Dr Benita Middleton

Senior Research Fellow
+44 (0)1483 689712
18 AY 02

Academic and research departments

School of Biosciences.


University roles and responsibilities

  • Senior Research Fellow
  • Member - Section of Chronbiology


    Harry A. Smith, Iain Templeman, Max Davis, Tommy Slater, David J. Clayton, Ian Varley, Lewis J. James, Benita Middleton, Jonathan D. Johnston, Leonidas G. Karagounis, Kostas Tsintzas, Dylan Thompson, Javier T Gonzalez, Jean-Philippe Walhin, James A. Betts (2024)Characterising 24-h skeletal muscle gene expression alongside metabolic & endocrine responses under diurnal conditions, In: The journal of clinical endocrinology and metabolismdgae350 Oxford University Press

    Skeletal muscle plays a central role in the storage, synthesis, and breakdown of nutrients, yet little research has explored temporal responses of this human tissue, especially with concurrent measures of systemic biomarkers of metabolism. To characterise temporal profiles in skeletal muscle expression of genes involved in carbohydrate metabolism, lipid metabolism, circadian clocks, and autophagy and descriptively relate them to systemic metabolites and hormones during a controlled laboratory protocol. Ten healthy adults (9M/1F, mean ± SD: age: 30 ± 10 y; BMI: 24.1 ± 2.7 kg·m-2) rested in the laboratory for 37 hours with all data collected during the final 24 hours of this period (i.e., 0800-0800 h). Participants ingested hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200-0700 h. Blood samples were collected hourly for endocrine and metabolite analyses, with muscle biopsies occurring every 4 h from 1200 h to 0800 h the following day to quantify gene expression. Plasma insulin displayed diurnal rhythmicity peaking at 1804 h. Expression of skeletal muscle genes involved in carbohydrate metabolism (Name - Acrophase; GLUT4 - 1440 h; PPARGC1A -1613 h; HK2 - 1824 h) and lipid metabolism (FABP3 - 1237 h; PDK4 - 0530 h; CPT1B - 1258 h) displayed 24 h rhythmicity that reflected the temporal rhythm of insulin. Equally, circulating glucose (0019 h), NEFA (0456 h), glycerol (0432 h), triglyceride (2314 h), urea (0046 h), CTX (0507 h) and cortisol concentrations (2250 h) also all displayed diurnal rhythmicity. Diurnal rhythms were present in human skeletal muscle gene expression as well systemic metabolites and hormones under controlled diurnal conditions. The temporal patterns of genes relating to carbohydrate and lipid metabolism alongside circulating insulin are consistent with diurnal rhythms being driven in part by the diurnal influence of cyclic feeding and fasting.

    Jonathan Johnston, Barbara Fielding, Alan Flanagan, Alexandra Johnstone, Claus-Dieter Mayer, Jeewaka Mendis, Benita Middleton, Peter Morgan, Victoria Revell, Leonie Ruddick-Collins, Michael Short, Johanna von Gerichten (2023)P15-013-23 Changes in Human Metabolism and Post-Prandial Responses Following a 5-Hour Simulated Jet-Lag, In: Current developments in nutrition7 Elsevier

    Objectives: Experimental inversion of circadian and behavioural rhythms by 12-hours adversely effects markers of metabolic health. The objective of this work was to investigate the effects of a more modest 5-hour delay in behavioural cycles (as observed in e.g. trans-Atlantic or trans-continental jetlag) on circadian and metabolic markers. Methods: Fourteen participants completed an 8-day inpatient laboratory protocol, with controlled sleep-wake opportunities, light-dark cycles, meal timing and diet composition. The 5-hour delay in sleep/wake and mealtimes was induced by delaying sleep opportunity. We measured melatonin to assess central circadian phase; markers of postprandial metabolism; subjective sleepiness, and appetite. Results: Melatonin rhythms gradually adapted to the new behavioural cycle. In the 4-hours after the phase delay, there was slower gastric emptying at breakfast, lower fasting plasma glucose, higher postprandial plasma glucose and triglycerides, and lower thermic effect of feeding (p < 0.05). These changes were abolished or attenuated within 48–72 hours of the phase delay. Conclusions: Moderate (5-hour) circadian desynchrony only transiently disrupts metabolism. Occasional jetlag may therefore pose low metabolic health risk

    Matt Spick, Thomas P.M. Hancox, Namrata R. Chowdhury, Benita Middleton, Debra J. Skene, Debra Jean Skene, A. Jennifer Morton (2023)Metabolomic Analysis of Plasma in Huntington’s Disease Transgenic Sheep (Ovis aries) Reveals Progressive Circadian Rhythm Dysregulation, In: Journal of Huntington's disease12(1)pp. 31-42 IOS Press

    Background: Metabolic abnormalities have long been predicted in Huntington’s disease (HD) but remain poorly characterized. Chronobiological dysregulation has been described in HD and may include abnormalities in circadian-driven metabolism. Objective: Here we investigated metabolite profiles in the transgenic sheep model of HD (OVT73) at presymptomatic ages. Our goal was to understand changes to the metabolome as well as potential metabolite rhythm changes associated with HD. Methods: We used targeted liquid chromatography mass spectrometry (LC-MS) metabolomics to analyze metabolites in plasma samples taken from female HD transgenic and normal (control) sheep aged 5 and 7 years. Samples were taken hourly across a 27-h period. The resulting dataset was investigated by machine learning and chronobiological analysis. Results: The metabolic profiles of HD and control sheep were separable by machine learning at both ages. We found both absolute and rhythmic differences in metabolites in HD compared to control sheep at 5 years of age. An increase in both the number of disturbed metabolites and the magnitude of change of acrophase (the time at which the rhythms peak) was seen in samples from 7-year-old HD compared to control sheep. There were striking similarities between the dysregulated metabolites identified in HD sheep and human patients (notably of phosphatidylcholines, amino acids, urea, and threonine). Conclusion: This work provides the first integrated analysis of changes in metabolism and circadian rhythmicity of metabolites in a large animal model of presymptomatic HD.

    Juliane Hannemann, Anika Laing, Karin Glismann, Debra Skene, BENITA ANNE MIDDLETON, B Staels, Nikolaus Marx, Peter J Grant, MO Federici, Josef Niebauer, Raina Boger (2020)Timed physical exercise does not influence circadian rhythms and glucose tolerance in rotating night shift workers: The EuRhythDia study, In: Diabetes and Vascular Disease Research17(5)pp. 1479164120950616-1479164120950616 SAGE Publications
    Yves Dauvilliers, Lucie Barateau, Benita Middleton, Daan R. van der Veen, Debra Skene (2021)Metabolomics Signature of Patients with Narcolepsy, In: Neurology98(5) Lippincott, Williams & Wilkin

    Background and Objectives Narcolepsy type 1 (NT1) is an orphan brain disorder caused by the irreversible destruction of orexin neurons. Metabolic disturbances are common in patients with NT1 who have a body mass index (BMI) 10% to 20% higher than the general population, with one-third being obese (BMI >30 kg/m2). Besides the destruction of orexin neurons in NT1, the metabolic alterations in obese and nonobese patients with NT1 remain unknown. The aim of this study was to identify possible differences in plasma metabolic profiles between patients with NT1 and controls as a function of their BMI status. Methods We used a targeted liquid chromatography–mass spectrometry metabolomics approach to measure 141 circulating, low-molecular-weight metabolites in drug-free fasted plasma samples from 117 patients with NT1 (including 41 obese individuals) compared with 116 BMI-matched controls (including 57 obese individuals). Results Common metabolites driving the difference between patients with NT1 and controls, regardless of BMI, were identified, namely sarcosine, glutamate, nonaylcarnitine (C9), 5 long-chain lysophosphatidylcholine acyls, 1 sphingolipid, 12 phosphatidylcholine diacyls, and 11 phosphatidylcholine acyl-akyls, all showing increased concentrations in NT1. Metabolite concentrations significantly affected by NT1 (n = 42) and BMI category (n = 40) showed little overlap (n = 5). Quantitative enrichment analysis revealed common metabolic pathways that were implicated in the NT1/control differences in both normal BMI and obese comparisons, namely glycine and serine, arachidonic acid, and tryptophan metabolism. The metabolites driving these differences were glutamate, sarcosine, and ornithine (glycine and serine metabolism); glutamate and PC aa C34:4 (arachidonic acid metabolism); and glutamate, serotonin, and tryptophan (tryptophan metabolism). Linear metabolite-endophenotype regression analyses highlight that as part of the NT1 metabolic phenotype, most of the relationships between the sleep parameters (i.e., slow-wave sleep duration, sleep latency, and periodic leg movement) and metabolite concentrations seen in the controls were lost. Discussion These results represent the most comprehensive metabolic profiling of patients with NT1 as a function of BMI and propose some metabolic diagnostic biomarkers for NT1, namely glutamate, sarcosine, serotonin, tryptophan, nonaylcarnitine, and some phosphatidylcholines. The metabolic pathways identified offer, if confirmed, possible targets for treatment of obesity in NT1. Classification of Evidence This study provides Class II evidence that a distinct metabolic profile can differentiate patients with NT1 from patients without the disorder.

    Francieli Ruiz Da Silva, Felipe Beijamini, Andrew Beale, Bruno da Silva B. Goncalves, Daniel Vartanian, Tamara P. Taporoski, Benita Middleton, Jose E. Krieger, Homero Vallada, Josephine Arendt, Alexandre C. Pereira, Kristen L. Knutson, Mario Pedrazzoli, Malcolm von Schantz (2020)Early chronotype with advanced activity rhythms and dim light melatonin onset in a rural population, In: Journal of Pineal Researche12675 Wiley

    Studying communities at different stages of urbanisation and industrialisation can teach us how timing and intensity of light affects the circadian clock under real-life conditions. We have previously described a strong tendency towards morningness in the Baependi Heart Study, located in a small rural town in Brazil. Here, we tested the hypothesis that this morningness tendency is associated with early circadian phase based on objective measurements (as determined by dim light melatonin onset, DLMO, and activity) and light exposure. We also analysed how well the previously collected chronotype questionnaire data was able to predict these DLMO values. The average DLMO observed in 73 participants (40 female) was 20:03±01:21, SD, with an earlier average onset in men (19:38±01:16) than in women (20:24±01:21; p≤0.01). However, men presented larger phase angle between DLMO and sleep onset time as measured by actigraphy (4.11 hours vs 3.16 hours; p≤0.01). Correlational analysis indicated associations between light exposure, activity rhythms, and DLMO, such that early DLMO was observed in participants with higher exposure to light, higher activity and earlier light exposure. The strongest significant predictor of DLMO was morningness-eveningness questionnaire (MEQ) (beta=-0.35, p≤0.05), followed by age(beta=-0.47, p≤0.01). Sex, light exposure, and variables derived from the Munich Chronotype Questionnaire were not significant predictors. Our observations demonstrate that both early sleep patterns and earlier circadian phase have been retained in this small rural town in spite of availability of electrification, in contrast to metropolitan post-industrial areas.

    Juliane Hannemann, Debra Jean Skene, Benita Middleton, Edzard Schwedhelm, Anika Laing, Rainer Böger (2023)Diurnal Variation of L-Arginine and the Cardiovascular Risk Markers Asymmetric and Symmetric Dimethylarginine and Homoarginine in Rotating Night Shift Workers and Controls, In: Biomolecules13(9)1282 MDPI

    Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) interfere with nitric oxide (NO) formation from L-arginine via different mechanisms. ADMA is a biomarker of cardiovascular disease and mortality, whilst SDMA is a biomarker of mortality after ischemic stroke. Homoarginine, another L-arginine-derived amino acid, is associated with stroke and congestive heart failure. Acute ischemic events like myocardial infarction show a time-of-day variation in the timing of their onset, as do NO-mediated vascular function and blood pressure. We studied whether the plasma concentrations of L-arginine-related amino acid metabolites show diurnal variation in a clinical study comparing 12 non-night shift workers with 60 rotating night shift workers. The plasma concentrations of L-arginine-related biomarkers, melatonin, and cortisol were measured every 3 h during a 24-h period. In addition, 24-h blood pressure recordings were performed. In non-night shift workers, L-arginine and homoarginine plasma concentrations showed diurnal variation with a 12-h period, which were both attenuated in night shift workers. ADMA and SDMA showed a 24-h rhythmicity with no significant differences in phase between night shift and non-night shift workers. The plasma profiles of melatonin and cortisol were not significantly different between both groups, suggesting that the rotating night shift work does not have a major influence on central suprachiasmatic nuclei clock timing. In addition, systolic and diastolic blood pressure patterns were similar between both groups. Our data show diurnal variation of dimethylarginines with the timing of their acrophases corresponding to the published timing of the peak incidence of cardiac ischemic events.

    Elise R Facer-Childs, Brunno M de Campos, Benita Middleton, Debra J Skene, Andrew P Bagshaw (2021)Temporal organisation of the brain's intrinsic motor network: The relationship with circadian phenotype and motor performance, In: NeuroImage (Orlando, Fla.)232117840 Elsevier Inc

    Functional connectivity (FC) of the motor network (MN) is often used to investigate how intrinsic properties of the brain are associated with motor abilities and performance. In addition, the MN is a key feature in clinical work to map the recovery after stroke and aid the understanding of neurodegenerative disorders. Time of day variation and individual differences in circadian timing, however, have not yet been considered collectively when looking at FC. A total of 33 healthy, right handed individuals (13 male, 23.1 ± 4.2 years) took part in the study. Actigraphy, sleep diaries and circadian phase markers (dim light melatonin onset and cortisol awakening response) were used to determine early (ECP, n =13) and late (LCP, n = 20) circadian phenotype groups. Resting state functional MRI testing sessions were conducted at 14:00 h, 20:00 h and 08:00 h and preceded by a maximum voluntary contraction test for isometric grip strength to measure motor performance. Significant differences in FC of the MN between ECPs and LCPs were found, as well as significant variations between different times of day. A higher amplitude in diurnal variation of FC and performance was observed in LCPs compared to ECPs, with the morning being most significantly affected. Overall, lower FC was significantly associated with poorer motor performance. Our findings uncover intrinsic differences between times of day and circadian phenotype groups. This suggests that central mechanisms contribute to diurnal variation in motor performance and the functional integrity of the MN at rest influences the ability to perform in a motor task.

