SMART Family and Friends: Feasibility and outcomes of a video-conference delivered intervention for families impacted by another's methamphetamine use.

INTRODUCTION: Families affected by another's substance use, including methamphetamine, experience harms to their mental and physical health. Yet, research has paid little attention to support and service needs of this population. This pilot study examines the feasibility and outcomes of SMART Family and Friends, a video-conference-delivered mutual-support group targeting families affected by another's methamphetamine use. METHODS: Recruitment for this study occurred between March-October 2021 via the SMART Recovery Australia website. Participants were English-speaking Australian residents, ≥18 years, affected by another's methamphetamine use, interested in participating in a manualised eight-module group delivered via video-conferencing. Feasibility was evaluated by attendance rates, participant satisfaction, fidelity ratings, and semi-structured interviews. Measures of distress, quality of life, and family functioning assessed outcomes at baseline and one-month post-treatment conclusion. RESULTS: Forty-three participants commenced SMART Family and Friends groups. 84 % (n = 36) completed ≥4 modules, 67 % (n = 29) completed ≥6, and 42 % (n = 18) completed all 8 modules. Participant satisfaction (M = 4.32, SD = 0.66, out of 5) and facilitator fidelity (>94 % for all modules) were high. A within-group analysis, without comparison condition demonstrated significant improvements in psychological distress (d = 0.38), family impact (d = 0.64), family strain symptoms (d = 0.48), and total family burden (d = 0.69) post-treatment. Qualitative findings illustrated the benefits and challenges of the video-conference-delivered group, as well as recommendations for improvement. CONCLUSIONS: Results provide initial support for the feasibility and positive outcomes of the SMART Family and Friends program. These findings demonstrate the successful provision of a mutual-support group for affected families delivered via video-conferencing, and merit further sufficiently powered randomised-control-trials to evaluate efficacy.

Nursing students' attitudes, behaviour, and knowledge toward smoking cessation: Results from a descriptive survey at a regional university.

BACKGROUND: Smoking is the largest single cause of lifestyle-related preventable morbidity and mortality. Nurses form the largest cohort of health professionals and are strategically placed to implement smoking cessation interventions. However their capacity is underutilised, particularly in rural and remote areas in countries such as Australia where the incidence of smoking in is higher than average and access to healthcare is limited. One strategy to address the underutilisation of nurses in smoking cessation interventions is to include training in the university/college nursing curriculum. To effectively implement this training, it is vital to have an in-depth knowledge of student nurses' attitudes towards smoking including the role of healthcare professionals in smoking cessation, their smoking behaviour and that of their peers, and knowledge regarding smoking cessation techniques and resources. OBJECTIVES: Investigate nursing students' attitudes, behaviour, and knowledge towards smoking cessation, determine the impact of demographics and educational experienced on these, and develop recommendations for future research and educational practice. DESIGN: Descriptive survey. PARTICIPANTS: Non-probability sample of undergraduate nursing students (n = 247) from a regional Australian university. RESULTS: Significantly more participants had tried smoking cigarettes than had not (p = 0.026). There were no significant relationships between gender and smoking (p = 0.169) or e-cigarette use (p = 0.200), but a significant relationship was found between age and smoking status where older participants (48-57 years) were more likely to smoke (p < 0.001). Most participants (70 %) were supportive of public health measures to reduce cigarette smoking but felt that they lacked specific knowledge to assist their patients to cease smoking. CONCLUSIONS: Within education there needs to be an emphasis on the central role that nurses play in smoking cessation with a greater focus on training nursing students about smoking cessation strategies and resources. There is also a need to ensure that students know it falls within their duty of care to address smoking cessation with patients.

Social prescribing of nature therapy for adults with mental illness living in the community: A scoping review of peer-reviewed international evidence.

