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Abstract Background There is little reliable information on the prevalence of fetal alcohol spectrum disorders (FASD) in Australia and no coordinated national approach to facilitate case detection. The aim of this study was to identify health professionals’ perceptions about screening for FASD in Australia. Method A modified Delphi process was used to assess perceptions of the need for, and the process of, screening for FASD in Australia. We recruited a panel of 130 Australian health professionals with experience or expertise in FASD screening or diagnosis. A systematic review of the literature was used to develop Likert statements on screening coverage, components and assessment methods which were administered using an online survey over two survey rounds. Results Of the panel members surveyed, 95 (73%) responded to the questions on screening in the first survey round and, of these, 81 (85%) responded to the second round. Following two rounds there was consensus agreement on the need for targeted screening at birth (76%) and in childhood (84%). Participants did not reach consensus agreement on the need for universal screening at birth (55%) or in childhood (40%). Support for targeted screening was linked to perceived constraints on service provision and the need to examine the performance, costs and benefits of screening. For targeted screening of high risk groups, we found highest agreement for siblings of known cases of FASD (96%) and children of mothers attending alcohol treatment services (93%). Participants agreed that screening for FASD primarily requires assessment of prenatal alcohol exposure at birth (86%) and in childhood (88%), and that a checklist is needed to identify the components of screening and criteria for referral at birth (84%) and in childhood (90%). Conclusions There is an agreed need for targeted but not universal screening for FASD in Australia, and sufficient consensus among health professionals to warrant development and evaluation of standardised methods for targeted screening and referral in the Australian context. Participants emphasised the need for locally-appropriate, evidence-based approaches to facilitate case detection, and the importance of ensuring that screening and referral programs are supported by adequate diagnostic and management capacity.

Abstract Background Despite the availability of five guidelines for the diagnosis of fetal alcohol spectrum disorders (FASD), there is no national endorsement for their use in diagnosis in Australia. In this study we aimed to describe health professionals’ perceptions about the adoption of existing guidelines for the diagnosis of FASD in Australia and identify implications for the development of national guidelines. Methods We surveyed 130 Australian and 9 international health professionals with expertise or involvement in the screening or diagnosis of FASD. An online questionnaire was used to evaluate participants’ familiarity with and use of five existing diagnostic guidelines for FASD, and to assess their perceptions about the adoption of these guidelines in Australia. Results Of the 139 participants surveyed, 84 Australian and 8 international health professionals (66.2%) responded to the questions on existing diagnostic guidelines. Participants most frequently reported using the University of Washington 4-Digit Diagnostic Code (27.2%) and the Canadian guidelines (18.5%) for diagnosis. These two guidelines were also most frequently recommended for adoption in Australia: 32.5% of the 40 participants who were familiar with the University of Washington 4-Digit Diagnostic Code recommended adoption of this guideline in Australia, and 30.8% of the 26 participants who were familiar with the Canadian guidelines recommended adoption of this guideline in Australia. However, for the majority of guidelines examined, most participants were unsure whether they should be adopted in Australia. The adoption of existing guidelines in Australia was perceived to be limited by: their lack of evidence base, including the appropriateness of established reference standards for the Australian population; their complexity; the need for training and support to use the guidelines; and the lack of an interdisciplinary and interagency model to support service delivery in Australia. Conclusions Participants indicated some support for the adoption of the University of Washington or Canadian guidelines for FASD diagnosis; however, concerns were raised about the adoption of these diagnostic guidelines in their current form. Australian diagnostic guidelines will require evaluation to establish their validity in the Australian context, and a comprehensive implementation model is needed to facilitate improved diagnostic capacity in Australia.

Abstract Background Recent developments have made screening tests for foetal abnormalities available earlier in pregnancy and women have a range of testing options accessible to them. It is now recommended that all women, regardless of their age, are provided with information on prenatal screening tests. General Practitioners (GPs) are often the first health professionals a woman consults in pregnancy. As such, GPs are well positioned to inform women of the increasing range of prenatal screening tests available. The aim of this study was to explore GPs experience of informing women of prenatal genetic screening tests for foetal abnormality. Methods A qualitative study consisting of four focus groups was conducted in metropolitan and rural Victoria, Australia. A discussion guide was used and the audio-taped transcripts were independently coded by two researchers using thematic analysis. Multiple coders and analysts and informant feedback were employed to reduce the potential for researcher bias and increase the validity of the findings. Results Six themes were identified and classified as 'intrinsic' if they occurred within the context of the consultation or 'extrinsic' if they consisted of elements that impacted on the GP beyond the scope of the consultation. The three intrinsic themes were the way GPs explained the limitations of screening, the extent to which GPs provided information selectively and the time pressures at play. The three extrinsic factors were GPs' attitudes and values towards screening, the conflict they experienced in offering screening information and the sense of powerlessness within the screening test process and the health care system generally. Extrinsic themes reveal GPs' attitudes and values to screening and to disability, as well as raising questions about the fundamental premise of testing. Conclusion The increasing availability and utilisation of screening tests, in particular first trimester tests, has expanded GPs' role in facilitating women's informed decision-making. Recognition of the importance of providing this complex information warrants longer consultations to respond to the time pressures that GPs experience. Understanding the intrinsic and extrinsic factors that impact on GPs may serve to shape educational resources to be more appropriate, relevant and supportive.

