Single cell RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes.

The establishment of left-right patterning in mice occurs at a transient structure called the embryonic node or left-right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0-1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.

Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.

The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.

Loss of Zic3 impairs planar cell polarity leading to abnormal left-right signaling, heart defects and neural tube defects.

Loss of function of ZIC3 causes heterotaxy (OMIM #306955), a disorder characterized by organ laterality defects including complex heart defects. Studies using Zic3 mutant mice have demonstrated that loss of Zic3 causes heterotaxy due to defects in establishment of left-right (LR) signaling, but the mechanistic basis for these defects remains unknown. Here, we demonstrate Zic3 null mice undergo cilia positioning defects at the embryonic node consistent with impaired planar cell polarity (PCP). Cell-based assays demonstrate that ZIC3 must enter the nucleus to regulate PCP and identify multiple critical ZIC3 domains required for regulation of PCP signaling. Furthermore, we show that Zic3 displays a genetic interaction with the PCP membrane protein Vangl2 and the PCP effector genes Rac1 and Daam1 resulting in increased frequency and severity of neural tube and heart defects. Gene and protein expression analyses indicate that Zic3 null embryos display disrupted expression of PCP components and reduced phosphorylation of the core PCP protein DVL2 at the time of LR axis determination. These results demonstrate that ZIC3 interacts with PCP signaling during early development, identifying a novel role for this transcription factor, and adding additional evidence about the importance of PCP function for normal LR patterning and subsequent heart development.

WNT-responsive SUMOylation of ZIC5 promotes murine neural crest cell development, having multiple effects on transcription.

Zinc finger of the cerebellum (Zic) proteins act as classic transcription factors to promote transcription of the Foxd3 gene during neural crest cell specification. Additionally, they can act as co-factors that bind proteins from the T-cell factor/lymphoid enhancing factor (TCF/LEF) family (TCFs) to repress WNT-ß-catenin-dependent transcription without contacting DNA. Here, we show that ZIC activity at the neural plate border is influenced by WNT-dependent SUMOylation. In the presence of high canonical WNT activity, a lysine residue within the highly conserved zinc finger N-terminally conserved (ZF-NC) domain of ZIC5 is SUMOylated, which reduces formation of the ZIC-TCF co-repressor complex and shifts the balance towards transcription factor function. The modification is crucial in vivo, as a ZIC5 SUMO-incompetent mouse strain exhibits neural crest specification defects. This work reveals the function of the ZF-NC domain within ZIC, provides in vivo validation of target protein SUMOylation and demonstrates that WNT-ß-catenin signalling directs transcription at non-TCF DNA-binding sites. Furthermore, it can explain how WNT signals convert a broad region of Zic ectodermal expression into a restricted region of neural crest cell specification.

Systematized reporter assays reveal ZIC protein regulatory abilities are Subclass-specific and dependent upon transcription factor binding site context.

The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding.

Experiences of older people following the introduction of consumer-directed care to home care packages: A qualitative descriptive study.

OBJECTIVE: To explore the experiences of older people receiving home care package (HCP) support following the introduction of consumer-directed care (CDC) by the Australian government on 1 July 2015. METHODS: Thirty-one older people with existing HCP support from two service providers in regional New South Wales, Australia, participated in a face-to-face interview and/or a qualitative survey. RESULTS: Analysis revealed the theme of Choices: Preferences, constraints, balancing and choosing. Participants described choosing to live at home with HCP support; however, they were constrained by poor communication and information about service changes and options, personal budgets and access to future care. HCP services remained largely unchanged during transition to CDC. CONCLUSION: Many aspects of the initial implementation of CDC were challenging for older people. Clear, relevant and timely communication and information about CDC and its consequences for consumers appear to be needed to enhance CDC.

ZIC3 in Heterotaxy.

