A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes.

OBJECTIVE: More than 300 genetic variants have been robustly associated with measures of human adiposity. Highly penetrant mutations causing human obesity do so largely by disrupting satiety pathways in the brain and increasing food intake. Most of the common obesity-predisposing variants are in, or near, genes expressed highly in the brain, but little is known of their function. Exploring the biology of these genes at scale in mammalian systems is challenging. We sought to establish and validate the use of a multicomponent screen for feeding behaviour phenotypes, taking advantage of the tractable model organism Drosophila melanogaster. METHODS: We validated a screen for feeding behaviour in Drosophila by comparing results after disrupting the expression of centrally expressed genes that influence energy balance in flies to those of 10 control genes. We then used this screen to explore the effects of disrupted expression of genes either a) implicated in energy homeostasis through human genome-wide association studies (GWAS) or b) expressed and nutritionally responsive in specific populations of hypothalamic neurons with a known role in feeding/fasting. RESULTS: Using data from the validation study to classify responses, we studied 53 Drosophila orthologues of genes implicated by human GWAS in body mass index and found that 15 significantly influenced feeding behaviour or energy homeostasis in the Drosophila screen. We then studied 50 Drosophila homologues of 47 murine genes reciprocally nutritionally regulated in POMC and agouti-related peptide neurons. Seven of these 50 genes were found by our screen to influence feeding behaviour in flies. CONCLUSION: We demonstrated the utility of Drosophila as a tractable model organism in a high-throughput genetic screen for food intake phenotypes. This simple, cost-efficient strategy is ideal for high-throughput interrogation of genes implicated in feeding behaviour and obesity in mammals and will facilitate the process of reaching a functional understanding of obesity pathogenesis.

Microbiological changes observed over 48 weeks of treatment with inhaled liposomal ciprofloxacin in individuals with non-cystic fibrosis bronchiectasis and chronic Pseudomonas aeruginosa lung infection.

OBJECTIVES: Non-cystic fibrosis bronchiectasis (NCFBE) with Pseudomonas aeruginosa has been associated with increased pulmonary exacerbation (PEx) and mortality risk. European Respiratory Society guidelines conditionally recommend inhaled antimicrobials for persons with NCFBE, P aeruginosa and three or more PEx/year. We report microbiological results of two randomized, 48-week placebo-controlled trials of ARD-3150 (inhaled liposomal ciprofloxacin) in individuals with NCFBE with P aeruginosa and PEx history [Lancet Respir Med 2019;7:213-26]. METHODS: Respiratory secretions from 582 participants receiving up to six 28-day on/off treatment cycles were analysed for sputum P. aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Escherichia coli densities, P. aeruginosa susceptibilities to ciprofloxacin and nine other antimicrobials, and prevalence of other bacterial opportunists. Associations between PEx risk and sputum density, antimicrobial susceptibility and opportunist prevalence changes were studied. RESULTS: Sputum P. aeruginosa density reductions from baseline after ARD-3150 treatments ranged from 1.77 (95% CI 2.13-1.40) versus 0.54 (95% CI 0.89-0.19) log10 CFU/g for placebo (second period) to 2.07 (95% CI 2.45-1.69) versus 0.70 (95% CI 1.11-0.29) log10 CFU/g for placebo (fourth period) with only modest correlation between density reduction magnitude and PEx benefit. ARD-3150 (but not placebo) treatment was associated with increased P. aeruginosa ciprofloxacin MIC but not emergence of other bacterial opportunists across the study; ciprofloxacin MIC50 increased from 0.5 to 1 mg/L, MIC90 increased from 4 to 16 mg/L. Other antimicrobial MIC were mostly unaffected. CONCLUSION: Microbiological changes over 48 weeks of ARD-3150 treatment appear modest. Ciprofloxacin susceptibility (but not other antimicrobial susceptibility) decreases were observed that did not appear to preclude PEx risk reduction benefit.

High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons.

