Facing up to Problem Gambling: Tracing the Emergence of Facial Recognition Technology as a means of Enforcing Voluntary Self-Exclusion.

Computer technology has long been touted as a means of increasing the effectiveness of voluntary self-exclusion schemes - especially in terms of relieving gaming venue staff of the task of manually identifying and verifying the status of new customers. This paper reports on the government-led implementation of facial recognition technology as part of an automated self-exclusion program in the city of Adelaide in South Australia-one of the first jurisdiction-wide enforcements of this controversial technology in small venue gambling. Drawing on stakeholder interviews, site visits and documentary analysis over a two year period, the paper contrasts initial claims that facial recognition offered a straightforward and benign improvement to the efficiency of the city's long-running self-excluded gambler program, with subsequent concerns that the new technology was associated with heightened inconsistencies, inefficiencies and uncertainties. As such, the paper contends that regardless of the enthusiasms of government, tech industry and gaming lobby, facial recognition does not offer a ready 'technical fix' to problem gambling. The South Australian case illustrates how this technology does not appear to better address the core issues underpinning problem gambling, and/or substantially improve conditions for problem gamblers to refrain from gambling. As such, it is concluded that the gambling sector needs to pay close attention to the practical outcomes arising from initial cases such as this, and resist industry pressures for the wider replication of this technology in other jurisdictions.

Cellular Autofluorescence in Mouse Embryonic Fibroblasts Interferes with Antigen Detection Using Flow Cytometry.

Immunostaining is one of the advantageous methods for the qualitative analysis of cellular markers of interest. Immuno-stained cells are typically analyzed by fluorescence microscopy or flow cytometry. Flow cytometry has the advantage of being able to process large numbers of cells in a short time thus enhancing its quantitative capacity. The staining protocol typically includes fixation of cells followed by permeabilization, blocking procedures to reduce non-specific binding of the label, and staining with specific antibodies labeled directly or indirectly with fluorescence-tags. Important controls include staining with a relevant non-immune antibody to identify any non-specific imminent globally binding and measurements in the absence of any fluorescent tag to detect non-specific sources of fluorescence. The most common source of non-specific fluorescence is caused by autofluorescence of naturally occurring chemicals within the cells of interest. In this study, we found high levels of cellular autofluorescence in mouse embryonic fibroblasts, at levels that interfered with the detection of a number of cellular antigens using common fluorophores. This autofluorescence was detected in three of the four fluorescence channels restricting useful analysis to only one channel (red) on the instrument. The study highlights an important limitation to immunostaining techniques and reinforces the need for the use of a thorough set of controls to ensure specificity of quantitative analysis.

The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts.

The methylation of cytosine at CpG dinucleotides (5 meC) is an important epigenetic mechanism that governs genome stability and gene expression. Important ontological and pathological transitions are associated with marked global changes in detectable levels of methylation. We have previously found two pools of immune-detectable 5 meC exist within cells, a pool that can be detected after acid treatment of fixed cells to denature chromatin and another large but variable pool that requires a further tryptic digestion step for complete epitope retrieval. The trypsin-sensitive pool has been shown to be largely associated with the heterochromatic fraction (by a heterochromatin marker, HP1-ß) of the genome, and the size of this pool varies with the growth disposition of cells. Since DNA damage imposes large changes on chromatin structure the present study analyzed how such changes influences the faithful immunological detection of 5 meC within mouse embryonic fibroblasts. DNA damage was induced by either UV-irradiation or doxorubicin treatment, each of which resulted in increased levels of immune-detectable 5 meC at 24-48 h after treatment. There was a marked trypsin-sensitive pool of 5 meC in these cells which was significantly increased after DNA damage. The increased levels of 5 meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-ß staining. The relative amount of masked 5 meC after DNA damage was positively associated with increased levels of HP1-ß. The methyl binding protein, MBD1, was a less reliable measure of changes in 5 meC, with a significant fraction of 5 meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5 meC within the nuclei of fibroblasts in response to DNA damage.

Common risky behaviours checklist: a tool to assist nurse supervisors to assess unsafe practice.

