Practical motives are prominent in test-ordering in the Emergency Department.

BACKGROUND: Laboratory test ordering under time pressure may impact test-ordering behavior. METHODS: To investigate the test-ordering behavior of doctors working under such pressure, we designed a questionnaire for trainees and staff in the Emergency Department (ED). This questionnaire addressed topics such as necessity of requested tests, time spent on ordering, costs and availability of tests, and the time of the day. We hypothesized that ordering behavior would be guided predominantly by the medical need of tests and aimed at identifying practical motives that also have an effect. RESULTS: Remarkably, two-third of the respondents (67%) admitted that tests were ordered that would not influence treatment policy directly and 48% of the doctors stated that tests were ordered that do not impact treatment at all. The frequency of such orders was "sometimes" and "frequent" in a 50:50 ratio. Interestingly, tests that could prove relevant at a later stage are often ordered simultaneously to reduce burden on the patient. None of the respondents spent more than 3 min on the ordering process and very few (8%) desired more time for ordering. Most respondents (81%) declared to have limited knowledge of the costs of laboratory tests. A random survey covering four tests confirmed this. Generally, turnaround time did influence ordering behavior while time of the day did not. CONCLUSIONS: In conclusion, doctors in an ED - besides first of all medical motives - heavily exploit practical (non-medical) reasoning for laboratory test ordering, e.g. taking availability of tests into account and ordering non-immediate tests.

A survey of doctors reveals that few laboratory tests are of primary importance at the Emergency Department.

BACKGROUND: Laboratory tests in hospitals are among the most important diagnostic tools for medical decision making at the Emergency Department. They are often ordered as part of extended test panels, which, although helpful and convenient for doctors, may lead to overuse of tests and overdiagnosis. To improve the ordering process, we investigated which laboratory tests are essential for optimal decision making at the Emergency Department of our hospital. METHODS: Forty-nine doctors regularly involved with the Emergency Department filled in a questionnaire asking for their opinions on laboratory test ordering and use. RESULTS: A limited number of laboratory tests are considered indispensable for the Emergency Department: CRP and leukocytes, urea and creatinin, sodium and potassium, and haemoglobin. Glucose and troponin should probably also be included in this list, but were not mentioned as glucose is measured using portable point-of-care devices in our hospital, while cardiac patients are referred directly to the cardiac care unit. CONCLUSIONS: Only a limited number of laboratory tests are essential for early medical decision making at the Emergency Department. Ordering facilities should be arranged such that these tests are permanently available, easy to order, and performed with short turnaround times. Test panels for the ED should incorporate these essential tests, with additional other tests so as to prevent essential tests from being forgotten, maintain convenience for doctors and promote sensible and effective use of diagnostic testing. The outcome of these conflicting aims is a compromise, as is discussed.

A multi-parameter imaging assay identifies different stages of ligand-induced androgen receptor activation.

Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands.

Reducing the number of clinical stat phlebotomy orders: feasible or not?

BACKGROUND: To manage the costs and performance of diagnostic services involving laboratory testing permanent evaluation is required. Among these, the ever increasing use of stat ordering, involving labour intensive phlebotomy,warrants explanation, especially for a phlebotomy service organised by the laboratory, not by those responsible for and/or carrying out the requests, such as doctors and nurses. METHODS: To explore the possibilities to reduce the number of stat phlebotomy requests, we conducted a survey among nurses and doctors of their motives in requesting 109 randomly selected stat orders. RESULTS: Fifty-fi ve percent of all stat phlebotomy orders were requested for immediate decision-making with respect to urgently required diagnosis and patient care, defined by us as medical reasons. The other 45 % of the stat orders were made for logistical reasons relating to the patient care or the hospital organisation. In total, 19 phlebotomy requests(17 % ) were unnecessary and could have been avoided.For most of the stat phlebotomy orders alternatives were not possible, as only 2 % of the requests could have been replaced by analysis in material that had been withdrawn earlier. CONCLUSIONS: The majority of the stat orders for phlebotomy were requested for good reasons, about equally distributed among the medical and logistical needs. This sets limits to the measures being feasible to further improve stat phlebotomy ordering efficiency, taking into account the way of functioning of modern hospital care.

Androgen receptor coregulators: recruitment via the coactivator binding groove.

Androgens are key regulators of male sexual differentiation and essential for development and maintenance of male reproductive tissues. The androgens testosterone and dihydrotestosterone mediate their effect by binding to, and activation of the androgen receptor (AR). Upon activation, the AR is able to recognize specific DNA sequences in gene promoters and enhancers from where it recruits coregulators to orchestrate chromatin remodeling and transcription regulation. The number of proteins that bind to the AR has surpassed 200 and many of them enhance (coactivator) or repress (corepressor) its transactivating capacity. For most of these coregulators, their AR binding interface and their exact mode of action still needs to be elucidated, but for some of the more classical coactivators and corepressors, we gained insight in their working mechanisms. Of particular interest are specific sequences (LxxLL and FxxLF-like motifs) in a subset of coactivators that interact with the AR via a coactivator binding groove in the ligand-binding domain. As compared to other steroid receptors, the conformation of the AR coactivator binding pocket is unique and preferentially binds FxxLF-like motifs. This predisposition is expected to contribute to the regulation of specific sets of target genes via recruitment of selected coregulators. This review provides an overview of these (inter)actions with a focus on the unique characteristics of the AR coactivator binding groove.

Inhibition of androgen receptor functions by gelsolin FxxFF peptide delivered by transfection, cell-penetrating peptides, and lentiviral infection.

BACKGROUND: Prostate cancer (PC) growth is dependent on the androgen-androgen receptor (AR) axis. Because current androgen ablation therapies of PC lead to resistance, novel approaches to block AR activity are urgently needed. METHODS: We inhibited AR function beyond the level of hormone binding by blockade of the coactivator groove in the ligand-binding domain (LBD) using a high-affinity gelsolin FxxFF peptide. Following peptide selection, the effect of the gelsolin FxxFF peptide on AR functions was determined in Hep3B cells that were transiently transfected with pM-peptide expression vectors or were incubated with synthetic gelsolin FxxFF peptide coupled to the TAT cell-penetrating peptide. Lentiviruses expressing the gelsolin FxxFF peptide were used to study endogenous AR target gene expression in LNCaP cells. RESULTS: pM-Gelsolin FxxFF efficiently interfered with AR N/C interaction and specifically inhibited AR-regulated reporter gene activity. The peptide did not inhibit progesterone receptor (PR) and glucocorticoid receptor (GR) activity, nor constitutively active gene promoters. The peptide also specifically blocked in vitro interactions of AR LBD with peptides. Like the gelsolin FxxFF peptide expressed by an expression vector, synthetic TAT-gelsolin FxxFF peptide efficiently blocked AR N/C interaction and inhibited full-length AR-regulated reporter gene activity. It hardly affected PR and GR activity, but the effect on constitutively active promoters was variable. Lentiviral gelsolin FxxFF peptide inhibited expression of KLK2 and NDRG1, but hardly affected PSA and TMPRSS2. CONCLUSIONS: Our results show that the AR coactivator groove may function as a target to overcome therapeutic failure that arises during current androgen ablation therapies.

Systematic structure-function analysis of androgen receptor Leu701 mutants explains the properties of the prostate cancer mutant L701H.

One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5alpha-dihydrotestosterone. Further analysis of the AR Leu(701) variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel of structurally related steroids to study which of the OH groups at positions 11beta, 17alpha, and 21, which discriminate cortisol from progesterone, underlie the differential responses to both hormones. The results showed that the 17alpha-OH group was essential for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated a conserved H-bonding network involving the steroidal 17alpha-OH group, His(701) or Gln(701), and the backbone of Ser(778). This network is absent in Leu(701) and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17alpha-OH group. Our results indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser(778), and the steroidal 17alpha-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone and cortisol of AR mutants, including the prostate cancer mutant L701H.

Functional screening of FxxLF-like peptide motifs identifies SMARCD1/BAF60a as an androgen receptor cofactor that modulates TMPRSS2 expression.

Androgen receptor (AR) transcriptional activity is tightly regulated by interacting cofactors and cofactor complexes. The best described cofactor interaction site in the AR is the hormone-induced coactivator binding groove in the ligand-binding domain, which serves as a high-affinity docking site for FxxLF-like motifs. This study aimed at identifying novel AR cofactors by in silico selection and functional screening of FxxLF-like peptide motifs. Candidate interacting motifs were selected from a proteome-wide screening and from a supervised screening focusing on components of protein complexes involved in transcriptional regulation. Of the 104 peptides tested, 12 displayed moderate to strong in vivo hormone-dependent interactions with AR. For three of these, ZBTB16/PLZF, SMARCA4/BRG1, and SMARCD1/BAF60a, the full-length protein was tested for interaction with AR. Of these, BAF60a, a subunit of the SWI/SNF chromatin remodeling complex, displayed hormone-dependent interactions with AR through its FxxFF motif. Vice versa, recruitment of BAF60a by the AR required an intact coactivator groove. BAF60a depletion by small interfering RNA in LNCaP cells demonstrated differential effects on expression of endogenous AR target genes. AR-driven expression of TMPRSS2 was almost completely blocked by BAF60a small interfering RNA. In summary, our data demonstrate that BAF60a directly interacts with the coactivator groove in the AR ligand-binding domain via its FxxFF motif, thereby selectively activating specific AR-driven promoters.

Truncated ETV1, fused to novel tissue-specific genes, and full-length ETV1 in prostate cancer.

In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin beta3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation.

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

Novel FXXFF and FXXMF motifs in androgen receptor cofactors mediate high affinity and specific interactions with the ligand-binding domain.

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.