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The ability to sustain attention is integral to healthy cognition in aging. The right PFC (rPFC) is critical for maintaining high levels of attentional focus. Whether plasticity of this region can be harnessed to support sustained attention in older adults is unknown. We used transcranial direct current stimulation to increase cortical excitability of the rPFC, while monitoring behavioral and electrophysiological markers of sustained attention in older adults with suboptimal sustained attention capacity. During rPFC transcranial direct current stimulation, fewer lapses of attention occurred and electroencephalography signals of frontal engagement and early visual attention were enhanced. To further verify these results, we repeated the experiment in an independent cohort of cognitively typical older adults using a different sustained attention paradigm. Again, prefrontal stimulation was associated with fewer attentional lapses. These experiments suggest the rPFC can be manipulated in later years to increase top-down modulation over early sensory processing and improve sustained attention performance. This holds valuable information for the development of neurorehabilitation protocols to ameliorate age-related deficits in this capacity.

Care and treatment in forensic mental health wards can present with challenges when loss of hope and freedom, and aggression are present, which can then influence ward atmosphere and feelings of safety. Safewards is a model designed to address a range of conflict (e.g., aggression and self-harm) and containment (e.g., use of restrictive interventions) events and may provide a suitable approach to delivery of care in a forensic setting, while also addressing aggression, restrictive interventions, and ward atmosphere. The aim of this study was to evaluate the introduction of Safewards to a forensic mental health ward to determine suitability, and to explore if changes to conflict, containment, and ward atmosphere occurred. A mixed methods approach was used involving the collection of incident data related to conflict and containment, an assessment of the degree to which interventions were implemented, and an assessment of the social climate before and after implementation. Results suggested that there were fewer conflict events after Safewards was introduced; however, there did not appear to be any changes in the already low use of restrictive interventions. The Safewards interventions were implemented to a high degree of fidelity, and there was indication of an increase in a positive perception of ward atmosphere, supported by themes of positive change, enhanced safety, and respectful relationships. Safewards may assist in contributing to an improvement in the perception of ward atmosphere. To enhance implementation in a forensic mental health setting, there may be a need to consider additional elements to Safewards, pertinent to this setting.

Allergic contact dermatitis (ACD) is an inflammatory disease that impacts 15-20% of the general population and accurate screening methods for chemical risk assessment are needed. However, most approaches poorly predict pre- and pro-hapten sensitizers, which require abiotic or metabolic conversion prior to inducing sensitization. We developed a tri-culture system comprised of MUTZ-3-derived Langerhans cells, HaCaT keratinocytes, and primary dermal fibroblasts to mimic the cellular and metabolic environments of skin sensitization. A panel of non-sensitizers and sensitizers was tested and the secretome was evaluated. A support vector machine (SVM) was used to identify the most predictive sensitization signature and classification trees identified statistical thresholds to predict sensitizer potency. The SVM computed 91% tri-culture prediction accuracy using the top 3 ranking biomarkers (IL-8, MIP-1β, and GM-CSF) and improved the detection of pre- and pro-haptens. This in vitro assay combined with in silico data analysis presents a promising approach and offers the possibility of multi-metric analysis for enhanced ACD sensitizer screening.

Aim and Objectives: To examine associations between risk of aggression and nursing interventions designed to prevent aggression., Background: There is scarce empirical research exploring the nature and effectiveness of interventions designed to prevent inpatient aggression. Some strategies may be effective when patients are escalating, whereas others may be effective when aggression is imminent. Research examining level of risk for aggression and selection and effectiveness of interventions and impact on aggression is necessary., Design: Archival case file., Methods: Data from clinical files of 30 male and 30 female patients across three forensic acute units for the first 60 days of hospitalisation were collected. Risk for imminent aggression as measured by the Dynamic Appraisal of Situational Aggression, documented nursing interventions following each assessment, and acts of aggression within the 24-hours following assessment were collected. Generalised estimating equations were used to investigate whether intervention strategies were associated with reduction in aggression., Results: When a Dynamic Appraisal of Situational Aggression assessment was completed, nurses intervened more frequently compared to days when no Dynamic Appraisal of Situational Aggression assessment was completed. Higher Dynamic Appraisal of Situational Aggression assessments were associated with a greater number of interventions. The percentage of interventions selected for males differed from females; males received more pro re nata medication and observation, and females received more limit setting, one-to-one nursing and reassurance. Pro re nata medication was the most commonly documented intervention (35.9%) in this study. Pro re nata medication, limit setting and reassurance were associated with an increased likelihood of aggression in some risk bands., Conclusions: Structured risk assessment prompts intervention, and higher risk ratings result in more interventions. Patient gender influences the type of interventions. Some interventions are associated with increased aggression, although this depends upon gender and risk level., Relevance to Clinical Practice: When structured risk assessments are used, there is greater likelihood of intervention. Intervention should occur early using least restrictive interventions., (© 2017 John Wiley & Sons Ltd.)

Mesenchymal stromal cell (MSC) therapies have become potential treatment options for multiple ailments and traumatic injuries. In the clinical setting, MSC are likely to be co-administered with local anesthetics (LA) which have been shown to have dose- and potency-dependent detrimental effects on the viability and function of cells. We previously developed and characterized a sustained-release LA delivery formulation comprised of alginate-encapsulated liposomal bupivacaine. The current studies were designed to evaluate the effect of this formulation on the secretion of three key MSC regulatory molecules, interleukin 6 (IL-6), prostaglandin E2 (PGE2), and transforming growth factor-beta 1 (TGF-β1). MSCs were treated with several bupivacaine formulations-bolus, liposome, or alginate-liposome construct (engineered construct)-in the presence or absence of inflammatory stimulus to stimulate an injured tissue environment. Our results indicated that compared to bolus or liposomal bupivacaine, the engineered construct preserved or promoted MSC anti-inflammatory PGE2 secretion; however, the engineered construct did not increase TGF-β1 secretion. Bupivacaine release profile analyses indicated that mode of drug delivery controlled the LA concentration over time and pathway analysis identified several shared and cytokine-specific molecular mediators for IL-6, PGE2, and TGF-β1 which could explain differential MSC secretion responses in the presence of bupivacaine. Collectively, these studies support the potential utility of alginate-encapsulated LA constructs for anti-inflammatory cell therapy co-administration and indicate that mode of local anesthetic delivery can significantly alter MSC secretome function.