Abstract Background Despite the wide availability of prenatal screening and diagnosis, a number of studies have reported no decrease in the rate of babies born with Down syndrome. The objective of this study was to investigate the geodemographic characteristics of women who have prenatal diagnosis in Victoria, Australia, by applying a novel consumer behaviour modelling technique in the analysis of health data. Methods A descriptive analysis of data on all prenatal diagnostic tests, births (1998 and 2002) and births of babies with Down syndrome (1998 to 2002) was undertaken using a Geographic Information System and socioeconomic lifestyle segmentation classifications. Results Most metropolitan women in Victoria have average or above State average levels of uptake of prenatal diagnosis. Inner city women residing in high socioeconomic lifestyle segments who have high rates of prenatal diagnosis spend 20% more on specialist physician's fees when compared to those whose rates are average. Rates of prenatal diagnosis are generally low amongst women in rural Victoria, with the lowest rates observed in farming districts. Reasons for this are likely to be a combination of lack of access to services (remoteness) and individual opportunity (lack of transportation, low levels of support and income). However, there are additional reasons for low uptake rates in farming areas that could not be explained by the behaviour modelling. These may relate to women's attitudes and choices. Conclusion A lack of statewide geodemographic consistency in uptake of prenatal diagnosis implies that there is a need to target health professionals and pregnant women in specific areas to ensure there is increased equity of access to services and that all pregnant women can make informed choices that are best for them. Equally as important is appropriate health service provision for families of children with Down syndrome. Our findings show that these potential interventions are particularly relevant in rural areas. Classifying data to lifestyle segments allowed for practical comparisons of the geodemographic characteristics of women having prenatal diagnosis in Australia at a population level. This methodology may in future be a feasible and cost-effective tool for service planners and policy developers.

Abstract Background By providing information on the relative merits and potential harms of the options available and a framework to clarify preferences, decision aids can improve knowledge and realistic expectations and decrease decisional conflict in individuals facing decisions between alternative forms of action. Decision-making about prenatal testing for fetal abnormalities is often confusing and difficult for women and the effectiveness of decision aids in this field has not been established. This study aims to test whether a decision aid for prenatal testing of fetal abnormalities, when compared to a pamphlet, improves women's informed decision-making and decreases decisional conflict. Methods/design A cluster designed randomised controlled trial is being conducted in Victoria, Australia. Fifty General Practitioners (GPs) have been randomised to one of two arms: providing women with either a decision aid or a pamphlet. The two primary outcomes will be measured by comparing the difference in percentages of women identified as making an informed choice and the difference in mean decisional conflict scores between the two groups. Data will be collected from women using questionnaires at 14 weeks and 24 weeks gestation. The sample size of 159 women in both arms of the trial has been calculated to detect a difference of 18% (50 to 68%) in informed choice between the two groups. The required numbers have been adjusted to accommodate the cluster design, miscarriage and participant lost – to – follow up. Baseline characteristics of women will be summarised for both arms of the trial. Similarly, characteristics of GPs will be compared between arms. Differences in the primary outcomes will be analysed using 'intention-to-treat' principles. Appropriate regression techniques will adjust for the effects of clustering and include covariates to adjust for the stratifying variable and major potential confounding factors. Discussion The findings from this trial will make a significant contribution to improving women's experience of prenatal testing and will have application to a variety of maternity care settings. The evaluation of a tailored decision aid will also have implications for pregnancy care providers by identifying whether or not such a resource will support their role in providing prenatal testing information.

Background: Management of type 1 diabetes (T1D) requires the use of insulin, which can cause hypoglycaemia (low blood glucose levels). While most hypoglycaemic episodes can be self-treated, all episodes can be sudden, inconvenient, challenging to prevent or manage, unpleasant and/or cause unwanted attention or embarrassment. Severe hypoglycaemic episodes, requiring assistance from others for recovery, are rare but potentially dangerous. Repeated exposure to hypoglycaemia can reduce classic warning symptoms ('awareness'), thereby increasing risk of severe episodes. Thus, fear of hypoglycaemia is common among adults with T1D and can have a negative impact on how they manage their diabetes, as well as on daily functioning, well-being and quality of life. While advances in glycaemic technologies and group-based psycho-educational programmes can reduce fear, frequency and impact of hypoglycaemia, they are not universally or freely available, nor do they fully resolve problematic hypoglycaemia or associated worries. This study aims to determine the effectiveness of a fully online, self-directed, scalable, psycho-educational intervention for reducing fear of hypoglycaemia: the Hypoglycaemia Prevention, Awareness of Symptoms, and Treatment (HypoPAST) programme., Methods: A 24-week, two-arm, parallel-group, hybrid type 1 randomised controlled trial, conducted remotely (online and telephone). Australian adults (≥ 18 years) with self-reported T1D and fear of hypoglycaemia will be recruited, and allocated at random (1:1) to HypoPAST or control (usual care). The primary outcome is the between-group difference in fear of hypoglycaemia (assessed using HFS-II Worry score) at 24 weeks. A sample size of N = 196 is required to detect a 9-point difference, with 90% power and allowing for 30% attrition. Multiple secondary outcomes include self-reported psychological, behavioural, biomedical, health economic, and process evaluation data. Data will be collected at baseline, 12 and 24 weeks using online surveys, 2-week ecological momentary assessments, website analytics and semi-structured interviews., Discussion: This study will provide evidence regarding the effectiveness, cost-effectiveness and acceptability of a novel, online psycho-educational programme: HypoPAST. Due to the fully online format, HypoPAST is expected to provide an inexpensive, convenient, accessible and scalable solution for reducing fear of hypoglycaemia among adults with T1D., Trial Registration: Australian and New Zealand Clinical Trials Registry (ANZCTR): ACTRN12623000894695 (21 August 2023)., (© 2024. The Author(s).)