BACKGROUND: Consumption of dietary fat is one of the key factors leading to obesity. High-fat diet (HFD)-induced obesity is characterized by induction of inflammation in the hypothalamus; however, the temporal regulation of proinflammatory markers and their impact on hypothalamic appetite-regulating neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons remains undefined. METHODS: Mice were injected with an acute lipid infusion for 24 h or fed a HFD over 8-20 weeks. Characterized mouse NPY/AgRP hypothalamic cell lines were used for in vitro experimentation. Immunohistochemistry in brain slices or quantitative real-time PCR in cell lines, was performed to determine changes in the expression of key inflammatory markers and neuropeptides. RESULTS: Hypothalamic inflammation, indicated by tumor necrosis factor (TNF)-α expression and astrocytosis in the arcuate nucleus, was evident following acute lipid infusion. HFD for 8 weeks suppressed TNF-α, while significantly increasing heat-shock protein 70 and ciliary neurotrophic factor, both neuroprotective components. HFD for 20 weeks induced TNF-α expression in NPY/AgRP neurons, suggesting a detrimental temporal regulatory mechanism. Using NPY/AgRP hypothalamic cell lines, we found that palmitate provoked a mixed inflammatory response on a panel of inflammatory and endoplasmic reticulum (ER) stress genes, whereas TNF-α significantly upregulated IκBα, nuclear factor (NF)-κB and interleukin-6 mRNA levels. Palmitate and TNF-α exposure predominantly induced NPY mRNA levels. Utilizing an I kappa B kinase ß (IKKß) inhibitor, we demonstrated that these effects potentially occur via the inflammatory IKKß/NF-κB pathway. CONCLUSIONS: These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

Impact of a short home-based yoga programme on blood pressure in patients with hypertension: a randomized controlled trial in primary care.

The present study was designed to evaluate yoga's impact on blood pressure (BP) and quality of life (QOL) and on stress, depression and anxiety in patients with hypertension in a primary care setting. We conducted a multi-centre randomized controlled trial with follow-up after 12-week intervention completion. Adult primary care patients diagnosed with hypertension were randomly allocated to yoga or usual care. The intervention group performed a short home-based Kundalini yoga programme 15 min twice-daily during the 12-week intervention period. At baseline and follow-up, the participants underwent standardized BP measurements and completed questionnaires on QOL, stress, anxiety and depression. Data obtained from 191 patients (mean age 64.7 years, s.d. 8.4) allocated to yoga intervention (n=96) and control group (n=95), with a total proportion of 52% women, showed a significant reduction in systolic and diastolic BP for both groups (-3.8/-1.7 mm Hg for yoga and -4.5/-3.0 mm Hg for control groups, respectively). However, the BP reduction for the yoga group was not significantly different from control. There were small but significant improvements for the yoga group in some of the QOL and depression measures (P<0.05, Hospital Anxiety and Depression scale, HADS-D) compared with control. The findings of our study, which is the largest study from an OECD country (Organization for Economic Co-operation and Development) to date, do not support the suggestion from previous smaller studies that yoga lowers the BP. Further clinical trials are needed to confirm these findings. However, the yoga patients had other health benefits.

A bioenergetic assessment of mitral regurgitation: a new tool to assess severity?

Mitral regurgitation is frequently classified as mild, moderate or severe based on echocardiography. Patients with mild mitral regurgitation are usually managed medically. We hypothesise that mild mitral regurgitation as assessed volumetrically can in fact be severe when analysed from a bioenergetics point of view. The conservation of energy predicts that any regurgitant volume will require the heart to provide more work energy to support the circulation. Mitral regurgitation involves the left ventricle imparting potential energy, via blood pressure, and kinetic energy, via regurgitant velocity, to the regurgitant blood volume. This implies that regurgitant volume, regurgitant velocity, systolic blood pressure, heart rate, regurgitant orifice area and cardiac output are all important factors. We present limited data to demonstrate our hypothesis. A bioenergetic analysis of mitral regurgitation, may identify patients whose mitral regurgitation, assessed via echocardiography as mild, is actually clinically significant. In addition we identify the importance of blood pressure and heart rate control in patients with mitral regurgitation. The concept that a bit of mitral regurgitation in patients with poor left ventricles is a good thing, as it helps offload the left ventricle is from an engineering point fundamentally flawed.

The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons.

