycemic control with insulin may improve outcomes -14. QuesStudies regarding hyperglycemia and glycemic control in pediatrics are limited. Those available demonstrate that high BG is prevalent and independently associated with increased morbidity and mortality -14. To dRecent studies indicate that hyperglycemia is a significant concern among physicians caring for critically ill children and suggest that glycemic management is routinely performed ,25. Our We conducted a survey to ascertain glycemic control beliefs and practice habits at different pediatric critical care centers in the United States. Participating centers were chosen in an effort to include institutions of varying size, geographic location, acuity, practice model , and type . Our pediatric ICU was not included in this survey. Request for participation was sent electronically to attending physicians (either Division Chiefs or other faculty) at different centers between January and May 2009. Surveys were conducted primarily by phone call between the investigators and participating attending physicians. Three centers chose to complete the survey electronically instead of by phone for convenience. One physician was chosen as the spokesperson to represent their institution. All participating individuals had the opportunity to review the survey with their coworkers and colleagues to ensure that their responses were representative of their center's beliefs and practices.The survey comprised a 22-point questionnaire. Questions were developed to investigate the actual practice habits of intensivists regarding glycemic control in non-diabetic hyperglycemic critically ill children. Sections within the survey included questions specific to pediatric ICU demographic and descriptive data, individual perceptions and beliefs regarding glycemic control in critically ill children, individual and center-specific threshold for treatment, method of treatment (if applicable), and safety and efficacy of management at each center.2 analysis (for categoric variables) and independent Student t-test (for continuous variables). A P value < 0.05 was considered statistically significant.Statistical analysis was conducted using a software package . We determined the significance of differences in responses between pediatric ICU centers with χOf 40 centers queried, 30 pediatric ICUs agreed to participate in our survey, equating to a response rate of 75%. Ten centers either did not respond to our electronic request for participation or were not able to respond in a timely manner. All participating centers responded to all items on the questionnaire. Table all critically ill adults with hyperglycemia should undergo some form of glycemic control, whereas only 3/30 (10%) stated that all critically ill children with hyperglycemia should be treated (P < 0.05). Almost all centers  believe that at least some subsets of adults with hyperglycemia should be routinely treated, while 20/30 (67%) stated that at least some subsets of children with hyperglycemia should routinely receive glycemic control (Table P < 0.05).Table  centers 9/30, 97%P < 0.05).To determine if there was a difference in attitude or practice habits based on ICU size, we analyzed responses based on ICU capacity. Significantly more  small ICUs (<12 beds) stated that subsets of critically ill children with hyperglycemia should be treated compared to large ICUs (>30 beds), in which only 55% (6/11) believed so  centers reported that their facility uses a standard approach to screen for and treat hyperglycemia. In addition, four other centers (13%) reported that they do have a standard approach to manage hyperglycemia despite no regular approach for screening  were more likely to have a standard protocol for hyperglycemic treatment compared to moderate (12 to 30 beds) and large (>30 beds) ICUs, 33%, 15%, and 18%, respectively. For centers that do employ a standard treatment approach, all (6/6) indicated they may use insulin infusions for glycemic control, while some also attempt to manage hyperglycemia using intermittent insulin (subcutaneous or intravenous) and/or modification of dextrose in fluids. Three of six centers that use a standard approach to treatment employ a written insulin infusion protocol.In contrast to other reports, our survey assessed centers 1% reporteany treatment, either via a standard protocol used by all physicians or based on individual physician discretion, most centers  reported that likely only a minority  of hyperglycemic patients receive any glycemic control. Figure or never practice glycemic control. Approximately 35% of centers reported that most of their physicians always practice glycemic control, while 7% reported that most of their physicians never practice glycemic control. When broken down by ICU size, a proportionately higher number of small ICUs (<12 beds) were more likely to report that all or most of their physicians practice some type of glycemic control all or most of the time, and were more likely to report that few or none of their physicians never practice glycemic control (P < 0.05)  regarding the definition of Centers were also asked what BG level they considered to be too low in critically ill children. The most common glucose level (47% centers) to define hypoglycemia was <2.2 mmol/L (40 mg/dL), followed by 37% of the centers using a BG <3.3 mmol/L (60 mg/dL), 10% using a BG of <4.4 mmol/L (80 mg/dL) and 3% using a cutoff of 2.8 mmol/L (50 mg/dL) or 5.5 mmol/L (100 mg/dL). Most centers (60%) believe that, in general, hypoglycemia is more dangerous than hyperglycemia. Although many centers have considered adopting a regular approach to glycemic management, 70% listed fear of management-induced hypoglycemia as a barrier to this practice in their unit.For over three years our group has practiced glycemic control in our pediatric ICU as standard care. We routinely screen patients for hyperglycemia and implement a center-developed algorithm to maintain BG 4.4 to 7.7 mmol/L (80 to 140 mg/dL). We have previously defined the incidence and risk factors for hyperglycemia, and have demonstrated what appears to be an effective and safe approach to hyperglycemic management ,13. DespOur survey suggests a considerable disparity between physician beliefs and actual practice habits among pediatric ICU practitioners, and is the first study to assess whether physician beliefs translate to practice strategies in pediatric ICUs in the United States. We find that beliefs and practice habits vary greatly between different centers, and even among practitioners