Social prescribing of nature therapy "green social prescribing" facilitates access to local nature-based activities that improve biopsychosocial wellbeing outcomes, are affordable, accessible, and can be adapted to context. These are becoming increasingly popular and gray literature is emerging, however, peer-reviewed scientific evidence is exiguous. This scoping review aimed to identify and critique peer-reviewed evidence for green social prescribing interventions and develop recommendations for research and clinical practice. Included studies were published in peer-reviewed journals in English on/after 1 January 2000. Participants were community-living adults with mental illness; Intervention was any green social prescribing program; Comparator was not restricted/required; Outcomes were any biopsychosocial measures; and any/all Study Designs were included. Twelve databases were searched on 15 October 2022; these were Academic Search Premier, APA PsycArticles, APA PsycINFO, CINAHL, Cochrane Library, Google Scholar, JSTOR, ProQuest, PubMed, Science Direct, Scopus, and Web of Science. The Mixed Methods Appraisal Tool was used to assess quality. Seven publications describing 6 unique studies (5 UK, 1 Australia) were identified including 3 mixed-methods, 2 qualitative, and 1 RCT. Participants included 334 adults (45% female, aged 35-70 years); sample sizes ranged from 9 to 164. All studies showed improvements in biopsychosocial wellbeing, and participants from most studies (n = 5) reported increased connection to the earth and intention to further access nature. Participant demographics and diagnoses were poorly reported, and intervention activities and assessments varied considerably. However, MMAT scores were good overall suggesting these studies may reliably demonstrate intervention outcomes. We conclude that socially prescribed nature therapy can improve biopsychosocial wellbeing and is a potentially important intervention for mental illness. Recommendations for research and clinical practice are provided.

Trauma informed care education for midwives: An integrative review.

BACKGROUND: Nearly half of new mothers describe their childbirth as traumatic. Perinatal trauma impacts both short and long-term biopsychosocial outcomes for mother and child. Midwife trauma-informed care education and practice is essential to mitigate this risk. OBJECTIVE: This review aimed to identify and describe the nature and extent of trauma informed care education provided for midwives and midwifery students. DESIGN: An integrative review. METHODS: Five databases (Medline, Embase, CINAHL, Psycinfo, and Emcare) were searched to identify primary research regarding trauma informed care education for midwives and midwifery students. Quality appraisal was conducted using the Mixed Methods Appraisal Tool. RESULTS: Three papers were identified. None of the papers were midwifery focused, with midwives representing a small proportion of the participants. Most midwives reported receiving no previous trauma informed care education and lacked confidence to provide quality care to women with lived trauma. Midwives reported trauma informed care education as essential and relevant for providing quality practice. Improvements in knowledge, skills and attitudes was demonstrated following trauma informed care education. More in-depth content and content delivered in multiple ways were recommended. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Midwives are well placed to deliver trauma informed care. Trauma informed care education for midwives is limited. Given the impact of perinatal trauma, further trauma informed care education and research is paramount.

N-acetylcysteine (NAC) for methamphetamine dependence: A randomised controlled trial.

BACKGROUND: Methamphetamine dependence is a significant global health concern for which there are no approved medications. The cysteine prodrug, N-acetylcysteine (NAC), has been found to ameliorate glutamate dysregulation in addiction, and to reduce craving for methamphetamine and other drugs. We evaluated the efficacy and safety of NAC as a pharmacotherapy for methamphetamine dependence. METHODS: A parallel double-blind randomised placebo-controlled trial of people dependent on methamphetamine recruited from Geelong, Melbourne and Wollongong, Australia, between July 2018 and December 2019. Participants were randomised to receive either 12 weeks of oral NAC (2400 mg/day) or matched placebo, delivered as a take-home medication. The primary outcome was methamphetamine use, measured in two ways: (a) change in days of use in the past 4 weeks from baseline to weeks 4, 8 and 12, assessed using the Timeline Followback; and (b) methamphetamine-positive oral fluid samples taken weekly. Analyses were intention-to-treat and based on imputed data. Secondary outcomes were craving, severity of dependence, withdrawal severity and psychiatric symptoms (depression, suicidality, hostility and psychotic symptoms). Significance levels were p < 0.025 for primary outcomes and p < 0.01 for secondary outcomes. Adverse events were compared between groups by system organ class. The study was prospectively registered, ACTRN12618000366257. RESULTS: Participants (N = 153; 59% male, mean [SD] age 38 [8]) were randomised to placebo (n = 77) or NAC (n = 76). Both groups had a median (IQR) of 24 (15-28) days of methamphetamine use in the 4 weeks prior to baseline. Both groups significantly reduced methamphetamine use (mean [SE] reduction of 7.3 [1.2]) days for placebo, 6.8 [1.2] for NAC) but NAC did not reduce days of methamphetamine use more than placebo (group difference of 0.5 days, 97.5% CI -3.4-4.3). There was no significant effect of NAC on methamphetamine-positive oral fluid samples (placebo 79%, NAC 76%; mean difference -2.6, 97.5% CI -12.6-7.4). NAC did not significantly reduce craving, severity of dependence, withdrawal, suicidality, depression, hostility or psychotic symptoms relative to placebo. Adverse events did not differ significantly between placebo and NAC groups. INTERPRETATION: These findings suggest that take-home oral NAC has no significant effect on methamphetamine use or most clinically related outcomes amongst people who are dependent on the drug.