Momentum has been building for professional development (PD) for all staff who teach in Irish higher education - they now have the national Professional Development Framework (PDF) to support them in planning and engaging with authentic, inclusive, scholarly, learner-centred and collaborative PD across their career. In mid-2016, the PD Framework was published by the National Forum for the Enhancement of Teaching and Learning. In 2017, the PDF was piloted with 215 individuals from 22 professional identity groups from across the sector, working with the framework to reflect on their professional practice and set goals for their future professional development. This initial implementation phase has now been fully evaluated and a robust evidence-base of 'what works, and why' in relation to staff engagement with the PDF is in place. Disseminating findings and continuing to engage with the sector through relevant platforms such as HEIT is key to advancing this work. Early 2018 witnessed the HEA publishing the HE System Performance Framework 2018-2020 with two recommendations which are key to the momentum of the PDF: • Implementation of the Continuous Professional Development Framework • Number of staff with "Digital Badges" for completed CPD by academic year Given these significant developments over a relatively short period, we are now asking the sector, what needs to happen next for the PDF to continue to drive progress for individual's careers and support them in dealing with change and transformation in their professional practice?

Seclusion and mechanical restraint are restrictive interventions that should be used only as a last resort and for the shortest possible time, yet little is known about duration of use in the broader context. Adult area mental health services throughout Victoria, Australia, were asked to complete a report form for prolonged episodes of seclusion (>8 hours) and mechanical restraint (>1 hour). The present, retrospective cohort study aimed to understand the individual (age, sex, type of service, duration of intervention) and contextual factors associated with prolonged use of restrictive interventions. Contextual factors describing the reasons for prolonged use of the restrictive interventions were captured qualitatively, and then coded using content analysis. Median duration was compared across individual factors using Mann-Whitney U-tests. During 2014, 690 episodes of prolonged restrictive intervention involving 311 consumers were reported. Close to half (n = 320, 46%) involved mechanical restraint. Seclusion episodes (n = 370) were longer in forensic mental health services compared to adult area mental health services (median: 24 hours and 18 min vs 16 hours and 42 min, P < 0.001). Mechanical restraint episodes (n = 320) were shorter in forensic mental health services compared to adult area mental health services (median: 3 hours and 25 min vs 4 hours and 15 min, P = 0.008). Some consumers were subject to multiple episodes of prolonged seclusion (55/206, 27%) and/or prolonged mechanical restraint (31/131, 24%). The most commonly occurring contextual factor for prolonged restrictive interventions was 'risk of harm to others'. Means for reducing the use of prolonged restrictive interventions are discussed in light of the findings., (© 2017 Australian College of Mental Health Nurses Inc.)

In the present study, we explored the predictive validity of the Dynamic Appraisal of Situational Aggression (DASA) assessment tool in male (n = 30) and female (n = 30) patients admitted to the acute units of a forensic mental health hospital. We also tested the psychometric properties of the original DASA bands and novel risk bands. The first 60 days of each patient's file was reviewed to identify daily DASA scores and subsequent risk-related nursing interventions and aggressive behaviour within the following 24 hours. Risk assessments, followed by documented nursing interventions, were removed to preserve the integrity of the risk-assessment analysis. Receiver-operator characteristics were used to test the predictive accuracy of the DASA, and generalized estimating equations (GEE) were used to account for repeated risk assessments, which occurs when analysing short-term risk-assessment data. The results revealed modest predictive validity for males and females. GEE analyses suggested the need to adjust the DASA risk bands to the following (with associated odds ratios (OR) for aggressive behaviour): 0 = low risk; 1, 2, 3 = moderate-risk OR, 4.70 (95% confidence interval (CI): 2.84-7.80); and 4, 5, 6, 7 = high-risk OR, 16.13 (95% CI: 9.71-26.78). The adjusted DASA risk bands could assist nurses by prompting violence-prevention interventions when the level of risk is elevated., (© 2017 Australian College of Mental Health Nurses Inc.)

Sensory modulation (SM) and trauma-informed-care (TIC) are therapeutic strategies which can help avoid incidents of aggression and thus reduce the use of restrictive interventions in mental health settings. In order to educate mental health nurses and allied health professionals in these strategies, a train-the-trainer intervention was developed and delivered to 19 area mental health services as a statewide, government funded program. This descriptive qualitative study evaluated the effectiveness of the intervention to: a) transfer knowledge; and, b) translate knowledge into practice. Semi-structured interviews were conducted with senior staff (n = 21); focus group discussions with trainees (n = 10); and, a paired in-depth interview with master trainers (n = 2). In total, 170 trainees attended two day train-the-trainer sessions. Many trainees were not in education roles. Most services facilitated further knowledge transfer to end-user clinicians, though training materials were often adapted. End-users' responses to SM/TIC training were generally positive to the training, but some were resistant to the change in practice. Limited anecdotal evidence of translation of SM/TIC into practice was provided. Ongoing support is required to maintain a focus on SM and TIC, sustain and encourage further knowledge transfer and translation, and assess the impact on consumer and staff health outcomes., (Copyright © 2017. Published by Elsevier Ltd.)

Psychiatric patients encounter many difficulties post-discharge reflected by quantitative data such as high suicide rates within the first 3 months. However, little is known about why discharge is so difficult. This article aimed to understand the lived experience of being discharged from a psychiatric unit. Eight participants were interviewed to explore their experiences of discharge. Interpretative phenomenological analysis was used to analyse data. Four superordinate themes emerged: leaving hospital; the outside world; the journey to health; and self-identity/beliefs. Clinical implications and suggestions for further research are discussed.