from the same center. Recently a study from the United Kingdom also reported a wide variation of beliefs regarding glycemic control when respondents were queried about potential clinical scenarios .adult patients 18 to 21 years old.The vast majority of adult ICUs have adopted regular approaches for glycemic control, and although the optimal goal BG target is unclear, there is little debate that glycemic control should be part of regular practice. Even following recent reports questioning outcome improvements and goal glycemic targets in adults, the American Diabetes Association, American College of Endocrinologist, and Institutes for Healthcare Improvements have all published recommendations that routine glycemic control be adopted in ICU-hospitalized adult patients -22. It iOur study illuminates a dichotomy between pediatric ICU practitioner beliefs and practice. Although many pediatric intensivists believe hyperglycemia may worsen outcome and at least some subsets of patients may benefit from glycemic control, a significant minority of centers have implemented a routine approach to identify or treat hyperglycemia, as only 7% of centers reported a regular approach for hyperglycemia screening and management.within the same practice group. This result raises concern on several levels. Although the particular glycemic metric for outcome improvement in adults with hyperglycemia is not clear, many reports suggest that in order to achieve clinical benefit, glycemic control must be maintained consistently throughout the ICU course [Admittedly there is little direct data indicating that glycemic control improves outcomes in critically ill children, yet a significant proportion of pediatric intensivists have apparently individually decided to incorporate glycemic control into practice while awaiting more definitive evidence. This has led to a wide variation in practice not only between centers, but frequently U course ,26,27. DU course -4,11,13.This study also highlights some notable differences regarding hyperglycemia beliefs and practice strategies and ICU size. We found that smaller pediatric ICUs, that is, those with fewer ICU beds, annual admissions, and number of attending physicians, were more likely to treat hyperglycemia than larger institutions. Small ICUs rarely reported that no or few intensivists treat hyperglycemia, and many reported that most physicians do employ glycemic control most of the time. Proportionately, smaller ICUs were more likely to have adopted a standard approach to hyperglycemia management as well. Further, smaller ICUs believe glycemic control should be instituted at a lower BG threshold compared to larger ICUs, and were more likely to report a lower BG definition for hypoglycemia. Previous studies have not reviewed or mentioned similar discrepancies, but these differences may likely be due to the less challenging nature of devising and agreeing upon practice policies in smaller groups compared to those with many practitioners.hypoglycemia is more dangerous than hyperglycemia in critically ill children [Similar to findings by others, we report that most pediatric ICU practitioners (60%) believe that children ,25. Althchildren ,26,27. Ichildren . These hchildren ,13. TherLimitations of our study should be noted. While we attempted to target centers of varying size, geographic location, acuity, practice model, and type, data obtained from this survey only represents a portion of pediatric critical care centers nationally. However, as there are approximately 340 pediatric critical care centers in the United States, our survey of 30 centers does represent approximately 9 to 10% of all centers, and thus we believe does include a respectable sample size of pediatric institutions . In addiet al, and more recently the results from the NICE-SUGAR investigators [Lastly, it is notable that results from at least two important studies in this field were published during the time this survey was conducted, specifically the aforementioned pediatric glycemic control trial by Vlasselaers tigators ,23. ThesIn summary, we find that there exists a significant awareness of hyperglycemia in pediatric ICU practice, but that few have modified their group practice to reflect their current beliefs. In general, pediatric intensivists may benefit from revisiting and staying abreast of the current state of literature regarding both hyper and hypoglycemia in critically ill children, and we recommend that all pediatric practitioners should consider treating hyperglycemia in their older, adult patients, such as those >18 years old, as suggested by multiple medical advisory groups. It may be premature to recommend the widespread adoption of glycemic control measures in all critically ill children on the basis of outcome studies, but for those centers that do practice glycemic control, there may be other quality and safety reasons to develop a center-consistent approach to this management. Support and encouragement of future studies to develop and validate safe and effective pediatric-specific approaches to glycemic control, and to assess whether this management impacts outcomes in critically ill children will be of utmost importance.• Hyperglycemia is common in critically ill patients, is associated with increased morbidity and mortality, and strict glycemic control with insulin may improve outcomes in some populations.• Most adult institutions have adopted regular approaches for glycemic control, and although the optimal goal BG target is unclear, many medical advisory committees recommend that at least some degree of glycemic control should be part of regular practice.• There is a paucity of direct evidence for glycemic control in children; however, the only randomized glycemic control trial conducted in critically ill children to date does suggest outcome improvement with this therapy in this population.• While most pediatric practitioners do believe hyperglycemia worsens outcomes in many of their patients, very few centers use a standard approach to treat hyperglycemia, and most that do attempt glycemic control use inconsistent, non-validated approaches.• Recommendations for routine glycemic control in all pediatric ICU patients may be premature at this time, but pediatric centers wishing to practice glycemic control in their patients based on the most recent literature and studies suggesting potential outcome improvement may benefit from adopting a routine, center-consistent approach at their institution to optimize effectiveness and safety of this therapy.BG: blood glucose; ICU: intensive care unit.The authors declare that they have no competing interests.Both authors of this manuscript contributed significantly and equally to this study, including study design, survey development, conduction of surveys, data gathering and analysis, and formal writing of this manuscript. All authors read and approved the final manuscript.