Significant information on reproductive function has been generated based on the rat model, including many seminal discoveries. Yet little is known about the molecular and cellular events involved in control of reproductive function, mainly due to the pervasive lack of cell models from rat. We have therefore generated a wide array of cell lines using primary cell culture from the rat hypothalamus. Immortalization of the primary cells was achieved through retroviral transfer of T-antigen, followed by selection with geneticin. The mixed cell populations were subcloned and each clonal cell line was analyzed for expression of specific cellular markers. Each line has a distinct phenotypic profile, with expression of key neuroendocrine markers. We have functionally analyzed two clonal cell lines, rHypoE-7 and rHypoE-8, for hormones implicated in the control of gonadotropin-releasing hormone neuronal function through melatonin, specifically kisspeptin (KISS) and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). We detected functional melatonin receptor activity, as each cell line exhibited inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation. Upon treatment with 10 nM melatonin, we found that KISS gene expression was decreased in the rHypoE-8 cell line, while RFRP-3 was increased in the rHypoE-7 cell line. These results are in accordance with the differential regulatory functions of these two peptides, particularly on GnRH neuronal control. These cell lines will serve as novel tools for the analysis of the cellular and molecular mechanisms involved in hypothalamic control of a number of physiological processes described in the rat animal model.

Visual compatibility of amiodarone hydrochloride injection with various intravenous drugs.

The Notes section welcomes the following types of contributions: (1) practical innovations or solutions to everyday practice problems, (2) substantial updates or elaborations on work previously published by the same authors, (3) important confirmations of research findings previously published by others, and (4) short research reports, including practice surveys, of modest scope or interest. Notes should be submitted with AJHP's manuscript checklist. The text should be concise, and the number of references, tables, and figures should be limited.

Effect of indomethacin on blood pressure control during treatment with nitrendipine.

This study tested the hypothesis that treatment with a nonsteroidal anti-inflammatory drug will not alter the hypotensive effect of a dihydropyridine calcium channel antagonist. Fifteen essential hypertensives (ages 58-80 years) had a supine diastolic blood pressure (DBP) < 100 mmHg after 4 weeks monotherapy with nitrendipine 5-20 mg twice daily. They entered a double-blind randomised crossover study in which the addition of indomethacin 25 mg three times daily was compared with placebo in treatment phases each of 4 weeks duration. Subjects were seen weekly and measurements in the last 2 weeks of each phase were compared. Supine blood pressure (mean +/- SE) was higher in the indomethacin phase (158 +/- 4/80 +/- 2) than in the placebo phase (154 +/- 4/76 +/- 3) (p < 0.01 for DBP). In 6/15 (40%) of subjects the increase in supine diastolic blood pressure with indomethacin was > 5 mmHg. Plasma urea was also increased in the indomethacin phase: 7.6 +/- 0.6 mmol/l compared with placebo: 6.3 +/- 0.5 mmol/l (p < 0.001). The study has demonstrated that concurrent treatment with the NSAID indomethacin impairs the blood pressure lowering effect of the dihydropyridine calcium channel antagonist nitrendipine. This increase in blood pressure with indomethacin in subjects treated with nitrendipine may represent either an independent pressor effect of indomethacin or a reduced vasodilator prostanoid contribution to the hypotensive effect of nitrendipine. This blood pressure increase may be sufficient to interfere significantly with clinical blood pressure control in some subjects.

Variations in breast feeding advice, a telephone survey of community midwives and health visitors.

Although 65% of mothers start breast feeding their baby, at six weeks after birth this proportion has dropped to 40%. It is postulated that one of the causes of this decrease might be that women receive conflicting advice from professionals. To examine this hypothesis, a telephone survey was designed to compare a random sample of community midwives (CMs) and a random sample of health visitors (HVs), in regard to the advice they would offer to breast feeding women, and the resources available to help them. On many topics, the advice likely to be offered by the two groups was markedly different. The CMs tended to be more uniform in their advice, which generally followed the Royal College of Midwives' booklet entitled Present Day Practice in Infant Feeding. Both groups recognised the value of lay breast-feeding counsellors, and there was much support for collaboration in the production of a guidance document on breast feeding.

Lack of effect of fish oil supplementation on blood pressure in treated hypertensives.