    Roselle A Herring, Fariba Shojaee-Moradie, Mary Stevenage, Iain Parsons , NICOLA JACKSON, JEEWAKA MENDIS, BENITA ANNE MIDDLETON, A. Margot Umpleby , BARBARA ANN FIELDING, Melanie Davies, David L Russell-Jones (2022)The SGLT2 Inhibitor Dapagliflozin Increases the Oxidation of Ingested Fatty Acids to Ketones in Type 2 Diabetes, In: Diabetes care American Diabetes Association

    Objective. To investigate the mechanism for increased ketogenesis following treatment with SGLT2 inhibitor, dapagliflozin in people with type 2 diabetes. Research, Design & Methods. This was a double-blind placebo-controlled crossover study with a 4-week washout period. Participants received dapagliflozin or placebo in random order for 4 weeks. After each treatment, they ingested 30ml of olive oil containing [U-13C] palmitate to measure ketogenesis with blood sampling for 480 min. Stable isotopes of glucose and glycerol were infused to measure glucose flux and lipolysis respectively at 450-480 min. Results. Glucose excretion rate was higher and peripheral glucose uptake lower with dapagliflozin than placebo. Plasma beta-hydroxybutyrate (BOHB) concentrations and [13C2] BOHB concentrations were higher and glucose concentrations lower with dapagliflozin than placebo. Non-esterified fatty acids (NEFA) were higher with dapagliflozin at 300 and 420 min but lipolysis at 450-480 min was not different. Triacylglycerol (TAG) at all time points and endogenous glucose production rate at 450-480 min were not different between treatments. Conclusions. The increase in ketone enrichment from the ingested palmitic acid tracer suggests meal derived fatty acids contribute to the increase in ketones during treatment with dapagliflozin. The increase in BOHB concentration with dapagliflozin, occurred with only minimal changes in plasma NEFA concentration and no change in lipolysis. This suggests a metabolic switch to increase ketogenesis within the liver.

    Juliane Hannemann, Anika Laing, Benita Middleton, Edzard Schwedhelm, Nikolaus Marx, Massimo Federici, Mariola Kastner, Debra J Skene, Rainer Böger (2024)Effect of oral melatonin treatment on insulin resistance and diurnal blood pressure variability in night shift workers. A double-blind, randomized, placebo-controlled study, In: Pharmacological research199

    BACKGROUNDNight shift work is associated with sleep disturbances, obesity, and cardiometabolic diseases. Disruption of the circadian clock system has been suggested to be an independent cause of type 2 diabetes and cardiovascular disease in shift workers. We aimed to improve alignment of circadian timing with social and environmental factors with administration of melatonin.METHODSIn a randomized, placebo-controlled, prospective study, we analysed the effects of 2mg of sustained-release melatonin versus placebo on glucose tolerance, insulin resistance indices, sleep quality, circadian profiles of plasma melatonin and cortisol, and diurnal blood pressure profiles in 24 rotating night shift workers during 12 weeks of treatment, followed by 12 weeks of wash-out. In a novel design, the time of melatonin administration (at night or in the morning) depended upon the shift schedule. We also compared the baseline profiles of the night shift (NS) workers with 12 healthy non-night shift (NNS)-working controls.RESULTSWe found significantly impaired indices of insulin resistance at baseline in NS versus NNS (p < 0.05), but no differences in oral glucose tolerance tests nor in the diurnal profiles of melatonin, cortisol, or blood pressure. Twelve weeks of melatonin treatment did not significantly improve insulin resistance, nor did it significantly affect diurnal blood pressure or melatonin and cortisol profiles. Melatonin administration, however, caused a significant improvement in sleep quality which was significantly impaired in NS versus NNS at baseline (p < 0.001).CONCLUSIONSRotating night shift work causes mild-to-moderate impairment of sleep quality and insulin resistance. Melatonin treatment at bedtime improves sleep quality, but does not significantly affect insulin resistance in rotating night shift workers after 12 weeks of administration.

    Kimberley F Prior, Benita Middleton, Alíz T.Y Owolabi, Mary L Westwood, Jacob Holland, Aidan J O'Donnell, Michael J Blackman, Debra J Skene, Sarah E Reece (2021)Synchrony between daily rhythms of malaria parasites and hosts is driven by an essential amino acid, In: Wellcome Open Research6186

    Background: Rapid asexual replication of blood stage malaria parasites is responsible for the severity of disease symptoms and fuels the production of transmission forms. Here, we demonstrate that a Plasmodium chabaudi’s schedule for asexual replication can be orchestrated by isoleucine, a metabolite provided to the parasite in a periodic manner due to the host’s rhythmic intake of food. Methods: We infect female C57BL/6 and Per1/2-null mice which have a disrupted canonical (transcription translation feedback loop, TTFL) clock with 1×105 red blood cells containing P. chabaudi (DK genotype). We perturb the timing of rhythms in asexual replication and host feeding-fasting cycles to identify nutrients with rhythms that match all combinations of host and parasite rhythms. We then test whether perturbing the availability of the best candidate nutrient in vitro changes the schedule for asexual development. Results: Our large-scale metabolomics experiment and follow up experiments reveal that only one metabolite - the amino acid isoleucine – fits criteria for a time-of-day cue used by parasites to set the schedule for replication. The response to isoleucine is a parasite strategy rather than solely the consequences of a constraint imposed by host rhythms, because unlike when parasites are deprived of other essential nutrients, they suffer no apparent costs from isoleucine withdrawal. Conclusions: Overall, our data suggest parasites can use the daily rhythmicity of blood-isoleucine concentration to synchronise asexual development with the availability of isoleucine, and potentially other resources, that arrive in the blood in a periodic manner due to the host’s daily feeding-fasting cycle. Identifying both how and why parasites keep time opens avenues for interventions; interfering with the parasite’s time-keeping mechanism may stall replication, increasing the efficacy of drugs and immune responses, and could also prevent parasites from entering dormancy to tolerate drugs.

    Tom Woelders, Victoria Louise Revell, Benita Middleton, Katrin Ackermann, Manfred Kayser, Florence I. Raynaud, Debra Jean Skene, Roelof A. Hut (2023)Machine learning estimation of human body time using metabolomic profiling, In: Proceedings of the National Academy of Sciences of the United States of America120(18)e2212685120 National Academy of Sciences

    Circadian rhythms influence physiology, metabolism, and molecular processes in the human body. Estimation of individual body time (circadian phase) is therefore highly relevant for individual optimization of behavior (sleep, meals, sports), diagnostic sampling, medical treatment, and for treatment of circadian rhythm disorders. Here, we provide a partial least squares regression (PLSR) machine learning approach that uses plasma-derived metabolomics data in one or more samples to estimate dim light melatonin onset (DLMO) as a proxy for circadian phase of the human body. For this purpose, our protocol was aimed to stay close to real-life conditions. We found that a metabolomics approach optimized for either women or men under entrained conditions performed equally well or better than existing approaches using more labor-intensive RNA sequencing-based methods. Although estimation of circadian body time using blood-targeted metabolomics requires further validation in shift work and other real-world conditions, it currently may offer a robust, feasible technique with relatively high accuracy to aid personalized optimization of behavior and clinical treatment after appropriate validation in patient populations.

    G Howatson, PG Bell, J Tallent, B Middleton, MP McHugh, J Ellis (2012)Melatonin And Sleep Quality Are Increased Following Tart Cherry Juice Consumption, In: MEDICINE AND SCIENCE IN SPORTS AND EXERCISE44pp. 315-315 LIPPINCOTT WILLIAMS & WILKINS
    B Middleton (2006)Measurement of melatonin and 6-sulphatoxymelatonin., In: Methods Mol Biol324pp. 235-254

    Melatonin and its major metabolite, 6-sulphatoxymelatonin, can routinely be measured by RIA or ELISA. Plasma, serum or saliva samples maybe used for melatonin measurement and urine for the metabolite. Melatonin is a hormone produced by the pineal gland witha clear circadian rhythm giving low levels by day and a peak during the night in normally entrained individuals. The timing of sample collection for measurement is therefore crucial. Melatonin levels are primarily assessed to determine the timing of an individual's internal body clock and therefore indicate any misalignment to the 24 h day.

    P.A. Nehme, F.G. Amaral, B. Middleton, A. Lowden, E. Marqueze, I. França-Junior, J.L.F. Antunes, J. Cipolla-Neto, D.J Skene, C.R.C. Moreno (2019)Melatonin profiles during the third trimester of pregnancy and health status in the offspring among day and night workers: A case series, In: Neurobiology of Sleep and Circadian Rhythms6pp. 70-76 Elsevier Inc.

    Successful pregnancy requires adaptation in maternal physiology. During intrauterine life the mother's circadian timing system supports successful birth and postnatal development. Maternal melatonin is important to transmit circadian timing and day length to the fetus. This study aims to describe the third trimester of pregnancy among day (n = 5) and night (n = 3) workers by assessing their melatonin levels in a natural environment. Additionally, we describe the worker's metabolic profiles and compare the health status of the newborns between groups of day and night working mothers. Our results indicate an occurrence of assisted delivery (caesarean and forceps) among night workers. Moreover, the newborns of night workers showed lower Apgar index and breastfeeding difficulty indicating a worse condition to deal with the immediate outside the womb environment.Additionally, there was lower night-time melatonin production among pregnant night workers compared to day workers. These findings may be related to light-induced suppression of melatonin that occurs during night work. We conclude that night work and consequent exposure to light at unconventional times might compromise the success of pregnancy and the health of the newborn. Further studies need to be carried out to monitor pregnancy and newborn health in pregnant night workers.

    Elise Facer-Childs, Benita Middleton, Andrew P. Bagshaw, Debra J. Skene (2019)Methods for Human Circadian Phenotyping and Diurnal Performance Testing in the Real World, In: Journal of Visualized Experiments Journal of Visualized Experiments (JoVE)

    In our continuously developing 'around the clock' society, there is a need to increase our understanding of how changes in biology, physiology and psychology influence our health and performance. Embedded within this challenge, is the increasing need to account for individual differences in sleep and circadian rhythms, as well as to explore the impact of time of day on performance in the real world. There are a number of ways to measure sleep and circadian rhythms from subjective questionnaire-based methods to objective sleep/wake monitoring, actigraphy and analysis of biological samples. This paper proposes a protocol that combines multiple techniques to categorize individuals into Early, Intermediate or Late circadian phenotype groups (ECPs/ICPs/LCPs) and recommends how to conduct diurnal performance testing in the field. Representative results show large differences in rest-activity patterns derived from actigraphy, circadian phase (dim light melatonin onset and peak time of cortisol awakening response) between circadian phenotypes. In addition, significant differences in diurnal performance rhythms between ECPs and LCPs emphasizes the need to account for circadian phenotype. In summary, despite the difficulties in controlling influencing factors, this protocol allows a real-world assessment of the impact of circadian phenotype on performance. This paper presents a simple method to assess circadian phenotype in the field and supports the need to consider time of day when designing performance studies.

    K Wulff, DJ Dijk, B Middleton, RG Foster, EM Joyce (2012)Sleep and circadian rhythm disruption in schizophrenia., In: Br J Psychiatry200(4)pp. 308-316

    Sleep disturbances comparable with insomnia occur in up to 80% of people with schizophrenia, but very little is known about the contribution of circadian coordination to these prevalent disruptions.

    Samantha Hopkins, Peter Lloyd Morgan, Luc J.M. Schlangen, Peter Williams, Debra Skene, Benita Middleton (2017)Blue-enriched Lighting for Older People Living in Care Homes: Effect on Activity, Actigraphic Sleep, Mood and Alertness, In: Current Alzheimer Research14(10)pp. 1053-1062 Bentham Science Publishers

    Objective: Environmental (little outdoor light; low indoor lighting) and age-related physiological factors (reduced light transmission through the ocular lens, reduced mobility) contribute to a light-deprived environment for older people living in care homes. Methods: This study investigates the effect of increasing indoor light levels with blue-enriched white lighting on objective (rest-activity rhythms, performance) and self-reported (mood, sleep, alertness) measures in older people. Eighty residents (69 female), aged 86 ± 8 yrs (mean ± SD), participated (MMSE 19 ± 6). Overhead fluorescent lighting was installed in communal rooms (n=20) of seven care homes. Four weeks of blue-enriched white lighting (17000 K ≅ 900 lux) were compared with four weeks of control white lighting (4000 K ≅ 200 lux), separated by three weeks wash-out. Participants completed validated mood and sleep questionnaires, psychomotor vigilance task (PVT) and wore activity and light monitors (AWL). Rest-activity rhythms were assessed by cosinor, non-parametric circadian rhythm (NPCRA) and actigraphic sleep analysis. Blue-enriched (17000 K) light increased wake time and activity during sleep decreasing actual sleep time, sleep percentage and sleep efficiency (p < 0.05) (actigraphic sleep). Compared to 4000 K lighting, blue-enriched 17000 K lighting significantly (p < 0.05) advanced the timing of participants’ rest-activity rhythm (cosinor), increased daytime and night-time activity (NPCRA), reduced subjective anxiety (HADA) and sleep quality (PSQI). There was no difference between the two light conditions in daytime alertness and performance (PVT). Conclusion: Blue-enriched lighting produced some positive (increased daytime activity, reduced anxiety) and negative (increased night-time activity, reduced sleep efficiency and quality) effects in older people.

    S Montagnese, B Middleton, DJ Skene, MY Morgan (2009)Sleep-wake patterns in patients with cirrhosis: All you need to know on a single sheet A simple sleep questionnaire for clinical use, In: JOURNAL OF HEPATOLOGY51(4)pp. 690-695 ELSEVIER SCIENCE BV
    Elise R Facer-Childs, Brunno M Campos, Benita Middleton, Debra J Skene, Andrew P Bagshaw (2019)Circadian phenotype impacts the brain’s resting state functional connectivity, attentional performance and sleepiness, In: Sleep42(5)zsz033 Oxford University Press (OUP)

    INTRODUCTION Functional connectivity (FC) of the human brain’s intrinsically connected networks underpins cognitive functioning and disruptions of FC are associated with sleep and neurological disorders. However, there is limited research on the impact of circadian phenotype and time of day on FC. STUDY OBJECTIVES The aim of this study was to investigate resting state FC of the default mode network (DMN) in Early and Late circadian phenotypes over a socially constrained day. METHODS 38 healthy individuals (14 male, 22.7 ± 4.2 years) categorised as Early (n =16) or Late (n = 22) using the Munich ChronoType Questionnaire took part. Following a two week baseline of actigraphy coupled with saliva samples for melatonin and cortisol rhythms, participants underwent testing at 14.00 h, 20.00 h and 08.00 h the following morning. Testing consisted of resting state functional MRI, a structural T1 scan, attentional cognitive performance tasks and self-reported daytime sleepiness. Seed based FC analysis from the medial prefrontal and posterior cingulate cortices of the DMN was performed, compared between groups and linked with behavioural data. RESULTS Fundamental differences in the DMN were observed between Early and Late circadian phenotypes. Resting state FC of the DMN predicted individual differences in attention and subjective ratings of sleepiness. CONCLUSION Differences in FC of the DMN may underlie the compromised attentional performance and increased sleepiness commonly associated with Late types when they conform to a societally constrained day that does not match their intrinsic circadian phenotype.

