Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats.

BACKGROUND AND OBJECTIVE: Maternal overnutrition has been implicated in affecting the offspring by programming metabolic disorders such as obesity and diabetes, by mechanisms that are not clearly understood. This study aimed to determine the long-term impact of maternal high-fat (HF) diet feeding on epigenetic changes in the offspring's hypothalamic Pomc gene, coding a key factor in the control of energy balance. Further, it aimed to study the additional effects of postnatal overnutrition on epigenetic programming by maternal nutrition. METHODS: Eight-week-old female Sprague-Dawley rats were fed HF diet or low-fat (LF) diet for 6 weeks before mating, and throughout gestation and lactation. At postnatal day 21, samples were collected from a third offspring and the remainder were weaned onto LF diet for 5 weeks, after which they were either fed LF or HF diet for 12 weeks, resulting in four groups of offspring differing by their maternal and postweaning diet. RESULTS: With maternal HF diet, offspring at weaning had rapid early weight gain, increased adiposity, and hyperleptinemia. The programmed adult offspring, subsequently fed LF diet, retained the increased body weight. Maternal HF diet combined with offspring HF diet caused more pronounced hyperphagia, fat mass, and insulin resistance. The ARC Pomc gene from programmed offspring at weaning showed hypermethylation in the enhancer (nPE1 and nPE2) regions and in the promoter sequence mediating leptin effects. Interestingly, hypermethylation at the Pomc promoter but not at the enhancer region persisted long term into adulthood in the programmed offspring. However, there were no additive effects on methylation levels in the regulatory regions of Pomc in programmed offspring fed a HF diet. CONCLUSION: Maternal overnutrition programs long-term epigenetic alterations in the offspring's hypothalamic Pomc promoter. This predisposes the offspring to metabolic disorders later in life.

Meeting the needs of four generations of nurses.

Every year increasing numbers of nurses and midwives leave the NHS. In the current climate of staff shortages it is crucial to understand why this is happening and try to resolve the problem; in Birmingham we have taken steps to do this. This article explores the different workforce generations, along with strategies that could enhance nursing and midwifery careers, ensuring they meet the needs of all generations, thereby encouraging practitioners to stay in their profession for longer.

Co-expression of protein phosphatases in insect cells affects phosphorylation status and expression levels of proteins.

The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.

Chronic glucocorticoid treatment induces hepatic lipid accumulation and hyperinsulinaemia in part through actions on AgRP neurons.

Glucocorticoids (GCs) are widely prescribed anti-inflammatory medicines, but their use can lead to metabolic side-effects. These may occur through direct actions of GCs on peripheral organs, but could also be mediated by the hypothalamic AgRP neurons, which can increase food intake and modify peripheral metabolism. Therefore, the aim of this study was to examine the metabolic effects of chronic treatment with the GC corticosterone (Cort, 75 µg/ml in drinking water) in mice lacking the glucocorticoid receptor (GR) on AgRP neurons. Female AgRP-GR KO mice had delayed onset of Cort-induced hyperphagia. However, AgRP-GR KO had little impact on the increased body weight or adiposity seen with 3 weeks Cort treatment. Cort caused hepatic steatosis in control mice, but in Cort treated female AgRP-GR KO mice there was a 25% reduction in liver lipid content and lower plasma triglycerides. Additionally, Cort treatment led to hyperinsulinaemia, but compared to controls, Cort-treated AgRP-GR KO mice had both lower fasting insulin levels and lower insulin levels during a glucose tolerance test. In conclusion, these data indicate that GCs do act through AgRP neurons to contribute, at least in part, to the adverse metabolic consequences of chronic GC treatment.

Links between granuloma annulare, necrobiosis lipoidica diabeticorum and childhood diabetes: a matter of time?

Diabetes mellitus is associated with a range of dermatologic presentations, including granuloma annulare and necrobiosis lipoidica diabeticorum. Granuloma annulare occurs earlier than necrobiosis lipoidica diabeticorum and the association with diabetes mellitus is much weaker. We describe two children with diabetes who both developed granuloma annulare and later, necrobiosis lipoidica diabeticorum. We postulate that the early onset and transient nature of granuloma annulare, compared with the later onset and persistence of necrobiosis lipoidica diabeticorum, might account for the different apparent rates of association with diabetes mellitus.

Nurses' early and ongoing encounters with the dying and the dead: a scoping review of the international literature.