Fish and fish oils have been reported to reduce blood pressure in normotensives and untreated hypertensives. The present study examined the effect of dietary supplementation with fish oil on blood pressure in 20 treated hypertensives with controlled blood pressures who continued their usual antihypertensive drug treatment throughout. A double-blind, randomized crossover design was used, with two phases, each of 8 weeks' duration. In one phase, subjects took fifteen 1 g fish oil capsules (Lipitac; Reckitt and Colman Pharmaceuticals, Sydney, Australia) daily, and in the other, 15 capsules of identical appearance containing 1 g olive oil daily. There was no difference between the treatment phases for any blood pressure parameter, heart rate or body weight, but blood pressure was lower in both phases compared with pretreatment values. The fasting plasma triglyceride concentration was 30% lower in the fish oil phase (P less than 0.001), but there was no difference between the phases for plasma concentrations of total or high-density lipoprotein (HDL) cholesterol. We conclude that, in treated hypertensives with controlled blood pressures, any additional fall in blood pressure produced by dietary supplementation with fish oil is so small that the requirement for antihypertensive drug therapy is unlikely to be reduced.

The effects of training in social perspective taking on socially maladjusted girls.

A paucity of research exists concerning training programs for the development of interpersonal functioning in socially maladjusted or delinquent adolescent females. Females in a residential institution participated in a role-play program designed to enhance social perspective-taking ability. In 15 sessions girls were coached in specific social skills and acted multiple role perspectives in typical problem situations. Compared to girls in a fitness training program, girls in the role-play training program showed enhanced performance on a measure of social perspective taking. Generalized effects were also found for performance on tests of interpersonal problem analysis, empathy, and the acceptance of individual differences. Additionally, observational data indicated that role-play training resulted in increased prosocial behaviors. Role-play training had no effect on a measure of referential communication.

Correlation between fall in blood pressure and in vivo amine release after alpha-methylDOPA.

Blood pressure (BP), hypothalamic tissue concentrations and the in vivo overflow of endogenous and alpha-methylated catecholamines were measured in urethane anaesthetised rats after alpha-methylDOPA (mDOPA) administration (200 mg/kg i.p.). Four hours after mDOPA, BP fell to its lowest value, 60% of control, and slowly returned towards control levels by 24 h. This was closely correlated with the evoked overflow of alpha-methylnoradrenaline (mNA, r = 0.9) and noradrenaline (NA, r = 0.7) but not dopamine (DA) or alpha-methyldopamine (mDA). However, the tissue content of mNA rose much more gradually and was not maximal until after 12 h while mDA content followed the development of the hypotension. The results provide direct evidence for a false transmitter role for mNA in the brain, and suggest that the release of newly synthesised mNA is responsible for the hypotensive effect of mDOPA. Differences in the time course of overflow and storage of NA and mNA suggest the presence of separate transmitter storage and releasable pools.

A simple, sensitive method for the determination of extracellular catecholamines in the rat hypothalamus using in vivo dialysis.

The design, construction and characterisation of a dialysis probe suitable for perfusing any deep brain structures is described. Using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) the effects of flow rate, concentration, dialysate composition and temperature on the recovery within the dialysate of authentic catecholamines are detailed. The dialysis probe was used to collect endogenous catecholamines from the anterior hypothalamus of urethane-anaesthetised rats. Following organic phase extraction of the in vivo samples, a small basal release of noradrenaline (NA) of 37 +/- 4 pg/30 min sample was found. Potassium stimulation markedly elevated the release of NA and dopamine from the anterior hypothalamus in a calcium ion and dose dependent manner. It appears therefore that the dialysis probe described here, in conjunction with HPLC-ECD, can be used to follow changes in neuronally released catecholamines within the anterior hypothalamus, providing a valuable tool to study the role of these neurotransmitters in physiological and pharmacological function.

Felodipine compared to prazosin as additional therapy to a beta-blocking drug.

Felodipine was compared with prazosin in patients with essential hypertension whose blood pressure was not controlled by a beta-blocking drug. One hundred patients with a supine diastolic blood pressure greater than or equal to mm Hg after 4 weeks or more on a beta-blocking drug and placebo were randomly assigned to felodipine or prazosin tablets. The drugs were titrated at 2-week intervals if diastolic BP was greater than or equal to 90 mm Hg. Titration steps of felodipine were 5, 10, 20 mg b.i.d. and of prazosin were 1, 2, 4 mg b.i.d. The fall in blood pressure with felodipine 32/21 mm Hg was greater than the fall with prazosin 16/12 mm Hg (p less than 0.001); 36 patients achieved a diastolic blood pressure of less than 90 mm Hg with felodipine, which was a significantly greater number than the 20 patients who obtained such a level with prazosin (p less than 0.01). Both drugs were well tolerated, but more patients complained of vascular type side effects (flushing, peripheral edema) with felodipine than with prazosin. There was significant weight gain with prazosin but not with felodipine. Felodipine was shown to be a well-tolerated, effective antihypertensive agent when used with a beta-blocking drug and to be suitable for people with hypertension who fail to be controlled with a beta-blocking drug.