Here we describe molecular and cellular responses, as well as survival, in AEscherichia coli or Staphylococcus aureus) or lipopolysaccharide (LPS). Mice inoculated with live bacteria were treated with an A2A AR agonist (ATL313) or phosphate buffered saline (PBS), with or without the addition of a dose of ceftriaxone. LPS inoculated mice were treated with ATL313 or PBS. Serum cytokines and chemokines were measured sequentially at 1, 2, 4, 8, and 24 hours after LPS was administered. In survival studies, mice were followed until death or for 7 days.Sepsis was induced in mice by intraperitoneal inoculation of live bacteria  or S. aureus  when treated with ATL313 in conjunction with an antibiotic versus antibiotic alone. ATL313 also improved survival from endotoxic shock when compared to PBS treatment . The serum concentrations of TNF-α, MIP-1α, MCP-1, IFN-γ, and IL-17 were decreased by ATL313 after LPS injection (p < 0.05). Additionally, ATL313 increased the concentration of IL-10 under the same conditions (p < 0.05). Circulating white blood cell concentrations were higher in ATL313 treated animals (p < 0.01).There was a significant survival benefit in mice infected with live Further studies are warranted to determine the clinical utility of ATL313 as a novel treatment for sepsis. Approximately 900,000 cases of sepsis occur annually in the United States, causing roughly 210,000 deaths and costing almost 17 billion dollars .1, A2A, A2B, and A3. The A2A adenosine receptor (AR) is present on inflammatory cells including neutrophils, mast cells, monocytes, macrophages, eosinophils, platelets, and T-cells, and is involved in anti-inflammatory activities [2A ARs results in an increase in cyclic AMP concentration in inflammatory cells which is increased further by concurrent type IV phosphodiesterase inhibitors. A2AAR agonists decrease superoxide production in neutrophils, degranulation of neutrophils, TNF-α production by monocytes and macrophages, and neutrophil-endothelial cell adherence [Adora2a, the A2A AR gene, after exposure to Concanavalin A liver injury within 8 hours compared to complete survival in wild-type mice. Pro-inflammatory cytokines are present in higher concentrations in Adora2a-/- mice when compared to wild-type mice. Similar findings are observed in experimental animals exposed to LPS [Tissue hypoxia, as occurs in sepsis, enhances breakdown of adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is then dephosphorylated by the cytosolic 5'nucloeotidase to adenosine . Adenosidherence . Death od to LPS .2A AR agonists, specifically ATL313, in the treatment of experimental sepsis due to different infections we contrasted survival results in mice challenged with a gram-negative (Escherichia coli) vs. gram-positive (Staphylococcus aureus) pathogen or purified LPS. T cells are increasingly recognized as important in the pathogenesis of experimental and clinical sepsis [To assess the broad applicability of Al sepsis . Cytokinl sepsis .2A AR agonists, we evaluated cytokines in animals undergoing experimental sepsis with and without the addition of ATL313. ATL313 is a hundred fold more selective for the A2A AR than for the A1 AR and twenty fold more selective than for the A3 AR. Furthermore, ATL313 is more selective and has a longer half-life (approximately 30 minutes in rodents) than its A2A AR agonist predecessors. The A2A AR agonists are potentially useful therapeutic agents because, unlike nonspecific AR agonists, A2A AR agonists do not induce hypotension [2A AR agonist on circulating white blood cell concentrations.Sepsis starts as a process of intravascular inflammation mediated by pro-inflammatory cytokines/chemokines including TNF-α, MIP-1α, MCP-1, IFN-γ, and IL-17 as well as anti-inflammatory cytokines, e.g. IL-10 [otension . We alsoad libitum, and were kept under specific pathogen-free conditions. The protocol used in this study was approved by the Animal Care and Use Committee of the University of Virginia.Female C57BL/6 and BALB/c mice  were housed at 68–72°F with a 12 h light/dark cycle, fed standard laboratory food and water E. coli O111:B4) was purchased from Sigma . The A2A AR agonist, ATL313, -9H-purin-2-yl)prop-2-ynyl}piperidine-1-carboxylic acid methyl ester) was supplied by Adenosine Therapeutics, LLC . Ketamine and xylazine were purchased from Vedco, Inc., . In all cases vehicle used was phosphate buffered saline (PBS).LPS  were administered every 6 hours to female BALB/c and C57BL/6 mice after the intraperitoneal (IP) injection of a lethal dose of O111:B4 LPS (20 mg/kg). A similar dose response curve was used to determine the optimal dose of LPS for BALB/c and C57BL/6 mice.8 colony forming units (CFU) live K12, O26:B6 E. coli or 8 × 108 CFU S. aureus at t = 0 and inoculated with the A2A AR agonist, ATL313 (5 μg/kg), or PBS, at t = 8, and then every 6 hours spanning 48 hours. To determine an optimal dosing interval, ATL313 was administered every 12 hours spanning 48 hours for S. aureus inoculated mice. A dose of ceftriaxone  was given to a subset of treated and control mice to create a total of four experimental groups . Female BALB/c mice were also tested in the E. coli model (data not shown). Female BALB/c mice undergoing endotoxemia were injected IP with LPS one half hour prior to injection with ATL313 (5 μg/kg) or PBS which occurred at t = 0 and every 6 hrs thereafter, for a total of eight doses spanning 48 hours (N = 20–29 per group). To provide equal fluid resuscitation in the setting of sepsis, all injections were I mL in volume. Moribund mice were anesthetized using ketamine and xylazine and euthanized via cervical dislocation.Female C57BL/6 mice were inoculated with 1 × 10Female C57BL/6 and BALB/c mice were injected intraperitoneally (IP) with O111:B4 LPS and ATL313 or PBS as described above. Experimental and control animals were sequentially sacrificed at times 1, 2, 4, 8, and 24 hours after LPS injection. The mice were anesthetized with ketamine and xylazine and killed by cervical dislocation. Immediately prior to sacrifice the animal underwent cardiac puncture and ventricular blood was aspirated for white blood cell analysis via the Hemavet 850 veterinary multi-species hematology system  