The aim of the present study was to describe incidences of restrictive interventions and the association of methamphetamine use at an acute adult inpatient mental health unit in metropolitan Melbourne, Victoria, Australia. A total of 232 consecutive consumer admissions to the inpatient unit across a 3-month period were described for illicit substance use and the use of restrictive interventions (seclusion, mechanical restraint, and physical restraint) prior to and during admission. Of all admissions, 25 (10.8%) involved consumers subjected to a restrictive intervention. Methamphetamine use was either self-reported or detected by saliva test for 71 (30.6%) consumers. Following multivariate analyses, methamphetamine use (odds ratio (OR): 7.83, 95% confidence interval (CI): 2.33-26.31) and restrictive intervention in the emergency department prior to admission (OR: 8.85, 95% CI: 2.83-27.70) were significant independent predictors of the use of restrictive interventions after inpatient admission. Anecdotal observations provided by clinical mental health staff that consumers intoxicated with methamphetamine appear to require restrictive intervention more frequently than other consumers was confirmed with the results of the current study. As the state of Victoria in Australia is on a pathway to the elimination of the use of restrictive interventions in mental health services, clinicians need to develop management strategies that provide specialist mental health care using the least-restrictive interventions. Although 26.8% of methamphetamine users were secluded after admission, restrictive interventions should not be the default management strategy for consumers who present with self-report or positive screen for methamphetamine use., (© 2016 Australian College of Mental Health Nurses Inc.)

Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro . Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro . The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro . SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo , SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds.

The aim of this paper was to propose key steps for community pharmacist integration into a patient care pathway for chronic obstructive pulmonary disease (COPD) management. A literature search was conducted to identify publications focusing on the role of the community pharmacist in identification and management of COPD. The literature search highlighted evidence supporting an important role for pharmacists at each of the four key steps in the patient care pathway for COPD management. Step 1 (primary prevention): pharmacists are ideally placed to provide information on disease awareness and risk prevention campaigns, and to encourage lifestyle interventions, including smoking cessation. Step 2 (early detection/case finding): pharmacists are often the first point of contact between the patient and the healthcare system and can therefore play an important role in the early identification of patients with COPD. Step 3 (management and ongoing support): pharmacists can assist patients by providing advice and education on dosage, inhaler technique, treatment expectations and the importance of adherence, and by supporting self-management, including recognition and treatment of COPD exacerbations. Step 4 (review and follow-up): pharmacists can play an important role in monitoring adherence and ongoing inhaler technique in patients with COPD. In summary, pharmacists are ideally positioned to play a vital role in all key stages of an integrated COPD patient care pathway from early disease detection to the support of management plans, including advice and counselling regarding medications, inhaler technique and treatment adherence. Areas requiring additional consideration include pharmacist training, increasing awareness of the pharmacist role, administration and reimbursement, and increasing physician-pharmacist collaboration., (© 2016 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Practicing with trauma informed care (TIC) can strengthen nurses' knowledge about the association of past trauma and the impact of trauma on the patient's current mental illness. An aim of TIC is to avoid potentially re-traumatising a patient during their episode of care. A TIC education package can provide nurses with content that describes the interplay of neurological, biological, psychological, and social effects of trauma that may reduce the likelihood of re-traumatisation. Although mental health nurses can be TIC leads in multidisciplinary environments, the translation of TIC into clinical practice by nurses working in emergency departments (EDs) is unknown. However, before ED nurses can begin to practice TIC, they must first be provided with meaningful and specific education about TIC. Therefore, the aims of this study were to; (1) evaluate the effectiveness of TIC education for ED nursing staff and (2) describe subsequent clinical practice that was trauma informed., Methods: This project was conducted as exploratory research with a mixed methods design. Quantitative data were collected with an 18-item pre-education and post-education questionnaire. Qualitative data were collected with two one-off focus groups conducted at least three-months after the TIC education. Two EDs were involved in the study., Results: A total of 34 ED nurses participated in the TIC education and 14 ED nurses participated in the focus groups. There was meaningful change (p < 0.01, r ≥ 0.35) in 9 of the 18-items after TIC education. Two themes, each with two sub-themes, were evident in the data. The themes were based on the perceived effectiveness of TIC education and the subsequent changes in clinical practice in the period after TIC education., Conclusion: Emergency department nurses became more informed of the interplay of trauma on an individual's mental health. However, providing care with a TIC framework in an ED setting was a considerable challenge primarily due to time constraints relative to the day-to-day ED environment and rapid turnover of patients with potentially multiple and complex presentations. Despite this, nurses understood the effect of TIC to reduce the likelihood of re-traumatisation and expressed a desire to use a TIC framework.

While general anesthetics control pain via consciousness regulation, local anesthetics (LAs) act by decreasing sensation in the localized area of administration by blocking nerve transmission to pain centers. Perioperative intra-articular administration of LAs is a commonly employed practice in orthopedic procedures to minimize patient surgical and post-surgical pain and discomfort. LAs are also co-administered with cellular mesenchymal stromal cell (MSC) therapies for a variety of tissue regenerative and inflammatory applications including osteoarthritis (OA) treatment; however, LAs can affect MSC viability and function. Therefore, finding an improved method to co-administer LAs with cells has become critically important. We have developed a sustained release LA delivery model that could enable the co-administration of LAs and MSCs. Encapsulation of liposomes within an alginate matrix leads to sustained release of bupivacaine as compared to bupivacaine-containing liposomes alone. Furthermore, drug release is maintained for a minimum of 4 days and the alginate-liposome capsules mitigated the adverse effects of bupivacaine on MSC viability.