AIM: To describe the development of the Common Risky Behaviour Checklist, a tool to aid nurse supervisors in determining when a nurse may be questionably fit to perform, particularly in cases of substance abuse. BACKGROUND: A significant number of nurses may have substance use disorders that could manifest as unsafe performance at work, and nurse supervisors lack the tools to assess a nurse's fitness to perform at work. METHOD: Job analysis techniques were used to identify the critical impairment behaviours for the tool. Job analysis is a legally defensible, multi-stage process used in the organisational psychology field to develop work performance assessments. RESULTS: A screening tool was developed for nurse supervisors to assess when a nurse may be questionably fit to perform. CONCLUSION: The development of this checklist is one of several needed advancements in order to address the issue of fitness to perform and patient safety. IMPLICATIONS FOR NURSING MANAGEMENT: The Common Risky Behaviour Checklist offers nurse managers assistance in protecting patient safety by providing a quick (one-page), systematic, behaviour-based method to collect information that can inform urgent decisions, trigger performance corrections and can complement formal organisational documentation processes in cases of unsafe practice due to substance abuse.

Paf receptor expression in the marsupial embryo and endometrium during embryonic diapause.

The control of reactivation from embryonic diapause in the tammar wallaby (Macropus eugenii) involves sequential activation of the corpus luteum, secretion of progesterone that stimulates endometrial secretion and subsequent changes in the uterine environment that activate the embryo. However, the precise signals between the endometrium and the blastocyst are currently unknown. In eutherians, both the phospholipid Paf and its receptor, platelet-activating factor receptor (PTAFR), are present in the embryo and the endometrium. In the tammar, endometrial Paf release in vitro increases around the time of the early progesterone pulse that occurs around the time of reactivation, but whether Paf can reactivate the blastocyst is unknown. We cloned and characterised the expression of PTAFR in the tammar embryo and endometrium at entry into embryonic diapause, during its maintenance and after reactivation. Tammar PTAFR sequence and protein were highly conserved with mammalian orthologues. In the endometrium, PTAFR was expressed at a constant level in the glandular epithelium across all stages and in the luminal epithelium during both diapause and reactivation. Thus, the presence of the receptor appears not to be a limiting factor for Paf actions in the endometrium. However, the low levels of PTAFR in the embryo during diapause, together with its up-regulation and subsequent internalisation at reactivation, supports earlier results suggesting that endometrial Paf could be involved in reactivation of the tammar blastocyst from embryonic diapause.

Systematic analysis of the factors that adversely affect the rate of cell accumulation in mouse embryos during their culture in vitro.

BACKGROUND: Retarded embryo growth is a pervasive effect of culture in vitro. METHODS: A systematic analysis of the interactions between media design, embryo culture density, oxygen tension, amino acids, trophic ligands and the genetic background of the mouse on embryo growth rates in vitro was performed. RESULTS: Growth retardation of mouse zygotes was greater in 20% O2 than 5%, a sequential media design was superior to static simple media designs, but the supplementation of simple media with mixed amino acids mitigated this difference. There was a beneficial effect of communal culture in small volumes, and supplementation with a trophic ligand (Paf) further enhanced growth rates. For hybrid strain zygotes (B6CBF1) communal culture in KSOM media supplemented with amino acids, albumin and Paf under 5% O2 resulted in complete rescue of their rate of accumulation of cells and blastocyst formation. Inbred strain (C57BL6/J) zygotes, however, still showed some retardation of development under these conditions. The additional supplementation of media with another trophic ligand (IGF1) showed a further additive beneficial effect on development of inbred strain embryos but they still showed a growth deficit of ~ 23% cell number. The results show that optimising the interactions between a range of culture conditions and media design can rescue hybrid strain embryos from a retarded rate of cell proliferation caused by culture in vitro, but this was incomplete for the B6 strain. CONCLUSIONS: The results indicate that the growth requirement of embryos in vitro varies depending upon their genetic background and provide models for the further genetic analysis of embryo growth.

Quasi-experimental evaluation of a substance use awareness educational intervention for nursing students.

This article reports on a study that evaluated the effectiveness of an educational intervention, Addressing Nurse Impairment, for addressing nursing students' knowledge acquisition, changes in self-efficacy to intervene, and changes in substance abuse stigma. A gap exists in nursing students' education regarding the risks of addiction within the profession and how to handle a colleague suspected of having a substance use disorder. The seminar was adapted from an existing evidence-based prevention program called Team Awareness, as well as information from focus groups and a pilot test. A quasi-experimental pretest-posttest design was used to evaluate the effect of the seminar. When the control and experimental groups were compared, the results indicated that the seminar significantly affected knowledge and self-efficacy to intervene but did not significantly affect stigma. This research contributes to the body of evidence related to educational interventions for nursing students regarding substance abuse in the nursing profession.

