Target-Based Discovery of an Inhibitor of the Regulatory Phosphatase PPP1R15B.

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

An inducible chaperone adapts proteasome assembly to stress.

The proteasome is essential for the selective degradation of most cellular proteins. To survive overwhelming demands on the proteasome arising during environmental stresses, cells increase proteasome abundance. Proteasome assembly is known to be complex. How stressed cells overcome this vital challenge is unknown. In an unbiased suppressor screen aimed at rescuing the defects of a yeast Rpt6 thermosensitive proteasome mutant, we identified a protein, hereafter named Adc17, as it functions as an ATPase dedicated chaperone. Adc17 interacts with the amino terminus of Rpt6 to assist formation of the Rpt6-Rpt3 ATPase pair, an early step in proteasome assembly. Adc17 is important for cell fitness, and its absence aggravates proteasome defects. The abundance of Adc17 increases upon proteasome stresses, and its function is crucial to maintain homeostatic proteasome levels. Thus, cells have mechanisms to adjust proteasome assembly when demands increase, and Adc17 is a critical effector of this process.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492.

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

The better sleep better well-being programme: Educating and training community healthcare nurses in developing interventions for families of infants with moderate sleep problems: a pilot study.

The main aim of this pilot study was to evaluate the benefit of the Better Sleep Better Well-being (BSBW) educational and training intervention programme regarding infants sleep problems for Community Health Care (CHC) nurses, on their perceptions on their family nursing practice skills and on their job demand, control and support. There were 6 CHC nurses who participated in the BSBW programme, and 26 nurses in the comparison group. The programme consisted of 4 sessions (8 hours per session) of lectures on the aetiology of infants sleep problems as well as on evidence-based and family relational practices and on 20 sessions of clinical cases, scenarios, discussions and reflections. The main finding indicated that the nurses in the intervention group reported significantly higher family nursing practices skills compared to the nurses in the comparison group. The findings are promising, since they offered additional resources to the CHC nurses, in their clinical practices.

ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion.

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

The Impact of Family Strengths Oriented Therapeutic Conversations on Parents of Children with a New Chronic Illness Diagnosis.

A growing number of families with children are dealing with a new diagnosis of chronic illnesses or health problems that are demanding. Nurses are in a prime position to provide support and empowerment to these families. The aim of the study was to evaluate the benefits of two sessions of a Family Strengths Oriented Therapeutic Conversation (FAM-SOTC) intervention, offered by advanced practice nurses (APNs) to mothers (N = 31) of children and adolescents in Iceland with newly diagnosed chronic illnesses/disorders. Families of children with Juvenile Idiopathic Arthritis (JIA), epilepsy, Type 1 diabetes (T1DM), or with sleep disturbance with attention-deficit/hyperactivity disorder (ADHD), reported significantly higher family support, greater conviction about their illness beliefs, increased quality of life, and greater satisfaction with health care services after receiving two sessions of the FAM-SOTC intervention (Time 2) compared to before the intervention (Time 1). The findings emphasize the importance of the APN's role and family nursing expertise in supporting families of children with a new diagnosis of chronic illnesses or disorders who are in active treatment.

The impact of nursing education and job characteristics on nurse's perceptions of their family nursing practice skills.

BACKGROUND: Implementing family system nursing in clinical settings is on the rise. However, little is known about the impact of graduate school education as well as continuing education in family systems nursing (FSN) on nurses' perceptions of their family nursing practice. AIMS: To evaluate the level of nursing education, having taken a continuing hospital educational course in family system nursing (FN-ETI programme), and the impact of job characteristics on nurses' perceptions of their family nursing practice skills. DESIGN AND METHODS: Participants were 436 nurses with either a BSc degree or graduate degree in nursing. The Job Demand, Control and Support model guided the study (R. Karasek and T. Theorell, 1992, Healthy Work: Stress, Productivity, and the Reconstruction of Working Life, Basic Books, New York, NY). Scores for the characteristics of job demands and job control were created to categorise participants into four job types: high strain (high demand, low control), passive (low demand, low control), low strain (low demand, high control) and active (high demand, high control). RESULTS: Nurses with a graduate education who had taken the FN-ETI programme scored significantly higher on the Family Nursing Practice Scale than nurses with an undergraduate education. Nurses who were characterised as low strain or active scored significantly higher on the Family Nursing Practice Scale than the nurses who were characterised as high strain. Further, the interaction of education by job type was significant regarding family nursing practice skills. Hierarchical regression revealed 25% of the variance in family nursing practice skills was explained by job control, family policy on the unit, graduate education and employment on the following divisions: Maternal-Child, Emergency, Mental Health or Internal Medicine. CONCLUSION: Graduate education plus continuing education in FSN can offer nurses increased job opportunities more control over one's work as well as increased skills working with families in clinical settings.

The importance of family support in pediatrics and its impact on healthcare satisfaction.