    S Montagnese, B Middleton, DJ Skene, MY Morgan (2009)Night-time sleep disturbance does not correlate with neuropsychiatric impairment in patients with cirrhosis, In: LIVER INTERNATIONAL29(9)pp. 1372-1382 WILEY-BLACKWELL PUBLISHING, INC
    M AlBreiki, B Middleton, S Hampton (2015)Are leptin responses influenced by bright light treatment in healthy young individuals, In: PROCEEDINGS OF THE NUTRITION SOCIETY74(OCE4)pp. E209-E209 CAMBRIDGE UNIV PRESS
    E Lowson, Benita Middleton, Sara Arber, Debra Skene (2013)Effects of night work on sleep, cortisol and mood of female nurses, their husbands and children, In: Sleep and Biological Rhythms11(1)pp. 7-13 Wiley-Blackwell

    Negative impacts of night work on employees are well documented, but little is known about immediate consequences for family members. This study examines how night work within a rotating shift pattern affects the sleep, mood and cortisol levels of female nurses, their husbands and children. Participants included twenty nurses (42.7 ± 6.5 years), their husbands and children (n=34, 8-18 years) who completed sleep diaries, rated their sleep quality, alertness and mood daily, and collected saliva samples each morning and evening for 14 days. Comparisons were made between night work and other shifts (Wilcoxon Signed Ranks test); and between periods preceding, during and following night shifts (repeated measures ANOVA with Tukey posthoc tests). Nurses’ sleep after the final night shift was significantly shorter (3h 58 mins ± 46 mins) and ended significantly earlier (13:28 ± 0:48h) than after the first night shift (sleep duration 5h 17 mins ± 1h 36 mins; wake time 14:58 ± 1:41h) (p

    JQM Ly, G Gaggioni, SL Chellappa, S Papachilleos, A Brzozowski, C Borsu, M Rosanova, S Sarasso, Benita Middleton, A Luxen, Simon Archer, C Phillips, Derk-Jan Dijk, P Maquet, M Massimini, G Vandewalle (2016)Circadian regulation of human cortical excitability, In: Nature Communications11828 Nature Publishing Group

    Prolonged wakefulness alters cortical excitability, which is essential for proper brain function and cognition. However, besides prior wakefulness, brain function and cognition are also affected by circadian rhythmicity. Whether the regulation of cognition involves a circadian impact on cortical excitability is unknown. Here, we assessed cortical excitability from scalp EEG-responses to transcranial magnetic stimulation in 22 participants during 29-h of wakefulness under constant conditions. Data reveal robust circadian dynamics of cortical excitability that were strongest in those individuals with highest endocrine markers of circadian amplitude. In addition, the time course of cortical excitability correlated with changes in EEG synchronization and cognitive performance. These results demonstrate that the crucial factor for cortical excitability, and basic brain function in general, is the balance between circadian rhythmicity and sleep need, rather than sleep homeostasis alone. These findings have implications for clinical applications such as noninvasive brain stimulation in neurorehabilitation.

    Jennifer Wild, Shama El-Salahi, Gabriella Tyson, Hjördis Lorenz, Carmine M Pariante, Andrea Danese, Apostolos Tsiachristas, Edward Watkins, Benita Middleton, Amanda Blaber, Anke Ehlers (2018)Preventing PTSD, depression and associated health problems in student paramedics: protocol for PREVENT-PTSD, a randomised controlled trial of supported online cognitive training for resilience versus alternative online training and standard practice, In: BMJ Open8(12)e022292pp. 1-10 BMJ Publishing Group

    Introduction Emergency workers dedicate their lives to promoting public health and safety, yet suffer higher rates of post-traumatic stress disorder (PTSD) and major depression (MD) compared with the general population. They also suffer an associated increased risk for physical health problems, which may be linked to specific immunological and endocrine markers or changes in relevant markers. Poor physical and mental health is costly to organisations, the National Health Service and society. Existing interventions aimed at reducing risk of mental ill health in this population are not very successful. More effective preventative interventions are urgently needed. We first conducted a large-scale prospective study of newly recruited student paramedics, identifying two cognitive factors (rumination and resilience appraisals) that predicted episodes of PTSD and MD over a 2-year period. We then developed internet-delivered cognitive training for resilience (iCT-R), a supported online intervention, to modify cognitive predictors. This protocol is for a randomised controlled trial to evaluate the efficacy of the resilience intervention. Methods and analysis 570 student paramedics will be recruited from participating universities. They will be randomly allocated to iCT-R or to supported online training of an alternative, widely available intervention or to training-as-usual. Follow-up will occur after the intervention/standard practice period and at 6, 12 and 24 months. Primary outcomes include rates of PTSD and MD and subsydnromal PTSD and MD, measured by the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, fifth edition, the Patient-Health Questionnaire-9 and the Post-traumatic Stress Disorder Checklist for Diagnostic and Statistical Manual of Mental Disorders, fifth edition. Secondary outcomes include measures of resilience, rumination, anxiety, psychological distress, well-being, salivary cortisol, plasma levels of C-reactive protein, smoking and alcohol use, weight gain, sleep problems, health-related quality of life, health resource utilisation and productivity. Ethics and dissemination The Medical Sciences Inter-Divisional Research Ethics Committee at the University of Oxford granted approval, reference: R44116/RE001. The results will be published in a peer-reviewed journal. Access to raw data and participant information will be available only to members of the research team.

    BA Middleton, J Arendt, B Stone (1996)Human circadian rhythms in constant dim light (8 lux) with knowledge of clock time, In: Journal of Sleep Research5(2)pp. 69-76 Wiley

    The light/dark (L/D) cycle is a major synchronizer of human circadian rhythms. In the absence of a strong L/D cycle, synchrony with 24 hours can nevertheless be maintained in a socially structured environment, as shown in Polar regions (Broadway et al. 1987) and by some blind subjects (Czeisler et al. 1995a). The relative contribution of other time cues to entrainment in dim light has not been fully explored. The present study investigated the behaviour of melatonin (assessed as 6-sulphatoxymelatonin); rectal temperature; activity and sleep (actigraphy and logs) in constant dim light (L/L) with access to a digital clock. 6 normal healthy males were maintained as a group in partial temporal isolation with attenuated sound and ambient temperature for 21 days. All 6 subjects showed free-running periodicity for 6-sulphatoxymelatonin and 5/6 subjects for temperature, activity and sleep offset. The average period (tau) was 24.26±0.049, substantially shorter than in previous experiments with a self selected L/D cycle but similar to a recent study conducted in very dim light. One subject maintained a rigid sleep/wake cycle throughout whilst his 6-sulphatoxymelatonin rhythm free-ran. Total sleep time, from actigraph data, did not change but sleep efficiency decreased during the experiment. The subjects did not show group synchronization. These results confirm previous data indicating the importance of the L/D cycle in human entrainment and underline the lesser role of social cues and knowledge of clock time. This particular approach will permit the administration of timed medication to sighted humans under free-running conditions.

    A Keyes, S Woerwag-Mehta, S Bartholdy, A Koskina, B Middleton, F Connan, P Webster, U Schmidt, IC Campbell (2015)Physical Activity and the Drive to Exercise in Anorexia Nervosa, In: INTERNATIONAL JOURNAL OF EATING DISORDERS48(1)pp. 46-54 WILEY-BLACKWELL
    M AlBreiki, B Middleton, J Ebajemito, S Hampton (2015)The effect of light on appetite in healthy young individuals, In: PROCEEDINGS OF THE NUTRITION SOCIETY74(OCE1)pp. E4-E4 CAMBRIDGE UNIV PRESS
    K Papantoniou, OJ Pozo, A Espinosa, J Marcos, G Castano-Vinyals, X Basagana, FC Ribas, J Mirabent, J Martin, G Carenys, CR Martin, B Middleton, DJ Skene, M Kogevinas (2014)Circadian Variation of Melatonin, Light Exposure, and Diurnal Preference in Day and Night Shift Workers of Both Sexes, In: CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION23(7)pp. 1176-1186 AMER ASSOC CANCER RESEARCH
    BA Middleton, BM Stone, J Arendt (2002)Human circadian phase in 12:12 h, 200: <8 lux and 1000: <8 lux light-dark cycles, without scheduled sleep or activity., In: Neuroscience Letters329(1)pp. 41-44 Elsevier

    The light levels required to maintain human circadian phase in the absence of other strong time cues are not defined. We investigated circadian phase in two groups of men, living in partial temporal isolation, exposed to 12 h:12 h light:dark cycles of: (A) 200:

    BA Middleton, J Arendt, BM Stone (1997)Complex effects of melatonin on human circadian rhythms in constant dim light., In: Journal of Biological Rhythms12(5)pp. 467-477 Sage

    In humans, the pineal hormone melatonin can phase shift a number of circadian rhythms (e.g., "fatigue," endogenous melatonin, core body temperature) together with the timing of prolactin secretion. It is uncertain, however, whether melatonin can fully entrain all human circadian rhythms. In this study, the authors investigated the effects of daily melatonin administration on sighted individuals kept in continuous very dim light. A total of 10 normal, healthy males were maintained in two separate groups in partial temporal isolation under constant dim light (< 8 lux) with attenuated sound and ambient temperature variations but with knowledge of clock time for two periods of 30 days. In these circumstances, the majority of individuals free run with a mean period of 24.3 h. In a double-blind, randomized crossover design, subjects received 5 mg melatonin at 20:00 h on Days 1 to 15 (Melatonin 1st) followed by placebo on Days 16 to 30 (Placebo 2nd) or vice versa (Placebo 1st, Melatonin 2nd) during Leg 1 with treatment reversed in Leg 2. The variables measured were melatonin (as 6-sulphatoxymelatonin), rectal temperature, activity, and sleep (actigraphy and logs). In the experiment, 9 of the 10 subjects free ran with Placebo 1st, whereas Melatonin 1st stabilized the sleep-wake cycle to 24 h in 8 of 10 individuals. In addition, 2 individuals showed irregular sleep with this treatment. In some subjects, there was a shortening of the period of the temperature rhythm without synchronization. Melatonin 2nd induced phase advances (5 of 9 subjects), phase delays (2 of 9 subjects), and stabilization (2 of 9 subjects) of the sleep-wake cycle with subsequent synchronization to 24 h in the majority of individuals (7 of 9). Temperature continued to free run in 4 subjects. Maximum phase advances in core temperature were seen when the first melatonin treatment was given approximately 2 h after the temperature acrophase. These results indicate that melatonin was able to phase shift sleep and core temperature but was unable to synchronize core temperature consistently. In the majority of subjects, the sleep-wake cycle could be synchronized.

    SMW Rajaratnam, D-J Dijk, B Middleton, BM Stone, J Arendt (2003)Melatonin phase-shifts human circadian rhythms with no evidence of changes in the duration of endogenous melatonin secretion or the 24-hour production of reproductive hormones, In: Journal of Clinical Endocrinology & Metabolism88(9)pp. 4303-4309 Endocrine Society

    The pineal hormone melatonin is a popular treatment for sleep and circadian rhythm disruption. Melatonin administered at optimal times of the day for treatment often results in a prolonged melatonin profile. In photoperiodic (day length-dependent) species, changes in melatonin profile duration influence the timing of seasonal rhythms. We investigated the effects of an artificially prolonged melatonin profile on endogenous melatonin and cortisol rhythms, wrist actigraphy, and reproductive hormones in humans. Eight healthy men took part in this double-blind, crossover study. Surge/sustained release melatonin (1.5 mg) or placebo was administered for 8 d at the beginning of a 16-h sleep opportunity (1600 h to 0800 h) in dim light. Compared with placebo, melatonin administration advanced the timing of endogenous melatonin and cortisol rhythms. Activity was reduced in the first half and increased in the second half of the sleep opportunity with melatonin; however, total activity during the sleep opportunities and wake episodes was not affected. Melatonin treatment did not affect the endogenous melatonin profile duration, pituitary/gonadal hormone levels (24-h), or sleepiness and mood levels on the subsequent day. In the short term, suitably timed sustained-release melatonin phase-shifts circadian rhythms and redistributes activity during a 16-h sleep opportunity, with no evidence of changes in the duration of endogenous melatonin secretion or pituitary/gonadal hormones.

    A. Jennifer Morton, Benita Middleton, Skye Rudiger, C. Simon Bawden, Timothy R. Kuchel, Debra Skene (2020)Increased plasma melatonin in presymptomatic Huntington disease sheep ( Ovis aries ): Compensatory neuroprotection in a neurodegenerative disease?, In: Journal of Pineal Research68(2)e12624 Wiley

    Melatonin is a pleiotrophic hormone, synthesised primarily by the pineal gland under the control of the suprachiasmatic nuclei (SCN). It not only provides a hormonal signal of darkness but also has neuroprotective properties. Huntington's disease (HD) is a progressive neurodegenerative disorder characterised by abnormal motor, cognitive and psychiatric symptoms. There is growing evidence, particularly from animal models, that circadian rhythms may also be disturbed in HD. We measured two circadian‐regulated hormones, melatonin and cortisol, in plasma samples collected around‐the‐clock from normal and presymptomatic transgenic HD sheep (Ovis aries) at 5 and 7 years of age, to assess SCN‐driven rhythms and the effect of genotype, sex and age. Melatonin‐related precursors and metabolites (tryptophan, serotonin, kynurenine) were also measured by liquid chromatography (LC)‐mass spectrometry (MS). At 5 years of age in both rams and ewes, plasma melatonin levels were significantly elevated in HD sheep. In ewes measured 2 years later, there was still a significant elevation of nocturnal melatonin. Furthermore, the daytime baseline levels of melatonin were significantly higher in HD sheep. Since increased melatonin could have global beneficial effects on brain function, we suggest that the increased melatonin measured in presymptomatic HD sheep is part of an autoprotective response to mutant huntingtin toxicity that may account, at least in part, for the late onset of disease that characterises HD.

    M Cropley, LW Rydstedt, JJ Devereux, B Middleton (2015)The Relationship Between Work-Related Rumination and Evening and Morning Salivary Cortisol Secretion, In: STRESS AND HEALTH31(2)pp. 150-157 WILEY-BLACKWELL
    Sophie Wehrens, Skevoulla Christou, Cheryl Isherwood, Benita Middleton, Michelle Gibbs, Simon Archer, Debra Skene, Jonathan Johnston (2017)Meal Timing Regulates the Human Circadian System, In: Current Biology27(12)pp. 1768-1775e3 Elsevier (Cell Press)

    Circadian rhythms, metabolism and nutrition are intimately linked [1, 2], although effects of meal timing on the human circadian system are poorly understood. We investigated the effect of a 5-hour delay in meals on markers of the human master clock and multiple peripheral circadian rhythms. Ten healthy young men undertook a 13-day laboratory protocol. Three meals (breakfast, lunch, dinner) were given at 5-hour intervals, beginning either 0.5 (early) or 5.5 (late) hours after wake. Participants were acclimated to early meals and then switched to late meals for 6 days. After each meal schedule, participants' circadian rhythms were measured in a 37-hour constant routine that removes sleep and environmental rhythms while replacing meals with hourly isocaloric snacks. Meal timing did not alter actigraphic sleep parameters before circadian rhythm measurement. In constant routines, meal timing did not affect rhythms of subjective hunger and sleepiness, master clock markers (plasma melatonin and cortisol), plasma triglycerides, or clock gene expression in whole blood. Following late meals, however, plasma glucose rhythms were delayed by 5.69 ± 1.29 hours (p < 0.001) and average glucose concentration decreased by 0.27 ± 0.05 mM (p < 0.001). In adipose tissue, PER2 mRNA rhythms were delayed by 0.97 ± 0.29 hours (p < 0.01), indicating that human molecular clocks may be regulated by feeding time and could underpin plasma glucose changes. Timed meals therefore play a role in synchronising peripheral circadian rhythms in humans, and may have particular relevance for patients with circadian rhythm disorders, shift workers, and transmeridian travellers.