Social Prescribing for Individuals Living with Mental Illness in an Australian Community Setting: A Pilot Study.

Social prescribing, also known as "community referral", is a means of referring individuals living in the community to existing local non-clinical health, welfare, and social support services. International evidence demonstrates that social prescribing improves biopsychosocial quality of life, and burden on health services. Australia's first social prescribing pilot program for individuals with mental illness (mood and psychotic spectrum disorders) was implemented in Sydney in 2016/2017; this study evaluates that program. Participants included 13 adults who were assessed at baseline and six-month follow-up. Outcomes included self-perceived quality of life, welfare needs, health status, loneliness, social participation, and economic participation. Results indicate significant improvements in quality of life and health status. This pilot program demonstrates that social prescribing may improve participant outcomes. It fits well within Australian health policy and funding models which focus on bolstering community care, and may be scalable, particularly in geographically isolated communities.

Clinical and demographic characteristics of people who smoke versus inject crystalline methamphetamine in Australia: Findings from a pharmacotherapy trial.

INTRODUCTION AND AIMS: There has been a rapid increase in smoking crystalline methamphetamine in Australia. We compare the clinical and demographic characteristics of those who smoke versus inject the drug in a cohort of people who use methamphetamine. DESIGN AND METHODS: Participants (N = 151) were dependent on methamphetamine, aged 18-60 years, enrolled in a pharmacotherapy trial for methamphetamine dependence, and reported either injecting (n = 54) or smoking (n = 97) methamphetamine. Measures included the Timeline Followback, Severity of Dependence Scale, Amphetamine Withdrawal Questionnaire, Craving Experience Questionnaire and the Brief Psychiatric Rating Scale (symptoms of depression, hostility, psychosis and suicidality). Simultaneous regression was used to identify independent demographic correlates of smoking methamphetamine and to compare the clinical characteristics of participants who smoked versus injected. RESULTS: Compared to participants who injected methamphetamine, those who smoked methamphetamine were younger and less likely to be unemployed, have a prison history or live alone. Participants who smoked methamphetamine used methamphetamine on more days in the past 4 weeks than participants who injected methamphetamine (26 vs. 19 days, P = 0.001); they did not differ significantly in their severity of methamphetamine dependence, withdrawal, craving or psychiatric symptoms (P > 0.05). After adjustment for demographic differences, participants who smoked had lower craving [b (SE) = -1.1 (0.5), P = 0.021] and were less likely to report psychotic symptoms [b (SE) = -1.8 (0.7), P = 0.013] or antidepressant use [b (SE) = -1.1 (0.5), P = 0.022]. DISCUSSION AND CONCLUSIONS: Smoking crystalline methamphetamine is associated with a younger less marginalised demographic profile than injecting methamphetamine, but a similarly severe clinical profile.

Stepped-Wedge Cluster Randomised Trial of Social Prescribing of Forest Therapy for Quality of Life and Biopsychosocial Wellbeing in Community-Living Australian Adults with Mental Illness: Protocol.

Social Prescribing (SP) involves linking individuals with mental illness to local health and welfare services to improve quality of life (QoL) and biopsychosocial wellbeing. SP programs address psychosocial wellbeing by linking individuals to group activities. Forest Therapy (FT) is a group nature walk with prescribed activities that promote mindfulness, relaxation, and shared experience. Improvements in psychological and physical wellbeing have been demonstrated in FT, but psychosocial impacts have not been widely investigated. This study will implement an SP FT intervention and assess the impacts on QoL and biopsychosocial wellbeing. Participants will include 140 community-living adults with mental illness at Sydney/Gold Coast, Australia. A stepped-wedge cluster randomised design will be used; each participant will complete a 10-week control period followed by a 10-week FT intervention. Weekly 90-min FT sessions will be conducted in groups of 6-10 in local nature reserves. Validated tools will measure self-report QoL and biopsychosocial wellbeing pre- and post-control and intervention periods, and 5-week follow-up. Blood pressure and heart rate will be measured pre- and post-FT sessions. Hypothesised outcomes include improvements in QoL and biopsychosocial wellbeing. This study is the first to assess SP FT, and may provide evidence for a novel, scalable mental illness intervention.