AIMS: To evaluate predictors of healthcare satisfaction for parents whose children received hospital-based healthcare services at the Children's hospital at Landspitali University Hospital. METHODS: In this cross-sectional study, data on perceived family support, family quality of life, expressive family functioning, coping strategies and healthcare satisfaction were collected from 159 mothers and 60 fathers (N = 177 families) of children and adolescents from 2011 to 2012. RESULTS: Logistic regression analysis revealed that, for mothers, 38.8% of the variance in satisfaction with healthcare services was predicted by perceived family support and their coping strategies, while for fathers, 59.9% of the variance of their satisfaction with healthcare service was predicted by perceived family support, family quality of life and whether the child had been hospitalised before. DISCUSSION: Perceived family support was the one factor that was found to predict both the mothers' and the fathers' satisfaction with healthcare services. Knowing which factors predict satisfaction with health care among parents of hospitalised children with different chronic illnesses and health issues can inform the delivery of effective family-focused interventions and evidence-based practice to families.

The process of translating family nursing knowledge into clinical practice.

PURPOSE: To report on approaches that were used to assist with implementation of family systems nursing (FSN) at a university hospital level in Northern Europe. DESIGN AND METHODS: A quasi-experimental research design was used for the first phase of the study. For the second phase, a cross-sectional research design was used. Data were collected in the first phase of the study from 457 nurses in all except one of the divisions of the hospital regarding their attitudes towards involving families into their care before and after having participated in the education and training intervention (ETI) program in FSN. Furthermore, in the second phase, data were collected from 812 nurses, after FSN had been implemented in all divisions at Landspitali University Hospital, regarding the nurses' knowledge of FSN and their evaluation of the quality of the ETI program (i.e., theoretical lectures on FSN as well as the benefit of the skill lab training regarding applying FSN into their clinical practices). Graham and colleagues' Knowledge to Action framework was used as the conceptual framework for the research. RESULTS: Nurses who had taken a course in FSN reported a significantly more positive attitude towards involving families in their care after the ETI program compared to those who had not taken such a course. Furthermore, a majority of the nurses who participated in the ETI program reported that the program was a favorable experience and indicated readiness for applying FSN in clinical practice. CONCLUSIONS: Further research is needed regarding the benefits of offering FSN at an institutional level, but focusing international attention on effective strategies to implement FSN into nursing practice may result in better health care for individuals and families around the globe. CLINICAL RELEVANCE: Providing clinically meaningful education and training in family nursing through programs such as the ETI program for practicing nurses at a university hospital is essential in supporting nurses applying new knowledge, when providing evidence-based health care services, to individuals and their family members. Such training can facilitate integration of new and needed information in clinical practice.

Biophysical and computational fragment-based approaches to targeting protein-protein interactions: applications in structure-guided drug discovery.

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein-protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids - so-called hotspots - may contribute the majority of interaction-free energy. The challenges posed by protein-protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein-protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods - nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry - to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein-protein interactions and have therapeutic value.

Strengths-oriented therapeutic conversations for families of children with chronic illnesses: findings from the Landspitali university hospital family nursing implementation project.

Psychosocial services have been recommended for families of children with chronic illnesses to assist them in adjusting to the illness experience. However, little is known about the benefit of psychosocial interventions in clinical practice. This study aimed at evaluating the benefits of a two-session family therapeutic conversation intervention (FAM-TCIs) for families of children diagnosed with asthma, cancer, or diabetes. A secondary data analysis was conducted on intervention data from 37 families of children with chronic illnesses. Mothers of the children/teenagers perceived significantly higher family support after the FAM-TCI compared with before; mothers reported significantly higher collaboration and problem-solving abilities on the expressive family functioning scale after the FAM-TCI. However, no significant differences were found on the fathers' perceived family support nor on their expressive family functioning after the FAM-TCI compared with that before the intervention. It is promising that mothers of children with chronic illness perceived the two-session FAM-TCI to be beneficial to them. More attention should be paid to psychosocial interventions for families of children with chronic illnesses.

Nurses' attitudes to family importance in nursing care: A two-sited cross-sectional study.