    Josephine Arendt, Benita Middleton (2017)Human seasonal and circadian studies in Antarctica (Halley, 75°S), In: General and Comparative Endocrinology258pp. 250-258 Elsevier

    Living for extended periods in Antarctica exposes base personnel to extremes of daylength (photoperiod) and temperature. At the British Antarctic Survey base of Halley, 75°S, the sun does not rise for 110 d in the winter and does not set for 100 d in summer. Photoperiod is the major time cue governing the timing of seasonal events such as reproduction in many species. The neuroendocrine signal providing photoperiodic information to body physiology is the duration of melatonin secretion which reflects the length of the night: longer in the short days of winter and shorter in summer. Light of sufficient intensity and spectral composition serves to suppress production of melatonin and to set the circadian timing and the duration of the rhythm. In humans early observations suggested that bright (>2000 lux) white light was needed to suppress melatonin completely. Shortly thereafter winter depression (Seasonal Affective Disorder or SAD) was described, and its successful treatment by an artificial summer photoperiod of bright white light, sufficient to shorten melatonin production. At Halley dim artificial light intensity during winter was measured, until 2003, at a maximum of approximately 500 lux in winter. Thus a strong seasonal and circadian time cue was absent. It seemed likely that winter depression would be common in the extended period of winter darkness and could be treated with an artificial summer photoperiod. These observations, and predictions, inspired a long series of studies regarding human seasonal and circadian status, and the effects of light treatment, in a small overwintering, isolated community, living in the same conditions for many months at Halley. We found little evidence of SAD, or change in duration of melatonin production with season. However the timing of the melatonin rhythm itself, and/or that of its metabolite 6-sulphatoxymelatonin (aMT6s), was used as a primary marker of seasonal, circadian and treatment changes. A substantial phase delay of melatonin in winter was advanced to summer phase by a two pulse 'skeleton' bright white light treatment. Subsequently a single morning pulse of bright white light was effective with regard to circadian phase and improved daytime performance. The circadian delay evidenced by melatonin was accompanied by delayed sleep (logs and actigraphy): poor sleep is a common complaint in Polar regions. Appropriate extra artificial light, both standard white, and blue enriched, present throughout the day, effectively countered delay in sleep timing and the aMT6s rhythm. The most important factor appeared to be the maximum light experienced. Another manifestation of the winter was a decline in self-rated libido (men only on base at this time). Women on the base showed lower aspects of physical and mental health compared to men. Free-running rhythms were seen in some subjects following night shift, but were rarely found at other times, probably because this base has strongly scheduled activity and leisure time. Complete circadian adaptation during a week of night shift, also seen in a similar situation on North Sea oil rigs, led to problems readapting back to day shift in winter, compared to summer. Here again timed light treatment was used to address the problem. Sleep, alertness and waking performance are critically dependent on optimum circadian phase. Circadian desynchrony is associated with increased risk of major disease in shift workers. These studies provide some groundwork for countering/avoiding circadian desynchrony in rather extreme conditions.

    Giulia Gaggioni, Julien Q.M. Ly, Sarah L. Chellappa, Dorothée Coppieters ‘t Wallant, Mario Rosanova, Simone Sarasso, André Luxen, Eric Salmon, Benita Middleton, Marcello Massimini, Christina Schmidt, Adenauer Casali, Christophe Phillips, Gilles Vandewalle (2018)Human fronto-parietal response scattering subserves vigilance at night., In: NeuroImage175pp. 354-364 Elsevier

    Lack of sleep has a considerable impact on vigilance: we perform worse, we make more errors, particularly at night, when we should be sleeping. Measures of brain functional connectivity suggest that decrease in vigilance during sleep loss is associated with an impaired cross-talk within the fronto-parietal cortex. However, fronto-parietal effective connectivity, which is more closely related to the causal cross-talk between brain regions, remains unexplored during prolonged wakefulness. In addition, no study has simultaneously investigated brain effective connectivity and wake-related changes in vigilance, preventing the concurrent incorporation of the two aspects. Here, we used electroencephalography (EEG) to record responses evoked by Transcranial Magnetic Stimulation (TMS) applied over the frontal lobe in 23 healthy young men (18–30 yr.), while they simultaneously performed a vigilance task, during 8 sessions spread over 29 h of sustained wakefulness. We assessed Response Scattering (ReSc), an estimate of effective connectivity, as the propagation of TMS-evoked EEG responses over the fronto-parietal cortex. Results disclose a significant change in fronto-parietal ReSc with time spent awake. When focusing on the night-time period, when one should be sleeping, participants with lower fronto-parietal ReSc performed worse on the vigilance task. Conversely, no association was detected during the well-rested, daytime period. Night-time fronto-parietal ReSc also correlated with objective EEG measures of sleepiness and alertness. These changes were not accompanied by variations in fronto-parietal response complexity. These results suggest that decreased brain response propagation within the fronto-parietal cortex is associated to increased vigilance failure during night-time prolonged wakefulness. This study reveals a novel facet of the detrimental effect on brain function of extended night-time waking hours, which is increasingly common in our societies.

    Juliane Fagotti, Adriano D. S. Targa, Lais S. Rodrigues, Ana Carolina D. Noseda, Flávia W. C. Dorieux, Franciele F. Scarante, Jessica L. Ilkiw, Fernando M. Louzada, Namrata Chowdhury, Daan R. van der Veen, Benita Middleton, Jeroen L. A. Pennings, Jonathan R. Swann, Debra Skene, Marcelo M. S. Lima (2018)Chronic sleep restriction in the rotenone Parkinson’s disease model in rats reveals peripheral early-phase biomarkers, In: Scientific Reports9(1)1898 Springer Nature Publishing

    Parkinson’s disease (PD) is a chronic disorder that presents a range of premotor signs, such as sleep disturbances and cognitive decline, which are key non-motor features of the disease. Increasing evidence of a possible association between sleep disruption and the neurodegenerative process suggests that sleep impairment could produce a detectable metabolic signature on the disease. In order to integrate neurocognitive and metabolic parameters, we performed untargeted and targeted metabolic profiling of the rotenone PD model in a chronic sleep restriction (SR) (6 h/day for 21 days) condition. We found that SR combined with PD altered several behavioural (reversal of locomotor activity impairment; cognitive impairment; delay of rest-activity rhythm) and metabolic parameters (branched-chain amino acids, tryptophan pathway, phenylalanine, and lipoproteins, pointing to mitochondrial impairment). If combined, our results bring a plethora of parameters that represents reliable early-phase PD biomarkers which can easily be measured and could be translated to human studies.

    MA Bonmati-Carrion, B Middleton, V Revell, DJ Skene, MA Rol, JA Madrid (2013)Circadian phase asessment by ambulatory monitoring in humans: Correlation with dim light melatonin onset., In: Chronobiol Int Informa Healthcare

    The increased prevalence of circadian disruptions due to abnormal coupling between internal and external time makes the detection of circadian phase in humans by ambulatory recordings a compelling need. Here, we propose an accurate practical procedure to estimate circadian phase with the least possible burden for the subject, that is, without the restraints of a constant routine protocol or laboratory techniques such as melatonin quantification, both of which are standard procedures. In this validation study, subjects (N = 13) wore ambulatory monitoring devices, kept daily sleep diaries and went about their daily routine for 10 days. The devices measured skin temperature at wrist level (WT), motor activity and body position on the arm, and light exposure by means of a sensor placed on the chest. Dim light melatonin onset (DLMO) was used to compare and evaluate the accuracy of the ambulatory variables in assessing circadian phase. An evening increase in WT: WTOnset (WTOn) and "WT increase onset" (WTiO) was found to anticipate the evening increase in melatonin, while decreases in motor activity (Activity Offset or AcOff), body position (Position Offset (POff)), integrative TAP (a combination of WT, activity and body position) (TAPOffset or TAPOff) and an increase in declared sleep propensity were phase delayed with respect to DLMO. The phase markers obtained from subjective sleep (R = 0.811), WT (R = 0.756) and the composite variable TAP (R = 0.720) were highly and significantly correlated with DLMO. The findings strongly support a new method to calculate circadian phase based on WT (WTiO) that accurately predicts and shows a temporal association with DLMO. WTiO is especially recommended due to its simplicity and applicability to clinical use under conditions where knowing endogenous circadian phase is important, such as in cancer chronotherapy and light therapy.

    Nicola J. Robertson, Kathryn Martinello, Ingran Lingam, Adnan Avdic-Belltheus, Christopher Meehan, Daniel Alonso-Alconada, Sara Ragab, Alan Bainbridge, Magdalena Sokolska, Mohamed Tachrount, Benita Middleton, David Price, Mariya Hristova, Xavier Golay, Annamaria Soliani Raschini, Giancarlo Aquino, Nicola Pelizzi, Fabrizio Facchinetti (2018)Melatonin as an adjunct to therapeutic hypothermia in a piglet model of neonatal encephalopathy: A translational study., In: Neurobiology of Disease121pp. 240-251 Elsevier

    Therapeutic hypothermia is only partially protective for neonatal encephalopathy; there is an urgent need to develop treatments that augment cooling. Our objective was to assess safety, efficacy and pharmacokinetics of 5 and 15 mg/kg/24 h melatonin (proprietary formulation) administered at 2 h and 26 h after hypoxia-ischemia (HI) with cooling in a piglet model. Following moderate cerebral HI, 30 piglets were eligible and randomized to: i) Hypothermia (33.5 °C, 2–26 h) and vehicle (HT + V;n = 13); b) HT and 5 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-5;n = 4); c) HT and 15 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-15;n = 13). Intensive care was maintained for 48 h; brain MRS was acquired and cell death (TUNEL) evaluated at 48 h. Comparing HT + V with HT + Mel-5 and HT + Mel-15, there was no difference in blood pressure or inotropic support needed, brain Lactate/N Acetylaspartate at 24 h and 48 h was similar, ATP/phosphate pool was higher for HT + Mel-15 versus HT + V at 24 h (p = 0.038) but not 48 h. A localized reduction in TUNEL positive cell death was observed in the sensorimotor cortex in the 15 mg/kg melatonin group (HT + Mel-15 versus HT + V; p 

    S Marini, O Santangeli, P Saarelainen, Benita Middleton, Namrata Roy Chowdhury, Debra Skene, R Costa, T Porkka-Heiskanen, S Montagnese (2017)Abnormalities in the Polysomnographic, Adenosine and Metabolic Response to Sleep Deprivation in an Animal Model of Hyperammonemia, In: Frontiers in Physiology8636 Frontiers Media

    Patients with liver cirrhosis can develop hyperammonemia and hepatic encephalopathy (HE), accompanied by pronounced daytime sleepiness. Previous studies with healthy volunteers show that experimental increase in blood ammonium levels increases sleepiness and slows the waking EEG. As ammonium increases adenosine levels in vitro, and adenosine is a known regulator of sleep/wake homeostasis, we hypothesized that the sleepiness-inducing effect of ammonium is mediated by adenosine. Eight adult male Wistar rats were fed with an ammonium-enriched diet for 4 weeks; eight rats on standard diet served as controls. Each animal was implanted with electroencephalography/electromyography (EEG/EMG) electrodes and a microdialysis probe. Sleep EEG recording and cerebral microdialysis were carried out at baseline and after 6 hours of sleep deprivation. Adenosine and metabolite levels were measured by HPLC and targeted LC/MS metabolomics, respectively. Baseline adenosine and metabolite levels (12 of 16 amino acids, taurine, t4-hydroxy-proline and acetylcarnitine) were lower in hyperammonemic animals, while putrescine was higher. After sleep deprivation, hyperammonemic animals exhibited a larger increase in adenosine levels, and a number of metabolites showed a different time-course in the two groups. In both groups the recovery period was characterized by a significant decrease in wakefulness/increase in NREM and REM sleep. However, while control animals exhibited a gradual compensatory effect, hyperammonemic animals showed a significantly shorter recovery phase. In conclusion, the adenosine/metabolite/EEG response to sleep deprivation was modulated by hyperammonemia, suggesting that ammonia affects homeostatic sleep regulation and its metabolic correlates.

    F Perrin, P Peigneux, S Fuchs, S Verhaeghe, S Laureys, B Middleton, C Degueldre, G Del Fiore, G Vandewalle, E Balteau, R Poirrier, V Moreau, A Luxen, P Maquet, D-J Dijk (2004)Nonvisual responses to light exposure in the human brain during the circadian night, In: Current Biology14(20)pp. 1842-1846 Elsevier (Cell Press)

    The brain processes light information to visually represent the environment but also to detect changes in ambient light level. The latter information induces non-image-forming responses and exerts powerful effects on physiology such as synchronization of the circadian clock and suppression of melatonin 1, 2 and 3. In rodents, irradiance information is transduced from a discrete subset of photosensitive retinal ganglion cells via the retinohypothalamic tract to various hypothalamic and brainstem regulatory structures including the hypothalamic suprachiasmatic nuclei, the master circadian pacemaker 4, 5 and 6. In humans, light also acutely modulates alertness 7 and 8, but the cerebral correlates of this effect are unknown. We assessed regional cerebral blood flow in 13 subjects attending to auditory and visual stimuli in near darkness following light exposures (>8000 lux) of different durations (0.5, 17, 16.5, and 0 min) during the biological night. The bright broadband polychromatic light suppressed melatonin and enhanced alertness. Functional imaging revealed that a large-scale occipito-parietal attention network, including the right intraparietal sulcus, was more active in proportion to the duration of light exposures preceding the scans. Activity in the hypothalamus decreased in proportion to previous illumination. These findings have important implications for understanding the effects of light on human behavior.