Background: Non-prescription (over-the-counter, or OTC) analgesics (painkillers) are used frequently. They are available in various brands, package sizes, formulations, and dose. They can be used for a range of different types of pain, but this overview reports on how well they work for acute pain (pain of short duration, usually with rapid onset). Thirty-nine Cochrane reviews of randomised trials have examined the analgesic efficacy of individual drug interventions in acute postoperative pain., Objectives: To examine published Cochrane reviews for information about the efficacy of pain medicines available without prescription using data from acute postoperative pain., Methods: We identified OTC analgesics available in the UK, Australia, Canada, and the USA by examining online pharmacy websites. We also included some analgesics (diclofenac potassium, dexketoprofen, dipyrone) of importance in parts of the world, but not currently available in these jurisdictions.We identified systematic reviews by searching the Cochrane Database of Systematic Reviews (CDSR) on The Cochrane Library through a simple search strategy. All reviews were overseen by a single review group, had a standard title, and had as their primary outcome numbers of participants with at least 50% pain relief over four to six hours compared with placebo. From individual reviews we extracted the number needed to treat for an additional beneficial outcome (NNT) for this outcome for each drug/dose combination, and also calculated the success rate to achieve at least 50% of maximum pain relief. We also examined the number of participants experiencing any adverse event, and whether the incidence was different from placebo., Main Results: We found information on 21 different OTC analgesic drugs, doses, and formulations, using information from 10 Cochrane reviews, supplemented by information from one non-Cochrane review with additional information on ibuprofen formulations (high quality evidence). The lowest (best) NNT values were for combinations of ibuprofen plus paracetamol, with NNT values below 2. Analgesics with values close to 2 included fast acting formulations of ibuprofen 200 mg and 400 mg, ibuprofen 200 mg plus caffeine 100 mg, and diclofenac potassium 50 mg. Combinations of ibuprofen plus paracetamol had success rates of almost 70%, with dipyrone 500 mg, fast acting ibuprofen formulations 200 mg and 400 mg, ibuprofen 200 mg plus caffeine 100 mg, and diclofenac potassium 50 mg having success rates above 50%. Paracetamol and aspirin at various doses had NNT values of 3 or above, and success rates of 11% to 43%. We found no information on many of the commonly available low dose codeine combinations.The proportion of participants experiencing an adverse event were generally not different from placebo, except for aspirin 1000 mg and (barely) ibuprofen 200 mg plus caffeine 100 mg. For ibuprofen plus paracetamol, adverse event rates were lower than with placebo., Authors' Conclusions: There is a body of reliable evidence about the efficacy of some of the most commonly available drugs and doses widely available without prescription. The postoperative pain model is predominantly pain after third molar extraction, which is used as the industry model for everyday pain. The proportion of people with acute pain who get good pain relief with any of them ranges from around 70% at best to less than 20% at worst; low doses of some drugs in fast acting formulations were among the best. Adverse events were generally no different from placebo. Consumers can make an informed choice based on this knowledge, together with availability and price. Headache and migraine were not included in this overview.

Induction of therapeutic mesenchymal stromal cell (MSC) function is dependent upon activating factors present in diseased or injured tissue microenvironments. These functions include modulation of macrophage phenotype via secreted molecules including prostaglandin E2 (PGE2). Many approaches aim to optimize MSC-based therapies, including preconditioning using soluble factors and cell immobilization in biomaterials. However, optimization of MSC function is usually inefficient as only a few factors are manipulated in parallel. We utilized fractional factorial design of experiments to screen a panel of 6 molecules (lipopolysaccharide [LPS], polyinosinic-polycytidylic acid [poly(I:C)], interleukin [IL]-6, IL-1β, interferon [IFN]-β, and IFN-γ), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-α, a pro-inflammatory molecule, by activated-MSC conditioned medium (CM). We used multivariable linear regression (MLR) and analysis of covariance to determine differences in functions of optimal factors on monolayer MSCs and alginate-encapsulated MSCs (eMSCs). The screen revealed that LPS and IL-1β potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-α. Activation by LPS and IL-1β together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-α. MLR and covariate analysis revealed that macrophage TNF-α was strongly dependent on the MSC activation factor, PGE2 level, and macrophage donor but not MSC culture format (monolayer versus encapsulated). The results demonstrate the feasibility and utility of using statistical approaches for higher throughput cell analysis. This approach can be extended to develop activation schemes to maximize MSC and MSC-biomaterial functions prior to transplantation to improve MSC therapies., (© 2015 American Institute of Chemical Engineers.)

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies - lidocaine, procaine, ropivacaine and bupivacaine - was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)- α and interferon (IFN)- γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF- α /IFN- γ -stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNF α /IFN γ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.

At least 70 US cities have now introduced ciclovías-large-scale street closures to promote physical activity-joining numerous other cities worldwide that have implemented ciclovías in efforts to improve population health. We assessed the impact of Summer Streets, a New York City program in which 6.9 contiguous miles of urban streets were closed to traffic and opened for walking, cycling, and group activities, such as dancing and yoga, on population physical activity levels. Screen line counts were used to estimate attendance, and a street intercept survey was conducted to assess demographic characteristics of participants, baseline adherence to physical activity recommendations, and type and duration of physical activity at Summer Streets. In addition, a traffic study was used to determine if there were vehicular traffic delays as a result of the program. About 50,000 people participated in Summer Streets; among participating New Yorkers, bicyclists averaged 6.7 miles, runners 4.3 miles, and walkers 3.6 miles, equivalent to 72-86 min of moderate physical activity. Among New Yorkers attending Summer Streets, 24 % reported that they did not routinely engage in moderate- or vigorous-intensity physical activity. These non-routine exercisers engaged in the equivalent of 26-68 min of moderate-intensity physical activity at Summer Streets. Summer Streets served as an enticement for New Yorkers, including those who did not ordinarily meet physical activity recommendations, to engage in physical activity. There were no significant vehicular traffic delays during the program.

To assess the public's propensity for allergic contact dermatitis (ACD), many alternatives to in vivo chemical screening have been developed which generally incorporate a small panel of cell surface and secreted dendritic cell biomarkers. However, given the underlying complexity of ACD, one cell type and limited cellular metrics may be insufficient to predict contact sensitizers accurately. To identify a molecular signature that can further characterize sensitization, we developed a novel system using RealSkin, a full thickness skin equivalent, in co-culture with MUTZ-3 derived Langerhan's cells. This system was used to distinguish a model moderate pro-hapten isoeugenol (IE) and a model strong pre-hapten p-phenylenediamine (PPD) from irritant, salicylic acid (SA). Commonly evaluated metrics such as CD86, CD54, and IL-8 secretion were assessed, in concert with a 27-cytokine multi-plex screen and a functional chemotaxis assay. Data were analyzed with feature selection methods using ANOVA, hierarchical cluster analysis, and a support vector machine to identify the best molecular signature for sensitization. A panel consisting of IL-12, IL-9, VEGF, and IFN-γ predicted sensitization with over 90% accuracy using this co-culture system analysis. Thus, a multi-metric approach that has the potential to identify a molecular signature may be more predictive of contact sensitization., (Copyright © 2014. Published by Elsevier Ltd.)