A study protocol for the N-ICE trial: A randomised double-blind placebo-controlled study of the safety and efficacy of N-acetyl-cysteine (NAC) as a pharmacotherapy for methamphetamine ("ice") dependence.

BACKGROUND: There are currently no approved pharmacotherapies for managing methamphetamine dependence. N-acetylcysteine (NAC) has been found to reduce the craving for methamphetamine and other drugs, but its effect on methamphetamine use and other clinically related endpoints are uncertain. The N-ICE trial is evaluating the safety and efficacy of NAC as a take-home pharmacotherapy for methamphetamine dependence. METHODS/DESIGN: This is a two-arm parallel double-blind placebo-controlled three-site randomised trial (ratio 1:1) using permuted block randomisation, with variable block sizes. It is stratified by site, sex and whether the methamphetamine is injected or not. Participants (N = 180; 60 per site) need to be dependent on methamphetamine, interested in reducing their methamphetamine use and not currently receiving treatment for substance use disorders. The trial is being conducted in outpatient settings in Melbourne, Geelong and Wollongong, Australia. Participants will receive either 2400 mg oral NAC or a matched placebo, delivered as a take-home medication for 12 weeks. Two 600 mg capsules are self-administered in the morning and two more in the evening. Adherence is being monitored using eCAP™ medication bottle lids, which record the date and time of each occasion the bottle is opened. The primary outcome is methamphetamine use during the 12-week trial medication period, measured as (a) days of use, assessed using the timeline followback, and (b) methamphetamine-positive saliva tests, taken weekly. Secondary measures include weekly assessment of methamphetamine craving, severity of methamphetamine dependence, methamphetamine withdrawal symptoms and psychiatric symptoms (depression, suicidality, psychotic symptoms and hostility). Adverse events are monitored at each weekly assessment. Tolerability is assessed using the Treatment Satisfaction Questionnaire for Medication. DISCUSSION: The N-ICE trial is the first clinical trial to assess whether NAC can reduce methamphetamine use. This trial will improve our understanding of the potential utility of NAC in managing methamphetamine dependence and clinically related outcomes. If found to be effective, take-home NAC could be a potentially scalable and affordable pharmacotherapy option for treating methamphetamine dependence. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry, ACTRN12618000366257 . Registered on 29 May 2018.

Evaluation of a community transition to professional practice program for graduate registered nurses in Australia.

Australia has an increasing demand for a sustainable primary health care registered nursing workforce. Targeting graduate registered nurses who typically begin their nursing career in acute-care hospital settings is a potential workforce development strategy. We evaluated a graduate registered nurse Community Transition to Professional Practice Program which was designed specifically to develop and foster skills required for primary health care. The aims of this study were to evaluate graduates' intention to remain in the primary health care nursing workforce, and graduate competency, confidence and experiences of program support; these were compared with graduates undertaking the conventional acute-care transition program. Preceptor ratings of graduate competence were also measured. All of the 25 graduates (n = 12 community, n = 13 acute-care) who completed the questionnaire at 6 and 12 months intended to remain in nursing, and 55% (n = 6) of graduates in the Community Transition Program intended to remain in the primary health care nursing workforce. There were no differences in graduate experiences, including level of competence, or preceptors' perceptions of graduate competence, between acute-care and Community Transition Programs. The Community Transition to Professional Practice program represents a substantial step towards developing the primary health care health workforce by facilitating graduate nurse employment in this area.

Factors associated with final year nursing students' desire to work in the primary health care setting: Findings from a national cross-sectional survey.

BACKGROUND: Registered nurses are under-represented in the primary health care setting both internationally and in Australia, and this shortage is predicted to worsen. To address the increasingly complex healthcare needs of an ageing population, it is vital to develop and sustain a primary health care nursing workforce, yet attracting nurses is challenging. In Australia, registered nurses graduating from university typically commence their careers in hospital-based transition to professional practice programs. Similar programs in primary health care settings may be a valuable strategy for developing the primary health care nursing workforce, yet little is known about nursing students desire to work in this setting, factors that influence this, or their expectations of primary health care-focused transition to professional practice programs. OBJECTIVES: This study sought to identify factors associated with final year nursing students' desire to work in primary health care setting including demographic factors, expectations of future employment conditions, and job content. It also explored expectations of graduate transition programs based in primary health care. DESIGN: A cross-sectional survey design comprising a quantitative online survey. SETTING: 14 Australian universities from all states/territories, both rural and urban. PARTICIPANTS: 530 final-year nursing students. METHODS: Binary logistic regression identifying factors contributing to desire to work in primary health care. RESULTS: The desire of nursing students to work in primary health care is associated with older age, greater perceived value of employment conditions including flexibility, and less perceived importance of workplace support. CONCLUSIONS: Collaborative efforts from primary health care nurses, health professionals, academics and policy makers are needed to attract new graduate nurses to primary health care.

