Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Stratum basale keratinocyte expression of the cell-surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration.

BACKGROUND: Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell-to-cell adhesion is crucial to the initiation and regulation of these processes. CUB-domain-containing protein (CDCP)1 is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling, and is expressed by keratinocytes in native human skin, as well as in primary cultures. OBJECTIVES: To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. METHODS: We examined both human skin tissue and an in vitro three-dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function-blocking anti-CDCP1 antibody and a real-time Transwell™ cell migration assay. RESULTS: Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a three-dimensional human skin equivalent model, CDCP1 was expressed only in the stratum basale, with localization restricted to the cell-cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Furthermore, an anti-CDCP1 function-blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. CONCLUSIONS: These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.

Experiences of technology integration in home care nursing.

The infusion of health care technologies into the home leads to substantial changes in the nature of work for home care nurses and their patients. Nurses and nursing practice must change to capitalize on these innovations. As part of a randomized field experiment evaluating web-based support for home care of patients with chronic heart disease, we engaged nine nurses in a dialogue about their experience integrating this modification of care delivery into their practice. They shared their perceptions of the work they needed to do and their perceptions and expectations for patients and themselves in using technologies to promote and manage self-care. We document three overarching themes that identify preexisting factors that influenced integration or represent the consequences of technology integration into home care: doing tasks differently, making accommodations in the home for devices and computers, and being mindful of existing expectations and skills of both nurses and patients.

The thyroid hormone receptor and the insulator protein CTCF: two different factors with overlapping functions.

Thyroid hormones and thyroid hormone receptors (TRs) confer a fundamental regulation of critical genes involved in metabolism, differentiation, and development. A similar role is attributed to the highly conserved zinc-finger factor CTCF. Furthermore, a potential role in tumour suppression has been attributed to CTCF. In addition to promoter regulation, CTCF has also been shown to be involved in chromatin insulation or enhancer blocking. In several cases, binding sites for TR and for CTCF have been found next to each other. Functionally, these sites mediate synergistic repression or induction dependent on the type of binding site and on the presence or absence of thyroid hormone. Here we discuss functional similarities between TR and CTCF and their roles within these composite elements.

Co-repressors 2000.

In the last 5 years, many co-repressors have been identified in eukaryotes that function in a wide range of species, from yeast to Drosophila and humans. Co-repressors are coregulators that are recruited by DNA-bound transcriptional silencers and play essential roles in many pathways including differentiation, proliferation, programmed cell death, and cell cycle. Accordingly, it has been shown that aberrant interactions of co-repressors with transcriptional silencers provide the molecular basis of a variety of human diseases. Co-repressors mediate transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin-modifying enzymes. Chromatin modification includes histone deacetylation, which is thought to lead to a compact chromatin structure to which the accessibility of transcriptional activators is impaired. In a general mechanistic view, the overall picture suggests that transcriptional silencers and co-repressors act in analogy to transcriptional activators and coactivators, but with the opposite effect leading to gene silencing. We provide a comprehensive overview of the currently known higher eukaryotic co-repressors, their mechanism of action, and their involvement in biological and pathophysiological pathways. We also show the different pathways that lead to the regulation of co-repressor-silencer complex formation.

Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains.

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13delta1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.

Transcriptional repression by the insulator protein CTCF involves histone deacetylases.

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.

Strategic planning for research use in nursing practice.

BACKGROUND/OBJECTIVE: To prepare for a culture change to integrate research utilization into daily nursing practice, the authors conducted a descriptive survey of all registered nurses (RNs) in an integrated healthcare delivery system. The purposes of this study were to assess RNs' knowledge, attitudes, and practices (KAP) of nursing research activities, assess factors that support a research environment, and determine facilitating and challenging factors related to conducting regional nursing research. METHODS: A 33-item survey based on the Iowa Model for Evidence-Based Practice was developed, validated, and determined to be reliable by the authors. Site coordinators organized and managed the orientation, administration, and collection of data from the 2,736 registered nurses who worked in 6 hospitals, 65 affiliated clinics, and 3 business units. Narrative notes taken by study investigators were analyzed for themes to determine challenging and facilitating factors for conducting regional research. RESULTS: Education and job title significantly predicted knowledge and ability to perform research activities but was not related to willingness to engage in research activities. Several environmental factors were associated with knowledge of, willingness to engage in, and ability to perform research utilization activities. Challenging and facilitating factors to conducting regional research were identified. CONCLUSIONS/IMPLICATIONS: Our research environment is changing to value research as shown in the philosophy, conceptual framework, and bylaws for the professional nursing staff. Novice-to-expert research utilization expectations are included in the promotional model for nursing. All RN job descriptions and the annual performance tool were revised to include responsibilities related to research activities. The Iowa Model for Evidence-Based Practice was adopted as the method for creating practice validation and change. Train-the-trainer educational and experiential sessions are being designed for nurse leaders; all new RN employees complete a self-assessment tool of research utilization knowledge and the nursing division strategic goals incorporate research utilization expectations. The elements of this plan may be useful for nurse executives. Healthcare systems are restructuring throughout the world and within the United States. These changes are occurring to better meet the evolving healthcare needs of the population through cost-effective approaches. Within the United States, emerging organized healthcare systems require research related to patient care outcomes and the health systems that can best address them.