and future cytokine analysis.Cytokine investigations were carried out using a protein bead-based multiplex immunoassay system . With this system we are able to measure IL-1α and β, TNF-α, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 p40 and p70, IL-17, KC, MIP-1α, and RANTES through use of pre-packaged bead arrays. This panel allows for evaluation of early and late appearing as well as pro- and anti-inflammatory cytokines.Statistical comparisons of cytokine values were done by a 2 tailed Student's t-Test . Survival data were plotted and the survival curves compared with a log-rank test . Data are displayed as means ± SEM unless otherwise stated. Differences were considered significant at p < 0.05.E. coli were: PBS alone, 0%; ATL313 alone, 0% ; Ceftriaxone alone, 20%; Ceftriaxone plus ATL313, 100%   and 13% of vehicle controls    Figure . A secon) Figure . Finally) Figure .Individual cytokine concentrations displayed statistically significant differences at different intervals after treatment with ATL313 when compared to controls. In plasma, at time (t) = 1 hour, TNF-α concentrations were significantly higher in untreated animals (N = 7) than treated animals (p = 0.041) Figure . ConversDuring experimental endotoxemia in BALB/c mice we found that the concentration of circulating white blood cells (WBCs) was higher in ATL313 treated animals when compared to untreated animals. This difference was consistent at all time points and statistically significant at t = 2 hours after LPS injection (p = 0.003) Figure . This fi2A AR agonists with a concordant increase in IL-10 concentrations; and 3) a relative increase in circulating peripheral blood leukocyte concentrations in mice receiving ATL313 when compared to controls. The first observation bears on the generalizability of our observations to different etiologies of sepsis, including both gram positive and gram negative infections, while the latter two emphasize the mechanism(s) of protection. While these results are encouraging, further work is needed to prove the efficacy of these compounds in clinical sepsis and other models of sepsis including cecal ligation and puncture.This study documents: 1) a survival benefit with administration of ATL313 during live E. coli and S. aureus sepsis (in conjunction with an antibiotic) and in LPS-induced sepsis; 2) a decrease of TNF-α, MIP-1α, MCP-1, IFN-γ, and IL-17 blood concentrations in animals receiving A2A AR agonist, ATL313. We have previously shown that the administration of an A2A AR agonist up to 24 hours after LPS challenge is still protective  when TNF-α concentrations in blood have returned to baseline; therefore the benefit cannot be attributed to effects on TNF-α alone [2A AR agonists in experimental murine sepsis. However, the effect of A2A AR agonists on multiple cytokines is encouraging, since the clinical presentation of sepsis in humans is highly variable with regard to the cytokine cascade, but other as yet undetermined targets may play a role as well.In our murine model of LPS-induced sepsis, TNF-α blood concentrations rise rapidly and are profoundly decreased by the co-administration of an A-α alone . Given t2A AR agonists may decrease neutrophil recruitment and therefore inflammatory damage to the endothelium. This conjecture is bolstered by the increased number of neutrophils in the circulation of mice treated with ATL313 after LPS challenge. Our findings are mirrored by in vitro and in vivo work which revealed that A2A AR agonists decreased neutrophil extravasation and their release of oxidative and non-oxidative products in experimental gram negative bacterial meningitis and S. aureus septic arthritis [Inoculation of experimental animals with TNF-α alone reproduces many of the fundamental pathophysiologic alterations typical of sepsis . In vivorthritis ,17.2A AR agonists down-regulate the expression of MIP-1α, MCP-1, IFN-γ, and IL-17 with a concurrent survival benefit.Cytokines can cause inflammation via upregulation of other pro-inflammatory cytokines in the case of TNF-α, MIP-1α and IL-17, modulation of T-cells by IFN-γ, and activation of monocytes by MCP-1 -21. ThesWhile pro-inflammatory cytokines such as TNF-α, MIP-1α, MCP-1, IFN-γ, and IL-17 increase the inflammatory response, anti-inflammatory cytokines such as IL-10 temper this response. IL-10 is produced primarily by Th-2 cells and decreases concentrations of TNF-α by degrading cytokine mRNA . IFN-γ p2A AR agonist in C57BL/6 mice, we studied survival in a predominantly Th2 T-lymphocyte populated mouse, e.g. BALB/c. Again, we showed a significant survival benefit in these animals treated with an A2A AR agonist after live E. coli or LPS O111:B4 injection.IL-10 reduces the release of TNF-α into the circulation which hinders the development of a systemic inflammatory response syndrome, and this correlates with an increase in survival -38. We h2A AR agonists may be more successful. The influence of A2A AR agonists on late mediators including triggering receptor expressed on myeloid cells (TREM-1) and High mobility group box 1 (HMGB1) should be investigated and is the subject of our ongoing research efforts.Therefore, the benefit of ATL313 in these sepsis models is equally beneficial in mice with a Th1 or Th2 phenotype, perhaps implying that ATL313 and similar compounds exert their anti-inflammatory effect through modification of the innate immune system. We are actively pursuing this line of investigation. Additionally, targeting individual cytokines alone has not been clinically successful, but a more pluripotent approach such as is offered by A2A AR agonists spans different mouse strains, different LPS preparations, as well as different formulations of A2A AR agonists. We also investigated a model of S. aureus sepsis and saw a survival benefit after treatment with ATL313 and ceftriaxone when compared to ceftriaxone alone. A2A AR agonists provide an avenue to study the inflammation due to sepsis and should be considered for clinical interventions in septic patients. Ultimately, it will be important to delineate which cytokines and cells are the effectors of this anti-inflammatory effect.Collectively, we have shown that the effect of A2A Adenosine Receptor agonist compound, ATL313WMS and JL have equity interests in Adenosine Therapeutics, LLC who provided the ACCM designed and performed all experiments and drafted the manuscript. ENM assisted in design and performance of some experiments. GL performed some experiments. TO assisted in design of some experiments. JL assisted in design of some experiments. WMS oversaw all elements of experimental design and execution and manuscript preparation.The pre-publication history for this paper can be accessed here:


Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies , and in the analysis of environmental endocrine disruptors.We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions.HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. The androgen receptor (AR), a member of the nuclear receptor superfamily, functions to regulate gene expression in response to androgens such as testosterone (T) and dihydrotestosterone (DHT). Several cell-based imaging models have been generated in recent years to study AR action, enabling researchers to correlate transcriptional competence of AR with some obligatory intracellular steps visible by fluorescence microscopy. These steps fit within the classical model of AR function: in response to ligands, AR sheds heat shock proteins, forms dimers, and translocates into the nucleus AR signaling leads to differentiation of the male sexual phenotype, and maturation of the secondary sex characteristics, as well to maintenance of male libido, muscle mass and bone density. Disruption of this signaling through inactivating mutations of AR can lead to androgen insensitivity syndromes (AIS), in which genotypic males are affected by a spectrum of developmental abnormalities of the genital apparatus and of the secondary sexual characteristics Some AR functions can now be investigated using automated single cell microscopy First, the results are intrinsically based on cumulative data derived from thousands/millions of cells that certainly vary in terms of cell cycle and/or AR expression level (either endogenous or transiently/stably introduced). These are both important shortcomings, as changes in the amount of expression of a transcription factor affect its intracellular mobility Second, luciferase assays provide single read-outs, yielding information only on the transcriptional reporter gene activity of AR, and are unable to contribute information on how AR cellular distribution, subnuclear organization and mobility, promoter occupancy and chromatin modeling are affected by various compounds. A single cell-based multiplex assay would have the ability to overcome some of these shortcomings, and more directly provide information on the mechanism of action of novel AR agonists or antagonists.Regarding drug discovery programs to identify the next generation of AR agonists or antagonists, a number of cell-based assays have been developed in the past, and most are based on use of a luciferase reporter gene. The reporter gene is placed under control of an AR-promoter or, in composite systems using Gal4-AR fusions, a UAS- promoter, either transiently or stably transfected into a selected cell lines. There are several limitations to this approach. The importance of analyzing receptor activity at the single cell level is highlighted by the role of cell cycle in nuclear receptor transcriptional reporter gene activity. Early studies of the glucocorticoid receptor indicated reduced receptor activity in G2 Here, we report the development and utilization of a novel high throughput image-based transcriptional assay to study multiple aspects of AR intracellular biology at the single cell level. The assay is based upon image acquisition using robotic fluorescent microscopy and automated image analysis, generally referred to as high content screening (HCS) To examine how wild-type and mutant ARs  respond to various experimental manipulations, HeLa cell lines were generated that stably express either wild type (GFP-AR), mutant GFP-ART877A  of the compounds of interest. The dsRED2skl gene encodes a red fluorescent protein that is targeted to the peroxisomes, which improves detection due to concentrating dsRED2skl in the small cytoplasmic organelles. When examining potential anti-androgenic activity, test compounds were titred against 10 nM R1881, a minimal dose found to be sufficient to generate a response in all key measurements. Cells were imaged using an automated microscope with a 40×/0.90 NA objective. For each field, three images were captured: DAPI , GFP , and dsRED2skl . Cytoshop (Beckman Coulter) or Pipeline Pilot (Accelrys) image analysis software was used to identify individual cells in each image. For each cell, the DAPI channel :nuclear variation of GFP signal intensity, NVAR):correlated channel 2/dsRED2skl signal, CORR2):To allow visualization of AR regulated transcriptional reporter gene activity, the HeLa cell lines were transiently transfected with the pARRpromoter , and the channel  was used channel . These m channel . Cell poTo determine the repeatability of the minimum and maximum responses (dynamic range) of the assay, we randomly selected and measured 50 cells treated with either the positive control  or the negative control (no ligand). We then calculated the mean, standard deviation (SD), and coefficient of variation (CV) for each measurement for all cells in each of the two wells . When the hyperspeckling response is examined , with GFP-AR reaching >55% of the maximal untagged AR response. The transcriptional reporter gene activity response . In all comparisons, the EC50 values were similar between tagged and untagged AR suggesting that the addition of the GFP tag does not significantly interfere with the C-terminal ligand binding domain.To determine the effects of the GFP fusion on AR function, we directly compared the responses of GFP-AR and untagged AR when transiently-transfected into HeLa cells along with the pARRThe ability to perform cell-by-cell analysis allowed us to test the hypothesis that expression level alters the observed responses. At higher levels of expression, an increased magnitude of hyperspeckling was observed with no effect on nuclear translocation . At thesIn addition to the response observed after 18–24 hrs of ligand treatment, we also quantified responses after a brief pulse of the agonist R1881. Surprisingly, after the HeLa GFP-AR cells were exposed to a 2 hr pulse of 1 nM R1881, the intracellular distribution of AR remained nuclear for up to 15 hr after treatment  assay. The principle of the assay is that if reiterative photobleaching in the cytoplasm results in loss of nuclear fluorescence, the fluorescent protein must be able to shuttle from the nucleus into the cytoplasm. We transfected HeLa GFP-AR cells with a plasmid encoding hcRED (a 25 kDa-red fluorescent protein) to allow visualization of the cytoplasmic region , progesterone (PRO), estrone (EST), corticosterone, and