AIM: This study aimed to describe nurses' attitudes and beliefs towards the importance of family in nursing care and explore differences in nurses' attitudes and beliefs towards family-centered care between different healthcare institutions, such as community healthcare centers and hospitals. BACKGROUND: Family significantly affects the well-being and health of individuals. Therefore, nurses should support family engagement in nursing care. In recent years, family nursing research has emphasized the importance of teaching family nursing skills in continued education in healthcare institutions. Research has indicated that nurses who believe that illness concerns the family as a whole are more likely to involve the family in patient care. DESIGN: A cross-sectional research design was used. METHOD: Data were collected at one timepoint between March and September 2019 from 425 nurses working at the Primary Health Care Centers of the Capital Area (n=112) and in clinical settings at the University Hospital in Iceland (n=313). RESULTS: The main findings indicated that nurses working in the women-and-child division at the University Hospital reported significantly more positive attitudes towards family evolvement in patient care than nurses working in the intensive care or surgical units. For nurses working at healthcare centers, a significant difference was also found in the nurses' attitudes towards involving families in patient care. The nurses who were working in home care had significantly more positive attitudes when compared to those working in the infant and young children health promotion units. CONCLUSIONS: Greater collaboration is required between healthcare providers and families to improve the quality of care and health-related outcomes. Therefore, it is crucial to enhance nurses' knowledge about the importance of families during patient care. TWEETABLE ABSTRACT: This study aimed to describe nurses' attitudes and beliefs towards family care. Differences were found between nurse's attitudes by units but not by institutions.

Proximity-driven site-specific cyclization of phage-displayed peptides.

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.

Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

Robust Prediction of Relative Binding Energies for Protein-Protein Complex Mutations Using Free Energy Perturbation Calculations.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how Protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

A simeprevir-inducible molecular switch for the control of cell and gene therapies.

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.

Knowledge translation in family nursing: does a short-term therapeutic conversation intervention benefit families of children and adolescents in a hospital setting? Findings from the Landspitali University Hospital Family Nursing Implementation Project.

In an effort to examine translation of family nursing knowledge to practice, the Landspitali University Hospital Family Nursing Implementation Project (2007-2011), was thoughtfully initiated in Reykjavik, Iceland and systematically evaluated. The mission was to implement family nursing in every department of the hospital. This publication is the first formal research report from this landmark project. The purpose of this research was to evaluate the effectiveness of a short-term therapeutic conversation intervention with families who were receiving health care services at the Children's Hospital at Landspitali University Hospital in Iceland related to childhood and adolescent acute and chronic illnesses. The therapeutic conversation was guided by Family Systems Nursing and used the Calgary Family Assessment and Intervention Models (Wright & Leahey, 2005, 2009). Families (N = 76) were randomly assigned to either an experimental group (short-term therapeutic conversation: n = 41) or to a control group (traditional care: n = 35). Parents in the experimental group reported significantly higher family support after the intervention, compared to the parents in the control group. Differences were noted between families experiencing acute versus chronic illnesses. Recommendations are made for conducting and designing intervention research with families experiencing the hospitalization of a child or adolescent.

Nursing students' perception of family importance in nursing care during the COVID-19 pandemic: A cross-sectional study.

BACKGROUND: Little is known about nursing students' illness beliefs and attitudes towards the involvement of families in nursing care during the COVID-19 epidemic. Focusing on family nursing throughout an undergraduate nursing education is not only appropriate or critical but also essential for advancing family nursing practice. OBJECTIVES: To evaluate the differences in undergraduate and graduate nursing students' perceptions of illness beliefs and their family nursing practice skills at the time of the COVID-19 pandemic. DESIGN: A cross-sectional study. SETTINGS: The Faculty of Nursing at the University of Iceland. PARTICIPANTS: Of the nursing and midwifery students, 109 participated in 2020 from one university. METHODS: Data was collected regarding illness beliefs and attitudes towards family involvement in nursing care, through questionnaires via the Red Cap software. RESULTS: The main finding indicated that the graduate students reported more confidence or reassurance regarding their knowledge of the cause of an illness, control, effect, suffering and what is the most and the least helpful in coping with an illness/health disorder when compared to the undergraduate students (t-value = -2.50, p-value = 0.014). Additionally, graduate nursing students also reported higher positive attitudes towards family importance in nursing care than undergraduate students (t-value = -2.16, p-value = 0.033). CONCLUSION: Even though the graduate students reported higher illness beliefs than undergraduate students, the undergraduate students reported a reasonably high or over medium high score, on the illness beliefs scale. University nursing educators need to be aware that nursing students' knowledge, skills and attitudes towards family nursing practice at the time of the COVID-19 pandemic shape clinical competence in family nursing within health care settings.

Application of 3D Culture Assays to Study Breast Morphogenesis, Epithelial Plasticity, and Cellular Interactions in an Epithelial Progenitor Cell Line.

Capturing breast morphogenesis and cancer progression in 3D culture using cell lines with stem cell properties can greatly increase understanding of the underlying mechanisms involved in these processes, highlighting the importance of the culture method. D492 is a breast epithelial progenitor cell line that provides a model for branching morphogenesis when cultured in 3D reconstituted basement membrane matrix (rBM). Along with its derivate cell lines D492M and D492HER2, D492 also serves as a robust model for epithelial to mesenchymal transition (EMT) and tumorigenicity, respectively. Here, we describe the routine maintenance and application of the D492 cell lines in 3D culture for the study of branching morphogenesis, EMT and epithelial-endothelial interaction.