Mutation of ZIC3 causes X-linked heterotaxy, a syndrome in which the laterality of internal organs is disrupted. Analysis of model organisms and gene expression during early development suggests ZIC3-related heterotaxy occurs due to defects at the earliest stage of left-right axis formation. Although there are data to support abnormalities of the node and cilia as underlying causes, it is unclear at the molecular level why loss of ZIC3 function causes such these defects. ZIC3 has putative roles in a number of developmental signalling pathways that have distinct roles in establishing the left-right axis. This complicates the understanding of the mechanistic basis of Zic3 in early development and left-right patterning. Here we summarise our current understanding of ZIC3 function and describe the potential role ZIC3 plays in important signalling pathways and their links to heterotaxy.

Home care packages: insights into the experiences of older people leading up to the introduction of consumer directed care in Australia.

This paper reports phase one, conducted from March to June 2015, of a two-phase, qualitative descriptive study designed to explore the perceptions and experiences of older people before and after the introduction of consumer directed care (CDC) to home care packages (HCP) in Australia. Eligible consumers with a local HCP provider were mailed information about the study. Data collection occurred before the introduction of CDC and included face-to-face, in-depth interviews, summaries of interviews, field notes and reflective journaling. Semi-structured questions and 'emotional touchpoints' relating to home care were used to guide the interview conversation. Line-by-line data analysis, where significant statements were highlighted and clustered to reveal emergent themes, was used. Five older people, aged 81 to 91 years, participated in the study. The four emergent themes were: seeking quality and reciprocity in carer relationships; patchworking services; the waiting game; and technology with utility. Continuity of carers was central to the development of a trusting relationship and perceptions of care quality among older consumers. Care coordinators and workers should play a key role in ensuring older people receive timely information about CDC and their rights and responsibilities. Participants' use of contemporary technologies suggests opportunities to improve engagement of HCP clients in CDC.

Socially disadvantaged women's views of barriers to feeling safe to engage in decision-making in maternity care.

BACKGROUND: Although midwifery literature suggests that woman-centred care can improve the birthing experiences of women and birth outcomes for women and babies, recent research has identified challenges in supporting socially disadvantaged women to engage in decision-making regarding care options in order to attain a sense of control within their maternity care encounters. OBJECTIVE: The objective of this paper is to provide an understanding of the issues that affect the socially disadvantaged woman's ability to actively engage in decision-making processes relevant to her care. RESEARCH DESIGN: The qualitative approach known as Interpretative Phenomenological Analysis was used to gain an understanding of maternity care encounters as experienced by each of the following cohorts: socially disadvantaged women, registered midwives and student midwives. This paper focuses specifically on data from participating socially disadvantaged women that relate to the elements of woman-centred care-choice and control and their understandings of capacity to engage in their maternity care encounters. FINDINGS: Socially disadvantaged women participants did not feel safe to engage in discussions regarding choice or to seek control within their maternity care encounters. Situations such as inadequate contextualised information, perceived risks in not conforming to routine procedures, and the actions and reactions of midwives when these women did seek choice or control resulted in a silent compliance. This response was interpreted as a consequence of women's decisions to accept responsibility for their baby's wellbeing by delegating health care decision-making to the health care professional. CONCLUSION: This research found that socially disadvantaged women want to engage in their care. However without adequate information and facilitation of choice by midwives, they believe they are outsiders to the maternity care culture and decision-making processes. Consequently, they delegate responsibility for maternity care choices to those who do belong; midwives. These findings suggest that midwives need to better communicate a valuing of the woman's participation in decision-making processes and to work with women so they do have a sense of belonging within the maternity care environment. Midwives need to ensure that socially disadvantaged women do feel safe about having a voice regarding their choices and find ways to give them a sense of control within their maternity care encounters.

A murine Zic3 transcript with a premature termination codon evades nonsense-mediated decay during axis formation.

The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.

Zinc fingers of the cerebellum (Zic): transcription factors and co-factors.