androstenedione. Consistent with previously published cytological and transcriptional results, our single cell analyses revealed that while EC50 values were significantly different, at high concentrations of these steroids, maximal effects rivaled known agonists . InteresWe and others have qualitatively shown that the anti-androgens o-hydroxyflutamide (OHF) and bicalutamide (CAS) can induce AR nuclear translocation, but not the hyperspeckling induced by AR agonists To characterize the potential antagonistic responses, each test compound was added to the cells approximately 15 minutes before adding 10 nM R1881. As expected, a significant dose-dependent effect on transcriptional reporter gene activity was observed, causing a 71–85% decrease in the dsRED2skl reporter signal . CalculaTo further understand the range of responses of AR trafficking and function potential mechanisms, we examined several environmental compounds previously characterized as having anti-androgenic activity. These compounds included Vinclozolin VNZ; , nitrofe50 = 13200±152 nM). Surprisingly, however, there was a decrease in R1881-induced nuclear translocation ; further, he small nuclear pool also exhibited a loss of hyperspeckling . These results are consistent with previous biochemical studies and demonstrate the utility of the single cell assay to identify and characterize novel cellular responses based on both cytological and functional readouts We also screened a small panel of novel compounds thought to interact with AR. While a majority of these compounds did show a response in terms of transcriptional reporter gene activity (data not shown), the compound Decursin demonstrated a unique response pattern. When observed in combination with 10 nM R1881 , the comTo further explore the utility of our assay, we examined compounds  that exhibit estrogenic activity, but have rather consistently been reported to lack androgenic activity To emphasize the multiplex nature of this assay further, we examined the relationship between the cell cycle and cellular responses observed with R1881, OHF, and thymidine. To perform these studies, we simplified the HCS cell cycle analysis techniques described by Gesparri et al. As expected, thymidine demonstrated a dose-dependent ability to block HeLa GFP-AR cells in G1 (EC50 = 2.6 mM±0.2 mM) and significantly reduced the occurrence of cells in S-phase . With an R1881 titration, this pattern persists with G2 cells having 8–12% more AR in the nucleus compared to G1 cellsp<0.05; . A similHaving determined both the agonist and antagonist responses in the assay with wild type AR, we next wanted to examine the assay performance with an AR mutant relevant to prostate cancer. The well-studied T877A mutation, found in the LNCaP cell line, effectively relaxes the stringency of the ligand binding pocket allowing the receptor to become “promiscuous” and respond to a variety of additional ligands Parallel to studying mutations associated with prostate cancer, we were also interested in characterizing the inactivating mutations associated with androgen insensitivity syndrome. In particular, we sought in compounds that are able to induce a normal response from the mutant receptor. The F764L mutation was isolated from a patient with complete AIS and was previously characterized as having an abnormally high ligand dissociation rate AR develops and maintains the male sexual phenotype under physiologic conditions, and abnormalities in AR function participate in the etiology of several diseases. In the ∼20 years that followed the molecular cloning of AR The work described here is advantageous over previous cell based assays in several ways and has provided useful insights on AR biology. First, this assay generates multiplex high throughput data that not only measures AR transcriptional reporter gene activity, but also two other upstream steps linked to transcriptional competence (nuclear translocation and hyperspeckling). By having the ability to compare how compounds affect each of the measurements, we were not only able to reiterate that compounds such as R1881 are powerful AR agonists, but also that formation of the hyperspeckled pattern associated with agonist bound nuclear receptors is a distinct mechanistic step from nuclear translocation and occurs only at ∼30-fold higher agonist concentrations. The correlation between formation of the hyperspeckled pattern and transcriptional reporter gene activity lends further support to the concept that this pattern represents an increased trend toward protein complex formation and transcriptional regulation. A similar link between hyperspeckling of other Type I nuclear receptors has been reported . While the speckles themselves appear to be only randomly associated with sites of transcription, and are demonstrated to be transient in the living cell by photobleaching methods, the degree of AR-AR or AR-coregulator interactions determined by FRET suggest they represent complexes at an ill-defined level of maturation . Alternately, the speckles simply represent the notion that the vast majority of complexes can form away from the very small portion of the nuclear volume that contains target genes. If the latter case were true, an underlying deterministic or stochastic mechanism(s) for AR dynamics would be necessary to facilitate a means for the complexes to find target genes.We were also able to quantitatively confirm that traditional AR antagonist  are able to induce nuclear translocation fail at forming a hyperspeckled pattern; furthermore, in the presence of agonist, transcription inhibition is linked to blocking the formation of the hyperspeckled pattern. The differences in the amount of speckling observed following exposure to the various compounds used could reflect differences in the identity of the recruited coregulators and/or the strength of AR-CoR binding We also tested compounds belonging to the rapidly-increasing group known as “endocrine or environmental disruptors,” electing to test agents with known ER or AR antagonistic activity. No induction of AR transcriptional reporter gene activity was detected with our small set of these substances, although both ER (at µM concentration) and AR (at sub µM concentrations) disruptors showed the ability to induce nuclear translocation and some minor hyperspeckling. That these compounds have some affect on two of the three agonist actions (translocation and hyperspeckling) may indicate a mechanistic action unlike the known antagonists. While failing to activate the probasin reporter, the moderate