Slow-release nifedipine as a single or additional agent in the treatment of essential hypertension--a placebo-controlled crossover study.

The efficacy and safety of a new slow-release formulation of nifedipine ("Adalat Retard") were assessed in a double-blind cross-over trial in 19 subjects with essential hypertension (14 male, 5 female--ages: 34-72 years), 14 of whom continued previous antihypertensive medication. There were two 6 week treatment phases in which nifedipine 20 mg twice daily and placebo tablets twice daily were administered in random order. Supine mean blood pressure was 115 +/- 2 mm Hg during the placebo phase and 105 +/- 2 mm Hg during the nifedipine phase (p less than 0.001); and standing mean blood pressure was 121 +/- 2 mm Hg after placebo and 110 +/- 2 mm Hg after nifedipine (p less than 0.001). The magnitude of the blood pressure difference between the two phases was not related either to age or to the placebo phase blood pressure. The hypotensive effect of nifedipine was observed when administered as a single agent or in combination with diuretic and/or beta blocker. Heart rate was increased after nifedipine--75 +/- 2 beats/minute compared with 71 +/- 2 beats/minute after placebo (p less than 0.01). In this dose nifedipine (as "Adalat Retard") is an effective hypotensive agent which is a useful addition to presently available therapy.

Embryonic hypothalamic tissue transplanted to the IVth ventricle of newborn Brattleboro rats.

The hypothalamic region, dissected from normal rat embryos, was transplanted into the IVth ventricle of newborn mutant Brattleboro rats. Water intake and urine osmolality were measured in both the recipient animals and unoperated littermate controls during a 7-week period following weaning. No differences were found between operated and unoperated animals. Ten weeks after transplantation, host animals were fixation perfused and the brains prepared for either catecholamine fluorescence or vasopressin immunohistochemistry. Well-developed grafts were found in the IVth ventricle of the hosts. They received innervation from the host locus coeruleus and contained many neurons with vasopressin-like immunoreactivity. Vasopressin-containing fibers were found running from the grafts into the host medulla.

Methods of selenium supplementation of ruminants.

Three methods of selenium supplementation, by subcutaneous injection, intraruminal pellet and addition to water, were tested in experiments with cattle and a fourth method, oral supplementation of a sodium selenite solution, was evaluated with lambs. All four methods worked effectively for periods ranging from four months to one year after treatment. It is suggested that choice of treatment will depend on the circumstances of each case, including cost, husbandry system and ease of administration.

Brainstem PNMT neurons and experimental hypertension in the rat.

The number of phenylethanolamine-N-methyl transferase (PNMT) cells visualised with immunohistochemical techniques in the medulla oblongata is increased by 20% in 4 week old spontaneously hypertensive rats (SHR) and stroke prone spontaneously hypertensive rats (SHR-SP). This is associated with a 50% increase in the activity of PNMT and a significant rise in the amount of PNMT enzyme protein present in the medulla and spinal cord of both 4 weeks old and 4 months old SHR and SHR-SP. Since previous experiments had demonstrated that sinoaortic denervation also increased spinal cord PNMT activity we subjected normotensive Wistar Kyoto control rats (WKY) and hypertensive SHR and SHR-SP to denervation and measured the changes in blood pressure and in PNMT activity. Mean arterial pressure rose immediately after denervation in all 3 strains of rats, with much greater rises in the SHR and SHR-SP than in WKY, but the increase in pressure was only sustained in the normotensive WKY, in which it remained elevated throughout the one week observation period. In a similar way, denervation of the arterial baroreceptors increased the activity of PNMT in the medulla and spinal cord of normotensive WKY controls, confirming the results of previous studies but was not able to increase the already elevated PNMT levels in the SHR and SHR-SP any further in these two tissues. We suggest that there is good evidence that PNMT neurons contribute to the maintenance and elevation of arterial pressure in both the neurogenic and genetic models of hypertension. It also seems likely that the activity of descending spinal PNMT neurons is more important in the maintenance of a sustained increase in pressure than in the induction of a transient rise.