Australia's first transition to professional practice in primary care program for graduate registered nurses: a pilot study.

BACKGROUND: Increases in ageing, chronic illness and complex co-morbidities in the Australian population are adding pressure to the primary care nursing workforce. Initiatives to attract and retain nurses are needed to establish a sustainable and skilled future primary care nursing workforce. We implemented a transition to professional practice program in general practice settings for graduate nurses and evaluated graduate nurse competency, the graduate nurse experience and program satisfaction. This study aimed to determine whether a transition to professional practice program implemented in the general practice setting led to competent practice nurses in their first year post-graduation. METHODS: A longitudinal, exploratory mixed-methods design was used to assess the pilot study. Data were collected at three times points (3, 6, 12 months) with complete data sets from graduate nurses (n = 4) and preceptors (n = 7). We assessed perceptions of the graduates' nursing competency and confidence, satisfaction with the preceptor/graduate relationship, and experiences and satisfaction with the program. Graduate nurse competency was assessed using the National Competency Standards for Nurses in General Practice. Semi-structured interviews with participants at Time 3 sought information about barriers, enablers, and the perceived impact of the program. RESULTS: Graduate nurses were found to be competent within their first year of clinical practice. Program perceptions from graduate nurses and preceptors were positive and the relationship between the graduate nurse and preceptor was key to this development. CONCLUSIONS: With appropriate support registered nurses can transition directly into primary care and are competent in their first year post-graduation. While wider implementation and research is needed, findings from this study demonstrate the potential value of transition to professional practice programs within primary care as a nursing workforce development strategy.

Cluster Randomized Controlled Trial of An Aged Care Specific Leadership and Management Program to Improve Work Environment, Staff Turnover, and Care Quality.

OBJECTIVE: To evaluate the effectiveness of a leadership and management program in aged care. DESIGN: Double-blind cluster randomized controlled trial. SETTING: Twelve residential and community-aged care sites in Australia. PARTICIPANTS: All care staff employed for 6 months or longer at the aged care sites were invited to participate in the surveys at 3 time points: baseline (time 1), 9 months from baseline (time 2), and 9 months after completion of time 2 (time 3) from 2011 to 2013. At each time point, at least 500 care staff completed a survey. At baseline (N = 503) the largest age group was 45 to 54 years (37%), and the majority of care staff were born in Australia (70%), spoke English (94%), and had at least completed secondary education (57%). INTERVENTION: A 12-month Clinical Leadership in Aged Care (CLiAC) program for middle managers, which aimed to further develop their leadership and management skills in creating positive workplace relationships and in enabling person-centered, evidence-based care. MAIN OUTCOME MEASURES: The primary outcomes were care staff ratings of the work environment, care quality and safety, and staff turnover rates. Secondary outcomes were care staff's intention to leave their employer and profession, workplace stress, job satisfaction, and cost-effectiveness of implementing the program. Absenteeism was excluded due to difficulty in obtaining reliable data. Managers' self-rated knowledge and skills in leadership and management are not included in this article, which focuses on care staff perceptions only. RESULTS: At 6 months after its completion, the CLiAC program was effective in improving care staff's perception of management support [mean difference 0.61, 95% confidence interval (CI) 0.04-1.18; P = .04]. Compared with the control sites, care staff at the intervention sites perceived their managers' leadership styles as more transformational (mean difference 0.30, 95% CI 0.09-0.51; P = .005), transactional (mean difference 0.22, 95% CI 0.05-0.39; P = .01), and less passive avoidant (mean difference 0.30, 95% CI 0.07-0.52; P = .01); and were rated higher on the overall leadership outcomes (mean difference 0.35, 95% CI 0.13-0.56; P = .001) as well as individual manager outcomes: extra effort (P = .004), effectiveness (P = .001), and satisfaction (P = .01). There was no evidence that CLiAC was effective in reducing staff turnover, or improving patient care quality and safety. CONCLUSIONS: While the CLiAC leadership program had direct impact on the primary process outcomes (management support, leadership actions, behaviors, and effects), this was insufficient to change the systems required to support care service quality and client safety. Nevertheless, the findings send a strong message that leadership and management skills in aged care managers can be nurtured and used to change leadership behaviors at a reasonable cost.