hyperspeckling observed may indicate transcriptional effects on other genes. Panels of AR sensitive transcriptional reporter genes and multi-color mRNA fluorescent in situ hybridization (FISH) experiments are in development to directly test this important question. In the R1881 competition assay, no AR antagonistic activity was detected for the ER disruptors; in contrast, the AR disruptors DDT, Vinclozolin and Nitrofen were effective in preventing hyperspeckling and transcriptional reporter gene activity at concentrations very similar (∼1.5-fold higher) to established AR antagonists . The presence of these substances in certain environments, and their recognized AR antagonistic activity at concentrations that may be achieved after chronic exposure is important, and according to some investigators may affect AR-mediated functions such as spermatogenesis The second advantage of our approach is that the microscopic nature of the assay permits the selection of individual healthy interphase cells expressing near endogenous levels of AR. This is an essential methodological characteristic because several critical functions of a nuclear receptor are affected when it is over-expressed, even if only to modestly higher levels Along with the ability to correlate responses to expression level, we were also able to link measurements to cell cycle subpopulations. Our results demonstrate that in S-phase and G2 cells, AR is significantly more nuclear than in G1 cells. With agonist treatment, the increased nuclear localization in G2 is associated with heightened nuclear hyperspeckling. In contrast, the S-phase cells demonstrate decreased hyperspeckling with agonist treatment. Although not directly studied here due to technical limitations, these results would predict AR to have the highest transcriptional reporter gene activity in G2 (increased nuclear translocation and hyperspeckling). However, others have found AR to have the highest transcriptional reporter gene activity in G0 and S phase Finally, the flexibility of the assay allowed us to examine disease-related AR mutations for altered compound responses. Especially important would be the analysis of AR mutations thought to be involved in prostate cancer treatment resistance Overall, the technology described in this paper represents a significant advance that builds upon our previous efforts to study AR at the single cell level by now allowing a quantitative assessment of multiple aspects of intracellular AR function. The technology is straightforward, reliable, reproducible, and is automated to the point where large libraries of compounds can now be tested to identify novel AR agonists and antagonist, including endocrine disruptors. Moreover, use of ARs harboring patient-related mutations will be amenable to agonist/antagonist screening for personalized patient drug selection. Finally, combined with current RNAi technologies, this multiplex HTM assay should also aid in the identification of proteins involved in pathways that regulate AR biology .All chemicals were obtained from Sigma-Aldrich  unless stated otherwise. Methyltrienolone (R1881) was obtained from NEN Life Science Products .Stable HeLa cell lines expressing GFP-AR (wild type and T887A) were generated to ensure GFP-AR protein was expressed in a high percentage of cells. HeLa cells were transfected with 1 µg/well plasmid DNA and 0.01 µg/well linear hygromycin marker (BD) using BioRad Transfectin reagent 1 day after plating in six-well plates. After 24 hours, cells were trypsinized and plated in medium supplemented with 500 µg/ml Hygromycin  in 10 cm tissue culture dishes. Clones were selected and checked for appropriate GFP-AR distribution and expression by widefield fluorescent microscopy and western blotting. Stable cell lines were maintained in DMEM/F12 medium supplemented with 5%FBS and 500 µg/ml hygromycin.−5 M to 10−14 M. Compound dilutions and final addition to multi-well plates were performed using a Beckman Biomek NX robotic platform to ensure repeatability from experiment to experiment. For competition studies, after experimental compounds were added to the cells, the competitor (10 nM R1881) was added to the wells approximately 15 minutes later. After incubation was complete, using the Biomek NX robot, plates were washed with PBS and fixed for 20 min at RT in 4% formaldehyde prepared in CSK buffer . After fixation, cells were briefly permeabilized (5 min) with 0.5% Triton-X and prepared for imaging by washing in PBS, aspirating the washed solution, and adding a 1 ng/ml DAPI solution. Cells were imaged in PBS.Twenty-four hours before transfection, cells were plated onto 100 mm plastic dishes in medium supplemented with charcoal stripped and dialyzed FBS (SD-FBS). Transient introduction of the pARR-2PB-dsRED2skl reporter construct was performed using 6.0 µg reporter plasmid and 6.0 µg carrier DNA  using Transfectin following standard protocols. After 8 hour incubation, DNA/lipid complexes were removed. Cells were then trypsinized and replated at 8500 cells per well in Matrical 384 poly-D-lysine treated 384 well optical glass bottom plates and incubated an additional 12 hours to allow for cell adhesion. Cells were then exposed to for 24 h to ligands at concentrations ranging from 10For experiments in which cell cycle effects were determined, cells were labeled using an Invitrogen Click-iT cell cycle analysis kit using supplied protocols. Prior to fixation, cells were exposed to 10 µM EdU for 30 minutes. Cells with EdU incorporation were labeled by an EdU specific antibody conjugated to an A647 florescent marker. Because the Click-iT labeling decreases GFP signal intensity, AR was labeled using an Anti-AR antibody (Dr. Nancy Weigel) and A488 secondary antibody (Molecular Probes).Chroma 82000 triple band filter set  3.) An imaging camera: Hamamatsu ORCA-ER Digital CCD camera  and 4.) A focusing camera: Photoonics COHU Progressive scan camera . The microscope was equipped with a Nikon S Fluor 40×/0.90NA objective and the imaging camera set to capture 8 bit images at 1×1 binning  with 4 images captured per field . In general, 49 images were captured per well for image analysis.Cells were imaged using the Cell Lab IC-100 Image Cytometer  platform which consists of 1.) Nikon Eclipse TE2000-U Inverted Microscope  2.) Images were analyzed using either Cytoshop Version 2.1 (Beckman Coulter) or Pipeline Pilot Version 6.1.5 (Scitegic) analysis software. Nuclear masks were generated by applying a non-linear least-squares image filter combined with automatic histogram based