    Karolina Lech, Fan Liu, Sarah K. Davies, Katrin Ackermann, Joo Ern Ang, Benita Middleton, Victoria L. Revell, Florence I. Raynaud, Igor Hoveijn, Roelof A. Hut, Debra J. Skene, Manfred Kayser (2017)investigation of metabolites for estimating blood deposition time, In: International Journal of Legal Medicine132(1)pp. 25-32 Springer Verlag

    Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor’s alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

    Debra Skene, Elena Skornyakov, Namrata Chowdhury, Rajendra P Gajula, Benita Middleton, Brieann C Satterfield, Kenneth I Porter, Hans P A Van Dongen, Shobhan Gaddameedhi (2018)Separation of circadian- and behavior-driven metabolite rhythms in humans provides a window on peripheral oscillators and metabolism, In: PNAS115(30)pp. 7825-7830 National Academy of Sciences

    Misalignment between internal circadian rhythmicity and externally imposed behavioral schedules, such as occurs in shift workers, has been implicated in elevated risk of metabolic disorders. To determine underlying mechanisms, it is esse ntial to assess whether and how peripheral clocks are disturbed during shift work and to what extent this is linked to the central suprachiasmatic nuclei (SCN) pacemaker and/or misaligned behavioral time cues. Investigating rhythms in circulating metabolites as biomarkers of peripheral clock distur- bances may offer new insight s. We evaluated the impact of misaligned sleep/wake and feeding/fasting cycles on circulating metabolites using a targeted metabolomics approach. Sequential plasma samples obtained during a 24-h constant routine that followed a 3-d simulated night-s hift schedule, compared with a simulated day-shift schedule, we re analyzed for 132 circulating metabolites. Nearly half of these metabolites showed a 24-h rhyth- micity under constant routine following either or both simulated shift schedules. However, while tradition al markers of the circadian clock in the SCN — melatonin, cortisol, and PER3 expression — maintained a stable phase alignment after both schedules, only a few metabo- lites did the same. Many showed reversed rhythms, lost their rhythms, or showed rhythmicity only under constant routine fol- lowing the night-shift schedule. Here, 95% of the metabolites with a 24-h rhythmicity showed rhythms that were driven by behavior- al time cues externally imposed during the preceding simulated shift schedule rather than being driven by the central SCN circa- dian clock. Characterization of these metabolite rhythms will pro- vide insight into the underlying mechanisms linking shift work and metabolic disorders

    Elise R. Facer-Childs, Benita Middleton, Debra J. Skene, Andrew P. Bagshaw (2019)Resetting the late timing of ‘night owls’ has a positive impact on mental health and performance, In: Sleep Medicine60pp. 236-247 Elsevier

    Background There is conflict between living according to our endogenous biological rhythms and our external environment, with disruptions resulting in negative consequences to health and performance. This is often documented in shift work and jet lag, but ‘societal norms’ (eg, typical working hours) can create profound issues for ‘night owls’, people whose internal biological timing predisposes them to follow an unusually late sleep-wake cycle. Night owls have also been associated with health issues, mood disturbances, poorer performance and increased mortality rates. Methods This study used a randomized control trial design aimed to shift the late timing of night owls to an earlier time (phase advance), using non-pharmacological, practical interventions in a real-world setting. These interventions targeted light exposure (through earlier wake up/sleep times), fixed meals times, caffeine intake and exercise. Results Overall, participants demonstrated a significant advance of ~2 h in sleep/wake timings as measured by actigraphy and circadian phase markers (dim light melatonin onset and peak time of the cortisol awakening response), whilst having no adverse effect on sleep duration. Notably, the phase advance was accompanied by significant improvements to self-reported depression and stress, as well as improved cognitive (reaction time) and physical (grip strength) performance measures during the typical 'suboptimal morning hours. Conclusions Our findings propose a novel strategy for shifting clock timing towards a pattern that is more aligned to societal demands that could significantly improve elements of performance, mental health and sleep timing in the real world.

    Mohammed S Albreiki, Benita Middleton, Shelagh Hampton (2017)A single night light exposure acutely alters hormonal and metabolic responses in healthy participants, In: Endocrine Connections6(2)pp. 100-110 BioScientifica

    Many animal studies have reported an association between melatonin suppression and the disturbance of metabolic responses; yet, few human studies have investigated bright light effects on metabolic and hormonal responses at night. This study investigated the impact of light on plasma hormones and metabolites prior to, and after, an evening meal in healthy participants. Seventeen healthy participants, 8 females (22.2 ± 2.59 years, mean ± s.d.) and 9 males (22.8 ± 3.5 years) were randomised to a two-way cross-over design protocol; dim light (DL) (500 lux) sessions, separated by at least seven days. Saliva and plasma samples were collected prior to and after a standard evening meal at specific intervals. Plasma non-esterified fatty acid (NEFA) levels were significantly higher pre-meal in DL compared to BL (P 

    S Mäntele, DT Otway, Benita Middleton, S Bretschneider, John Wright, Margaret Robertson, Debra Skene, Jonathan Johnston (2012)Daily Rhythms of Plasma Melatonin, but Not Plasma Leptin or Leptin mRNA, Vary between Lean, Obese and Type 2 Diabetic Men., In: PLoS One7(5)pp. e37123-? Public Library of Science

    Melatonin and leptin exhibit daily rhythms that may contribute towards changes in metabolic physiology. It remains unclear, however, whether this rhythmicity is altered in obesity or type 2 diabetes (T2DM). We tested the hypothesis that 24-hour profiles of melatonin, leptin and leptin mRNA are altered by metabolic status in laboratory conditions. Men between 45-65 years old were recruited into lean, obese-non-diabetic or obese-T2DM groups. Volunteers followed strict sleep-wake and dietary regimes for 1 week before the laboratory study. They were then maintained in controlled light-dark conditions, semi-recumbent posture and fed hourly iso-energetic drinks during wake periods. Hourly blood samples were collected for hormone analysis. Subcutaneous adipose biopsies were collected 6-hourly for gene expression analysis. Although there was no effect of subject group on the timing of dim light melatonin onset (DLMO), nocturnal plasma melatonin concentration was significantly higher in obese-non-diabetic subjects compared to weight-matched T2DM subjects (p

    AJ Bradley, R Webb-Mitchell, A Hazu, N Slater, BA Middleton, P Gallagher, H McAllister-Williams, KN Anderson (2017)Sleep and circadian rhythm disturbance in bipolar disorder, In: Psychological Medicine47(9)pp. 1678-1689 Cambridge University Press

    Background. Subjective reports of insomnia and hypersomnia are common in bipolar disorder (BD). It is unclear to what extent these relate to underlying circadian rhythm disturbance (CRD). In this study we aimed to objectively assess sleep and circadian rhythm in a cohort of patients with BD compared to matched controls. Method. Forty-six patients with BD and 42 controls had comprehensive sleep/circadian rhythm assessment with respiratory sleep studies, prolonged accelerometry over 3 weeks, sleep questionnaires and diaries, melatonin levels, alongside mood, psychosocial functioning and quality of life (QoL) questionnaires. Results. Twenty-three (50%) patients with BD had abnormal sleep, of whom 12 (52%) had CRD and 29% had obstructive sleep apnoea. Patients with abnormal sleep had lower 24-h melatonin secretion compared to controls and patients with normal sleep. Abnormal sleep/CRD in BD was associated with impaired functioning and worse QoL. Conclusions. BD is associated with high rates of abnormal sleep and CRD. The association between these disorders, mood and functioning, and the direction of causality, warrants further investigation.

    I Gogenur, B Middleton, VB Kristiansen, DJ Skene, J Rosenberg (2007)Disturbances in melatonin and core body temperature circadian rhythms after minimal invasive surgery, In: ACTA ANAESTHESIOLOGICA SCANDINAVICA51(8)pp. 1099-1106 BLACKWELL PUBLISHING
    M De Rui, Benita Middleton, A Sticca, A Gatta, P Amodio, Debra Skene, S Montagnese (2015)Sleep and Circadian Rhythms in Hospitalized Patients with Decompensated Cirrhosis: Effect of Light Therapy, In: NEUROCHEMICAL RESEARCH40(2)pp. 284-292 SPRINGER/PLENUM PUBLISHERS
    S Montagnese, B Middleton, AR Mani, DJ Skene, MY Morgan (2010)On the origin and the consequences of circadian abnormalities in patients with cirrhosis., In: Am J Gastroenterol105(8)pp. 1773-1781

    OBJECTIVES: Plasma melatonin profile abnormalities have been described in patients with cirrhosis and generally attributed to impaired hepatic melatonin metabolism. The possibility that they might reflect circadian clock dysfunction has not been explored. In addition, the relationship between plasma melatonin profiles and the sleep disturbances observed in these patients remains unclear. The aims of this study were: (i) to evaluate circadian clock function and hepatic melatonin metabolism in cirrhotic patients, and (ii) to study the relationship between plasma melatonin profiles and sleep-wake behavior. METHODS: The study population comprised 20 patients with cirrhosis (mean (range) age, 59 (39-77) years) and 9 healthy volunteers (60 (38-84) years). Plasma melatonin/cortisol concentrations were measured hourly, for 24 h, in light/posture-controlled conditions. Urinary 6-sulfatoxymelatonin, the main melatonin metabolite, was measured simultaneously to determine clearance. The ability of light to suppress nocturnal melatonin synthesis was assessed. Habitual sleep quality/timing was evaluated using a questionnaire, actigraphy, and sleep diaries. RESULTS: There was evidence of central circadian disruption in patients compared with healthy controls: peak plasma melatonin/cortisol times were delayed (04:48+/-02:36 vs. 02:48+/-00:54, P=0.01; 10:18+/-02:54 vs. 08:54+/-01:24, P=0.06) and the plasma melatonin response to light was reduced (12%+/-19% vs. 24%+/-15%, P=0.09). However, the mean 24 h plasma melatonin clearance did not differ significantly between patients and healthy volunteers (0.22+/-0.10 vs. 0.28+/-0.17 l/kg per h, P=0.36). Finally, although patients showed a degree of misalignment between sleep and circadian timings, there was no association between circadian abnormalities and impaired sleep quality. CONCLUSIONS: Plasma melatonin profile abnormalities, predominantly central in origin, are observed in patients with mild to moderately decompensated cirrhosis. However, they are substantially unrelated to the sleep disturbances prevalent in this population.

    K Ackermann, R Plomp, O Lao, B Middleton, VL Revell, DJ Skene, M Kayser (2013)Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans., In: Chronobiol Int30(7)pp. 901-909 Informa Healthcare

    This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48 h (sleep vs. no-sleep nights) in 12 young males (mean ± SD: 23 ± 5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation.

    SW Lockley, DJ Skene, K Thapan, J English, D Ribeiro, I Haimov, S Hampton, B Middleton, M von Schantz, J Arendt (1998)Extraocular light exposure does not suppress plasma melatonin in humans, In: J CLIN ENDOCR METAB83(9)pp. 3369-3372 ENDOCRINE SOC

    Light affects the circadian axis in at least two ways. It can cause the acute suppression of pineal melatonin synthesis, and/or a phase-shift of the circadian oscillator. As recent evidence has suggested that extraocular light exposure may cause phase-shifts of the circadian clock, we have investigated whether suppression of melatonin can be induced by the same type of light exposure. In the first study subjects’ eyes were exposed to white light (2250 lux for 30 min) via a fibre optic cable. As expected, suppression of nighttime plasma melatonin levels (61 ± 6%) was observed. In the second study, light of the same quality but higher intensity (14,000 or 67,500 lux for 180 mins) was delivered in the same manner to the popliteal region behind the subjects’ knees, whilst shielding their eyes. No suppression of plasma melatonin levels (4 ± 7%) was detected in any of the subjects. Thus, extraocular photoreception, if it exists in mammals, does not affect the suprachiasmatic nuclei-pineal pathway.

    I Gogenur, U Ocak, O Altunpinar, B Middleton, DJ Skene, J Rosenberg (2007)Disturbances in melatonin, cortisol and core body temperature rhythms after major surgery, In: WORLD JOURNAL OF SURGERY31(2)pp. 290-298 SPRINGER
    Matteo Turco, Nora Cazzagon, Irene Franceschet, Chiara Formentin, Giovanni Frighetto, Francesca Giordani, Nicola Cellini, Gabriella Mazzotta, Rodolfo Costa, Benita Middleton, Debra Skene, Annarosa Floreani, Sara Montagnese (2018)Morning Bright Light Treatment for Sleep-Wake Disturbances in Primary Biliary Cholangitis: A Pilot Study, In: Frontiers in Physiology91530 Frontiers Media

    Patients with Primary Biliary Cholangitis (PBC) exhibit delayed sleep-wake habits, disturbed night sleep and daytime sleepiness/fatigue. Such combination of symptoms is reminiscent of delayed sleep-wake phase disorder (DSPD), which benefits from morning light treatment. The aim of the present pilot study was to test the effect of morning light treatment in a group of 13 well-characterized patients with PBC [all females; (mean ± SD) 53 ± 10 years]. Six healthy individuals (4 females, 57 ± 14 years) and 7 patients with cirrhosis (1 female, 57 ± 12 years) served as controls and diseased controls, respectively. At baseline, all participants underwent an assessment of quality of life, diurnal preference, sleep quality/timing (subjective plus actigraphy), daytime sleepiness, and urinary 6-sulphatoxymelatonin (aMT6s) rhythmicity. Then they underwent a 15-day course of morning bright light treatment, immediately after getting up (light box, 10,000 lux, 45 min) whilst monitoring sleep-wake patterns and aMT6s rhythmicity. At baseline, both patients with PBC and patients with cirrhosis had significantly worse subjective sleep quality compared to controls. In patients with PBC, light treatment resulted in an improvement in subjective sleep quality and a reduction in daytime sleepiness. In addition, both their sleep onset and get-up time were significantly advanced. Finally, the robustness of aMT6s rhythmicity (i.e., strength of the cosinor fit) increased after light administration but post-hoc comparisons were not significant in any of the groups. In conclusion, a brief course of morning bright light treatment had positive effects on subjective sleep quality, daytime sleepiness, and sleep timing in patients with PBC. This unobtrusive, side-effect free, non-pharmacological treatment is worthy of further study.

    I Gogenur, B Middleton, S Burgdorf, LS Rasmussen, DJ Skene, J Rosenberg (2007)Impact of sleep and circadian disturbances in urinary 6-sulphatoxymelatonin levels, on cognitive function after major surgery, In: JOURNAL OF PINEAL RESEARCH43(2)pp. 179-184 BLACKWELL PUBLISHING
    P Gringras, B Middleton, DJ Skene, VL Revell (2015)Bigger, Brighter, Bluer-Better? Current Light-Emitting Devices - Adverse Sleep Properties and Preventative Strategies., In: Frontiers in public health3 Frontiers Media

    OBJECTIVE: In an effort to enhance the efficiency, brightness, and contrast of light-emitting (LE) devices during the day, displays often generate substantial short-wavelength (blue-enriched) light emissions that can adversely affect sleep. We set out to verify the extent of such short-wavelength emissions, produced by a tablet (iPad Air), e-reader (Kindle Paperwhite 1st generation), and smartphone (iPhone 5s) and to determine the impact of strategies designed to reduce these light emissions. SETTING: University of Surrey dedicated chronobiology facility. METHODS: First, the spectral power of all the LE devices was assessed when displaying identical text. Second, we compared the text output with that of "Angry Birds" - a popular top 100 "App Store" game. Finally, we measured the impact of two strategies that attempt to reduce the output of short-wavelength light emissions. The first strategy employed an inexpensive commercially available pair of orange-tinted "blue-blocking" glasses. The second strategy tested an app designed to be "sleep-aware" whose designers deliberately attempted to reduce short-wavelength light emissions. RESULTS: All the LE devices shared very similar enhanced short-wavelength peaks when displaying text. This included the output from the backlit Kindle Paperwhite device. The spectra when comparing text to the Angry Birds game were also very similar, although the text emissions were higher intensity. Both the orange-tinted glasses and the "sleep-aware" app significantly reduced short-wavelength emissions. CONCLUSION: The LE devices tested were all bright and characterized by short-wavelength enriched emissions. Since this type of light is likely to cause the most disruption to sleep as it most effectively suppresses melatonin and increases alertness, there needs to be the recognition that at night-time "brighter and bluer" is not synonymous with "better." Ideally future software design could be better optimized when night-time use is anticipated, and hardware should allow an automatic "bedtime mode" that shifts blue and green light emissions to yellow and red as well as reduce backlight/light intensity.