Limit setting is an intervention that is frequently used by mental health nurses. However, limit setting is poorly conceptualized, its purpose is unclear, and there are few evidence-based guidelines to assist nurses to set limits in a safe and effective manner. What is known is that the manner in which nurses set limits influences patients' perceptions of the interactions and their emotional and behavioural responses. In this qualitative study, 12 nurses and 12 patients participated in personal, semistructured interviews that aimed to explore limit setting and to propose principles to guide practice. The findings suggested that: (i) limit setting is important to safety in mental health hospitals; (ii) engaging patients in an empathic manner is necessary when setting limits (when nurses engage in an empathic manner, the therapeutic relationship is more likely to be preserved and the risk of aggressive responses is reduced); and (iii) an authoritative (fair, respectful, consistent, and knowledgeable), rather than authoritarian (controlling and indifferent), limit-setting style enhances positive outcomes with regards to adherence, reduced likelihood of aggression, and preservation of the therapeutic relationship. In conclusion, a limit-setting style characterized by empathic responding and an authoritative, rather than authoritarian interpersonal, style is recommended. Elucidating the components of this style is critical for effective training and best practice of mental health nurses, and to reduce aggressive responses from limit setting., (© 2013 Australian College of Mental Health Nurses Inc.)

Background: Surgery for pelvic organ prolapse is a common surgical procedure. There is little research studying post-operative pain, contrasting with extensive literature on pain after childbirth trauma which shows that suture method has a significant effect on pain. We designed a protocol for a trial comparing suture techniques and post-operative pain, and conducted a pre-protocol pilot to inform our trial design., Methods: Routine surgical data, post-operative pain scores (from 10 cm Visual Analogue Scales, VAS) and analgesic requirements were obtained from the notes of a cohort of women undergoing vaginal prolapse surgery. Median VAS scores at 4, 12 and 24 hours were compared by suture material used and method of closure (single continuous suture or interrupted sutures). The women whose data were obtained were invited to attend focus groups of up to six people in the twelve weeks following surgery. A semi structured question guide was used, and interviews were recorded, transcribed anonymously and analysed using the constant comparative method of grounded theory. Ethical approval was not sought because formal pre-protocol work is exempt for UK ethical requirements, but formal written consent on standard forms was obtained for publishing outcomes and anonymised comments from participants., Results: Complete VAS data and operative details were available from 41 women. Surgery was completed using absorbable polyglactin sutures with continuous suturing (17 women) or interrupted sutures (24 women). Pain scores at 4 and 12 hours were similar; pain at 24 hours was greater in the women with interrupted sutures (median VAS 3 (range 0-8) versus 1.5 (0-8) (p = 0.0513). Analgesic needs were similar. Two focus groups (nine participants) revealed that women regarded post-operative pain as insignificant and not a topic worthy of formal research. It was apparent that the use, and especially removal of, vaginal packs was a practice associated with pain which women remembered as a significant part of their post-operative experience., Conclusions: Formal pre-protocol work is informative; we found a moderate difference in our proposed outcomes, suggesting a trial was feasible but women themselves were unconvinced of the need for formal research into pain following vaginal surgery.

Torg and Winchester syndromes and patients reported by Al-AqeelSawairi as well as nodulosis-arthropathy-osteolysis (NAO) patients, patients with multicentric NAO share autosomal recessive inheritance. The common presenting symptomatology includes progressive osteolysis chiefly affecting the carpal, tarsal and interphalangeal joints. Here, we report a patient with Torg syndrome. Torg syndrome is caused by homozygous or compound heterozygous mutations in the matrix metalloproteinase 2 (MMP2) gene. MMP2 codes for a gelatinase that cleaves type IV collagen, a major component of basement membrane. The clinical presentation of our patient included moderate osteolysis of the small joints of the hands and knees, hirsutism, nodulosis sparing the palms and soles, corneal opacities and mild facial dysmorphism without gum hypertrophy. Genetic analysis showed that the patient was homozygous for a novel base variant c538 G>A (p.D180N) in the MMP2 gene. Both parents were carriers of the same mutated variant. Our patient had some previously unreported endocrine manifestations such as premature thelarche and elevated follicle-stimulating hormone levels.

Background & Aims: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes., Methods: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured., Results: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion., Conclusions: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Dendritic cells are the principal antigen presenting cells that are responsible for acquiring and transporting antigen from the peripheral tissue to the secondary lymphoid tissue. There they present it to T cells which ultimately initiate an antigen specific immune response. In vivo , the migration of dendritic cells (DCs) and T cell activation are intimately linked. However, ex vivo systems that facilitate integrated evaluation of DC chemotaxis and resulting T cell activation by migrated DCs are lacking. In this work, we have developed a microfabricated platform that integrates DC chemotaxis with T cell activation. The basic design of the microdevice includes two layers of PDMS, with the top layer comprising the chemotaxis compartment and the bottom layer containing a T cell compartment. In the chemotaxis compartment, the DCs are subjected to a chemokine gradient, and their migratory response is evaluated. In the T cell compartment, rapid DC-induced activation of T cells is evaluated by measuring the level of calcium in T cells. We demonstrate the efficacy of our approach by evaluating the integrated response of mature DCs, whereby the overall T cell activation response is governed both by the chemotaxis and the T cell activation potential of mature DCs relative to immature DCs. Our system provides a powerful platform for systematically probing various aspects of antigen induced immune responses - DC maturation, migration and T cell activation - in an integrated fashion.

Venipuncture is pivotal to a wide range of clinical interventions and is consequently the leading cause of medical injury in the U.S. Complications associated with venipuncture are exacerbated in difficult settings, where the rate of success depends heavily on the patient's physiology and the practitioner's experience. In this paper, we describe a device that improves the accuracy and safety of the procedure by autonomously establishing a peripheral line for blood draws and IV's. The device combines a near-infrared imaging system, computer vision software, and a robotically driven needle within a portable shell. The device operates by imaging and mapping in real-time the 3D spatial coordinates of subcutaneous veins in order to direct the needle into a designated vein. We demonstrate proof of concept by assessing imaging performance in humans and cannulation accuracy on an advanced phlebotomy training model.

