The post-illumination pupil response of melanopsin expressing retinal ganglion cells

Purpose The post-illumination pupil response (PIPR) has been quantified in the literature by four metrics. The spectral sensitivity of only one metric is known and this study quantifies the other three. To optimize the measurement of the PIPR in humans, we also determine the stimulus protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation. Methods The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer (35.6° diameter stimulus). - Experiment 1: Spectral sensitivity of four PIPR metrics [plateau, 6 s, area under curve (AUC) early and late recovery] was determined from a criterion PIPR (n = 2 participants) to a 1 s pulse at five wavelengths (409-592nm) and fitted with Vitamin A nomogram (ƛmax = 482 nm). - Experiment 2: The PLR was measured in five healthy participants [29 to 42 years (mean = 32.6 years)] as a function of three stimulus durations (1 s, 10 s, 30 s), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8 to 14.8 log quanta.cm-2.s-1), and two wavelengths, one with high (465 nm) and one with low (637 nm) melanopsin excitation. Intra and inter-individual coefficients of variation (CV) were calculated. Results The melanopsin (opn4) photopigment nomogram adequately described the spectral sensitivity derived from all four PIPR metrics. The largest PIPR amplitude was observed with 1 s short wavelength pulses (retinal irradiance ≥ 12.8 log quanta.cm-2.s-1). Of the 4 PIPR metrics, the plateau and 6 s PIPR showed the least intra and inter-individual CV (≤ 0.2). The maximum duration of the sustained PIPR was 83.4 ± 48.0 s (mean ± SD) for 1 s pulses and 180.1 ± 106.2 s for 30 s pulses (465 nm; 14.8 log quanta.cm-2.s-1). Conclusions All current PIPR metrics provide a direct measure of intrinsic melanopsin retinal ganglion cell function. To measure progressive changes in melanopsin function in disease, we recommend that the intrinsic melanopsin response should be measured using a 1 s pulse with high melanopsin excitation and the PIPR should be analyzed with the plateau and/or 6 s metrics. That the PIPR can have a sustained constriction for as long as 3 minutes, our PIPR duration data provide a baseline for the selection of inter-stimulus intervals between consecutive pupil testing sequences.