The Zic genes encode zinc finger containing proteins that can bind proteins and DNA. The understanding of Zic molecular networks has been hampered by functional redundancy amongst family members, and because their loss-of-function phenotypes are indicative of a role in many signalling pathways. Recently molecular evidence has emerged confirming the pleiotropic nature of these proteins: they act both as classical transcription factors and as co-factors to directly and indirectly influence gene expression. It has long been known that germ-line mutation of the Zic genes in human and mouse causes a range of congenital disorders. Recently connections between Zic proteins and stem cell function have also emerged suggesting a role in adult onset diseases. The immediate challenge is to determine when and where these proteins act as transcription factors/co-factors during development and disease and how the switch between these roles is controlled.

Efficacy of the use of evidence-based algorithmic guidelines in the acute care setting for pain assessment and management in older people: a critical review of the literature.

AIM: The aim of the literature review was to identify all examples of primary research using an algorithmic approach for the implementation of a clinical practice guideline relating to pain assessment and/or management within acute care, with a specific focus on older people. DESIGN: Critical literature review. DATA SOURCES: Inclusion criteria were; English language publications within the past 13 years; peer reviewed; research conducted within a hospital; about adult inpatients. Exclusion criteria; research located outside of a hospital context; quality improvement studies; rehabilitation studies and literature reviews. REVIEW METHODS: Critical appraisal of the literature by using a qualitative interpretation of a translational approach. The literature was thematically mapped according to the criteria of credibility, transferability, plausibility and applicability. RESULTS: No clinical practice guideline was found that directly related to both assessment and management of pain using an algorithm in acute care for older people. Five studies were found to have relevance and were critically evaluated. CONCLUSION: A critique of the literature shows that an algorithmic approach is feasible for translation into a clinical practice guideline for assessment and management of pain in older people within the acute care setting. IMPLICATIONS FOR PRACTICE: Implementation of any algorithmic approach requires consideration of the environment, culture and availability of resources.

Examination of the utility of the promoting action on research implementation in health services framework for implementation of evidence based practice in residential aged care settings.

AIM: This study examined the relevance and fit of the PARiHS framework (Promoting Action on Research Implementation in Health Services) as an explanatory model for practice change in residential aged care. BACKGROUND: Translation of research knowledge into routine practice is a complex matter in health and social care environments. Examination of the environment may identify factors likely to support and hinder practice change, inform strategy development, predict and explain successful uptake of new ways of working. Frameworks to enable this have been described but none has been tested in residential aged care. METHODS: This paper reports preliminary qualitative analyses from the Encouraging Best Practice in Residential Aged Care Nutrition and Hydration project conducted in New South Wales in 2007-2009. We examined congruence with the PARiHS framework of factors staff described as influential for practice change during 29 digitally recorded and transcribed staff interviews and meetings at three facilities. FINDINGS: Unique features of the setting were flagged, with facilities simultaneously filling the roles of residents' home, staff's workplace and businesses. Participants discussed many of the same characteristics identified by the PARiHS framework, but in addition temporal dimensions of practice change were flagged. CONCLUSION: Overall factors described by staff as important for practice change in aged care settings showed good fit with those of the PARiHS framework. This framework can be recommended for use in this setting. Widespread adoption will enable cross-project and international synthesis of findings, a major step towards building a cumulative science of knowledge translation and practice change.

The effectiveness of using human patient simulation manikins in the teaching of clinical reasoning skills to undergraduate nursing students: a systematic review.

