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OBJECTIVESHigh concern about child’s health is a common reason parents of children with medical complexity (CMC) seek care in emergency departments and hospitals. Factors driving parental concern are unknown. This study explores associations of parent’s sociodemographic and child’s clinical factors with high parental concern.PATIENT AND METHODSSecondary analysis of a pilot study of CMC and parents who used daily for 3 months MyChildCMC, a home monitoring app. Parents recorded their child’s vital signs (temperature, heart rate, respiratory rate, oximetry), symptoms (pain, seizures, fluid intake/feeding, mental status), and oxygen use, and received immediate feedback. Parents rated their child’s health concern on a 4-point Likert scale. Concern scores were dichotomized (3–4 = high, 1–2 = low) and modeled in a mixed-effects logistic regression to explore important associations.RESULTSWe analyzed 1223 measurements from 24 CMC/parents, with 113 (9.24%) instances of high concern. Child factors associated with high parental concern were increased pain (odds ratio [OR], 5.10; 95% confidence interval [CI], 2.53–10.29; P < .01), increased oxygen requirement (OR, 28.91; 95% CI, 10.07–82.96; P < .01), reduced nutrition/fluid intake (OR, 71.58; 95% CI, 13.01–393.80; P < .01), and worsened mental status (OR, 2.15; 95% CI, 1.10–4.17, P = .02). No other associations existed.CONCLUSIONSChanges in CMC’s clinical parameters were associated with high concern, which may be an early indicator of acute illness in CMC when it is the primary complaint. Monitoring and responding to high parental concerns may support CMC care at home.

In 1960s and 1970s singer-songwriter music, some artists used a malleable approach to meter in performance that resulted in songs with extremes of self-expressive timing flexibility that cannot be accounted for by using a single conception of meter. As a solution, this article draws together theories of metric hierarchy, metrical reinterpretation, and metric process to develop the theory of flexible meter. This approach recasts meter as able to encompass the variety of metric scenarios presented by these singer-songwriters, from metric regularity to metric ambiguity and vacillations between these two possibilities. Performances by Bob Dylan, Joni Mitchell, Buffy Sainte-Marie, Paul Simon, and Cat Stevens are discussed to investigate their levels of engagement with meter—the degree to which their performances are regular or ambiguous—and how the individual metric style of each artist contributes to the self-expressive singer-songwriter performance practice.

Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

The coronavirus (COVID-19) pandemic has caused a shift in education. Nursing education must respond to these changing times by redesigning classroom instruction. Flipped learning (FL) is a proposed method for teaching advanced practice nurses that is student-centered and engaging, both critical areas for contemporary learners. This qualitative study explored the perspectives and experiences of the family nurse practitioner faculty and the family nurse practitioner nursing students who participated in FL. The type and level of FL practices faculty can apply to support students coming to class prepared to engage in active learning activities was also investigated. The study used an insider action research design. There were two arms of the study, faculty and student. Ten faculty-led learning circles with four faculty and three family nurse practitioner student focus groups, each with six students, were held over the course of the study. Thematic findings were developed from both student and faculty data. The results revealed that family nurse practitioner students had a positive attitude toward FL. Many expressed FL provided perceptions of gained confidence, personalized learning, increased engagement, and preparedness for classroom instruction. Overall, faculty preferred FL over traditional teaching approaches. Several active learning activities were used by faculty with clinical case studies being the most preferred by student participants. This study confirms FL is a plausible and promising approach to traditional classroom instruction. However, more robust studies are needed to determine its benefits in advanced practice nursing education.

The purpose of this article is to investigate challenges that complex care programs face in caring for children with medical complexity (CMC), as well as to explore whether identified challenges could be met through collaboration with pediatric palliative care or additional training for complex care teams.Medical providers who self-identified as providing clinical care to children with medical complexity were asked to complete an online anonymous survey. Subjects were recruited through a Complex Care listerv. Data were analyzed using descriptive statistics.85 subjects completed the survey, of whom 87.1% (n= 74) were physicians, and 12.0% (n=11) were nurse practitioners. Subjects reported several challenges in caring for CMC, including symptom management, establishing goals of care, advance care planning, and coordination of care. A majority of subjects reported benefitting from palliative care consultative assistance in each subject area. Most subjects described their relationship with palliative care as a close partnership with frequent overlap.The evolving field of pediatric complex care is associated with an array of challenges in caring for CMC. Many of these challenges include competency areas where palliative care providers receive concerted training. Our research suggests greater palliative care involvement in the CMC population can benefit complex care teams and patients, given the expertise palliative providers can bring to the population and the discipline of complex care.