    SM Rajaratnam, B Middleton, BM Stone, J Arendt, D-J Dijk (2004)Melatonin advances the circadian timing of EEG sleep and directly facilitates sleep without altering its duration in extended sleep opportunities in humans., In: Journal of Physiology561(Pt 1)pp. 339-351 Wiley

    The rhythm of plasma melatonin originating from the pineal gland and driven by the circadian pacemaker located in the suprachiasmatic nucleus is closely associated with the circadian (approximately 24 h) variation in sleep propensity and sleep spindle activity in humans. We investigated the contribution of melatonin to variation in sleep propensity, structure, duration and EEG activity in a protocol in which sleep was scheduled to begin during the biological day, i.e. when endogenous melatonin concentrations are low. The two 14 day trials were conducted in an environmental scheduling facility. Each trial included two circadian phase assessments, baseline sleep and nine 16 h sleep opportunities (16.00–08.00 h) in near darkness. Eight healthy male volunteers (24.4 ± 4.4 years) without sleep complaints were recruited, and melatonin (1.5 mg) or placebo was administered at the start of the first eight 16 h sleep opportunities. During melatonin treatment, sleep in the first 8 h of the 16 h sleep opportunities was increased by 2 h. Sleep per 16 h was not significantly different and approached asymptotic values of 8.7 h in both conditions. The percentage of rapid eye movement (REM) sleep was not affected by melatonin, but the percentage of stage 2 sleep and sleep spindle activity increased, and the percentage of stage 3 sleep decreased. During the washout night, the melatonin-induced advance in sleep timing persisted, but was smaller than on the preceding treatment night and was consistent with the advance in the endogenous melatonin rhythm. These data demonstrate robust, direct sleep-facilitating and circadian effects of melatonin without concomitant changes in sleep duration, and support the use of melatonin in the treatment of sleep disorders in which the circadian melatonin rhythm is delayed relative to desired sleep time.

    Debra Skene, Benita Middleton, CK Fraser, JLA Pennings, TR Kuchel, SR Rudiger, CS Bawden, AJ Morton (2017)Metabolic profiling of presymptomatic Huntington’s disease sheep reveals novel biomarkers, In: Scientific Reports743030 Nature Publishing Group

    The pronounced cachexia (unexplained wasting) seen in Huntington’s disease (HD) patients suggests that metabolic dysregulation plays a role in HD pathogenesis, although evidence of metabolic abnormalities in HD patients is inconsistent. We performed metabolic profiling of plasma from presymptomatic HD transgenic and control sheep. Metabolites were quantified in sequential plasma samples taken over a 25h period using a targeted LC/MS metabolomics approach. Significant changes with respect to genotype were observed in 89/130 identified metabolites, including sphingolipids, biogenic amines, amino acids and urea. Citrulline and arginine increased significantly in HD compared to control sheep. Ten other amino acids decreased in presymptomatic HD sheep, including branched chain amino acids (isoleucine, leucine and valine) that have been identified previously as potential biomarkers of HD. Significant increases in urea, arginine, citrulline, asymmetric and symmetric dimethylarginine, alongside decreases in sphingolipids, indicate that both the urea cycle and nitric oxide pathways are dysregulated at early stages in HD. Logistic prediction modelling identified a set of 8 biomarkers that can identify 80% of the presymptomatic HD sheep as transgenic, with 90% confidence. This level of sensitivity, using minimally invasive methods, offers novel opportunities for monitoring disease progression in HD patients.

    M Herljevic, Benita Middleton, K Thapan, Debra Skene (2005)Light-induced melatonin suppression: age-related reduction in response to short wavelength light, In: Experimental Gerontology40(3)pp. 237-242 Elsevier

    One of the possible causes of disturbed circadian rhythms and sleep in the elderly may be impaired photic input to the circadian clock. Age-related changes in lens density are known to reduce the transmission of short wavelength light, which has been shown to be most effective in suppressing nocturnal melatonin. The aim of the study therefore was to investigate age-related changes in melatonin suppression in response to short and medium wavelength light. Young premenopausal (n=13) and postmenopausal (n=21) women were exposed to 30 min of monochromatic light at two different wavelengths and irradiances (λmax 456 nm: 3.8 and 9.8 μW/cm2; λmax 548 nm: 28 and 62 μW/cm2). Melatonin suppression was compared across light treatments and between age groups. Significantly reduced melatonin suppression was noted in the elderly subjects following exposure to short wavelength (456 nm) light compared to the young subjects. These results are likely to reflect age-related changes in lens density.

    CR Moreno, S Vasconcelos, EC Marqueze, A Lowden, B Middleton, FM Fischer, FM Louzada, DJ Skene (2015)Sleep patterns in Amazon rubber tappers with and without electric light at home., In: SCIENTIFIC REPORTS5 NATURE PUBLISHING GROUP

    Today's modern society is exposed to artificial electric lighting in addition to the natural light-dark cycle. Studies assessing the impact of electric light exposure on sleep and its relation to work hours are rare due to the ubiquitous presence of electricity. Here we report a unique study conducted in two phases in a homogenous group of rubber tappers living and working in a remote area of the Amazon forest, comparing those living without electric light (n = 243 in first phase; n = 25 in second phase) to those with electric light at home (n = 97 in first phase; n = 17 in second phase). Questionnaire data (Phase 1) revealed that rubber tappers with availability of electric light had significantly shorter sleep on work days (30 min/day less) than those without electric light. Analysis of the data from the Phase 2 sample showed a significant delay in the timing of melatonin onset in workers with electric light compared to those without electric light (p 

    DJ Skene, SE Timbers, B Middleton, J English, C Kopp, I Tobler, C Ioannides (2006)Mice convert melatonin to 6-sulphatoxymelatonin, In: GENERAL AND COMPARATIVE ENDOCRINOLOGY147(3)pp. 371-376 ACADEMIC PRESS INC ELSEVIER SCIENCE
    S Hampton, B Middleton (2011)Validation of electronic diary (PRO-Diary) compared to validated paper questionnaires in normal individuals, In: PROCEEDINGS OF THE NUTRITION SOCIETY70(OCE3)pp. E67-E67 CAMBRIDGE UNIV PRESS
    G Francis, L Bishop, C Luke, B Middleton, P Williams, J Arendt (2008)Sleep during the Antarctic winter: preliminary observations on changing the spectral composition of artificial light, In: JOURNAL OF SLEEP RESEARCH17(3)pp. 354-360 WILEY-BLACKWELL
    M De Rui, S Gaiani, B Middleton, DJ Skene, S Schiff, A Gatta, C Merkel, P Amodio, S Montagnese (2011)Bright times for patients with cirrhosis and delayed sleep habits: a case report on the beneficial effect of light therapy., In: Am J Gastroenterol106(11)pp. 2048-2049
    TL Sletten, VL Revell, B Middleton, KA Lederle, DJ Skene (2009)Age-Related Changes in Acute and Phase-Advancing Responses to Monochromatic Light, In: JOURNAL OF BIOLOGICAL RHYTHMS24(1)pp. 73-84 SAGE PUBLICATIONS INC
    V Mottram, B Middleton, P Williams, J Arendt (2011)The impact of bright artificial white and 'blue-enriched' light on sleep and circadian phase during the polar winter., In: J Sleep Res20(1 Pt 2)pp. 154-161

    Delayed sleep phase (and sometimes free-run) is common in the Antarctic winter (no natural sunlight) and optimizing the artificial light conditions is desirable. This project evaluated sleep when using 17,000 K blue-enriched lamps compared with standard white lamps (5000 K) for personal and communal illumination. Base personnel, 10 males, five females, 32.5±8 years took part in the study. From 24 March to 21 September 2006 light exposure alternated between 4-5-week periods of standard white (5000 K) and blue-enriched lamps (17,000 K), with a 3-week control before and after extra light. Sleep and light exposure were assessed by actigraphy and sleep diaries. General health (RAND 36-item questionnaire) and circadian phase (urinary 6-sulphatoxymelatonin rhythm) were evaluated at the end of each light condition. Direct comparison (rmanova) of blue-enriched light with white light showed that sleep onset was earlier by 19 min (P=0.022), and sleep latency tended to be shorter by 4 min (P=0.065) with blue-enriched light. Analysing all light conditions, control, blue and white, again provided evidence for greater efficiency of blue-enriched light compared with white (P

    S Montagnese, B Middleton, M Corrias, AR Mani, DJ Skene, MY Morgan (2015)Assessment of 6-sulfatoxymelatonin rhythms and melatonin response to light in disease states: Lessons from cirrhosis, In: CHRONOBIOLOGY INTERNATIONAL32(2)pp. 187-194 INFORMA HEALTHCARE

    Circadian rhythmicity and non-visual sensitivity to light can be assessed, in healthy subjects, by measuring the rhythm of the urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) and by determining the response of plasma melatonin to nocturnal retinal light exposure, respectively. However, the validity of these techniques has not been assessed in disease states in which disruption of the circadian rhythm is known or suspected to occur. Thus, the aims of this study were as follows: (i) to assess the reliability of circadian aMT6s profile estimates derived from 36 h versus 56 h urine collections and (ii) to test different models for calculating melatonin suppression in response to light in healthy volunteers and patients with cirrhosis. Twenty patients with biopsy-proven cirrhosis and 10 matched healthy volunteers undertook: (i) separate 36 - and 56-h urine collections, under controlled conditions, for cosinor analysis of the urinary aMT6s profile; (ii) a melatonin suppression test, comprising of a baseline night, during which subjects were woken and asked to sit in front of a switched off light sphere, and an experimental night, identically executed, except that the light sphere was switched on and the subjects were exposed to white light (4.1 × 10(14) photons/cm(2)/s) for 30 min. Alternative approaches to the calculation of melatonin suppression were taken, with/without inclusion of the baseline night. Eighteen patients and eight healthy volunteers had matched analysable 36 - and 56-h urinary samples. Cosinor analysis showed a significant fit in 88% of the remaining 56 h collections, and 48% of the remaining 36-h collections. Thus, eight patients and five healthy volunteers had matched analysable samples for cosinor analysis. In the healthy volunteers, aMT6s profile indices obtained using the 36 - and the 56-h collections did not differ significantly. In contrast, considerably more variability was observed in patients [i.e. the difference in the aMT6s peak time was 0.5 ± 1.7 h (limits of agreement: -3.9; +2.9 h)]. No difficulties were encountered in obtaining suppression estimates by use of the experimental night only. In contrast, suppression estimates obtained by use of both nights were considered inaccurate in one (11%) healthy volunteer and in 5 (28%) patients, primarily because: (i) melatonin concentrations at the beginning of light administration were significantly different on baseline and experimental night; (ii) the rise in melatonin was inconsistent on baseline night; and (iii) the shape of the rising phase of melatonin was different on baseline and experimental night. In conclusion, shorter urine collections lead to a higher number of profiles with no significant cosinor fit, and differences in cosinor indices obtained from the 36 - and 56-h collections were considerable, especially in patients. Thus, 56-h collections are probably advisable. Use of both baseline and experimental nights to calculate melatonin suppression often resulted in increased variation and confounding, due to point oscillations in melatonin concentration and lack of repeatability of the melatonin profiles on the two nights. Thus, use of the experimental night only is probably advisable.

    G Vandewalle, B Middleton, SMW Rajaratnam, BM Stone, B Thorleifsdottir, J Arendt, D-J Dijk (2007)Robust circadian rhythm in heart rate and its variability: influence of exogenous melatonin and photoperiod, In: JOURNAL OF SLEEP RESEARCH16(2)pp. 148-155 BLACKWELL PUBLISHING
    SL Chellappa, G Gaggioni, JQM Ly, S Papachilleos, C Borsu, A Brzozowski, M Rosanova, S Sarasso, A Luxen, Benita Middleton, Simon Archer, Derk-Jan Dijk, M Massimini, P Maquet, C Phillips, RJ Moran, G Vandewalle (2016)Circadian dynamics in measures of cortical excitation and inhibition balance, In: Scientific Reports633661 Nature Publishing Group

    Several neuropsychiatric and neurological disorders have recently been characterized as dysfunctions arising from a ‘final common pathway’ of imbalanced excitation to inhibition within cortical networks. How the regulation of a cortical E/I ratio is affected by sleep and the circadian rhythm however, remains to be established. Here we addressed this issue through the analyses of TMS-evoked responses recorded over a 29h sleep deprivation protocol conducted in young and healthy volunteers. Spectral analyses of TMS-evoked responses in frontal cortex revealed non-linear changes in gamma band evoked oscillations, compatible with an influence of circadian timing on inhibitory interneuron activity. In silico inferences of cell-to-cell excitatory and inhibitory connectivity and GABA/Glutamate receptor time constant based on neural mass modeling within the Dynamic causal modeling framework, further suggested excitation/inhibition balance was under a strong circadian influence. These results indicate that circadian changes in EEG spectral properties, in measure of excitatory/inhibitory connectivity and in GABA/glutamate receptor function could support the maintenance of cognitive performance during a normal waking day, but also during overnight wakefulness. More generally, these findings demonstrate a slow daily regulation of cortical excitation/inhibition balance, which depends on circadian-timing and prior sleep-wake history.