thresholding. General background signal in the GFP channel was corrected by automatic mean background subtraction. Total area of GFP-AR image extraction was determined by intersection of a chosen extraction radius (approximately 25% larger than average nucleus radius) and a Voroni tessellation polygon. Cell populations were filtered to achieve a uniform population of cells without cell aggregates, mitotic cells, apoptotic cells, and cellular debris. Applied gates were based upon 1.) nuclear area 2.) nuclear wiggle (AREA/PERIMETER) 3.) DNA content (DAPI INTENSITY). AR cytoplasm to nuclear translocation, AR nuclear variation/hyperspeckling, and transcriptional reporter gene activity were determined using algorithms within the image analysis software , Nuclear Variation (NVAR), and total channel 2 intensity (CORR2)). For cell cycle analysis, two additional parameters were collected; total channel 0 nuclear intensity (TOTAL_NUC_DAPI) and average channel 3 nuclear intensity (AVG_NUC_Ch03). All data was exported to Pipeline Pilot and responses were normalized to a 0 to 1 range based on (−) and (+) controls and quadruplicates averaged. EC50 values were calculated by plotting a simple scatter plot of response vs. ligand concentration and using SigmaPlot four parameter logistic curve fitting algorithm. Due to the nature of the curve fitting algorithm, for those responses that did not plateau the response observed at the highest concentration was assumed maximal.For live cell FLIP experiments, HeLa GFP-AR cells grown on 23 mm glass bottom Delta T dishes (Bioptechs), transfected with 0.4 µg of pCMV-hcRED plasmid plus 0.8 µg of carrier DNA using Transfectin (BioRad), and allowed to recovery for 24 hrs before being placed onto a LSM 510 confocal microscope  equipped with a 63× (NA 1.4) objective. Cells were maintained at 37°C using a Bioptechs Delta Controller and fresh media containing the appropriate ligand was cycled over the cells. A single Z-section was imaged before and at time intervals following each bleach. The bleach was performed using the laser set 488 nm for GFP at maximum power for 10 iterations (∼1 sec) in a circular region contained within the cytoplasm of the cell. Bleaches were repeated every 10 seconds for the duration of the experiment (700 s) Fluorescent intensities of regions of interest were determined using LSM software and data was exported to Excel  to normalize intensity to the pre-bleach image. LSM images were exported as TIF files and final figures were generated using Adobe Photoshop.Overall assay quality was determined using the Z' calculation, a dimensionless measurement determined using the following equation:α and zβ relate to type 1 and type 2 errors was used to construct a plot that represents the number of cells needed for the desired minimum significant response. For the assay described here, acceptable type 1 and type 2 errors were set at 0.01 and 0.20  of R1881, (A) nuclear translocation (FLIN), (B) nuclear speckling (NVAR), and (C) AR transcriptional reporter gene activity (CORR2) are minimal. As R1881 concentration increases, a dramatic increase in the measurements is observed with saturation of response observed at ≈100 nM. The color scale represents a range of response from maximal (white) to minimal response (black) for each measurement. All results shown are from a single plate of the set.(2.38 MB TIF)Click here for additional data file.Figure S2GFP-AR responds in a similar manner to untagged AR but with diminished maximal responses. HeLa cells were transiently transfected with either untagged AR or GFP-AR plasmids in addition to the pARR-2PB-dsRED reporter plasmid. Twenty-four hours after transfection, cells were exposed to multiple concentrations of DHT ranging from 0.002 nM to 200 nM for 18 hr. Cells were then fixed and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in images captured by the automated IC100 microscope. Images were analyzed using Pipeline Pilot software and the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) responses quantified.(0.83 MB TIF)Click here for additional data file.Figure S3GFP-AR retains a nuclear distribution with decreased hyperspeckling but maintains the ability to shuttle into the cytoplasm after agonist removal. A. HeLa GFP-AR cells were treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was removed by serial washes with ligand free media containing cyclohexamide to prevent new protein synthesis. Cells were then fixed, imaged, and examined for the localization of the receptor at 3, 6, 9, 12, and 15 hrs using previously described image analysis tools. Responses were normalized to untreated controls and response seen with 1 nM R1881 treatment for 2 hrs. An additional experiment using untagged AR was also performed to ensure response was not due to the inclusion of the GFP tag on the receptor. The ability of GFP-AR to shuttle between the nuclear and cytoplasmic compartments during and after ligand treatment was analyzed using the FLIP photobleaching technique where a region in the cytoplasm is repeatedly bleached. B. A graph comparing the rate at which nuclear GFP-AR fluorescence is lost in the absence of ligand , in the presence of 10 nM R1881 , and after ligand withdrawal . To ensure results were not due to general photobleaching during imaging, cells were examined where the targeted photobleaching region was outside of the cellular area (Photobleach Control). Both R1881 treatment and withdrawal significantly slow but does not stop the rate that the receptor shuttles between the nucleus and the cytoplasm. C. Selected images from FLIP experiment.(2.19 MB TIF)Click here for additional data file.Figure S4Differential responses of the T877A AR mutation. The differential effects of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with selected compounds. Cells stably expressing either WT (unhatched) or T877A (hatched) forms of AR were transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound either alone (grey bars) or with 10 nM R1881 (white bars) for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error. Data represents average of 4 experiments.(1.51 MB TIF)Click here for additional data file.Table S1(0.05 MB PDF)Click here for additional data file.Table S2(0.03 MB PDF)Click here for additional data file.Table S3(0.04 MB PDF)Click here for additional data file.Table S4(0.03 MB PDF)Click here for additional data file.Table S5(0.56 MB TIF)Click here for additional data file.Table S6(0.06 MB PDF)Click here for additional data file.Table S7(0.04 MB PDF)Click here for additional data file.