Bivalent small-molecule degraders, or proteolysis targeting chimeras (PROTACs), work by simultaneously binding a target protein and E3 ubiquitin ligase to produce a ternary complex. To drive target ubiquitination and degradation at low catalytic concentrations, degraders must form appropriately positioned complexes of sufficient stability, aided by intra-complex interactions. We hypothesized these molecular recognition features could be enhanced by increasing binding valency. Here we present trivalent PROTACs as a strategy to boost protein degradation. Our design for a trivalent PROTAC consisted of two BET bromodomain inhibitors and an E3 ligase ligand, each separately tethered via a branched linker. In screening, we identified SIM1, a VHL-based PROTAC, as a highly potent BET degrader, capable of low picomolar degradation for all family members, with preference for BRD2. In functional comparison studies to bivalent PROTACs or inhibitors, SIM1 showed more sustained anti-cancer activity across numerous therapeutically relevant cell lines. Biophysical, biochemical, and cellular mechanistic studies showed SIM1 induces conformational changes upon binding to the BET protein to simultaneously engage with high avidity both its bromodomains in a cis intramolecular fashion. The resulting 1:1:1 complex showed positive cooperativity, high stability and prolonged cellular residence time. We provide proof-of-concept for augmenting the binding valency of proximity-induced modalities as a strategy to leverage both cooperativity and avidity within the ternary complex to advance functional outcomes.

Pediatric health care providers are frequently responsible for prescribing physical, occupational, and speech therapies and monitoring therapeutic progress for children with temporary or permanent disabilities in their practices. This clinical report will provide pediatricians and other pediatric health care providers with information about how best to manage the therapeutic needs of their patients in the medical home by reviewing the International Classification of Functioning, Disability and Health; describing the general goals of habilitative and rehabilitative therapies; delineating the types, locations, and benefits of therapy services; and detailing how to write a therapy prescription and include therapists in the medical home neighborhood.

Estimates indicate 70% of all individuals with HIV will be age 50 or older by 2030. Chronic conditions, including cardiovascular disease, diabetes mellitus, kidney disease, malignancies, neurocognitive disorders, and osteopenia or osteoporosis, occur more frequently in patients with HIV and have become the leading cause of morbidity in this population. NPs play an integral role in helping this population age healthfully.

People living with HIV can experience the full benefits of retention when they are continuously engaged in care. Continuous engagement in care promotes improved adherence to ART and positive health outcomes. An infectious disease clinic has implemented a protocol to primarily improve patient retention. The retrospective, facility-based, costing study took place in an infectious disease clinic in Washington DC. Retention was defined in two ways and over a 12-month period. Micro-costing direct measurement methods were used to collect unit costs in time series. Return on investment accounted for the cost of treatment based on CD4 strata. ROI was expressed in 2016USD. The difference in CD4 and viral load levels between the two periods of analysis were determined for active patients, infected with HIV. The year before the intervention was compared to the year of the intervention. Total treatment expenditure decreased from $2,435,653.00 to $2,283,296.23, resulting in a $152,356.77 gain from investment for the healthcare system over a 12-month investment period. The viral load suppression rate increased from 81 to 95 (p = 0.04) over the investment period. The number of patients in need of HIV related opportunistic infection prophylaxis decreased from 21 to 13 (p = 0.06). Improved immunologic, virologic and healthcare expenditure outcomes can be linked to the quality of retention practice.

Problem Current research suggests behavioral and environmental interventions to prevent neonatal pain prior to an invasive procedure are rarely administered and seldom documented. The aim of this study was to systematically review findings from published randomized controlled trials that tested the effects of behavioral and environmental procedural pain management interventions on behavioral pain response in preterm infants. Eligibility Criteria Randomized controlled trials examining the effects of behavioral and environmental pain management interventions on behavioral pain response in preterm infants were identified. Articles accepted for inclusion met the following criteria: English language, original, peer refereed, randomized controlled clinical trials published within the past 5 years, study sample: preterm infants, setting: neonatal intensive care units, study intervention behavioral and environmental, outcome pain measurement score from valid and reliable pain scale. Sample Fourteen randomized controlled trials from a literature search of PubMed and Medline databases were included in this review. Results Across all age groups, facilitated tucking, oral sucrose, and kangaroo care decreased behavioral and physiologic pain response alone and in combination with other behavioral and environmental interventions. Conclusion Among preterm infants, facilitated tucking, oral sucrose, and kangaroo care significantly mitigates biobehavioral pain response associated with acutely painful procedures. Implications Evidence suggests that behavioral and environmental interventions can decrease biobehavioral pain response associated with acutely painful procedures in preterm infants. This review highlights the need for rigorous studies to help healthcare providers to build a tailored pain treatment plan for preterm infants.