Aims: The concept of the "learning curve" is a term that has become increasingly prevalent in medical literature. Using a unique Female Urology fellowship program running over the last 3 years, we set out to better define the learning process for mid-urethral slings., Methods: We examined surgical outcomes for six urology trainees who participated in the 6-month program from 2006 to 2011. We identified all retropubic mid-urethral sling procedures they had performed. Demographics included age, BMI, and smoking status. Outcomes focused on complication rates, as well as a subjective patient assessment. Analysis was by the cumulative sum method., Results: Six trainees performed 187 retropubic slings during their fellowships. Mean age was 54 (SD ± 12.7), mean BMI was 29.5 (SD ± 5.5). One hundred sixty-five (88%) patients only underwent a mid-urethral tape with 22 (12%) undergoing a concomitant procedure. There were 5 cases of bladder perforation, 1 case of urethral injury, 25 cases of voiding dysfunction, and 8 cases of mesh exposure. One hundred sixty-seven out of 180 patients reported a cure or improvement. All complications occurred in the first 4 months of training. CUSUM analysis of voiding dysfunction showed that four out of six trainees did not reach the expected incidence of voiding dysfunction within the completed fellowship. Bladder perforation showed a similar trend., Conclusion: CUSUM analysis is an underused tool for the analysis of surgical competence. The learning curve for retropubic sling surgery is variable and may be longer than is often acknowledged. We suggest the focus of surgical training should move away from absolute numbers to look at training in an individualized manner., (Copyright © 2013 Wiley Periodicals, Inc.)

Forensic mental health nursing is a recognized field of nursing in most countries. Despite a growing body of literature describing aspects of practice, no publication has been found that captures the core knowledge, skills, and attitudes of forensic mental health nurses. One group of nurses in Australia have pooled their knowledge of relevant literature and their own clinical experience and have written standards of practice for forensic mental health nursing. This paper identifies the need for standards, provides a summary of the standards of practice for forensic mental health nurses, and concludes with how these standards can be used and can articulate to others the desired and achievable level of performance in the specialty area.

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46, and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype, and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

There is a large emphasis within the pharmaceutical industry to provide tools that will allow early research and development groups to better predict dose ranges for and metabolic responses of candidate molecules in a high throughput manner, prior to entering clinical trials. These tools incorporate approaches ranging from PBPK, QSAR, and molecular dynamics simulations in the in silico realm, to micro cell culture analogue (CCAs)s in the in vitro realm. This paper will serve to review these areas of high throughput predictive research, and highlight hurdles and potential solutions. In particular we will focus on CCAs, as their incorporation with PBPK modeling has the potential to replace animal testing, with a more predictive assay that can combine multiple organ analogs on one microfluidic platform in physiologically correct volume ratios. While several advantages arise from the current embodiments of CCAS in a microfluidic format that can be exploited for realistic simulations of drug absorption, metabolism and action, we explore some of the concerns with these systems, and provide a potential path forward to realizing animal-free solutions. Furthermore we envision that, together with theoretical modeling, CCAs may produce reliable predictions of the efficacy of newly developed drugs.

Osteolysis syndromes are rare hereditary disorders characterized by destruction and resorption of affected bones. The current study adds three new patients from two unrelated consanguineous families with a severe form of inherited osteolysis. Clinical examination, radiological, biochemical, ultrastructural and molecular studies were conducted. Clinical and radiological studies suggested the diagnosis of Torg-Winchester syndrome. The three affected patients were homozygous for novel MMP2 gene mutations which confirmed the diagnosis. Our patients are the first to be reported from Egypt thus, supporting the pan ethnic nature of the disease.

Reactive oxygen species (ROS) are generated as by-products of many cellular processes and can modulate cellular signaling pathways. However, high ROS levels are toxic; thus, intracellular ROS need to be tightly controlled. Therefore, cells use a group of antioxidant molecules and detoxifying enzymes that remove or detoxify reactive species. We found that the level of the antioxidant glutathione is greatly increased in human cytomegalovirus (HCMV)-infected cells due to activation of glutathione synthetic enzymes. In addition, our data suggest that virus-specific mechanisms are used to induce the expression of target antioxidant and detoxifying enzymes critical for the success of the infection. As a result of this virus-induced anti-ROS environment, key signaling kinases, such as the mammalian target of rapamycin (mTOR) kinase in mTOR complex 1 (mTORC1), are protected from inhibition by exogenous hydrogen peroxide (H(2)O(2)). In this regard, we found that phosphorylation of mTOR kinase at serine 2448 (suggested to be activating) was maintained during infection even under ROS stress conditions that inhibited it in uninfected cells. We also show that AMP-dependent kinase (AMPK)-mediated phosphorylation of serine 792 of raptor, the specificity subunit of mTORC1, increases in infected cells after H(2)O(2) treatment. This phosphorylation is normally inhibitory for mTORC1. However, in infected cells this did not result in inhibition of mTORC1 activity, suggesting that inhibitory effects of raptor phosphorylation are circumvented. Overall, our data suggest that HCMV utilizes virus-specific mechanisms to activate a variety of means to protect the cell and mTORC1 from the effects of ROS.

Previously we have used human hepatocytes in suspension by measuring the parent loss for prediction of metabolic clearance according to a 1(st)-order kinetic model. In this study, we evaluated a novel integrative approach using plated human hepatocytes to include both uptake processes and metabolism in a single assay. Test articles were added in the medium, and the intrinsic clearance was determined based on the disappearance of the parent compound from the medium. Three different methods: direct, well-stirred, and parallel tube were tested for scaling purpose. With 30 randomly selected compounds with clinical clearance data, the scaled clearance showed reasonable linear correlation with r(2) values of 0.67, 0.72, and 0.70 for direct, well-stirred and parallel tube models, respectively. When human serum albumin (HSA) was added to the incubation medium a shift to lower in vitro clearance was observed for most of the compounds, suggesting that protein binding may have an effect on the metabolic clearance. In the presence of 4% of HSA, which is equivalent to the albumin concentration in the human plasma, the in vitro clearance data have the best prediction of human clearance when using the well-stirred method, followed by the parallel tube method and direct method. This study demonstrates the utility of using plated human hepatocyte as an integrated system for the prediction of human metabolic clearance. In addition, evaluation of the protein binding shift in the clearance showed that a significant number of compounds may not follow the equilibrium assumption according to the well-stirred model.