The corepressor N-CoR and its variants RIP13a and RIP13Delta1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70.

Repression of transcription by the classical nuclear receptors (e.g. TR, RAR), the orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mxi-1 and Mad bHLH-zip proteins and the oncoproteins PLZF and LAZ3/BCL6 is mediated by the corepressors N-CoR and SMRT. The interaction of the corepressors with the components involved in chromatin remodelling, such as the recruiting proteins Sin3A/B and the histone deacteylases HDAc-1 and RPD3, has been analysed in detail. The N-CoR/Sin3/HDAc complexes have a key role in the regulation of cellular proliferation and differentiation. However, the interaction of these corepressors with the basal transcriptional machinery has remained obscure. In this study we demonstrated that the N-terminalrepression domains and the receptor interactiondomains (RID) of N-CoR and its splice variants, RIP13a and RIP13Delta1, directly interact with TAFII32 in vivo and in vitro . We show that interaction domain II within the N-CoR and RIP13a RID is required for the interaction with TAFII32. We also observed that N-CoR directly interacts with each of the basal factors, TFIIB and TAFII70, and can simultaneously interact with all three basal factors in a non-competitive manner. Furthermore, we provide evidence that suggests the RVR/Rev-erbbeta-corepressor complex also interacts with the general transcriptional machinery, and that the physicalassociation of TFIIB with N-CoR also occurs in the presence of Sin3B and HDAc-1. Interestingly, we observed that N-CoR expression ablated the functional interaction between TFIIB and TAFII32 that is critical to the initiation of transcription. In conclusion, this study demonstrates that the N-terminal repressor region and the C-terminal RIDs are part of the corepressor contact interface that mediates the interaction with the general transcription factors, and demonstrates that TAFs can also directly interact with corepressors to mediate signals from repressors to the basal machinery. We also suggest that N-CoR interacts with the central components of the transcriptional initiation process (TFIIB, TAFs) and locks them into a non-functional complex or conformation that is not conducive to transcription.

Implementing the differentiated pay structure model. Process and outcomes.

OBJECTIVE: A salaried Differentiated Pay Structure (DPS) model based on the work of Dr. Virginia Cleland was tested on two units. The project objectives were to: 1) create a budget-neutral compensation distinction for different competencies and educational levels; 2) evaluate the effect of the new salaried model on unit costs and pay; 3) determine the effect of the DPS model on job satisfaction, organizational commitment, and anticipated turnover; and 4) assess the impact of professional commitment, professional practice climate, perception of staffing adequacy, and dispositional optimism on job satisfaction, organizational commitment, and anticipated turnover. BACKGROUND: Although there has been long-standing interest in salaried models and reward methodologies, there is a dearth of systematic research associated with specific compensation models. METHODS: A quasi-experimental, non-equivalent control group design was used to examine the effects of the DPS model. RESULTS: Findings demonstrated that nurses were paid more under the DPS model, and that they were paid for more hours than they actually worked (N = 68). No difference in job satisfaction was found between experimental and control groups. For all nurses (N = 232) dispositional optimism was associated with all job satisfaction subscales except pay. Organizational commitment, professional commitment, professional practice climate, and staffing adequacy were also correlated with job satisfaction, perceptions of care quality and anticipated turnover. Older nurses who had worked longer in nursing, and who had more tenure were less satisfied with their coworkers and care quality. CONCLUSION: Further longitudinal research with larger experimental samples is required in order to fully understand the effects of the DPS model in nursing.

Identification and characterization of a novel corepressor interaction region in RVR and Rev-erbA alpha.

Rev-erbA alpha and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA alpha and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13 delta 1. The physical association between the corepressors and Rev-erbA alpha and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. Our previous study demonstrated that the E region of RVR and Rev-erbA alpha is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13 delta 1. The present investigation demonstrates that two corepressor interaction regions, CIR-1 and CIR-2, separated by approximately 150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13 delta A. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA alpha were necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13 delta 1 and N-CoR/RIP13a differentially interact with Rev-erbA alpha. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA alpha, demonstrated that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13 delta 1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA alpha, respectively, were critical residues in supporting corepressor interaction. Cotransfection studies demonstrated that repression of a physiological target, the human Rev-erbA alpha promoter, by RVR was significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13 delta 1-RIDs alleviated RVR-mediated repression of the Rev-erbA alpha promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapositioned CIR-1 and CIR-2 was sufficient for corepressor binding and transcriptional repression. In conclusion, our study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA alpha, that is required for transcriptional silencing. Furthermore, we provide evidence that CIR-1 and CIR-2 may form a single corepressor interaction interface.

Research-based planning for change: assessing nurses' attitudes toward governance and professional practice autonomy after hospital acquisition.

OBJECTIVE: This article describes one medical center's experience in using research to plan for nursing staff integration after hospital acquisition. BACKGROUND: Resistance to new policies, procedures, and standards; passive acceptance of new leadership; limited support for management plans; and failure to integrate with new nursing units are common staff reactions after acquisitions. Little has been written regarding which key staff variables to assess after acquisitions and how to use this data to plan for change. Structural contingency and attribution theory were used to guide leadership staff's assessment of acquired staff attributes to determine their congruence with concepts valued by the acquiring organization. METHODS: Qualitative and quantitative data were collected using a survey method. All 141 registered nurses and licensed practical nurses of the acquired medical center received a mailed survey. Sixty-six completed surveys were returned through the U.S. mail. No identifying information was placed on the survey to assure anonymity. RESULTS: The survey results described nurses perceptions of the advantages, concerns, and suggestions for a smooth transition after acquisition. In addition, the results clarified that nurses in the newly acquired hospital preferred a shared governance structure (congruent with the acquiring medical center's values) and the nurses perceived professional nursing autonomy was similar to that of nurses who worked at the acquiring medical center. CONCLUSIONS: By sharing the findings, both staffs were sensitized to the similarities among the staff as well as to their differences. Transition strategies were planned to capitalize on this knowledge. This process may be useful for other nurse executives to replicate as they guide their organizations through similar transitions.

Transcriptional repression by COUP-TF II is dependent on the C-terminal domain and involves the N-CoR variant, RIP13delta1.

COUP-TF II/ARP-1 is an 'orphan' steroid receptor that inhibits basal transcription, and represses trans-activation by the vitamin D, thyroid hormone and retinoid receptors. The molecular basis of repression by COUP-TF II remains obscure. In this study we utilized the GAL4 hybrid system to demonstrate that COUP-TF II contains sequences within the C-terminal region that encode a dominant transcriptional repressor that inhibits the ability of the potent chimeric transactivator GAL4VP16 to induce transcription. Mammalian two hybrid analysis demonstrated that COUP-TF II did not efficiently interact with either interaction domains I or II from N-CoR and RIP13. However, COUP-TF II efficiently interacts with a region comprised of interaction domains I + II from the corepressor, RIP13delta1. Efficient interaction of the orphan receptor with the corepressor was critically dependent on a large region comprised of the C, D and E domains of COUP-TF II, which correlated with the domain that maximally represses transcription. This investigation suggested that the N-CoR variant, RIP13delta1 interacts with a region of COUP-TF II that functions as a dominant transcriptional repressor.

The clinical practice developmental model: the transition process.

The authors report their hospital's experience in replicating Benner's novice-to-expert clinical nursing practice model, called the Clinical Practice Developmental Model. The authors describe the outcomes of an exploratory, qualitative study conducted to understand staff nurses' perceptions of their transition experience from a traditional clinical ladder for advancement and recognition to the theoretically based clinical practice developmental model. The findings of this study identify critical factors that influenced nurses' perceptions and describe positive and negative outcomes of transition. Specific recommendations to facilitate organizational changes for the nurse executive and the individual nurse are discussed.

Two receptor interaction domains in the corepressor, N-CoR/RIP13, are required for an efficient interaction with Rev-erbA alpha and RVR: physical association is dependent on the E region of the orphan receptors.