Aims: To examine the psychometric properties of the Diabetes Management Experiences Questionnaire (DME-Q). Adapted from the validated Glucose Monitoring Experiences Questionnaire, the DME-Q captures satisfaction with diabetes management irrespective of treatment modalities., Methods: The DME-Q was completed by adults with type 1 diabetes as part of a randomized controlled trial comparing hybrid closed loop (HCL) to standard therapy. Most psychometric properties were examined with pre-randomization data (n = 149); responsiveness was examined using baseline and 26-week follow-up data (n = 120)., Results: Pre-randomization, participants' mean age was 44 ± 12 years, 52% were women. HbA1c was 61 ± 11 mmol/mol (7.8 ± 1.0%), diabetes duration was 24 ± 12 years and 47% used an insulin pump prior to the trial. A forced three-factor analysis revealed three expected domains, that is, 'Convenience', 'Effectiveness' and 'Intrusiveness', and a forced one-factor solution was also satisfactory. Internal consistency reliability was strong for the three subscales ( α range = 0.74-0.84) and 'Total satisfaction' ( α  = 0.85). Convergent validity was demonstrated with moderate correlations between DME-Q 'Total satisfaction' and diabetes distress (PAID: r s  = -0.57) and treatment satisfaction (DTSQ; r s  = 0.58). Divergent validity was demonstrated with a weak correlation with prospective/retrospective memory (PRMQ: r s  = -0.16 and - 0.13 respectively). Responsiveness was demonstrated, as participants randomized to HCL had higher 'Effectiveness' and 'Total satisfaction' scores than those randomized to standard therapy., Conclusions: The 22-item DME-Q is a brief, acceptable, reliable measure with satisfactory structural and construct validity, which is responsive to intervention. The DME-Q is likely to be useful for evaluation of new pharmaceutical agents and technologies in research and clinical settings., (© 2023 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

People with diabetes often encounter stigma (ie, negative social judgments, stereotypes, prejudice), which can adversely affect emotional, mental, and physical health; self-care, access to optimal health care; and social and professional opportunities. To accelerate an end to diabetes stigma and discrimination, an international multidisciplinary expert panel (n=51 members, from 18 countries) conducted rapid reviews and participated in a three-round Delphi survey process. We achieved consensus on 25 statements of evidence and 24 statements of recommendations. The consensus is that diabetes stigma is driven primarily by blame, perceptions of burden or sickness, invisibility, and fear or disgust. On average, four in five adults with diabetes experience diabetes stigma and one in five experience discrimination (ie, unfair and prejudicial treatment) due to diabetes, such as in health care, education, and employment. Diabetes stigma and discrimination are harmful, unacceptable, unethical, and counterproductive. Collective leadership is needed to proactively challenge, and bring an end to, diabetes stigma and discrimination. Consequently, we achieved unanimous consensus on a pledge to end diabetes stigma and discrimination., Competing Interests: Declaration of interests In the past 3 years, JSp and EH-T have received competitive research grants from the Diabetes Australia Research Program, the Medical Research Future Fund Targeted Translational Research Accelerator, and the National Health and Medical Research Council of Australia. JSp and EH-T have received an investigator-initiated research grant from Sanofi Diabetes and an unrestricted educational grant from Diabetes Australia and are supported by core funding to the Australian Centre for Behavioural Research in Diabetes derived from the collaboration between Diabetes Victoria and Deakin University. JSp has also received a competitive research grant from the Ian Potter Foundation; a research contract from the Australian Government Department of Health; a consulting fee from Diabetes Canada; honoraria to present at educational meetings from Novo Nordisk and Sanofi Diabetes; honoraria for participating in advisory boards from Insulet and Sanofi Diabetes; and support for attending meetings (including travel) from the Novo Nordisk Foundation. EH-T received honoraria for presentation at educational meetings from Roche Diabetes Care. All these payments have been made directly to the Australian Centre for Behavioural Research in Diabetes; this research centre owns the copyright of the type 1 and type 2 Diabetes Stigma Assessment Scales. JSp is the Chair, JSt the Vice-chair, and EH-T website redevelopment lead of the international PsychoSocial Aspects of Diabetes Study Group (unpaid roles). MG has received project funding from One Drop, Eli Lilly, Boehringer Ingelheim and Eli Lilly Alliance, Abbott, and Genentech and honorarium from Sanofi and Diabetes Sisters (all paid to the diaTribe Foundation). IW has shares in Novo Nordisk. ASe has received competitive research grants from the Juvenile Diabetes Research Foundation and Auckland Medical Research Foundation (all paid to the University of Auckland) and conference attendance support (as an invited speaker) from the International Society for Pediatric and Adolescent Diabetes. BA received conference funding support to present at a RAPID conference hosted by PsychoSocial Aspects of Diabetes and Steno. MdG has received competitive research grants from the American Diabetes Association and National Institute on Aging and received consulting fees from Mediflix and Kenner Family Foundation. SH has received consulting fees from Novo Nordisk, Lilly Diabetes Care, Medtronic Diabetes Care, and Dexcom Germany; received payments of honoraria from Novo Nordisk and Ascensia Diabetes Care; payment for expert testimony from VitalAire; received support for attending meetings from Ascensia, Diabetes Center Berne, Diabeloop, Dexcom, Lilly, Medtronic, Novo Nordisk, Sanofi, Tandem, embecta, and dotcool; received medical equipment from Abbott Diabetes Care, Dexcom, and Ypsomed; and is Head of Communications for #dedoc°, Dedoc Labs. RIGH has received research support from Novo Nordisk; speaker honoraria from EASD, Eli Lilly, Encore, Liberum, Novo Nordisk, and ROVI; and conference attendance funding from Novo Nordisk and Eli Lilly. KK is supported by the UK National Institute for Health Research's Applied Research Collaboration East Midlands and Leicester Biomedical Research Centre; has received research funding from AstraZeneca, Boehringer Ingelheim, Eli Lilly, MSD, Novartis, Novo Nordisk, Roche, Oramed Pharmaceuticals, and Sanofi; received consultancy fees from AstraZeneca, Boehringer Ingelheim, Eli Lilly, MSD, Novo Nordisk, Roche, Sanofi, and Servier; and received payments of honoraria from AstraZeneca, Boehringer Ingelheim, Eli Lilly, Novartis, Novo Nordisk, Roche, and Sanofi. RLP has received research funding from the US National Institutes of Health (paid to institution); receieved presentation honorarium from the Diabetes Leadership Council; and is Chair of Policy Track for The Obesity Society Annual Meeting Program Committee (unpaid role). RMP has received research funding and consultancy fees from Eli Lilly. MSe has received conference funding support from the International Diabetes Federation and the International Society for Pediatric and Adolescent Diabetes. MV has received investigator-initiated research funding from Abbott, Bausch, and Novo Nordisk; received consulting feeds from Abbott, Abbvie, Boehringer Ingelheim, and Novo Nordisk; received payments and travel support from Abbott, Abbvie, Boehringer Ingelheim, Novo Nordisk, Eli Lilly, Merck, and Pfizer; has a pending patent with AmpHealth for a digital health intervention; and is a Diabetes Canada Mission Committee member (unpaid role). All other authors report no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Diabetes distress is a commonly experienced negative emotional response to the ongoing burden of diabetes. Holistic diabetes care, including attention to diabetes distress, is recommended in clinical guidelines, yet not routinely implemented. Diabetes health professionals have highlighted lack of training as a barrier to implementation of psychological care. Therefore, we developed an e-learning: 'Diabetes distress e-learning: A course for diabetes educators' to address this need. This pilot study aimed to examine the feasibility of evaluating the e-learning in a randomised controlled trial study, the acceptability of the e-learning to credentialled diabetes educators (CDEs); and preliminary evidence of its effect upon CDEs' diabetes distress-related knowledge, motivation, confidence, behavioural skills, and barriers to implementation., Methods: A pilot, unblinded, 2-armed, parallel group randomised controlled trial. Participants were recruited during a 4-month timeframe. Eligible participants were CDEs for ≥ 1 year providing care to ≥ 10 adults with type 1 or type 2 diabetes per week. Participants were randomly allocated (1:1 computer automated) to 1 of 2 learning activities: diabetes distress e-learning (intervention) or diabetes distress chapter (active control). They had 4 weeks to access the activity. They completed online surveys at baseline, 2-week and 12-week follow-up., Results: Seventy-four eligible CDEs (36 intervention, 38 active control) participated. At baseline, recognition of the clinical importance of diabetes distress was high but knowledge and confidence to provide support were low-to-moderate. Engagement with learning activities was high (intervention: 83%; active control: 92%). Fifty-five percent returned at least 1 follow-up survey. All 30 intervention participants who returned the 2-week follow-up survey deemed the e-learning high quality and relevant. Systemic barriers (e.g., financial limitations and access to mental health professionals) to supporting people with diabetes distress were common at baseline and follow-up.  CONCLUSIONS: The e-learning was acceptable to CDEs. The study design was feasible but needs modification to improve follow-up survey return. The e-learning showed potential for improving diabetes distress-related knowledge, confidence and asking behaviours, but systemic barriers to implementation remained. Systemic barriers need to be addressed to facilitate implementation of support for diabetes distress in clinical practice. Future larger-scale evaluation of the e-learning is warranted., (© 2022. The Author(s).)