This essay reframes the interdisciplinary collaborative health team model by proposing the application of 3 foundational pillars-democratic professionalism, implementation science, and therapeutic alliance to advance this practice. The aim was to address challenges to the model, enhance their functional capacity, and explicate and enact social justice practices to affect individual health outcomes while simultaneously addressing health inequities. The pillars are described and examples from the author's dissertation research illustrate how the pillars were used to bring about action. Related theories, models, and frameworks that have negotiation, capacity building, collaboration, and knowledge/task/power sharing as central concepts are presented under each of the pillars.

Cervical mobilization and manipulation have been shown to improve cervical range of motion and pain. Rotatory thrust manipulation applied to the lower cervical segments is associated with controversy and the potential for eliciting adverse reactions (AR). The purpose of this clinical trial was to describe two translatory non-thrust mobilization techniques and evaluate their effect on cervical pain, motion restriction, and whether any adverse effects were reported when applied to the C7 segment.This trial included 30 participants with painful and restricted cervical rotation. Participants were randomly assigned to receive one of the two mobilization techniques. Active cervical rotation and pain intensity measurements were recorded pre- and post-intervention. Within group comparisons were determined using the Wilcoxon signed-rank test and between group comparisons were analyzed using the Mann-Whitney U test. Significance was set at P = 0.05.Thirty participants were evaluated immediately after one of the two mobilization techniques was applied. There was a statistically significant difference (improvement) for active cervical rotation after application of the C7 facet distraction technique for both right (P = 0.022) and left (P = 0.022) rotation. Statistically significant improvement was also found for the C7 facet gliding technique for both right (P = 0.022) and left rotation (P = 0.020). Pain reduction was statistically significant for both right and left rotation after application of both techniques. Both mobilization techniques produced similar positive effects and one was not statistically superior to the other.A single application of both C7 mobilization techniques improved active cervical rotation, reduced perceived pain, and did not produce any AR in 30 patients with neck pain and movement limitation. These two non-thrust techniques may offer clinicians an additional safe and effective manual intervention for patients with limited and painful cervical rotation. A more robust experimental design is recommended to further examine these and similar cervical translatory mobilization techniques.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Routine typing of a potential bone marrow donor by sequence-specific oligonucleotide probes and sequence based typing produced inconclusive subtyping results, suggesting a new allele. A magnetic bead-based method, haplotype specific extraction (HSE), was used to separate the diploid sample into its haploid components. The sample was then re-typed using standard sequence-based typing (SBT), revealing a new human leukocyte antigen (HLA) allele, since named B*1576. HSE used in conjunction with standard SBT is a convenient and simple tool for resolving ambiguous and novel allele combinations without the need for amplification or subcloning.

Recent years have witnessed an increased interest in the role of faith-based services to meet the social and practical needs of the American populace. The research upon which this paper is based represent the first ever attempt to document empirically the characteristic of men who sought assistance from a faith-based batterers' intervention program in the United States. Analyzing closed case files from 1059 men who have been involved in the batterers' intervention program since its inception over ten years ago revealed a number of differences between faith-based clients and those who seek help from a secular agency. The implications for interventions with batterers are explored.

Open architecture is the technology that provides the means to open up a world of investment choice, options and ease in reporting and administration for company retirement plans and participants by enabling multiple retirement plan service providers to communicate and share data electronically. This ability to provide data to and receive data from a variety of investment providers means that the plan sponsor has more options and control in the setup and administration of its retirement plan. Perhaps the most important function that open-architecture systems provide the plan sponsor is the ability to work with multiple, independent service providers, commonly known as unbundling. This article outlines the features and benefits of open architecture and discusses how to find an open-architecture provider and why open-architecture systems make unbundling a primary choice for plan sponsors today.

This chapter presents an overview of ways in which Pentecostal and charismatic spirituality interface with domestic violence. Pentecostal experiences and practices are significant forces in the world. Understanding texts, practices, beliefs, attitudes, and values of Pentecostalism can help practitioners offer healing to survivors and perpetrators from this spiritual background. Understanding biblical perspectives that address dignity of relational partners can help Pentecostal and Charismatic pastoral counselors offer help to congregants suffering from domestic abuse. The authors offer historical background and insights into Pentecostal practices, including women’s roles from early days to current situations in some of the largest Pentecostal and Charismatic denominations. We make recommendations for interactions with clients or congregants, emphasizing safety for victims and accountability for perpetrators’ behaviors, taking into consideration the Holy Spirit’s power to redeem, forgive, heal, and transform. We also discuss the context of perpetrators’ actions within global practices of commodification, and scriptural precedence for respect accorded to all humanity.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1. These examples demonstrate the power of this technology in enabling the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics.