Validation of a clinical leadership qualities framework for managers in aged care: a Delphi study.

AIMS AND OBJECTIVES: To establish validity of a clinical leadership framework for aged care middle managers (The Aged care Clinical Leadership Qualities Framework). BACKGROUND: Middle managers in aged care have responsibility not only for organisational governance also and operational management but also quality service delivery. There is a need to better define clinical leadership abilities in aged care middle managers, in order to optimise their positional authority to lead others to achieve quality outcomes. DESIGN: A Delphi method. METHODS: Sixty-nine experts in aged care were recruited, representing rural, remote and metropolitan community and residential aged care settings. Panellists were asked to rate the proposed framework in terms of the relevance and importance of each leadership quality using four-point Likert scales, and to provide comments. Three rounds of consultation were conducted. The number and corresponding percentage of the relevance and importance rating for each quality was calculated for each consultation round, as well as mean scores. Consensus was determined to be reached when a percentage score reached 70% or greater. RESULTS: Twenty-three panellists completed all three rounds of consultation. Following the three rounds of consultation, the acceptability and face validity of the framework was confirmed. CONCLUSIONS: The study confirmed the framework as useful in identifying leadership requirements for middle managers in Australian aged care settings. The framework is the first validated framework of clinical leadership attributes for middle managers in aged care and offers an initial step forward in clarifying the aged care middle manager role. RELEVANCE TO CLINICAL PRACTICE: The framework provides clarity in the breadth of role expectations for the middle managers and can be used to inform an aged care specific leadership program development, individuals' and organisations' performance and development processes; and policy and guidelines about the types of activities required of middle managers in aged care.

Differentiation of organotypic epidermis in the presence of skin disease-linked dominant-negative Cx26 mutants and knockdown Cx26.

In this study, we chose a differentiation-competent rat epidermal keratinocyte (REK) cell line to examine the role of Cx26 and disease-linked Cx26 mutants in organotypic epidermal differentiation. First, we generated stable REK cell lines expressing three skin disease-linked mutants (G59A, D66H and R75W). Second, we used an RNAi approach to knock down the expression of Cx26 in REKs. Interestingly, the three-dimensional (3D) architecture of the organotypic epidermis altered the intracellular spatial distribution of the mutants in comparison to 2D cultured REKs, highlighting the importance of using organotypic cultures. Unexpectedly, the presence of disease-linked mutants or the overexpression of wild-type Cx26 had little effect on the differentiation of the organotypic epidermis as determined by the architecture of the epidermis, expression of molecular markers indicative of epidermis differentiation (keratin 10, keratin 14, involucrin, loricrin) and stratification/cornification of the epidermis. Likewise, organotypic epidermis continued to differentiate normally upon Cx26 knockdown. While Cx26 has been reported to be upregulated during wound healing, no reduction in wound closure was observed in 2D REK cultures that expressed loss-of-function, dominant Cx26 mutants. In conclusion, we demonstrate that gain or loss of Cx26 function does not disrupt organotypic epidermal differentiation and offer insights into why patients harboring Cx26 mutations do not frequently present with more severe disease that encompasses thin skin.

Cx26 inhibits breast MDA-MB-435 cell tumorigenic properties by a gap junctional intercellular communication-independent mechanism.