    C Meyer, V Muto, M Jaspar, C Kusse, E Lambot, SL Chellappa, C Degueldre, E Balteau, A Luxen, B Middleton, SN Archer, F Collette, D-J Dijk, C Phillips, P Maquet, G Vandewalle (2016)Seasonality in human cognitive brain responses, In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA113(11)pp. 3066-3071 NATL ACAD SCIENCES
    Shanaz Diessler, Maxime Jan, Yann Emmenegger, Nicolas Guex, Benita Middleton, Debra J. Skene, Mark Ibberson, Frederic Burdet, Lou Götz, Marco Pagni, Martial Sankar, Robin Liechti, Charlotte N. Hor, Ioannis Xenarios, Paul Franken (2018)A systems genetics resource and analysis of sleep regulation in the mouse, In: PLOS Biology16(8)e2005750pp. 1-39 Public Library of Science

    Sleep is essential for optimal brain functioning and health, but the biological substrates through which sleep delivers these beneficial effects remain largely unknown. We used a systems genetics approach in the BXD genetic reference population (GRP) of mice and assembled a comprehensive experimental knowledge base comprising a deep "sleep-wake" phenome, central and peripheral transcriptomes, and plasma metabolome data, collected under undisturbed baseline conditions and after sleep deprivation (SD). We present analytical tools to interactively interrogate the database, visualize the molecular networks altered by sleep loss, and prioritize candidate genes. We found that a one-time, short disruption of sleep already extensively reshaped the systems genetics landscape by altering 60%–78% of the transcriptomes and the metabolome, with numerous genetic loci affecting the magnitude and direction of change. Systems genetics integrative analyses drawing on all levels of organization imply α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking and fatty acid turnover as substrates of the negative effects of insufficient sleep. Our analyses demonstrate that genetic heterogeneity and the effects of insufficient sleep itself on the transcriptome and metabolome are far more widespread than previously reported.

    B Middleton (2013)Measurement of melatonin and 6-sulphatoxymelatonin, In: Methods in Molecular Biology1065pp. 171-199

    Melatonin is an indole hormone secreted by the pineal gland during the hours of darkness in a normally entrained individual. There is a clear circadian rhythm in its production with low levels during the day and a peak in the early hours of the morning. The timing of sample collection is crucial and single time point measurements are of little use. Measurement of melatonin or its major metabolite, 6-sulphatoxymelatonin, is normally carried out to determine the timing of an individual's internal body clock and whether it is synchronized to the 24 h day. Misalignment of the clock or disruption of the rhythm can lead to difficulties in sleeping and health problems such as are associated with jet-lag or shift work. Both melatonin and 6-sulphatoxymelatonin can be measured by RIA or ELISA. Details of sample collection and preparation and the assay procedures are described. © 2013 Springer New York.

    PJ Gunn, Benita Middleton, SK Davies, Victoria Revell, Debra Skene (2016)Sex differences in the circadian profiles of melatonin and cortisol in plasma and urine matrices under constant routine conditions, In: CHRONOBIOLOGY INTERNATIONAL33(1)pp. 39-50 TAYLOR & FRANCIS INC

    Conflicting evidence exists as to whether there are differences between males and females in circadian timing. The aim of the current study was to assess whether sex differences are present in the circadian regulation of melatonin and cortisol in plasma and urine matrices during a constant routine protocol. Thirty-two healthy individuals (16 females taking the oral contraceptive pill (OCP)), aged 23.8 ± 3.7 (mean ± SD) years, participated. Blood (hourly) and urine (4-hourly) samples were collected for measurement of plasma melatonin and cortisol, and urinary 6-sulfatoxymelatonin (aMT6s) and cortisol, respectively. Data from 28 individuals (14 females) showed no significant differences in the timing of plasma and urinary circadian phase markers between sexes. Females, however, exhibited significantly greater levels of plasma melatonin and cortisol than males (AUC melatonin: 937 ± 104 (mean ± SEM) vs. 642 ± 47 pg/ml.h; AUC cortisol: 13581 ± 1313 vs. 7340 ± 368 mmol/L.h). Females also exhibited a significantly higher amplitude rhythm in both hormones (melatonin: 43.8 ± 5.8 vs. 29.9 ± 2.3 pg/ml; cortisol: 241.7 ± 23.1 vs. 161.8 ± 15.9 mmol/L). Males excreted significantly more urinary cortisol than females during the CR (519.5 ± 63.8 vs. 349.2 ± 39.3 mol) but aMT6s levels did not differ between sexes. It was not possible to distinguish whether the elevated plasma melatonin and cortisol levels observed in females resulted from innate sex differences or the OCP affecting the synthetic and metabolic pathways of these hormones. The fact that the sex differences observed in total plasma concentrations for melatonin and cortisol were not reproduced in the urinary markers challenges their use as a proxy for plasma levels in circadian research, especially in OCP users.

    V Muto, M Jaspar, C Meyer, C Kussé, SL Chellappa, C Degueldre, E Balteau, A Shaffii-Le Bourdiec, A Luxen, B Middleton, SN Archer, C Phillips, F Collette, G Vandewalle, D Dijk, P Maquet (2016)Local Modulation of Human Brain Responses by Circadian Rhythmicity and Sleep Debt, In: Science353(6300)

    Human performance results from an interaction between circadian rhythmicity and homeostatic sleep pressure. Whether and how this interaction is represented at the regional brain level is not established. We quantified changes in brain responses to a sustained-attention task during 13 functional magnetic resonance imaging (fMRI) sessions scheduled across the circadian cycle during 42h of wakefulness and following recovery sleep, in 33 healthy participants. Cortical responses showed significant circadian rhythmicity, the phase of which varied across brain regions. Cortical responses also significantly decreased with accrued sleep debt. Subcortical areas exhibited primarily a circadian modulation, which closely followed the melatonin profile. These findings expand our understanding of the mechanisms involved in maintaining cognition during the day and its deterioration during sleep deprivation and circadian misalignment.

    SK Davies, JE Ang, VL Revell, B Holmes, A Mann, FP Robertson, N Cui, B Middleton, K Ackermann, M Kayser, AE Thumser, FI Raynaud, DJ Skene (2014)Effect of sleep deprivation on the human metabolome, In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA111(29)pp. 10761-10766 NATL ACAD SCIENCES
    MA Bonmati-Carrion, B Middleton, VL Revell, DJ Skene, MA Rol, JA Madrid (2015)Validation of an innovative method, based on tilt sensing, for the assessment of activity and body position, In: CHRONOBIOLOGY INTERNATIONAL32(5)pp. 701-710 INFORMA HEALTHCARE
    K Wulff, E Joyce, B Middleton, DJ Dijk, RG Foster (2006)The suitability of actigraphy, diary data, and urinary melatonin profiles for quantitative assessment of sleep disturbances in schizophrenia: A case report, In: CHRONOBIOLOGY INTERNATIONAL23(1-2)pp. 485-495 TAYLOR & FRANCIS INC
    Aya Honma, Victoria L. Revell, Pippa J. Gunn, Sarah K. Davies, Benita Middleton, Florence I. Raynaud, Debra J. Skene (2019)Effect of Acute Total Sleep Deprivation on Plasma Melatonin, Cortisol and Metabolite Rhythms in Females, In: European Journal of Neuroscience Wiley

    Disruption to sleep and circadian rhythms can impact on metabolism. The study aimed to investigate the effect of acute sleep deprivation on plasma melatonin, cortisol and metabolites, to increase understanding of the metabolic pathways involved in sleep/wake regulation processes. Twelve healthy young female subjects remained in controlled laboratory conditions for ~92 h with respect to posture, meals and environment light (18:00‐23:00 h and 07:00‐09:00 h

    J Arendt, B Middleton, P Williams, G Francis, C Luke (2006)Sleep and circadian phase in a ships crew, In: JOURNAL OF BIOLOGICAL RHYTHMS21(3)pp. 214-221 SAGE PUBLICATIONS INC
    A.L. Darling, K.H. Hart, S. Arber, J.L. Berry, P.L. Morgan, B.A. Middleton, S. Lanham-New, D.J. Skene (2019)25-Hydroxyvitamin D status, light exposure and sleep quality in UK dwelling South Asian and Caucasian postmenopausal women, In: The Journal of Steroid Biochemistry and Molecular Biology189pp. pp 265-273 Elsevier

    There is a lack of research into 25-hydroxyvitamin D (25(OH)D) status, light exposure and sleep patterns in South Asian populations. In addition, results of research studies are conflicting as to whether there is an association between 25(OH)D status and sleep quality. We investigated 25(OH)D status, self-reported and actigraphic sleep quality in n = 35 UK dwelling postmenopausal women (n = 13 South Asians, n = 22 Caucasians), who kept daily sleep diaries and wore wrist-worn actiwatch (AWL-L) devices for 14 days. A subset of n = 27 women (n = 11 South Asian and n = 16 Caucasian) also wore a neck-worn AWL-L device to measure their light exposure. For 25(OH)D concentration, South Asians had a median ± IQR of 43.8 ± 28.2 nmol/L, which was significantly lower than Caucasians (68.7 ± 37.4 nmol/L)(P = 0.001). Similarly, there was a higher sleep fragmentation in the South Asians (mean ± SD 36.9 ± 8.9) compared with the Caucasians (24.7 ± 7.1)(P = 0.002). Non-parametric circadian rhythm analysis of rest/activity patterns showed a higher night-time activity (L5) (22.6 ± 14.0 vs. 10.5 ± 4.4; P = 0.0008) and lower relative amplitude (0.85 ± 0.07 vs. 0.94 ± 0.02; P ˂ 0.0001) in the South Asian compared with the Caucasian women. More South Asians (50%) met the criteria for sleep disorders (PSQI score ˃5) than did Caucasians (27%) (P = 0.001, Fishers Exact Test). However, there was no association between 25(OH)D concentration and any sleep parameter measured (P ˃ 0.05) in either ethnic group. South Asians spent significantly less time in illuminance levels over 200 lx (P = 0.009) than did Caucasians. Overall, our results show that postmenopausal South Asian women have lower 25(OH)D concentration than Caucasian women. They also have higher sleep fragmentation, as well as a lower light exposure across the day. This may have detrimental implications for their general health and further research into sleep quality and light exposure in the South Asian ethnic group is warranted.

    S Montagnese, B Middleton, AR Mani, DJ Skene, MY Morgan (2009)Sleep and circadian abnormalities in patients with cirrhosis: features of delayed sleep phase syndrome?, In: METAB BRAIN DIS24(3)pp. 427-439 SPRINGER/PLENUM PUBLISHERS

    Sleep disturbances are common in patients with cirrhosis but their origins are unknown. The aim of this study was to investigate possible involvement of the circadian system. Sleep was monitored for two weeks, in the home environment, using sleep diaries and actigraphy, in 35 patients with cirrhosis (21 men; mean age [+/- 1SD] 58 +/- 10 yr) and 12 matched healthy controls (eight men; mean age 56 +/- 15 yr); urinary 6-sulphatoxymelatonin (aMT6s), the major metabolite of melatonin, was measured over 56 h, to assess circadian rhythmicity. The patients woke up and got up significantly later than the healthy volunteers and their sleep was significantly more fragmented. Mean 24-hour urinary aMT6s outputs were comparable in the patients and controls (15.5 +/- 13.1 vs. 20.3 +/- 13.8 mu g/24 h) but were significantly lower in the decompensated patients (9.8 +/- 11.3 vs. 17.0 +/- 13.3 mu g/24 h; p=0.03). Significant 24-hour urinary aMT6s rhythms were observed in 26 (79%) of the 33 patients with complete urine collections; 20 patients had a normally timed (midnight-06:00) urinary aMT6s peak, while it was delayed (>= 06:00) in the remainder. Significant correlations were observed between abnormalities in the urinary aMT6s profile (delays and/or lack of a 24-hour rhythm) and indices of sleep timing; parallel delays were observed in sleep habits and urinary aMT6s peaks. The association between delayed circadian rhythms and delayed sleep habits observed in approximately one-third of the patients with cirrhosis is reminiscent of 'delayed sleep phase syndrome'; this condition is managed by attempting to resynchronise the circadian clock by exposure to bright light shortly after morning awakening.

    NM Merchant, DV Azzopardi, AF Hawwa, JC McElnay, B Middleton, J Arendt, T Arichi, P Gressens, AD Edwards (2013)Pharmacokinetics of melatonin in preterm infants, In: BRITISH JOURNAL OF CLINICAL PHARMACOLOGY76(5)pp. 725-733 WILEY-BLACKWELL
    M Turco, A Biscontin, M Corrias, L Caccin, M Bano, F Chiaromanni, M Salamanca, D Mattei, C Salvoro, G Mazzotta, C De Pittà, Benita Middleton, Debra Skene, S Montagnese, R Costa (2017)Diurnal preference, mood and the response to morning light in relation to polymorphisms in the human clock gene PER3, In: Scientific Reports7(6967)pp. 1-10 Nature Publishing Group

    PER3 gene polymorphisms have been associated with differences in human sleep-wake phenotypes, and sensitivity to light. The aims of this study were to assess: i) the frequency of allelic variants at two PER3 polymorphic sites (rs57875989 length polymorphism: PER34, PER35; rs228697 SNP: PER3C, PER3G) in relation to sleep-wake timing; ii) the effect of morning light on behavioural/circadian variables in PER34/PER34 and PER35/PER35 homozygotes. 786 Caucasian subjects living in Northern Italy donated buccal DNA and completed diurnal preference, sleep quality/timing and sleepiness/ mood questionnaires. 19 PER34/PER34 and 11 PER35/PER35 homozygotes underwent morning light administration, whilst monitoring sleep-wake patterns and the urinary 6-sulphatoxymelatonin (aMT6s) rhythm. No significant relationship was observed between the length polymorphism and diurnal preference. By contrast, a significant association was observed between the PER3G variant and morningness (OR = 2.10), and between the PER3G-PER34 haplotype and morningness (OR = 2.19), for which a mechanistic hypothesis is suggested. No significant differences were observed in sleep timing/ aMT6s rhythms between PER35/PER35 and PER34/PER34 subjects at baseline. After light administration, PER34/PER34 subjects advanced their aMT6s acrophase (p < 0.05), and showed a trend of advanced sleep-wake timing. In conclusion, significant associations were observed between PER3 polymorphic variants/their combinations and both diurnal preference and the response to light.

    K Papantoniou, OJ Pozo, A Espinosa, J Marcos, G Castano-Vinyals, X Basagana, E Juanola Pages, J Mirabent, J Martin, P Such Faro, A Gasco Aparici, B Middleton, DJ Skene, M Kogevinas (2015)Increased and Mistimed Sex Hormone Production in Night Shift Workers, In: CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION24(5)pp. 854-863 AMER ASSOC CANCER RESEARCH

    BACKGROUND: Night shift work has been associated with an increased risk for breast and prostate cancer. The effect of circadian disruption on sex steroid production is a possible underlying mechanism, underinvestigated in humans. We have assessed daily rhythms of sex hormones and melatonin in night and day shift workers of both sexes. METHODS: We recruited 75 night and 42 day workers, ages 22 to 64 years, in different working settings. Participants collected urine samples from all voids over 24 hours on a working day. Urinary concentrations of 16 sex steroid hormones and metabolites (estrogens, progestagens, and androgens) and 6-sulfatoxymelatonin were measured in all samples. Mean levels and peak time of total and individual metabolite production were compared between night and day workers. RESULTS: Night workers had higher levels of total progestagens [geometric mean ratio (GMR) 1.65; 95% confidence intervals (CI), 1.17-2.32] and androgens (GMR: 1.44; 95% CI, 1.03-2.00), compared with day workers, after adjusting for potential confounders. The increased sex hormone levels among night shift workers were not related to the observed suppression of 6-sulfatoxymelatonin. Peak time of androgens was significantly later among night workers, compared with day workers (testosterone: 12:14 hours; 10:06-14:48 vs. 08:35 hours; 06:52-10:46). CONCLUSIONS: We found increased levels of progestagens and androgens as well as delayed peak androgen production in night shift workers compared with day workers. IMPACT: The increase and mistiming of sex hormone production may explain part of the increased risk for hormone-related cancers observed in night shift workers.

    Andreas Psomas, Namrata R. Chowdhury, Benita Middleton, Raphaelle Winsky-Sommerer, Debra J. Skene, Menno P. Gerkema, Daan R van der Veen (2023)Co-expression of diurnal and ultradian rhythms in the plasma metabolome of common voles (Microtus arvalis), In: The FASEB JournalIn press(In press) Wiley

    Metabolic rhythms include rapid, ultradian (hourly) dynamics, but it is unclear what their relationship to circadian metabolic rhythms is, and what role meal timing plays in coordinating these ultradian rhythms in metabolism. Here, we characterised widespread ultradian rhythms under ad libitum feeding conditions in the plasma metabolome of the vole, the gold standard animal model for behavioural ultradian rhythms, naturally expressing ~2-hour foraging rhythms throughout the day and night. These ultradian metabolite rhythms co-expressed with diurnal 24-hour rhythms in the same metabolites and did not align with food intake patterns. Specifically, under light-dark entrained conditions we showed twice daily entrainment of phase and period of ultradian behavioural rhythms associated by phase adjustment of the ultradian cycle around the light-dark and dark-light transitions. These ultradian activity patterns also drove an ultradian feeding pattern. We used a unique approach to map this behavioural activity/feeding status to high temporal resolution (every 90 minutes) measures of plasma metabolite profiles across the 24-hour light-dark cycle. A total of 148 known metabolites were detected in vole plasma. Supervised, discriminant analysis did not group metabolite concentration by feeding status, instead, unsupervised clustering of metabolite time courses revealed clusters of metabolites that exhibited significant ultradian rhythms with periods different from the feeding cycle. Two clusters with dissimilar ultradian dynamics, one lipid-enriched (period = 3.4 h) and one amino acid-enriched (period = 4.1 h), both showed co-expression with diurnal cycles. A third cluster solely comprised of glycerophospholipids (specifically ether-linked phosphatidylcholines) and expressed an 11.9 h ultradian rhythm without co-expressed diurnal rhythmicity. Our findings show coordinated co-expression of diurnal metabolic rhythms with rapid dynamics in feeding and metabolism. These findings reveal that ultradian rhythms are integral to biological timing of metabolic regulation, and will be important in interpreting the impact of circadian desynchrony and meal timing on metabolic rhythms.

    Barbara N. Harding, Debra J. Skene, Ana Espinosa, Benita Middleton, Gemma Castaño-Vinyals, Kyriaki Papantoniou, José Maria Navarrete, Patricia Such, Antonio Torrejón, Manolis Kogevinas, Marissa G. Baker (2022)Metabolic profiling of night shift work - The HORMONIT study, In: Chronobiology international Taylor & Francis

    Mechanistic studies are needed to understand how rotating shift work perturbs metabolic processing. We collected plasma samples (n = 196) from 49 males, rotating car factory shift workers at the beginning and end of a night-shift (22:00-06:00 h) and day-shift (06:00 h-14:00 h). Samples underwent targeted LC-MS/MS metabolomics and concentrations of 130 metabolites were log 2 -transformed and pareto-scaled. An elastic net selected the most influential metabolites for linear mixed models examining within-person variation in metabolite levels at night-shift end (06:00 h) compared to day-shift start (06:00 h). Quantitative enrichment analysis explored differentially enriched biological pathways between sample time points. We included 20 metabolites (amino acids, biogenic amines, acylcarnitines, glycerophospholipids) in mixed models. Night-shift was associated with changes in concentrations of arginine (geometric mean ratio [GMR] 2.30, 95%CI 1.25, 4.23), glutamine (GMR 2.22, 95%CI 1.53, 3.24), kynurenine (GMR 3.22, 95%CI 1.05, 9.87), lysoPC18:2 (GMR 1.86, 95%CI 1.11, 3.11), lysoPC20:3 (GMR 2.48, 95%CI 1.05, 5.83), PCaa34:2 (GMR 2.27, 95%CI 1.16, 4.44), and PCae38:5 (GMR 1.66, 95%CI 1.02, 2.68). Tryptophan metabolism, glutathione metabolism, alanine metabolism, glycine and serine metabolism, and urea cycle were pathways differing between shifts. Night shift work was associated with changes in metabolites and the perturbation of metabolic and biochemical pathways related to a variety of health outcomes.

    Juliane Hannemann, Anika Laing, Benita Middleton, Jonathan Cridland, Bart Staels, Nikolaus Marx, Peter J Grant, Massimo Federici, Tarja Stenberg, Debra J Skene, Rainer Böger (2021)Light therapy improves diurnal blood pressure control in night shift workers via reduction of catecholamines: the EuRhythDia study, In: Journal of hypertension39(8)pp. 1678-1688

    Objectives: Night shift work is associated with high rates of hypertension and cardiometabolic disease, which are linked to disrupted circadian rhythms. We hypothesized that timed light therapy might improve disrupted circadian rhythms and stabilize diurnal control of blood pressure and glucose in night shift workers. Mthods: We randomized 24 rotating night shift workers (mean age, 36 ± 13 years, 7 men) who had spent a median of 6 years on rotating night shifts (median, six night shifts per month) to 12 weeks of light therapy or no intervention and compared them with 12 daytime workers (37 ± 11 years, 6 men). We measured oral glucose tolerance (OGTT), 24-h blood pressure and arterial stiffness, and the circadian profiles of melatonin, cortisol, metanephrine and nor-metanephrine at baseline, after 12  weeks of intervention, and 12 weeks after the end of intervention. Results: At baseline, fewer night shift workers showed dipper status as compared with daytime workers (29 vs. 58%; P 

    SW Lockley, DJ Skene, K Thapan, J English, D Ribeiro, I Haimov, S Hampton, B Middleton, M von Schantz, J Arendt (1998)Extraocular light exposure does not suppress plasma melatonin in humans., In: The Journal of Clinical Endocrinology & Metabolism83(9)pp. 3369-3372 Endocrine Society

    Light affects the circadian axis in at least two ways. It can cause the acute suppression of pineal melatonin synthesis, and/or a phase-shift of the circadian oscillator. As recent evidence has suggested that extraocular light exposure may cause phase-shifts of the circadian clock, we have investigated whether suppression of melatonin can be induced by the same type of light exposure. In the first study subjects’ eyes were exposed to white light (2250 lux for 30 min) via a fibre optic cable. As expected, suppression of nighttime plasma melatonin levels (61 ± 6%) was observed. In the second study, light of the same quality but higher intensity (14,000 or 67,500 lux for 180 mins) was delivered in the same manner to the popliteal region behind the subjects’ knees, whilst shielding their eyes. No suppression of plasma melatonin levels (4 ± 7%) was detected in any of the subjects. Thus, extraocular photoreception, if it exists in mammals, does not affect the suprachiasmatic nuclei-pineal pathway.

    PD Lewis, BA Middleton, RM Gous (2006)Exogenous melatonin modifies rate of sexual maturation in domestic pullets, In: POULTRY SCIENCE85(1)pp. 117-122 POULTRY SCIENCE ASSOC INC
    J Arendt, B Middleton, B Stone, D Skene (1999)Complex effects of melatonin: Evidence for photoperiodic responses in humans?, In: SLEEP22(5)pp. 625-635 AMER SLEEP DISORDERS ASSOC
    S Montagnese, B Middleton, AR Mani, DJ Skene, MY Morgan (2006)Evidence of central circadian disruption in patients with cirrhosis, In: JOURNAL OF HEPATOLOGY44pp. S276-S276
    C Isherwood, DT Otway, S Maentele, B Middleton, J Wright, MD Robertson, DJ Skene, M Gibbs, JD Johnston (2015)Daily rhythms in hormonal markers of diabetes and obesity: effect of weight and Type 2 diabetes, In: PROCEEDINGS OF THE NUTRITION SOCIETY74(OCE1)pp. E37-E37
    PD Lewis, BA Middleton, RM Gous (2005)Supplementary radio noise advances sexual maturity in domestic pullets exposed to 7-h photoperiods, In: SOUTH AFRICAN JOURNAL OF ANIMAL SCIENCE35(3)pp. 180-185 SOUTH AFRICAN JOURNAL OF ANIMAL SCIENCES
    K Ackermann, O Lao, VL Revell, R Plomp, B Middleton, DJ Skene, M Kayser (2012)Effect of total sleep deprivation on clock gene expression and melatonin rhythms in human peripheral blood cells, In: JOURNAL OF SLEEP RESEARCH21pp. 39-39
    I Gogenur, B Middleton, VB Kristiansen, DJ Skene, J Rosenberg (2006)Disturbances in melatonin and core body temperature circadian rhythms after minimal invasive surgery, In: JOURNAL OF SLEEP RESEARCH15pp. 95-95
    KA Lederle, B Middleton, TL Sletten, VL Revell, DJ Skene (2008)Subjective and actigraphic sleep in older people with control and 'blue-enriched' white light, In: JOURNAL OF SLEEP RESEARCH17pp. 120-120
    K Wulff, E Joyce, B Middleton, R Foster, D-J Dijk (2008)Circadian activity and sleep cycle disturbances in schizophrenia patients in comparison to unemployed healthy controls, In: INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY11pp. 150-150
    PL Morgan, S Hampton, A Karatziotou, J Zaslona, B Middleton (2012)Validation of two new activity monitors: motionwatch 8 and pro-diary motion, In: JOURNAL OF SLEEP RESEARCH21pp. 217-218
    SMW Rajaratnam, DJ Dijk, B Middleton, BM Stone, J Arendt (2004)Melatonin phase-shifts human circadian rhythms with no evidence of changes in the duration of endogenous melatonin secretion or the 24-hour production of reproductive hormones (vol 88, pg 4303, 2003), In: JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM89(6)pp. 2997-2997 ENDOCRINE SOC
    PL Morgan, S Hopkins, LJM Schlangen, DJ Skene, B Middleton (2012)Effects of light supplementation on mood, alertness and rest-activity rhythms in older people living in care homes, In: JOURNAL OF SLEEP RESEARCH21pp. 370-371
    L Gribble, A Korimbocus, B Middleton, R Gouni, D Kerr, D Coppini, J Boyle (2008)Sleep in painful diabetic peripheral neuropathy: Subjective measures and actigraphy analysis, In: SLEEP31pp. A299-A299
    C Meyer, M Jaspar, V Muto, C Kusse, SL Chellappa, C Degueldre, E Balteau, A Luxen, F Collette, B Middleton, C Phillips, SN Archer, D-J Dijk, G Vandewalle, P Maquet (2014)Seasonal variation in human executive brain responses, In: JOURNAL OF SLEEP RESEARCH23pp. 171-171
    K Wulff, EM Joyce, B Middleton, RG Foster, D Dijk (2008)Sleep and rest/activity cycle disturbances in schizophrenia patients in comparison to unemployed healthy controls, In: JOURNAL OF SLEEP RESEARCH17pp. 76-77
    V Muto, M Jaspar, C Meyer, AS LeBourdiec, C Kussee, SL Chellappa, G Vandewalle, C Degueldre, A Luxen, F Collette, C Phillips, B Middleton, SN Archer, D-J Dijk, P Maquet (2014)Neural correlates of sustained attention under sleep deprivation during a constant routine: circadian and homeostatic interaction, In: JOURNAL OF SLEEP RESEARCH23pp. 61-61
    K Wulff, EM Joyce, B Middleton, RG Foster, DJ Dijk (2007)Sleep and circadian activity/rest disturbances in schizophrenia patients in comparison to unemployed healthy controls, In: EUROPEAN NEUROPSYCHOPHARMACOLOGY17pp. S415-S416
    M Turco, L Caccin, M Corrias, A Biscontin, C De Pitta, B Middleton, DJ Skene, R Costa, S Montagnese (2014)Length polymorphism in the human clock gene Period3 and diurnal preference, subjective sleepiness and the response to morning light, In: JOURNAL OF SLEEP RESEARCH23pp. 55-56
    TL Sletten, VL Revell, B Middleton, KA Lederle, DJ Skene (2008)Short wavelength light exposure in the elderly: acute and phase shifting effects, In: JOURNAL OF SLEEP RESEARCH17pp. 79-79
    S Montagnese, B Middleton, DJ Skene, MY Morgan (2007)Sleep-wake abnormalities do not correlate with neuropsychiatric performance in patients with cirrhosis, In: HEPATOLOGY46(4)pp. 562A-563A
    K Wulff, EM Joyce, B Middleton, DJ Dijk, RG Foster (2007)Sleep in schizophrenia, In: EUROPEAN NEUROPSYCHOPHARMACOLOGY17pp. S136-S137
    S Benloucif, HJ Burgess, EB Klerman, AJ Lewy, B Middleton, PJ Murphy, BL Parry, VL Revell (2008)Measuring melatonin in humans, In: Journal of Clinical Sleep Medicine4(1)pp. 66-69
    SM Montagnese, M De Rui, M Corrias, M Turco, P Amodio, C De Pitta, R Costa, B Middleton, DJ Skene (2014)Sleep-wake abnormalities in patients with cirrhosis, In: JOURNAL OF SLEEP RESEARCH23pp. 146-146
    DJ Skene, SK Davies, JE Ang, VL Revell, B Holmes, A Mann, R Robertson, N Cui, B Middleton, K Ackermann, M Kayser, AE Thumser, FI Raynaud (2014)Effect of sleep deprivation on human plasma metabolome rhythms, In: JOURNAL OF SLEEP RESEARCH23pp. 36-37
    S Montagnese, B Middleton, DJ Skene, MY Morgan (2008)Sleep-wake disturbances in patients with cirrhosis: relations to neuropsychiatric performance and health-related quality of life, In: JOURNAL OF SLEEP RESEARCH17pp. 76-76
    S Montagnese, B Middleton, DJ Skene, MY Morgan (2008)Sleep disturbances are not a feature of hepatic encephalopathy, In: JOURNAL OF HEPATOLOGY48pp. S40-S40
    M Jaspar, C Meyer, V Muto, A Shaffii-LeBourdiec, SL Chellappa, C Kussee, G Vandewalle, F Collette, B Middleton, S Archer, DJ Dijk, P Maquet (2014)Sleep loss changes executive brain responses in the wake maintenance zone, In: JOURNAL OF SLEEP RESEARCH23pp. 61-61