BACKGROUND: Human patient simulation manikins are being used extensively both nationally (in Australia) and internationally in the education of health professionals. There is evidence suggesting that these types of technologies are effective in teaching psychomotor skills. Furthermore student satisfaction with simulation approaches is generally high. However, the extent to which human patient simulation manikins are effective in the teaching of clinical reasoning skills to undergraduate nursing students is less clear. OBJECTIVE: The aim of this systematic review was to identify the best available evidence for the effectiveness of using whole-body high-fidelity human patient simulation manikins to teach clinical reasoning skills to undergraduate nursing students. INCLUSION CRITERIA: The review included all randomised controlled trials that assessed the effectiveness of high fidelity human patient manikins in educating undergraduate nursing students. Studies that included health professionals were excluded unless data for nursing students were analysed separately. The primary outcome measure was clinical reasoning. Other outcome measures included critical thinking, student satisfaction, knowledge acquisition, confidence levels, and skill performance as assessed by methods such as objective structured clinical examinations, and questionnaires. SEARCH STRATEGY: Using a systematic search strategy designed for each database, the following electronic sources were searched for the period 1999 -2009: AMED, CINAHL, Cochrane Database, Dissertation and Theses, EMBASE, ERIC, Journals@Ovid, MEDLINE, Proquest Nursing Journals, PsycINFO. Hand searching of the reference lists of included studies and conference proceedings was undertaken to identify further studies. METHODOLOGICAL VALIDITY: Two independent reviewers assessed the methodological quality of each study selected for retrieval prior to inclusion using a critical appraisal tool from the Joanna Briggs Institute. DATA COLLECTION AND SYNTHESIS: Data were extracted from studies using the standardised data extraction tool from the Joanna Briggs Institute. Statistical pooling was not possible and the findings are therefore presented in narrative form. RESULTS: Eight studies were selected for inclusion in this review. The results indicate that the use of human patient simulation manikins improved knowledge acquisition and critical thinking; and enhanced students' satisfaction with learning. There is lack of unequivocal evidence on the effectiveness of using high-fidelity human patient simulation manikins in the teaching of clinical reasoning skills to undergraduate nursing students. CONCLUSION: Further research is required to ascertain the effectiveness of the use of human patient simulation manikins as an educational strategy to improve clinical reasoning skills of undergraduate nursing students. The importance of this research is underscored by the potential for patient outcomes to be improved by enhancing the clinical reasoning skills of undergraduate graduate nursing students and graduates. IMPLICATIONS FOR EDUCATION: This review presents evidence to suggest that using HPSMs significantly improve learning outcomes related to clinical reasoning namely: critical thinking; clinical skill performance and knowledge acquisition. In addition, results indicate high student satisfaction with HPSMs simulation experience. IMPLICATIONS FOR RESEARCH: Future priorities for research include undertaking larger more robust pre-test and post-test multisite experimental studies with reliable and valid instruments that measure clinical reasoning in undergraduate nursing students.

The critical elements within a journey towards the achievement of quality use of medicines.

In Australia, as in much of the rest of the Western world, the changing demographics of the population has resulted ina need for the aged care industry and the nursing profession tomodify their traditional skill mix in order to better respond to changes in the needs of older Australians. In particular, the role of the Enrolled Nurse (EN) in New South Wales (NSW) Australia has been under considerable scrutiny for the last decade resulting in a number of changes in the EN's scope of practice; notably the 2004 amendment to legislation and education that enabled ENs to administer medication. This changed scope of EN practice has resulted in a need to reconsider the role of all members of the aged care medication team. Quality Use of Medicines (QUM) within a Residential Aged Care (RAC) facility is dependent upon systematic and evidence-based approaches underpinning the relevant structures and processes involved in the management of medications. Implementation of effective QUM systems and processes is particularly dependent upon the nature of relationships among members within the aged care medication team. Although there are numerous mechanisms to support QUM by nurses, the RAC sector in Australia lacks a tool that is both contemporaneous and context-specific and supports an expanded scope of practice for ENs who work in the RAC sector. This paper reports on an action research project that aimed to enhance of the health of older Australians in one RAC facility by developing an implementation framework that supports ENs as practitioners involved in QUM. The research identified the enabling factors and barriers to the achievement of QUM within the aged care medication team. The project encompassed a re-conceptualization and reconfiguration of local nursing medication-related practices and culminated in a conceptual framework to support quality medicine outcomes for older Australians. The outcomes of the research included a set of principles to guide the expanded role of the EN in SRAC.