Diabetes distress is a common negative emotional response to the ongoing burden of living with diabetes. Elevated diabetes distress is associated with impaired diabetes self-management and quality of life yet rarely identified and addressed in clinical practice. Health professionals report numerous barriers to the provision of care for diabetes distress, including lack of skills and confidence, but few diabetes distress training opportunities exist. The purpose of this paper is to describe how we utilized Intervention Mapping to plan the development, implementation, and evaluation of a novel diabetes distress e-learning program for diabetes educators, to meet a well-documented need and significant gap in diabetes care. A multidisciplinary team (combining expertise in research, health and clinical psychology, diabetes education, nursing, tertiary education, and website architecture) developed a diabetes distress e-learning program. We followed a six-step process (logic model of the problem, program outcomes and objectives, program design, program production, program implementation plan, and evaluation plan) known as Intervention Mapping. The program is underpinned by educational and psychological theory, including Bloom's Taxonomy of Educational Objectives and social cognitive theory. We developed a short (estimated 4 h) e-learning program for diabetes educators, which draws on the content of the Diabetes and Emotional Health handbook and toolkit. It integrates a 7As model, which provides a stepwise approach to identifying and addressing diabetes distress. Our diabetes distress e-learning program has been developed systematically, guided by an Intervention Mapping approach. In the next phase of the project, we will trial the e-learning., (© Society of Behavioral Medicine 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: In an unselected clinical sample, we aimed to: 1) investigate the willingness of adults with diabetes to talk with their health professional(s) about their feelings and experiences living with diabetes, 2) assess the prevalence of impaired general emotional well-being and severe diabetes distress and 3) examine whether willingness to talk related to general and/or diabetes-specific emotional well-being., Methods: Unselected adults with type 1 diabetes (T1D) or type 2 diabetes (T2D) attending 4 Australian specialist diabetes clinics completed surveys about their experiences of, and preferences for, talking with their diabetes health professional(s) about their feelings and personal experiences of diabetes. They indicated preferred topics to discuss from a list and completed validated measures of emotional well-being (World Health Organisation-5 Well-being Index) and diabetes distress (Problem Areas In Diabetes scale)., Results: Among 682 participants (T1D, n=440; T2D, n=142), one-fourth of adults with T1D and nearly half with T2D wanted to talk with their health professional about their "feelings and personal experience of living with diabetes," with >50% reported having been asked. The most commonly selected topic was "How diabetes affects my mood" (T1D, 35%; T2D, 37%). Impaired emotional well-being (T1D, 33%; T2D, 39%) and severe diabetes distress (T1D, 17%; T2D, 25%) were prevalent. Those willing to talk had greater diabetes distress., Conclusions: In this study we show that many adults with T1D and T2D both need and want to talk to their diabetes health professionals about the emotional impact of diabetes. Those who were most willing to have this conversation were most in need of emotional support., (Copyright © 2020 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.)

Background: Health professionals have expressed unmet needs, including lacking the skills, confidence, training, and resources needed to properly attend to the psychological needs of people with diabetes., Objective: Informed by needs assessments, this study aimed to develop practical, evidence-based resources to support health professionals to address the emotional needs of adults with type 1 or type 2 diabetes., Methods: We developed a new handbook and toolkit informed by formative evaluation, including literature reviews, stakeholder consultation and review, and a qualitative study. In the qualitative study, health professionals participated in interviews after reading sections of the handbook and toolkit., Results: The literature review uncovered that psychological problems are common among adults with diabetes, but health professionals lack resources to provide related support. We planned and drafted resources to fill this unmet need, guided by stakeholder consultation and an Expert Reference Group (ERG). Before finalizing the resources, we implemented feedback received from stakeholders (ERG, health professionals, academics, and people with diabetes). The resulting resources were the practical, evidence-based Diabetes and Emotional Health handbook and toolkit. A total of 19 health professionals took part in the qualitative study about the handbook and toolkit. They viewed the resources favorably, felt empowered to support people with diabetes experiencing psychological problems, and felt motivated to share the resources with others. Some gave examples of how they had used the handbook in clinical practice. A perceived highlight was the inclusion of a process model outlining 7 steps for identifying and supporting people with emotional problems: the 7 A's model. With funding from the National Diabetes Services Scheme (NDSS), more than 2400 copies of Diabetes and Emotional Health have been distributed. It is freely available on the Web. The NDSS is an initiative of the Australian Government administered with the assistance of Diabetes Australia., Conclusions: The new evidence-based resources are perceived by stakeholders as effective aids to assist health professionals in providing emotional support to adults with diabetes. The 7 A's model may have clinical utility for routine monitoring of other psychological and health-related problems, as part of person-centered clinical care., (©Jennifer A Halliday, Jane Speight, Andrea Bennet, Linda J Beeney, Christel Hendrieckx. Originally published in JMIR Formative Research (http://formative.jmir.org), 21.02.2020.)

RNA transcription errors are transient, yet frequent, events that do have consequences for the cell. However, until recently we lacked the tools to empirically measure and study these errors. Advances in RNA library preparation and next generation sequencing (NGS) have allowed the spectrum of transcription errors to be empirically measured over the entire transcriptome and in nascent transcripts. Combining these powerful methods with forward and reverse genetic strategies has refined our understanding of transcription factors known to enhance RNA accuracy and will enable the discovery of new candidates. Furthermore, these approaches will shed additional light on the complex interplay between transcription fidelity and other DNA transactions, such as replication and repair, and explore a role for transcription errors in cellular evolution and disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transcription is a fundamental cellular process and the first step in gene regulation. Although RNA polymerase (RNAP) is highly processive, in growing cells the progression of transcription can be hindered by obstacles on the DNA template, such as damaged DNA. The authors recent findings highlight a trade-off between transcription fidelity and DNA break repair. While a lot of work has focused on the interaction between transcription and nucleotide excision repair, less is known about how transcription influences the repair of DNA breaks. The authors suggest that when the cell experiences stress from DNA breaks, the control of RNAP processivity affects the balance between preserving transcription integrity and DNA repair. Here, how the conflict between transcription and DNA double-strand break (DSB) repair threatens the integrity of both RNA and DNA are discussed. In reviewing this field, the authors speculate on cellular paradigms where this equilibrium is well sustained, and instances where the maintenance of transcription fidelity is favored over genome stability., (© 2018 WILEY Periodicals, Inc.)

It was recently shown that removal of GreA, a transcription fidelity factor, enhances DNA break repair. This counterintuitive result, arising from unresolved backtracked RNA polymerase impeding DNA resection and thereby facilitating RecA-loading, leads to an interesting corollary: error-free full-length transcripts and broken chromosomes. Therefore, transcription fidelity may compromise genomic integrity.

σ S is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σ S general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σ S levels transcriptionally, translationally, and posttranslationally in Escherichia coli It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σ S synthesis, thus playing an important role in activation of the σ S stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σ S levels. In addition, based on effects of DksA N88I , a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σ S activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σ S and also increase transcription from other promoters, including P dsrA and P iraP By improving the translation and stabilization of σ S , as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σ S Understanding the factors governing σ S activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σ S levels., (Copyright © 2017 American Society for Microbiology.)

Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.

Aims: Screening for depression is recommended internationally. The World Health Organization's 5-item Well-being Index (WHO-5) is used clinically to screen for depression but its empirical suitability for this purpose is not well documented. We investigated the psychometric properties of the WHO-5 and its suitability for identifying likely depression in Australian adults with diabetes., Methods: The Diabetes MILES - Australia study dataset provided a sample of N=3249 who completed the WHO-5 (positively-worded 5-item measure of emotional well-being) and the PHQ-9 (9-item measure of depressive symptoms). Analyses were conducted for the full sample, and separately by diabetes type and treatment (type 1, non-insulin-treated type 2, and insulin-treated type 2 diabetes). Construct (convergent and factorial) validity and reliability of the WHO-5 were examined. ROC analyses were used to examine the sensitivity and specificity of the WHO-5 as a depression screening instrument, comparing two commonly used WHO-5 cut-off values (≤7 and <13) with the PHQ-9., Results: For the whole sample, the WHO-5 demonstrated satisfactory internal consistency reliability (α=0.90) and convergent validity with the PHQ-9 (r=-0.73, p<0.001). Confirmatory factor analysis partially supported factorial validity: Χ 2 (5)=834.94, p<0.001; RMSEA=0.23, 90% CI 0.21-0.24; CFI=0.98, TLI=0.96; factor loadings=0.78-0.92. The AUC was 0.87 (95% CI: 0.86-0.89, p<0.001). The sensitivity/specificity of the WHO-5 for detecting likely depression was 0.44/0.96 for the ≤7 cut-off, and 0.79/0.79 for the <13 cut-off, with similar findings by diabetes type and treatment., Conclusions: These findings support use of a WHO-5 cut-point of <13 to identify likely depression in Australian adults with diabetes, regardless of type/treatment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli . We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Errors in information transfer from DNA to RNA to protein are inevitable. Here, we focus on errors that occur in nascent transcripts during transcription, epimutations. Recent approaches using novel cDNA library preparation and next-generation sequencing begin to directly determine the rate of epimutation and allow analysis of the epimutational spectrum of transcription errors, the type and sequence context of the errors produced in a transcript by an RNA polymerase. The phenotypic consequences of transcription errors have been assessed using both forward and reverse epimutation systems. These studies reveal that transient transcription errors can produce a modification of cell phenotype, partial phenotypic suppression of a mutant allele, and a heritable change in cell phenotype, epigenetic switching in a bistable gene network., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Although obesity among immigrants remains an important area of study given the increasing migrant population in Australia and other developed countries, research on factors amenable to intervention is sparse. The aim of the study was to develop a culturally-competent obesity prevention program for sub-Saharan African (SSA) families with children aged 12-17 years using a community-partnered participatory approach., Methods: A community-partnered participatory approach that allowed the intervention to be developed in collaborative partnership with communities was used. Three pilot studies were carried out in 2008 and 2009 which included focus groups, interviews, and workshops with SSA parents, teenagers and health professionals, and emerging themes were used to inform the intervention content. A cultural competence framework containing 10 strategies was developed to inform the development of the program. Using findings from our scoping research, together with community consultations through the African Review Panel, a draft program outline (skeleton) was developed and presented in two separate community forums with SSA community members and health professionals working with SSA communities in Melbourne., Results: The 'Healthy Migrant Families Initiative (HMFI): Challenges and Choices' program was developed and designed to assist African families in their transition to life in a new country. The program consists of nine sessions, each approximately 1 1/2 hours in length, which are divided into two modules based on the topic. The first module 'Healthy lifestyles in a new culture' (5 sessions) focuses on healthy eating, active living and healthy body weight. The second module 'Healthy families in a new culture' (4 sessions) focuses on parenting, communication and problem solving. The sessions are designed for a group setting (6-12 people per group), as many of the program activities are discussion-based, supported by session materials and program resources., Conclusion: Strong partnerships and participation by SSA migrant communities enabled the design of a culturally competent and evidence-based intervention that addresses obesity prevention through a focus on healthy lifestyles and healthy families. Program implementation and evaluation will further inform obesity prevention interventions for ethnic minorities and disadvantaged communities.

Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in β-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The lysis/lysogeny switch of bacteriophage lambda serves as a paradigm for binary cell fate decision, long-term maintenance of cellular state and stimulus-triggered switching between states. In the literature, the system is often referred to as "bistable." However, it remains unclear whether this term provides an accurate description or is instead a misnomer. Here we address this question directly. We first quantify transcriptional regulation governing lysogenic maintenance using a single-cell fluorescence reporter. We then use the single-cell data to derive a stochastic theoretical model for the underlying regulatory network. We use the model to predict the steady states of the system and then validate these predictions experimentally. Specifically, a regime of bistability, and the resulting hysteretic behavior, are observed. Beyond the steady states, the theoretical model successfully predicts the kinetics of switching from lysogeny to lysis. Our results show how the physics-inspired concept of bistability can be reliably used to describe cellular phenotype, and how an experimentally-calibrated theoretical model can have accurate predictive power for cell-state switching.

There is mounting evidence that family functioning is linked to childhood overweight and obesity, and that both of these are associated with health-related behaviours and adverse health outcomes in children and adolescents. This paper systematically examines the peer-reviewed evidence regarding the relationship between child and adolescent overweight and obesity and family functioning. Peer-reviewed literature published between 1990 and 2011 hosted in Scopus, Pub Med or Psyc INFO were searched, in addition to the reference lists of included papers. Twenty-one studies met the selection criteria. Of the 17 identified cross-sectional and longitudinal studies, 12 reported significant associations between family functioning and childhood overweight and obesity. The instruments used to measure family functioning in the identified studies were heterogeneous. Poor family functioning was associated with increased risk of obesity and overweight in children and adolescents, and obese children and adolescents were more likely to come from families with poor family functioning. Aspects of family functioning which were associated with increased risk of child and adolescent obesity included poor communication, poor behaviour control, high levels of family conflict and low family hierarchy values. Half (2/4) of the identified intervention studies showed a significant relationship between family functioning and changes in child weight. The results demonstrate that family functioning is linked to obesity; however, higher level evidence and greater understanding of the mechanisms behind this relationship are required. The results indicate a need for a standardised family functioning measure applicable across populations. The results provide evidence of the value of considering family functioning in childhood obesity research and intervention.

Aim: To investigate severe hypoglycaemia (SH) in adults with type 1 diabetes and its associations with impaired awareness of hypoglycaemia (IAH), clinical, psychological and socio-demographic factors., Methods: Attendees of three specialist diabetes clinics in Melbourne, Australia completed questions about frequency of SH in the past six months; impaired awareness of hypoglycaemia (Gold score); and measures of general emotional well-being (WHO-5), diabetes-specific positive well-being (subscale of W-BQ28), diabetes-related distress (PAID) and fear of hypoglycaemia (HFS)., Results: Of 422 participants (mean ± SD age 37.5 ± 15.0 years; 54% women), 78 (18.5%) reported at least one SH event and 86 (20.5%) had IAH. SH and IAH frequencies were similar at all clinics. In total, 194 SH events were reported, with 10 people experiencing 40% of events. Compared with those without SH, participants with SH had longer diabetes duration, were younger at diabetes onset and more likely to have IAH (p<0.01). Those with SH had greater fear of hypoglycaemia and diabetes-related distress, poorer general emotional well-being, and lower diabetes-specific positive well-being, (p<0.01). There were no associations with age, gender, insulin regimen or HbA1c., Conclusions: This study has identified that SH and IAH in Australian adults with type 1 diabetes exist at similar levels to those reported in US and European research. SH was significantly associated with IAH and fear of hypoglycaemia. Assessment of hypoglycaemia, IAH and psychological well-being as part of a routine diabetes clinic visit was well accepted by attendees and enabled identification of those who may benefit from medical, educational or therapeutic interventions., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Obesity is an emerging problem for African migrants in Australia, but few prevention programs incorporate their cultural beliefs and values. This study reports on the application of community capacity-building and empowerment principles in 4 workshops with Sudanese families in Australia. Workshop participants prioritized health behaviors, skill and knowledge gaps, and environments for change to identify culturally centered approaches to health promotion. The workshops highlighted a need for culturally and age-appropriate interventions that build whole-of-family skills and knowledge around the positive effects of physical activity and nutrition to improve health within communities while reducing intergenerational and gender role family conflicts.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.

The auxiliary factor DksA is a global transcription regulator and, with the help of ppGpp, controls the nutritional stress response in Escherichia coli. Although the consequences of its modulation of RNA polymerase (RNAP) are becoming better explained, it is still not fully understood how the two proteins interact. We employed a series of genetic suppressor selections to find residues in RNAP that alter its sensitivity to DksA. Our approach allowed us to identify and genetically characterize in vivo three single amino acid substitutions: β' E677G, β V146F, and β G534D. We demonstrate that the mutation β' E677G affects the activity of both DksA and its homolog, TraR, but does not affect the action of other secondary interactors, such as GreA or GreB. Our mutants provide insight into how different auxiliary transcription factors interact with RNAP and contribute to our understanding of how different stages of transcription are regulated through the secondary channel of RNAP in vivo.

Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimutations') remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the lac operon, a memory-module, to capture the consequences of transient transcription errors in living Escherichia coli cells. We engineered an error-prone transcription sequence (A9 run) in the gene encoding the lac repressor and show that this 'slippery' sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease., Competing Interests: The authors have declared that no competing interests exist.

Objective: To assess the association between 25-hydroxyvitamin D (25[OH]D) status and obesity, cardiovascular diseases (CVDs), the metabolic syndrome, and type 2 diabetes mellitus (T2DM) in ethnic minorities., Methods: Databases searched were CINHAL with full text, Global Health, MEDLINE with full text, and PsycINFO from 1980 through 2010 (February). Studies were included if they 1) targeted immigrants from low- to high-income countries or ethnic minorities, 2) focused primarily on 25(OH)D and its relation to obesity, T2DM, and/or CVDs, and 3) were published in peer-reviewed journals. The influences of key confounders such as age, gender, and ethnicity on any observed relations were also assessed. Due to the heterogeneity of study characteristics, only a narrative synthesis was undertaken., Results: Ethnic minorities had significantly higher rates of vitamin D insufficiency (25[OH]D <50 nmol/L; children 43.6-48.7% versus 10%; adults 30.3-53% versus 13.7-26%) than their white counterparts. None of the studies reported a prevalence of obesity stratified by ethnicity. There was evidence supporting links between vitamin D deficiency and obesity-related chronic diseases, with 14 of 14 studies reporting a statistically significant result with a measurement of obesity, four of five for T2DM, four of five for CVDs, and one of one for the metabolic syndrome. However, the strength of the association varied across ethnic groups depending on the index used to measure adiposity, T2DM, and CVDs. Because most of the included studies were cross-sectional and there were variations in outcome measurements, it was not possible to determine the relative contributions of obesity or vitamin D insufficiency to CVD risk and risk of T2DM or which is the initial driver It is possible both have a role to play., Conclusion: Further research specific to migrant populations using randomized controlled trials are required to establish whether causal links between 25(OH)D and obesity-related chronic disease exist, and whether vitamin D supplementation could be valuable in the prevention or treatment of obesity-related diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Unicellular organisms are constantly subject to sudden changes in environment. Here, we describe recent progress in understanding how epigenetic mechanisms can generate differentiation within genetically identical single cells of a clonal population. Such intrinsic phenotypic heterogeneity within a population may be considered as a bet-hedging strategy in fluctuating environments. One aspect we highlight is how transient errors in information transfer, be it errors in transcription or translation, or alternatives in protein folding, can influence the quantity and the quality of the resulting proteins, and therefore, contribute to genetic noise within individual cells. These stochastic events can provide the impetus for heritable phenotypic change in bistable epigenetic regulatory networks that are susceptible to noise and proteins capable of dominant variant conformations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Migration to industrialised countries poses a "double whammy" for type 2 diabetes among sub-Saharan African migrant and refugee adults. This population group has been found to be at an increased risk of obesity and type 2 diabetes, which may be further aggravated by inadequate vitamin D status. Thus, this study aimed to describe the demographics of vitamin D insufficiency, obesity, and risk factors for type 2 diabetes among sub-Saharan African migrants and refugees aged 20 years or older living in Melbourne, Australia (n=49). Data were obtained by a questionnaire, medical assessment, and fasting blood samples. The mean serum 25-hydroxyvitamin D level was 27.3 nmol/L (95% CI: 22.2, 32.4 nmol/L); with 25-hydroxyvitamin D levels <50 nmol/L occurring in 88% of participants. Participants displayed a cluster of risk factors for type 2 diabetes and cardiovascular disease: 62% were overweight or obese, 47% had insulin resistance (HOMA-IR >=2), 25% had low density lipoprotein cholesterol levels >=3.5 mmol/L, 24.5% had high density lipoprotein cholesterol levels <=1.03 mmol/L, 34.6% had borderline or high levels of total cholesterol (>=5.2 mmol/L), 18.2% had borderline or high levels of triglyceride (>=1.7 mmol/L), and 16% had hypertension (systolic blood pressure >=140 mmHg or diastolic blood pressure >=90 mmHg). These findings suggest that sub-Saharan African migrants and refugees may be at risk of type 2 diabetes and atherosclerosis-related diseases such as ischemic heart disease, stroke, and peripheral vascular disease. Well-designed vitamin D interventions that incorporate lifestyle changes are urgently needed in this sub-population.

Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA-damage response and recruits the main recombination protein RecA. This function of DksA is independent of its transcription initiation activity but requires its less-studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Objectives: To assess the efficacy of a novel hydrogel polymer buccal insert containing 5 mg pilocarpine in releasing the pilocarpine in a controlled fashion over a three hour period, and to assess the effects of this on quantitative tear and saliva production and the acceptability of the insert to the patient., Design: This was an open, uncontrolled pilot study for which Ethics Committee approval was obtained prior to starting. Hydrogel buccal inserts containing 5 mg pilocarpine were used three times a day for seven days., Setting: The Department of Oral Medicine, Glasgow Dental Hospital & School., Subjects: Eight patients with Sjögren's syndrome., Main Outcome Measures: Changes over baseline in (1) Schirmer test, (2) whole saliva flow rate, (3) oral comfort score (VAS), (4) ocular comfort score (VAS), (5) patient acceptability., Results: The buccal inserts successfully released in excess of 85% of their 5 mg pilocarpine load over three hours. There was a general improvement in oral and ocular comfort scores assessed by visual linear analogue scale, and saliva and tear production generally increased. The inserts were well tolerated by all patients except one (who wore dentures). Adverse events were few and none was serious., Conclusion: This novel form of buccal pilocarpine delivery demonstrated potential for use in treating patients with Sjögren's syndrome.

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.

Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 A. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents.

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.

The major proteins of baboon milk were identified as beta-lactoglobulin (beta LG), alpha-lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon beta LG are identical to those of macaque (Macaca fasicularis) beta LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)+ mRNA purified from lactating mammary gland. Baboon beta LG consists of 168 amino acid residues (M(r) 20,750) and is the longest beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Monogeneans (flatworms) are among the most host-specific of parasites in general and may be the most host-specific of all fish parasites. Specificity, in terms of a restricted spatial distribution within an environment, is not unique to parasites and is displayed by some fungi, insects, birds, symbionts and pelagic larvae of free-living marine invertebrates. The nature of cues, how "habitats" are recognised and how interactions between partners are mediated and maintained is of interest across these diverse "associations". We review some experiments that demonstrate important factors that contribute to host-specificity at the level of infective stages (larvae of oviparous monogeneans; juveniles of viviparous gyrodactylids) and adult parasites. Recent research on immune responses by fish to monogenean infections is considered. We emphasise the critical importance of host epidermis to the Monogenea. Monogeneans live on host epidermis, they live in its products (e.g. mucus), monopisthocotyleans feed on it, some of its products are "attractants" and it may be an inhospitable surface because of its immunological activity. We focus attention on fish but reference is made to amphibian hosts. We develop the concept for a potential role in host-specificity by the anterior adhesive areas, either the specialised tegument and/or anterior secretions produced by monogeneans for temporary but firm attachment during locomotion on host epithelial surfaces. Initial contact between the anterior adhesive areas of infective stages and host epidermis may serve two important purposes. (1) Appropriate sense organs or receptors on the parasite interact with a specific chemical or chemicals or with surface structures on host epidermis. (2) A specific but instant recognition or reaction occurs between component(s) of host mucus and the adhesive(s) secreted by monogeneans. The chemical composition of fish skin is known to be species-specific and our preliminary analysis of the chemistry of some monogenean adhesives indicates they are novel proteins that display some differences between parasite families and species.

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.