It has been well established that the restoration of connexin expression in tumor cells often leads to a partial reversion of tumor cell phenotypes and increased growth control. In this study, a less-aggressive variant of MDA-MB-435 cells obtained from the MDA-MB-435 cell line was engineered to express gap junctional intercellular communication (GJIC)-competent Cx26, a GJIC-incompetent cell surface transported GFP-Cx26 chimera, or a Golgi apparatus-localized, disease-linked Cx26 mutant (D66H). Collectively, these cell lines were designed to establish whether Cx26 regulates tumor properties, such as migration, invasion and growth, by (i) a GJIC-dependent pathway; (ii) a mechanism requiring Cx26 transport to the cell surface; or (iii) a mechanism where Cx26 expression alone was sufficient. The expression of Cx26 and green fluorescent protein (GFP)-Cx26 decreased cell proliferation while all three Cx26 variants inhibited anchorage-independent cell growth. All three Cx26 variants also altered the distribution of filamentous actin and significantly reduced cell migration, while only the D66H mutant failed to inhibit cell invasion through matrigel. Furthermore, expression of all the Cx26 variants reduced the levels of total beta1 integrin, and decreased the activity of matrix metalloproteinase-9 (MMP-9) while increasing tissue inhibitors of MMP-1 (TIMP-1) activity. Interestingly, the expression of Cx43 regulated the same gene products without significantly affecting the tumorigenic properties of the MDA-MB-435 cells. Together, these results suggest that Cx26 expression, independent of the necessity for gap junctional intercellular communication, partially reverted MDA-MB-435 cell properties associated with tumorigenesis, and regulated the expression of genes important in cell migration and invasion.

Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration.

Previous reports have suggested that Cx26 exhibits unique intracellular transport pathways en route to the cell surface compared with other members of the connexin family. To directly examine and compare nascent and steady-state delivery of Cx43 and Cx26 to the plasma membrane and gap junction biogenesis we expressed fluorescent-protein-tagged Cx43 and Cx26 in BICR-M1Rk and NRK cells. Static and time-lapse imaging revealed that both connexins were routed through the Golgi apparatus prior to being transported to the cell surface, a process inhibited in the presence of brefeldin A (BFA) or the expression of a dominant-negative form of Sar1 GTPase. During recovery from BFA, time-lapse imaging of nascent connexin Golgi-to-plasma membrane delivery revealed many dynamic post-Golgi carriers (PGCs) originating from the distal side of the Golgi apparatus consisting of heterogeneous vesicles and long, tubular-like extensions. Vesicles and tubular extensions were also observed in HBL-100 cells expressing a human, disease-linked, Golgi-localized Cx26 mutant, D66H-GFP. A diffuse cell surface rim of fluorescent-protein-tagged wild-type connexins was observed prior to the appearance of punctate gap junctions, which suggests that random fusion of PGCs occurred with the plasma membrane followed by lateral diffusion of connexins into clusters. Fluorescence recovery after photobleaching studies revealed that Cx26-YFP was more mobile within gap junction plaques compared with Cx43-GFP. Intriguingly, Cx43-GFP delivery and gap junction regeneration was inhibited by BFA and nocodazole, whereas Cx26-GFP delivery was prevented by BFA but not nocodazole. Collectively, these studies suggest that during gap junction biogenesis two phylogenetically distinct members of the connexin family, Cx43 and Cx26, share common secretory pathways, types of transport intermediates and turnover dynamics but differ in their microtubule-dependence and mobility within the plasma membrane, which might reflect differences in binding to protein scaffolds.

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation.

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.

Functional domain mapping and selective trans-dominant effects exhibited by Cx26 disease-causing mutations.

Mutations in Cx26 are a major cause of autosomal dominant and recessive forms of sensorineural deafness. Some mutations in Cx26 are associated not only with deafness but also with skin disease. We examined the subcellular localization and function of two green fluorescent protein (GFP)-tagged Cx26 point mutants that exhibit both phenotypes, G59A-GFP and D66H-GFP. D66H-GFP was retained within the brefeldin A-insensitive trans-Golgi network, whereas a population of G59A-GFP was transported to the cell surface. Neither G59A nor D66H formed gap junctions that were permeable to small fluorescent dyes, suggesting they are loss-of-function mutations. When co-expressed with wild-type Cx26, both G59A and D66H exerted dominant-negative effects on Cx26 function. G59A also exerted a trans-dominant negative effect on co-expressed wild type Cx32 and Cx43, whereas D66H exerted a trans-dominant negative effect on Cx43 but not Cx32. We propose that the severity of the skin disease is dependent on the specific nature of the Cx26 mutation and the trans-dominant selectivity of the Cx26 mutants on co-expressed connexins. Additional systematic mutations at residue D66, in which the overall charge of this motif was altered, suggested that the first extracellular loop is critical for Cx26 transport to the cell surface as well as function of the resulting gap junction channels.

Transport and function of cx26 mutants involved in skin and deafness disorders.

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and DeltaE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and DeltaE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor DeltaE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and DeltaE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.