Integral to the discovery of new pharmaceutical entities is the ability to predict in vivo pharmacokinetic parameters from early stage in vitro data generated prior to the onset of clinical testing. Within the pharmaceutical industry, a whole host of assay methods and mathematical models exist to predict the in vivo pharmacokinetic parameters of drug candidates. One of the most important pharmacokinetic properties of new drug candidates predicted from these methods and models is the hepatic clearance. Current methods, while useful, are still limited in their predictive efficacy. In order to address this issue, we have established a novel microfluidic in vitro culture system, the patented HmuREL device. The device comprises multiple compartments that are designed to be proportional to the physiological architectures and enhanced with the consideration of flow. Here we demonstrate the functionality of the liver-relevant chamber in the HmuREL device, and the feasibility of utilizing our system for predicting hepatic clearance. Cryopreserved human hepatocytes from a single donor were seeded within the HmuREL device to predict the in vivo hepatic clearance (CL(H)) of six marketed model compounds (carbamazepine, caffeine, timolol, sildenafil, imipramine, and buspirone). The intrinsic clearance rates from static culture controls, as well as clearance rates from the HmuREL device were subsequently compared to in vivo data available from the literature.

The generation of a large number of fully functional hepatocytes from a renewable cell source can provide an unlimited resource for bioartificial liver devices and cell replacement therapies. We have established a directed differentiation system using sodium butyrate treatment to generate an enriched population of hepatocyte-like cells from embryonic stem cells. A metabolic analysis of the hepatocyte populations revealed glycolytic and mitochondrial phenotypes similar to mouse hepatoma cells, implying that these cells represent an immature hepatocyte phenotype. To mediate further differentiation, S-NitrosoAcetylPenicillamine (SNAP), a nitric oxide donor, was utilized to induce mitochondrial development in the precursor populations. A comparative analysis of the different treated populations showed that 500microM SNAP treatment resulted in the generation of an enriched population of metabolically mature hepatocyte-like cells with increased differentiated function. Specifically, 500microM SNAP treatment increased glucose consumption, lactate production rates, mitochondrial mass, and potential as compared to untreated populations. In addition, functional analysis revealed that intracellular albumin content, urea secretion rates, and cytochrome P450 7a1 promoter activity were increased in the treated population. The methodology described here to generate an enriched population of metabolically and functionally mature hepatocyte-like cells may have potential implications in drug discovery and regenerative medicine.

The inflammatory response initiated upon burn injury is also associated with extensive metabolic adjustments. While there is a significant body of literature on the characterization of these changes at the metabolite level, little is known on the mechanisms of induction, especially with respect to the role of gene expression. We have comprehensively analyzed changes in gene expression in rat livers during the first 7 d after 20% total body surface area burn injury using Affymetrix microarrays. A total of 740 genes were significantly altered in expression at 1, 2, 4, and 7 d after burn injury compared to sham-burn controls. Functional classification based on gene ontology terms indicated that metabolism, transport, signaling, and defense/inflammation response accounted for more than 70% of the significantly altered genes. Fisher least-significant difference post-hoc testing of the 740 differentially expressed genes indicated that over 60% of the genes demonstrated significant changes in expression either on d 1 or on d 7 postburn. The gene expression trends were corroborated by biochemical measurements of triglycerides and fatty acids 24 h postburn but not at later time points. This suggests that fatty acids are used, at least in part, in the liver as energy substrates for the first 4 d after injury. Our data also suggest that long-term regulation of energy substrate utilization in the liver following burn injury is primarily at the posttranscriptional level. Last, relevance networks of significantly expressed genes indicate the involvement of key small molecules in the hepatic response to 20% total body surface area burn injury.

Integral to the development of embryonic stem cell therapeutic strategies for hepatic disorders is the identification and establishment of a controllable hepatic differentiation strategy. In order to address this issue we have established an alginate microencapsulation approach which provides a means to modulate the differentiation process through changes in key encapsulation parameters. We report that a wide array of hepatocyte specific markers is expressed by cells differentiated during a 23-day period within an alginate bead microenvironment. These include urea and albumin secretion, glycogen storage, and cytochrome P450 transcription factor activity. In addition, we demonstrate that cellular aggregation is integral to the control of differentiation within the bead environment and this process is mediated by the E-cadherin protein. The temporal expression of surface E-cadherin and hepatocyte functional expression occur concomitantly and both cellular aggregation and albumin synthesis are blocked in the presence of anti E-cadherin immunoglobulin. Furthermore, by establishing a compartmental model of differentiation, which incorporates this aggregation phenomenon, we can optimize key encapsulation parameters., (Copyright 2007 Wiley Periodicals, Inc.)

Background: Thermal injury is among the most severe forms of trauma and its effects are both local and systemic. Response to thermal injury includes cellular protection mechanisms, inflammation, hypermetabolism, prolonged catabolism, organ dysfunction and immuno-suppression. It has been hypothesized that gene expression patterns in the liver will change with severe burns, thus reflecting the role the liver plays in the response to burn injury. Characterizing the molecular fingerprint (i.e., expression profile) of the inflammatory response resulting from burns may help elucidate the activated mechanisms and suggest new therapeutic intervention. In this paper we propose a novel integrated framework for analyzing time-series transcriptional data, with emphasis on the burn-induced response within the context of the rat animal model. Our analysis robustly identifies critical expression motifs, indicative of the dynamic evolution of the inflammatory response and we further propose a putative reconstruction of the associated transcription factor activities., Results: Implementation of our algorithm on data obtained from an animal (rat) burn injury study identified 281 genes corresponding to 4 unique profiles. Enrichment evaluation upon both gene ontologies and transcription factors, verifies the inflammation-specific character of the selections and the rationalization of the burn-induced inflammatory response. Conducting the transcription network reconstruction and analysis, we have identified transcription factors, including AHR, Octamer Binding Proteins, Kruppel-like Factors, and cell cycle regulators as being highly important to an organism's response to burn response. These transcription factors are notable due to their roles in pathways that play a part in the gross physiological response to burn such as changes in the immune response and inflammation., Conclusion: Our results indicate that our novel selection/classification algorithm has been successful in selecting out genes with play an important role in thermal injury. Additionally, we have demonstrated the value of an integrative approach in identifying possible points of intervention, namely the activation of certain transcription factors that govern the organism's response.