Emergency surgery has become a rare event after percutaneous coronary intervention (PCI). Whether having cardiac-surgery services available on-site is essential for ensuring the best possible outcomes during and after PCI remains uncertain.We enrolled patients with indications for nonemergency PCI who presented at hospitals in Massachusetts without on-site cardiac surgery and randomly assigned these patients, in a 3:1 ratio, to undergo PCI at that hospital or at a partner hospital that had cardiac surgery services available. A total of 10 hospitals without on-site cardiac surgery and 7 with on-site cardiac surgery participated. The coprimary end points were the rates of major adverse cardiac events--a composite of death, myocardial infarction, repeat revascularization, or stroke--at 30 days (safety end point) and at 12 months (effectiveness end point). The primary end points were analyzed according to the intention-to-treat principle and were tested with the use of multiplicative noninferiority margins of 1.5 (for safety) and 1.3 (for effectiveness).A total of 3691 patients were randomly assigned to undergo PCI at a hospital without on-site cardiac surgery (2774 patients) or at a hospital with on-site cardiac surgery (917 patients). The rates of major adverse cardiac events were 9.5% in hospitals without on-site cardiac surgery and 9.4% in hospitals with on-site cardiac surgery at 30 days (relative risk, 1.00; 95% one-sided upper confidence limit, 1.22; P0.001 for noninferiority) and 17.3% and 17.8%, respectively, at 12 months (relative risk, 0.98; 95% one-sided upper confidence limit, 1.13; P0.001 for noninferiority). The rates of death, myocardial infarction, repeat revascularization, and stroke (the components of the primary end point) did not differ significantly between the groups at either time point.Nonemergency PCI procedures performed at hospitals in Massachusetts without on-site surgical services were noninferior to procedures performed at hospitals with on-site surgical services with respect to the 30-day and 1-year rates of clinical events. (Funded by the participating hospitals without on-site cardiac surgery; MASS COM ClinicalTrials.gov number, NCT01116882.).

SummaryAcquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation., JOURNAL ARTICLE, FLWOA, SCOPUS: re.j, info:eu-repo/semantics/published

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.

In the current U.S. health care system, both good health and a higher quality of health care are more likely to be experienced by those who have access to money, power, and privilege. Consequently, serious health disparities exist between the rich and poor, White people and people of color, and men and women. The American Association of Colleges of Nursing (AACN) has made explicit the values that form the cornerstone of professional nursing: altruism, autonomy, human dignity, integrity, and social justice. In this article, the authors explore the interconnections between values explicated by the AACN and nursing practices and policies. The authors draw on work in the field of pain management, cultural competency, and harm reduction as exemplars of this interconnection. The authors propose that through political action, nurses can strive for congruence between their professional values, practice, and policies and subsequently effect change and improve health outcomes.

This article describes the Village AIDS Day Treatment Program, a program for people living with HIV/AIDS that provides health care by using a full range of interdependent services. Opened in 1988, this program was the first of its kind in the country. It has provided leadership in developing a model of care that addresses the full spectrum of health care—promotion, prevention, maintenance, and treatment. Along with describing the program and its services, this article includes the program’s history and its influencing philosophies.

Epidermal growth factor (EGF), a potent mitogen and tumor promoter, is excreted in urine permitting it to incubate with urothelial cells. We have previously shown that the distribution of receptors for EGF (EGF-Rs) on malignant urothelium changes to favor contact between EGF-Rs and intraluminal EGF. OBJECTIVES. To determine if concentrations of EGF in voided urine are different in patients with transitional cell carcinoma (TCC) of the bladder than in those without this condition. METHODS. EGF was measured by radioimmunoassay in the urine of 54 patients with newly diagnosed TCC (16 with grade 1, Stage Ta lesions, and 38 with grade 3, Stage Tor = 2 lesions) and 66 pathologic and normal controls without TCC. Subjects were matched for age and good renal function. RESULTS. EGF concentrations were significantly reduced in patients with TCC compared with controls either when uncorrected (p0.0001) or corrected (p0.000000002) for urinary creatinine excretion. CONCLUSIONS. The reduced concentration of EGF in voided urine supports previous evidence that the urinary EGF/urothelial EGF-R interaction is important for TCC development and growth, and has the potential to serve as a marker of tumor persistence, recurrence, and therapeutic response.

Background Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells. Results In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1. Conclusion These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.

New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.