Evaluation of a workplace alcohol and other drug policy

BACKGROUND The current impetus for developing alcohol and/or other drugs (AODs) workplace policies in Australia is to reduce workplace AOD impairment, improve safety, and prevent AOD-related injury in the workplace. For these policies to be effective, they need to be informed by scientific evidence. Evidence to inform the development and implementation of effective workplace AOD policies is currently lacking. There does not currently appear to be conclusive evidence for the effectiveness of workplace AOD policies in reducing impairment and preventing AOD-related injury. There is also no apparent evidence regarding which factors facilitate or impede the success of an AOD policy, or whether, for example, unsuccessful policy outcomes were due to poor policy or merely poor implementation of the policy. It was the aim of this research to undertake a process, impact, and outcome evaluation of a workplace AOD policy, and to contribute to the body of knowledge on the development and implementation of effective workplace AOD policies. METHODS The research setting was a state-based power-generating industry in Australia between May 2008 and May 2010. Participants for the process evaluation study were individuals who were integral to either the development or the implementation of the workplace AOD policy, or both of these processes (key informants), and comprised the majority of individuals who were involved in the process of developing and/or implementing the workplace AOD policy. The sample represented the two main groups of interest—management and union delegates/employee representatives—from all three of the participating organisations. For the impact and outcome evaluation studies, the population included all employees from the three participating organisations, and participants were all employees who consented to participate in the study and who completed both the pre-and post-policy implementation questionnaires. Qualitative methods in the form of interviews with key stakeholders were used to evaluate the process of developing and implementing the workplace AOD policy. In order to evaluate the impact of the policy with regard to the risk factors for workplace AOD impairment, and the outcome of the policy in terms of reducing workplace AOD impairment, quantitative methods in the form of a non-randomised single group pre- and post-test design were used. Changes from Time 1 (pre) to Time 2 (post) in the risk factors for workplace AOD impairment, and changes in the behaviour of interest—(self-reported) workplace AOD impairment—were measured. An integration of the findings from the process, impact, and outcome evaluation studies was undertaken using a combination of qualitative and quantitative methods. RESULTS For the process evaluation study Study respondents indicated that their policy was developed in the context of comparable industries across Australia developing workplace AOD policies, and that this was mainly out of concern for the deleterious health and safety impacts of workplace AOD impairment. Results from the process evaluation study also indicated that in developing and implementing the workplace AOD policy, there were mainly ‗winners', in terms of health and safety in the workplace. While there were some components of the development and implementation of the policy that were better done than others, and the process was expensive and took a long time, there were, overall, few unanticipated consequences to implementing the policy and it was reported to be thorough and of a high standard. Findings also indicated that overall the policy was developed and implemented according to best-practice in that: consultation during the policy development phase (with all the main stakeholders) was extensive; the policy was comprehensive; there was universal application of the policy to all employees; changes in the workplace (with regard to the policy) were gradual; and, the policy was publicised appropriately. Furthermore, study participants' responses indicated that the role of an independent external expert, who was trusted by all stakeholders, was integral to the success of the policy. For the impact and outcome evaluation studies Notwithstanding the limitations of pre- and post-test study designs with regard to attributing cause to the intervention, the findings from the impact evaluation study indicated that following policy implementation, statistically significant positive changes with regard to workplace AOD impairment were recorded for the following variables (risk factors for workplace AOD impairment): Knowledge; Attitudes; Perceived Behavioural Control; Perceptions of the Certainty of being punished for coming to work impaired by AODs; Perceptions of the Swiftness of punishment for coming to work impaired by AODs; and Direct and Indirect Experience with Punishment Avoidance for workplace AOD impairment. There were, however, no statistically significant positive changes following policy implementation for Behavioural Intentions, Subjective Norms, and Perceptions of the Severity of punishment for workplace AOD impairment. With regard to the outcome evaluation, there was a statistically significant reduction in self-reported workplace AOD impairment following the implementation of the policy. As with the impact evaluation, these findings need to be interpreted in light of the limitations of the study design in being able to attribute cause to the intervention alone. The findings from the outcome evaluation study also showed that while a positive change in self-reported workplace AOD impairment following implementation of the policy did not appear to be related to gender, age group, or employment type, it did appear to be related to levels of employee general alcohol use, cannabis use, site type, and employment role. Integration of the process, impact, and outcome evaluation studies There appeared to be qualitative support for the relationship between the process of developing and implementing the policy, and the impact of the policy in changing the risk factors for workplace AOD impairment. That is, overall the workplace AOD policy was developed and implemented well and, following its implementation, there were positive changes in the majority of measured risk factors for workplace AOD impairment. Quantitative findings lend further support for a relationship between the process and impact of the policy, in that there was a statistically significant association between employee perceived fidelity of the policy (related to the process of the policy) and positive changes in some risk factors for workplace AOD impairment (representing the impact of the policy). Findings also indicated support for the relationship between the impact of the policy in changing the risk factors for workplace AOD impairment and the outcome of the policy in reducing workplace AOD impairment: positive changes in the risk factors for workplace AOD impairment (impact) were related to positive changes in self reported workplace AOD impairment (representing the main goal and outcome of the policy). CONCLUSIONS The findings from the research indicate support for the conclusion that the policy was appropriately implemented and that it achieved its objectives and main goal. The Doctoral research findings also addressed a number of gaps in the literature on workplace AOD impairment, namely: the likely effectiveness of AOD policies for reducing AOD impairment in the workplace, which factors in the development and implementation of a workplace AOD policy are likely to facilitate or impede the effectiveness of the policy to reduce workplace AOD impairment, and which employee groups are less likely to respond well to policies of this type. The findings from this research not only represent an example of translational, applied research—through the evaluation of the study industry's policy—but also add to the body of knowledge on workplace AOD policies and provide policy-makers with evidence which may be useful in the development and implementation of effective workplace AOD policies. Importantly, the findings espouse the importance of scientific evidence in the development, implementation, and evaluation of workplace AOD policies.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.