Rev-erbA alpha and RVR/Rev-erb beta/BD73 are orphan steroid receptors that have no known ligands in the 'classical sense'. These 'orphans' do not activate transcription, but function as dominant transcriptional silencers. The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional silencers by binding corepressors (e.g. N-CoR/RIP13 and SMRT/TRAC-2) in the absence of ligands. The molecular basis of repression by orphan receptors, however, remains obscure, and it is unclear whether these corepressors mediate transcriptional silencing by Rev-erbA alpha and RVR. Recently, two new variants of N-CoR have been described, RIP13a and RIP13delta1. The characterisation of these splice variants has identified a second receptor interaction domain (ID-II), in addition to the previously characterised interaction domain (ID-I). This investigation utilised the mammalian two hybrid system and transfection analysis to demonstrate that Rev-erbA alpha and RVR will not efficiently interact with either ID-I or ID-II separately from RIP13a or RIP13delta1. However, they interact efficiently with a domain composed of ID-I and ID-II from RIP13a. Interestingly, the interaction of Rev-erbA alpha and RVR is strongest with ID-I and ID-II from RIP13delta1. Detailed deletion analysis of the orphan receptor interaction with RIP13/N-CoR rigorously demonstrated that the physical association was critically dependent on an intact E region of Rev-erbA alpha and RVR. Over-expression of the corepressor interaction domains (i.e. dominant negative forms of N-CoR/RIP13) could alleviate orphan receptor-mediated repression of transactivation by GALVP16. This demonstrated that these regions could function as anti-repressors. In conclusion, these data from two independent approaches demonstrate that repression by Rev-erbA alpha and RVR is mediated by an interaction of ID-I and ID-II of N-CoR, RIP13a and delta1 with the putative ligand binding domain of the orphan receptors.

Transcriptional repression by Rev-erbA alpha is dependent on the signature motif and helix 5 in the ligand binding domain: silencing does not involve an interaction with N-CoR.

Rev-erbA alpha is an orphan nuclear receptor that functions as a dominant transcriptional repressor. Tissue culture and in situ hybridisation studies indicated that Rev-erbA alpha plays an important role in mammalian differentiation and development. Previous studies have localised the silencing domain of Rev-erbA alpha to the D/E region of the orphan receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains the ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxxLL] and a region that, according to the recently published crystal structures of steroid receptors, would be predicted to form helix 5 of the canonical LBD structure. Fine deletions and site-specific mutagenesis indicated that both the LBD signature motif and helix 5 were necessary for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepressor, N-CoR, that is known to interact with the thyroid hormone and retinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. In conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this orphan receptor.

Securing life through technology acceptance: the first six months after transvenous internal cardioverter defibrillator implantation.

PURPOSE: To understand the experience of living with a transvenous internal cardioverter defibrillator (ICD) during the first 6 months after implantation. DESIGN: A grounded theory approach was used to gain an understanding of the antecedents, conditions, and consequences of the core process. SETTING: Two midwestern community medical centers and patients' homes within a 250-mile radius from the medical centers. SAMPLE: Ten women and 14 men between 22 and 78 years of age, who had undergone a new transvenous ICD implantation within the last week. RESULTS: The core process that described the experience of living with a transvenous ICD during the first 6 months after implantation was the process of "securing life through technology acceptance." This core process was characterized by three major categories: choosing life with technology, integrating technology into life, and living life through technology. IMPLICATIONS: Nursing interventions related to individualized assessment, education, and counseling are warranted to facilitate patient and family understanding and to plan for the cyclic process involved in integrating a transvenous ICD into their lives. Directions for further research are provided.

Living with recurrent ventricular dysrhythmias.

The number of patients who live with recurrent ventricular dysrhythmias is increasing as medical advances such as pharmacologic and electrical therapies decrease mortality rates in this population. As a result, nurses frequently encounter patients who are trying to learn to live with chronic aspects of recurrent life-threatening dysrhythmias. The findings of this study provide an important beginning description of strategies used by patients to address their concerns. The RVD patients in this study reported using a variety of strategies to manage their physiologic and psychosocial concerns. Their responses described a continuum of strategies to handle concerns that ranged from "compensate for the concern" to "unable to handle the concern." These findings suggest several implications for nursing. Careful assessment of all patients with RVD is appropriate to detect areas where patients perceive a need for additional strategies to manage their concerns, require support for existing self-reliance strategies, and need identification of additional nursing interventions. Individualized or group programs may be appropriate interventions to assist some patients. Strategies to support patients who report an inability to manage specific concerns need clarification. Ideally, as further research defines the processes patients with RVD use to manage their concerns, high-risk patients can be discovered and provided with interventions to promote their adjustment to living with life-threatening dysrhythmias.