Transformation-related protein 53 expression in the early mouse embryo compromises preimplantation embryonic development by preventing the formation of a proliferating inner cell mass.

The developmental viability of the preimplantation embryo requires the successful formation of a cluster of pluripotent stem cells called the inner cell mass. Development is variably compromised by a range of exogenous stressors (including their production by assisted reproductive technologies). Inbred C57BL/6 strain embryos are particularly susceptible to the stresses associated with embryo culture, whereas hybrid embryos are more resistant, and this is accounted for in part by the overexpression of transformation-related protein 53 in cultured inbred embryos compared with similarly treated hybrid embryos or embryos not subjected to culture. We show here that this loss of viability is a consequence of the Trp53-dependent reduction in the capacity of blastocysts to form a proliferating inner cell mass. Formation of the trophectodermal line was not adversely affected by these stresses.

Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro.

This study investigates the suitability of surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) and electrospray ionization (ESI) mass spectrometry for analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) protein chips detected a protein peak at m/z approximately 8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the presence of the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo-conditioned media identified a total of 20 proteins released during development from the zygote to blastocyst stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more proteins were in at least one culture. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modifications of proteins. This study shows the feasibility of ESI mass spectrometry for identifying the proteins secreted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of early embryo development.

Activation of a chloride channel by a trophic ligand is required for development of the mouse preimplantation embryo in vitro.

Platelet-activating factor (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine [PAF]) is one of several autocrine trophic factors supporting the development of the preimplantation embryo. PAF acts on the embryo to induce receptor-mediated intracellular calcium (Ca(2+))(i) transients, and these coincide with a marked membrane hyperpolarization. Patch-clamp analysis of 2-cell embryos showed that these Ca(2+)(i) transients resulted in an outward membrane current. The present study characterizes this current and assesses its role in embryo development. The outward current was dependent upon the presence of anions in the extracellular medium and occurred as a consequence of the PAF-induced Ca(2+)(i) transients. The anion current induced by PAF was inhibited by niflumic acid (NFA), a selective blocker of Ca(2+)-activated Cl(-) channels, but this drug did not block the PAF-induced Ca(2+)(i) transients. Voltage ramp analysis showed that the Cl(-) conductance was outwardly rectifying and inactivated at holding potentials more positive than +30 mV. Culture in NFA or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (a broad-specificity anion channel blocker) from the zygote stage significantly reduced development to blastocysts, with most arresting at the 4-cell and 8-cell stages. Niflumic acid exposure only from the zygote to the late 2-cell stage also reduced the subsequent development to blastocysts. By contrast, treatment from the late 2-cell stage or the 8-cell stage had no effect on development to the blastocyst stage. This study demonstrates the activation of a Ca(2+)-sensitive Cl(-) channel in the 2-cell embryo by PAF and shows that this current activity during the zygote to 2-cell stage is required for normal embryo development in vitro.

Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development.

The development of the preimplantation mammalian embryo is an autopoietic process; once initiated development proceeds without an absolute requirement for external information or growth cues. This developmental autonomy is partly explained by the generation of autocrine trophic ligands that are released and act back on the embryo via specific receptors. Several embryotrophic ligands cause receptor-dependent activation of 1-o-phosphatidylinositol 3-kinase. This enzyme phosphorylates phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. Genetic or pharmacological ablation of this enzyme activity disrupts normal development of preimplantation embryos. Phosphatidylinositol-3,4,5-trisphosphate is a membrane lipid that acts as a docking site for a wide range of proteins possessing the pleckstrin homology (PH) domain. Such proteins are important regulators of cell survival, proliferation, and differentiation. RAC-alpha serine/threonine protein kinase is an important PH domain protein and its activity is required for normal preimplantation embryo development and survival. The activity of a range of PH domain proteins is also implicated in the normal development of the embryo. This review critically examines the evidence for the activation of 1-o-phosphatidylinositol 3-kinase in the generation of pleiotypic trophic response to embryotrophins in the autopoietic development of the preimplantation embryo.

Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts.

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.

Measurement of global DNA methylation levels by flow cytometry in mouse fibroblasts.