BACKGROUND: End-of-life care is high on policy and political agendas in the UK and internationally. Nurses are at the forefront of this, caring for dying patients, 'managing' the dead body, and dealing with the corporeal, emotional and relational dimensions of death. Little is known about nurses' prior or early professional experiences of and reactions to death, dying and the corpse and how these might influence practice. AIMS: To appraise the international literature on nurses' early experiences of death, dying and the dead body, to better understand how these might influence subsequent practice, and how this might inform our teaching of death, dying and last offices. METHODS: A scoping review was undertaken of peer-reviewed publications between, 2000 and 2019, which included nurses working in hospital, care homes and the community. Medline, PubMed, PsychINFO and CINAHL databases were searched and 23 papers meeting the inclusion criteria were read. Arksey and O'Malley's (2005) five-stage approach was adopted to scope the relevant international literature, using where relevant the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Selected papers were independently reviewed and subjected to thematic analysis, leading to the generation of five overarching themes. RESULTS: The five themes were: different philosophies of care; relationships; knowledge; impact of death; and giving care. The studies came from diverse geographical locations across different settings and were primarily qualitative in design. CONCLUSIONS: Students and registered nurses are impacted both positively and negatively by their early encounters with death and dying. Good communication with patients, families and between professionals, understanding of what constitutes a 'good' death, and high-quality mentorship and support were of particular importance.

A collaborative approach to facilitate professionals to support the breathless patient.

OBJECTIVES: Breathlessness is a major problem for people in their last weeks of life. Breathlessness is considered to be multidimensional with physical, psychological, emotional, social and spiritual factors all playing a part. It has been recognised that specific training to health professionals is beneficial in order to improve the care for patients with breathlessness.Breathlessness courses have tended to focus on senior nurses. A new flexible and collaborative training course was designed to include a wider range of nurses and other health professionals in hospital, hospice, primary care and community settings. The aim of the 'Practical Skills to Support the Breathless Patient' programme was to make patients and carers feel better supported in their breathlessness, for health professionals to develop confidence and skills in using proven interventions, and to adopt a flexible educational design that could be adapted to different contexts. METHODS: The course is learner-centred and teaching methods encourage interaction and participation via a mix of lectures and discussions with practical skills-focused, experiential workshops in smaller groups. Case study work was included to integrate learning with participants' practice environment. Evaluation is built in during the course, so adaptations can be made throughout to respond to changing learner needs. RESULTS: Participants reported increased confidence in terms of knowledge and applying this within everyday practice. The theory-practice dynamic worked well within each participant' specific work context in particular through the case study approach. CONCLUSIONS: The course developed a number of innovative approaches, such as multi-disciplinary learning groups, regular feedback loops, reflexive learning about putting theory into practice and long-term follow-up. Combining these elements increases professionals' confidence and sustains new clinical practice.

New understandings of fathers' experiences of grief and loss following stillbirth and neonatal death: A scoping review.

OBJECTIVE: To report on research conducted on men's experiences of grief and loss following stillbirth and neonatal death in high-income, Western countries. DESIGN: This review was guided by the following research questions: 1. The impact of perinatal death for men 2. The meaning of the loss for a father's sense of identity 3. The extent to which men were able to express grief while supporting their partners and, 4. how men's experience of grief was mediated by the support and care received by health professionals. DATA SOURCES: We searched the following databases: Medline; PsychINFO; CINAHL to identify relevant articles published from the year 2000 onwards. The searches were run between 1/04/2018 and 8/4/2018. REVIEW METHODS: A scoping review was conducted of nursing, psychological, medical and social science databases using these key words: fathers' grief, men's grief, perinatal loss and death, stillbirth and neonatal death. RESULTS: Studies indicated that men reported less intense and enduring levels of psychological outcomes than women but were more likely to engage in avoidance and coping behaviours such as increased alcohol consumption. Men felt that their role was primarily as a 'supportive partner' and that they were overlooked by health professionals. CONCLUSIONS: Further research is needed on men's experience of grief following perinatal death, especially on their physical and mental well-being. IMPACT: This review addressed the problem of the lack of knowledge around paternal needs following perinatal death and highlighted areas which researchers could usefully investigate with the eventual aim of improving care for fathers.

Role of peer support for people with a spinal cord injury.

Spinal cord injuries can result in significant physiological and psychological challenges for patients. Nurses have an important role in the rehabilitation of people with a spinal cord injury, as does the provision of peer support by people who are 'living well' after experiencing a spinal cord injury. AIM: To explore peer support and whether it can have an effective role in a multidisciplinary team approach to supporting a patient with a spinal cord injury. METHOD: This was an independent evaluation that used an online survey, a focus group and telephone interviews to elicit the views of respondents about the Spinal Injury Association (SIA) peer support service in England and Wales. There were four groups of respondents: people with a spinal cord injury; their family and friends; peer support officers; and nurse specialists and other healthcare practitioners. FINDINGS: It was identified that peer support officers were valued for their ability to provide a positive role model for people with a spinal cord injury. Healthcare practitioners involved in the care of people with a spinal cord injury also appreciated the training and support they received from peer support officers. CONCLUSION: This evaluation demonstrated that peer support officers were regarded as an important element of a multidisciplinary team approach to managing the care of those who had experienced a spinal cord injury. Peer support officers were also considered a valuable source of information and education, particularly for healthcare practitioners working in settings where spinal cord injury was not a common presentation.

Metabolic Abnormalities of Chronic High-Dose Glucocorticoids Are Not Mediated by Hypothalamic AgRP in Male Mice.

Glucocorticoids are potent and widely used medicines but often cause metabolic side effects. A murine model of corticosterone treatment resulted in increased hypothalamic expression of the melanocortin antagonist AgRP in parallel with obesity and hyperglycemia. We investigated how these adverse effects develop over time, with particular emphasis on hypothalamic involvement. Wild-type and Agrp-/- male mice were treated with corticosterone for 3 weeks. Phenotypic, biochemical, protein, and mRNA analyses were undertaken on central and peripheral tissues, including white and brown adipose tissue, liver, and muscle, to determine the metabolic consequences. Corticosterone treatment induced hyperphagia within 1 day in wild-type mice, which persisted for 3 weeks. Despite this early increase in food intake, the body weight only started to increase after 10 days. Hyperinsulinemia occurred at day 1. Also, although after 2 days, alterations were present in the genes often associated with insulin resistance in several peripheral tissues, hyperglycemia only developed at 3 weeks. Throughout, sustained elevation in hypothalamic Agrp expression was present. Mice with Agrp deleted [using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, Agrp-/-] were partially protected against corticosterone-induced hyperphagia. However, Agrp-/- mice still had corticosterone-induced increases in body weight and adiposity similar to those of the Agrp+/+ mice. Loss of Agrp did not diminish corticosterone-induced hyperinsulinemia or correct changes in hepatic gluconeogenic genes. Chronic glucocorticoid treatment in mice mimics many of the metabolic side effects seen in patients and leads to a robust increase in Agrp. However, AgRP does not appear to be responsible for most of the glucocorticoid-induced adverse metabolic effects.

Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment.

OBJECTIVE: Glucocorticoids (GCs) are widely prescribed medications that are well recognized to cause adverse metabolic effects including hyperphagia, obesity, and hyperglycemia. These effects have been recapitulated in a murine model of GC excess, and we hypothesize that they are mediated, in part, through central mechanisms. This study aimed to identify genes in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment and evaluate their contribution to GC-induced metabolic abnormalities. METHODS: Corticosterone (Cort; 75 µg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Phenotypic analysis of each group was undertaken and central and peripheral tissues were collected for biochemical and mRNA analyses. Arcuate nuclei were isolated by laser capture microdissection and tissue analyzed by RNA-seq. RESULTS: RNA-seq analysis of ARC tissue from 4 week Cort treated mice revealed 21 upregulated and 22 downregulated genes at a time when mice had increased food intake, expansion of adipose tissue mass, and insulin resistance. In comparison, after 2 days Cort treatment, when the main phenotypic change was increased food intake, RNA-seq identified 30 upregulated and 16 downregulated genes. Within the genes altered at 2 days were a range of novel genes but also those known to be regulated by GCs, including Fkbp5, Mt2, Fam107a, as well as some involved in the control of energy balance, such as Agrp, Sepp1, Dio2, and Nmb. Of the candidate genes identified by RNA-seq, type-II iodothyronine deiodinase (Dio2) was chosen for further investigation as it was increased (2-fold) with Cort, and has been implicated in the control of energy balance via the modulation of hypothalamic thyroid hormone availability. Targeted knockdown of Dio2 in the MBH using AAV-mediated CRISPR-Cas9 produced a mild attenuation in GC-induced brown adipose tissue weight gain, as well as a 56% reduction in the GC-induced increase in Agrp. However, this conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia. CONCLUSIONS: This study identified a comprehensive set of genes altered by GCs in the ARC and enabled the selection of key candidate genes. Targeted knockdown of hypothalamic Dio2 revealed that it did not mediate the chronic GC effects on hyperphagia and hyperglycemia.

Elevated Hypothalamic Glucocorticoid Levels Are Associated With Obesity and Hyperphagia in Male Mice.

Glucocorticoid (Gc) excess, from endogenous overproduction in disorders of the hypothalamic-pituitary-adrenal axis or exogenous medical therapy, is recognized to cause adverse metabolic side effects. The Gc receptor (GR) is widely expressed throughout the body, including brain regions such as the hypothalamus. However, the extent to which chronic Gcs affect Gc concentrations in the hypothalamus and impact on GR and target genes is unknown. To investigate this, we used a murine model of corticosterone (Cort)-induced obesity and analyzed Cort levels in the hypothalamus and expression of genes relevant to Gc action. Mice were administered Cort (75 µg/mL) or ethanol (1%, vehicle) in drinking water for 4 weeks. Cort-treated mice had increased body weight, food intake, and adiposity. As expected, Cort increased plasma Cort levels at both zeitgeber time 1 and zeitgeber time 13, ablating the diurnal rhythm. Liquid chromatography dual tandem mass spectrometry revealed a 4-fold increase in hypothalamic Cort, which correlated with circulating levels and concentrations of Cort in other brain regions. This occurred despite decreased 11ß-hydroxysteroid dehydrogenase (Hsd11b1) expression, the gene encoding the enzyme that regenerates active Gcs, whereas efflux transporter Abcb1 mRNA was unaltered. In addition, although Cort decreased hypothalamic GR (Nr3c1) expression 2-fold, the Gc-induced leucine zipper (Tsc22d3) mRNA increased, which indicated elevated GR activation. In keeping with the development of hyperphagia and obesity, Cort increased Agrp, but there were no changes in Pomc, Npy, or Cart mRNA in the hypothalamus. In summary, chronic Cort treatment causes chronic increases in hypothalamic Cort levels and a persistent elevation in Agrp, a mediator in the development of metabolic disturbances.

Maternal undernutrition programs tissue-specific epigenetic changes in the glucocorticoid receptor in adult offspring.

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids.

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Galpha(i). Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Galpha(i) signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Galpha(i) signalling mediated by GPR43 in SCFA-stimulated leptin secretion.

Australian general practice and pandemic influenza: models of clinical practice in an established pandemic.

To minimise the health impact of pandemic influenza, general practice will need to provide influenza-related and non-influenza primary health care, as well as contribute to the public health goal of disease control. Through interviews and workshops with general practitioners, nurses and policy leaders between March and July 2006, and literature analysis, we identified potential models of general practice in an established pandemic, and assessed their strengths and weaknesses. Three possible clinical models were identified: a default model of no change to service delivery; a streamed services model, where general practices reorganise themselves to take on either influenza-specific care or other clinical services; and a staff-determined mixed model, where staff move between different types of services. No single model or set of strategies meets the needs of all general practices to deliver and sustain the essential functions of primary health care during an established pandemic. Governments, general practice and the relevant peak professional bodies should decide before a pandemic on the suite of measures needed to support the models most suitable in their regions. Effective participation by general practice in a pandemic requires supplementary infrastructure support, changes to financial and staffing patterns, a review of legislation on medicolegal implications during an emergency, and intensive collaboration between general practices.

Molecular genetic analysis of chromosome 9 candidate tumor-suppressor loci in bladder cancer cell lines.

Underrepresentation of chromosome 9 is a common finding in bladder cancer. Frequent loss of the whole chromosome suggests the presence of at least one relevant tumor suppressor gene on each arm. Candidate regions identified by loss of heterozygosity (LOH) analysis include a region at 9p21 containing CDKN2A, which encodes p16 and p14(ARF), a large region at 9q12-31 including PTCH and many other genes, a small region at 9q32-33, which includes the DBCCR1 gene, and a region at 9q34 including the TSC1 gene. Experimental replacement of genes or chromosomes into tumor cells with appropriate deletions or mutations represents an important approach to test the functional significance of candidate tumor suppressor genes. Loss of an entire copy of chromosome 9 in many bladder tumor cell lines provides no indication of which gene or genes are affected, and selection of appropriate recipient cells for gene replacement is difficult. We have investigated three candidate tumor suppressor genes on chromosome 9 (CDKN2A, DBCCR1, and TSC1), at the DNA level and by expression analysis in a panel of bladder tumor cell lines, many of which have probable LOH along the length of the chromosome, as indicated by homozygosity for multiple polymorphic markers. Cytogenetically, we found no reduction in the numbers of chromosomes 9 relative to total chromosome count. Homozygous deletion of the CDKN2A locus was frequent but homozygous deletion of TSC1 was not found. A new cell line, DSH1, derived from a pT1G2 transitional cell carcinoma with known homozygous deletion of DBCCR1, is described. This study identifies suitable cell lines for future functional analysis of both CDKN2A and DBCCR1.