Erythroid Kruppel-like factor (EKLF, KLF1) plays an important role in definitive erythropoiesis and beta-globin gene regulation but failure to rectify lethal fetal anemia upon correction of globin chain imbalance suggested additional critical EKLF target genes. We employed expression profiling of EKLF-null fetal liver and EKLF-null erythroid cell lines containing an inducible EKLF-estrogen receptor (EKLF-ER) fusion construct to search for such targets. An overlapping list of EKLF-regulated genes from the 2 systems included alpha-hemoglobin stabilizing protein (AHSP), cytoskeletal proteins, hemesynthesis enzymes, transcription factors, and blood group antigens. One EKLF target gene, dematin, which encodes an erythrocyte cytoskeletal protein (band 4.9), contains several phylogenetically conserved consensus CACC motifs predicted to bind EKLF. Chromatin immunoprecipitation demonstrated in vivo EKLF occupancy at these sites and promoter reporter assays showed that EKLF activates gene transcription through these DNA elements. Furthermore, investigation of EKLF target genes in the yolk sac led to the discovery of unexpected additional defects in the embryonic red cell membrane and cytoskeleton. In short, EKLF regulates global erythroid gene expression that is critical for the development of primitive and definitive red cells.

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies., (Copyright 2005 Wiley Periodicals, Inc.)

In this study, four inhibitor of apoptosis genes (iaps) in the genome of Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) that are homologous to iap-1, iap-2, iap-3 and iap-4 genes of other baculoviruses have been identified. All four iap genes were sequenced and the iap-1 and iap-2 genes were shown to be functional inhibitors of apoptosis. The iap-1, iap-2 and iap-3 genes contain two baculovirus apoptosis inhibitor repeat motifs and a C(3)HC(4) RING finger-like motif. The activity of the iap genes was tested by transient expression in Spodoptera frugiperda (Sf-21) cells treated with the apoptosis-inducing agents actinomycin D, cycloheximide, anisomycin, tumour necrosis factor-alpha and UV light. The iap-2 gene prevented apoptosis induced by all agents tested, indicating activity towards a conserved component(s) of multiple apoptotic pathways. However, the iap-2 gene was unable to function in the absence of a gene immediately upstream of iap-2 that has homology to the orf69 gene of Autographa californica MNPV. The use of a CMV promoter rescued the apoptosis inhibition activity of the iap-2 gene, indicating that the upstream orf69 homologue is associated with expression of iap-2. The iap-1 gene was able to delay the onset of apoptosis caused by all of the induction agents tested but, unlike iap-2, was unable to prevent the development of an apoptotic response upon prolonged exposure of cells to the apoptosis induction agents. No anti-apoptotic activity was observed for the iap-3 and iap-4 genes of EppoMNPV.

Basal cell carcinoma of the skin (BCC) is the most common cancer worldwide. Unlike most other human malignancies, BCCs rarely metastasise. In this investigation, we show that the serine protease urokinase plasminogen activator (u-PA), which is causally involved in metastasis, is expressed at lower levels in BCCs compared to other skin cancers, such as squamous-cell carcinomas (SCCs) or malignant melanomas. Similarly, the u-PA receptor as well as the inhibitor PAI-1 were present at lower levels in BCCs relative to both SCCs and melanomas. In contrast to u-PA, tissue-plasminogen activator, which is not thought to be involved in metastasis, was present at similar levels in the different types of skin lesion investigated. We conclude that the failure of BCCs to metastasise may at least be partially related to low expression of components of the u-PA system., (Copyright 2000 Wiley-Liss, Inc.)

The estrogen receptor (ER) is the most useful marker currently available for breast cancer, being used both to predict response to therapy and assess prognosis. Recently, a new form of the ER, known as ER-beta, was identified. In this preliminary study we show that ER-beta mRNA was expressed less frequently in breast cancers than ER-alpha. ER-alpha but not ER-beta levels correlated with ER protein as determined by ELISA. We conclude that ER-beta is expressed in approximately 50% of breast cancers but it does not appear to be detected by a widely available ELISA.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.

Background: Controversy persists over the educational value of student clerkship clinical activities., Methods: Students (109) from the class of 1995 recorded their clinical experiences in a logbook during their surgical clerkship at one of four affiliated teaching hospitals. The influence of clinical experience on examination scores and on correlations between prerotation and postrotation examination performance was determined., Results: Between sites, marked variation in clinical experience was observed but postrotation scores were similar. High-volume experience in emergency admissions and feedback was associated with better objective structured clinical examination (OSCE) performance, but high-volume outpatient clinic experience was associated with less satisfactory OSCE performance. Correlations between prerotation examination performance and the OSCE was increased by feedback on emergency and elective admissions, in a positive and negative direction, respectively., Conclusions: These data show that surgical clerks' clinical skills were enhanced by an increased volume of some but not all clinical experiences and that feedback does not necessarily enhance performance. These data suggest that both the volume of clinical experience and the quality of feedback should be carefully monitored by surgical clerkship directors.

Urokinase plasminogen activator (uPA) is a multifunctional protein involved in both extracellular proteolysis and signal transduction. uPA usually mediates its actions while attached to a membrane-bound receptor, termed uPAR. In this study, uPA and its receptor were measured at both protein and mRNA levels in breast cancer. At both levels, concentrations of uPA were significantly correlated with those for uPAR. uPA levels also correlated significantly with cathepsin B and cathepsin D but not with cathepsin L, MMP-8 or MMP-9 levels. Irrespective of the cut-off point used (e.g., median, tertile or quartile values), uPA was a significant prognostic marker for breast cancer.