DNA Methylation, 5meC, is an epigenetic modification that acts as an important regulator of genomic stability and gene expressivity. Genome-wide changes in methylation have been associated with lineage-specific changes in gene expression profiles during development and in some cell-based pathologies, including oncogenesis. Cost-effective and rapid platforms for the detection of changes in the global levels of methylation are of value for the investigation of the processes that regulate methylation. Flow cytometry allows rapid and quantitative analysis of epitopes within a large number of cells. We have recently optimised the conditions required for valid detection of 5meC by immunofluorescence microscopy. These studies showed that immunological detection of 5meC requires the sequential denaturation of chromatin by a brief period of acidification followed by a partial tryptic digestion step. We have assessed the reliability of flow cytometry for the detection of changes in 5meC when coupled with this optimised epitope retrieval strategy. This study provides support for the use of high throughput screening of 5meC by flow cytometry for the analysis of the epigenetic regulation of important cell transitions.

The incidence of dissociated liners in 4,751 consecutive total hip arthroplasties using Pinnacle polyethylene acetabular liners.

INTRODUCTION: Acetabular liner dissociation is a complication exclusive to modular designs. We present a single surgeon series of 8 polyethylene liner dissociations with the Pinnacle Acetabular System (DePuy Orthopaedics) from over 4,750 cases. We also present a review of the literature and data from the UK National Joint Registry (NJR) on dissociation in total hip arthroplasty (THA). METHODS: The Pinnacle Acetabular System has been used exclusively by the senior author since April 2003, and to date 5,882 have been implanted (837 ceramic liners, 4,751 polyethylene liners (1,606 Enduron/3,145 Marathon) and 294 metal liners). We reviewed all cases of liner dissociation from this cohort to determine an overall incidence with polyethylene liners, identify associated risk factors, and report the outcome following revision surgery. RESULTS: Our incidence of this complication is 8 out of 4,751 cases (0.17%). Review of these cases and the literature suggests that femoral neck impingement against the polyethylene liner and/or edge loading may produce fatigue failure of the locking mechanism and subsequent dissociation. CONCLUSIONS: Ensuring correct liner seating/locking, minimising impingement, achieving appropriate component version and avoiding radiographic cup inclinations >50° should minimise the risk of liner dissociation. Any new noise or squeaking from a polyethylene liner should undergo radiographic investigation to exclude dissociation. We recommend managing late cases of liner dissociation with revision of the acetabular shell if the cup orientation could be improved or if there is any damage to the liner-locking groove, to reduce the risk of recurrence.

Regulation of cellular adhesion molecule expression in murine oocytes, peri-implantation and post-implantation embryos.

Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, alpha chain), and CD11a (LFA-1, alpha chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophectoderm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophectoderm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.

The exit of mouse embryonic fibroblasts from the cell-cycle changes the nature of solvent exposure of the 5'-methylcytosine epitope within chromatin.

The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.

5'-Methylcytosine and 5'-hydroxymethylcytosine each provide epigenetic information to the mouse zygote.

Covalent modification of cytosine nucleotides within the genome encode essential epigenetic information, with methylation (5meC) and hydroxymethylation (5hmC) having received most attention. It has been proposed that the formation of 5hmC is an intermediate in the active demethylation of 5meC. Some reports show that global loss of 5meC in the newly fertilised embryo is accompanied by increased 5hmC, but others have failed to confirm this finding. These analyses have relied on immuno-localization of these modifications. In this study we have established the conditions required for equilibrium binding of antibodies to 5meC and 5hmC in zygotes. Simultaneous detection of these antigens required denaturation of chromatin by acid treatment followed by antigen retrieval by tryptic digestion. Equilibrium binding then required incubation at 4°C for greater than 6 h. These are more demanding conditions than generally reported and resulted in the consistent detection of 5meC and 5hmC in both male and female pronuclei throughout zygotic maturation. No dynamic reciprocal change in the level of 5meC relative to 5hmC was observed. Both 5meC and 5hmC accumulated within the peri-nucleolar regions and this was more pronounced in the male pronucleus. Staining of 5meC was relatively more intense within the cortical and 5hmC in the central regions of pronuclei. The results are not consistent with a role for 5hmC in global demethylation in the zygote. The persistence of both modifications throughout zygotic maturation, and their differing patterns of localization and solvent exposure infer each modification provides its own epigenetic information to the early embryo.

Persistence of cytosine methylation of DNA following fertilisation in the mouse.

Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote.