Circadian regulation of dentate gyrus excitability mediated by G-protein signaling.

The central circadian regulator within the suprachiasmatic nucleus transmits time of day information by a diurnal spiking rhythm driven by molecular clock genes controlling membrane excitability. Most brain regions, including the hippocampus, harbor similar intrinsic circadian transcriptional machinery, but whether these molecular programs generate oscillations of membrane properties is unclear. Here, we show that intrinsic excitability of mouse dentate granule neurons exhibits a 24-h oscillation that controls spiking probability. Diurnal changes in excitability are mediated by antiphase G-protein regulation of potassium and sodium currents that reduce excitability during the Light phase. Disruption of the circadian transcriptional machinery by conditional deletion of Bmal1 enhances excitability selectively during the Light phase by removing G-protein regulation. These results reveal that circadian transcriptional machinery regulates intrinsic excitability by coordinated regulation of ion channels by G-protein signaling, highlighting a potential novel mechanism of cell-autonomous oscillations.

Examination of Diurnal Variation and Sex Differences in Hippocampal Neurophysiology and Spatial Memory.

Circadian rhythms are biological processes that cycle across 24 h and regulate many facets of neurophysiology, including learning and memory. Circadian variation in spatial memory task performance is well documented; however, the effect of sex across circadian time (CT) remains unclear. Additionally, little is known regarding the impact of time-of-day on hippocampal neuronal physiology. Here, we investigated the influence of both sex and time-of-day on hippocampal neurophysiology and memory in mice. Performance on the object location memory (OLM) task depended on both circadian time and sex, with memory enhanced at night in males but during the day in females. Long-term synaptic potentiation (LTP) magnitude at CA3-CA1 synapses was greater at night compared with day in both sexes. Next, we measured spontaneous synaptic excitation and inhibition onto CA1 pyramidal neurons. Frequency and amplitude of inhibition was greater during the day compared with night, regardless of sex. Frequency and amplitude of excitation was larger in females, compared with males, independent of time-of-day, although both time-of-day and sex influenced presynaptic release probability. At night, CA1 pyramidal neurons showed enhanced excitability (action potential firing and/or baseline potential) that was dependent on synaptic excitation and inhibition, regardless of sex. This study emphasizes the importance of sex and time-of-day in hippocampal physiology, especially given that many neurologic disorders impacting the hippocampus are linked to circadian disruption and present differently in men and women. Knowledge about how sex and circadian rhythms affect hippocampal physiology can improve the translational relevancy of therapeutics and inform the appropriate timing of existing treatments.

Circadian disruption of hippocampus in an early senescence male mouse model.

Age-related cognitive decline and disruptions in circadian rhythms are growing problems as the average human life span increases. Multiple strains of the senescence-accelerated mouse (SAM) show reduced life span, and the SAMP8 strain in particular has been well documented to show cognitive deficits in behavior as well as a bimodal pattern of circadian locomotor activity. However, little is known about circadian regulation within the hippocampus of these strains of mice. Here we test the hypothesis that in this early senescence model, disruption of the molecular circadian clock in SAMP8 animals drives disrupted behavior and physiology. We found normal rhythms in PER2 protein expression in the SCN of SAMP8 animals at 4 months, despite the presence of disrupted wheel-running activity rhythms at this age. Interestingly, a significant rhythm in PER2 expression was not observed in the hippocampus of SAMP8 animals, despite a significant 24-h rhythm in SAMR1 controls. We also examined time-restricted feeding as a potential strategy to rescue disrupted hippocampal plasticity. Time-restricted feeding increased long-term potentiation at Schaffer collateral-CA1 synapses in SAMP8 mice (compared to SAMR1 controls). Overall, we confirm disrupted circadian locomotor rhythms in this early senescence model (as early as 4 months) and discovered that this disruption is not due to arrhythmic PER2 levels in the SCN; however, other extra-SCN circadian oscillators (i.e., hippocampus) are likely impaired with accelerated aging.

Dysregulated clock gene expression and abnormal diurnal regulation of hippocampal inhibitory transmission and spatial memory in amyloid precursor protein transgenic mice.

Patients with Alzheimer's disease (AD) often have fragmentation of sleep/wake cycles and disrupted 24-h (circadian) activity. Despite this, little work has investigated the potential underlying day/night disruptions in cognition and neuronal physiology in the hippocampus. The molecular clock, an intrinsic transcription-translation feedback loop that regulates circadian behavior, may also regulate hippocampal neurophysiological activity. We hypothesized that disrupted diurnal variation in clock gene expression in the hippocampus corresponds with loss of normal day/night differences in membrane excitability, synaptic physiology, and cognition. We previously reported disrupted circadian locomotor rhythms and neurophysiological output of the suprachiasmatic nucleus (the primary circadian clock) in Tg-SwDI mice with human amyloid-beta precursor protein mutations. Here, we report that Tg-SwDI mice failed to show day/night differences in a spatial working memory task, unlike wild-type controls that exhibited enhanced spatial working memory at night. Moreover, Tg-SwDI mice had lower levels of Per2, one of the core components of the molecular clock, at both mRNA and protein levels when compared to age-matched controls. Interestingly, we discovered neurophysiological impairments in area CA1 of the Tg-SwDI hippocampus. In controls, spontaneous inhibitory post-synaptic currents (sIPSCs) in pyramidal cells showed greater amplitude and lower inter-event interval during the day than the night. However, the normal day/night differences in sIPSCs were absent (amplitude) or reversed (inter-event interval) in pyramidal cells from Tg-SwDI mice. In control mice, current injection into CA1 pyramidal cells produced more firing during the night than during the day, but no day/night difference in excitability was observed in Tg-SwDI mice. The normal day/night difference in excitability in controls was blocked by GABA receptor inhibition. Together, these results demonstrate that the normal diurnal regulation of inhibitory transmission in the hippocampus is diminished in a mouse model of AD, leading to decreased daytime inhibition onto hippocampal CA1 pyramidal cells. Uncovering disrupted day/night differences in circadian gene regulation, hippocampal physiology, and memory in AD mouse models may provide insight into possible chronotherapeutic strategies to ameliorate Alzheimer's disease symptoms or delay pathological onset.

Circadian regulation of membrane physiology in neural oscillators throughout the brain.

Twenty-four-hour rhythmicity in physiology and behavior are driven by changes in neurophysiological activity that vary across the light-dark and rest-activity cycle. Although this neural code is most prominent in neurons of the primary circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus, there are many other regions in the brain where region-specific function and behavioral rhythmicity may be encoded by changes in electrical properties of those neurons. In this review, we explore the existing evidence for molecular clocks and/or neurophysiological rhythms (i.e., 24 hr) in brain regions outside the SCN. In addition, we highlight the brain regions that are ripe for future investigation into the critical role of circadian rhythmicity for local oscillators. For example, the cerebellum expresses rhythmicity in over 2,000 gene transcripts, and yet we know very little about how circadian regulation drives 24-hr changes in the neural coding responsible for motor coordination. Finally, we conclude with a discussion of how our understanding of circadian regulation of electrical properties may yield insight into disease mechanisms which may lead to novel chronotherapeutic strategies in the future.

Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function.

The in-depth understanding of skin resident memory CD8+ T lymphocytes (TRM ) may help to uncover strategies for their manipulation during disease. We investigated isolated TRM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8+ T cell populations from the same donors. There were significantly increased proportions of CD8+ CD45RA- CD27- T cells in the skin that expressed low levels of killer cell lectin-like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8+ TRM in skin were therefore phenotypically distinct from circulating CD8+ CD45RA- CD27- T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8+ TRM with T cell receptor (TCR)/CD28 or interleukin (IL)-2 or IL-15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)-1 further boosted granzyme B expression. A unique feature of some CD8+ TRM cells was their ability to secrete high levels of tumour necrosis factor (TNF)-α and IL-2, a cytokine combination that was not seen frequently in circulating CD8+ T cells. The cutaneous CD8+ TRM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous TRM appears to be stringently controlled by environmental signals in situ.

A novel processed food classification system applied to Australian food composition databases.

BACKGROUND: The extent of food processing can affect the nutritional quality of foodstuffs. Categorising foods by the level of processing emphasises the differences in nutritional quality between foods within the same food group and is likely useful for determining dietary processed food consumption. The present study aimed to categorise foods within Australian food composition databases according to the level of food processing using a processed food classification system, as well as assess the variation in the levels of processing within food groups. METHODS: A processed foods classification system was applied to food and beverage items contained within Australian Food and Nutrient (AUSNUT) 2007 (n = 3874) and AUSNUT 2011-13 (n = 5740). The proportion of Minimally Processed (MP), Processed Culinary Ingredients (PCI) Processed (P) and Ultra Processed (ULP) by AUSNUT food group and the overall proportion of the four processed food categories across AUSNUT 2007 and AUSNUT 2011-13 were calculated. RESULTS: Across the food composition databases, the overall proportions of foods classified as MP, PCI, P and ULP were 27%, 3%, 26% and 44% for AUSNUT 2007 and 38%, 2%, 24% and 36% for AUSNUT 2011-13. Although there was wide variation in the classifications of food processing within the food groups, approximately one-third of foodstuffs were classified as ULP food items across both the 2007 and 2011-13 AUSNUT databases. CONCLUSIONS: This Australian processed food classification system will allow researchers to easily quantify the contribution of processed foods within the Australian food supply to assist in assessing the nutritional quality of the dietary intake of population groups.

Mediating effects of dietary intake on associations of TV viewing, body mass index and metabolic syndrome in adolescents.

OBJECTIVE: Evidence suggests that TV viewing is associated with body mass index (BMI) and metabolic syndrome (MetS) in adolescents. However, it is unclear whether dietary intake mediates these relationships. METHODS: A cross-sectional analysis was conducted in adolescents (12-19 years) participating in the 2003-2006 United States National Health and Nutrition Examination Survey. BMI z scores (zBMI) (n = 3,161) and MetS (n = 1,379) were calculated using age- and sex-specific criteria for adolescents. TV viewing (h/day) was measured via a self-reported questionnaire, and dietary intake was assessed using two 24-h recalls. Using the MacKinnon method, a series of mediation analyses were conducted examining five dietary mediators (total energy intake, fruit and vegetable intake, discretionary snacks, sugar-sweetened beverages and diet quality) of the relationships between TV viewing and zBMI and MetS. RESULTS: Small positive relationships were observed between TV viewing and zBMI (ß = 0.99, p < 0.001) and TV viewing and MetS (OR = 1.18, p = 0.046). No dietary element appeared to mediate the relationship between TV viewing and zBMI. However, sugar-sweetened beverage consumption and fruit and vegetable intake partially mediated the relationship between TV viewing and MetS, explaining 8.7% and 4.1% of the relationship, respectively. CONCLUSIONS: These findings highlight the complexity of the relationships between TV viewing, dietary intake and cardiometabolic health outcomes, and that TV viewing should remain a target for interventions.

The importance of taste on dietary choice, behaviour and intake in a group of young adults.

The 'taste of food' plays an important role in food choice. Furthermore, foods high in fat, sugar and salt are highly palatable and associated with increased food consumption. Research exploring taste importance on dietary choice, behaviour and intake is limited, particularly in young adults. Therefore, in this study a total of 1306 Australian university students completed questionnaires assessing dietary behaviors (such as how important taste was on food choice) and frequency of food consumption over the prior month. Diet quality was also assessed using a dietary guideline index. Participants had a mean age of 20 ± 5 years, Body Mass Index (BMI) of 22 ± 3 kg/m(2), 79% were female and 84% Australian. Taste was rated as being a very or extremely important factor for food choice by 82% of participants. Participants who rated taste as highly important, had a poorer diet quality (p = 0.001) and were more likely to consume less fruit (p = 0.03) and vegetables (p = 0.05). Furthermore, they were significantly more likely to consume foods high in fat, sugar and salt, including chocolate and confectionary, cakes and puddings, sweet pastries, biscuits, meat pies, pizza, hot chips, potato chips, takeaway meals, soft drink, cordial and fruit juice (p = 0.001-0.02). They were also more likely to consider avoiding adding salt to cooking (p = 0.02) and adding sugar to tea or coffee (p = 0.01) as less important for health. These findings suggest that the importance individuals place on taste plays an important role in influencing food choice, dietary behaviors and intake.

Is the relationship between sedentary behaviour and cardiometabolic health in adolescents independent of dietary intake? A systematic review.

Screen time, but not overall sedentary behaviour, is consistently related to cardiometabolic health in adolescents. Because of the associations screen time has with dietary intake, diet may be an important factor in the screen time and health relationship; however, evidence has not previously been synthesized. Thus, the aim of this systematic review was to explore whether the associations between various sedentary behaviours and cardiometabolic risk markers are independent of dietary intake in adolescents. Online databases and personal libraries were searched for peer-reviewed original research articles published in English before March 2014. Included studies assessed associations between sedentary behaviour and cardiometabolic markers in 12- to 18-year-olds and adjusted for dietary intake. Twenty-five studies met the inclusion criteria. From the 21 studies examining sedentary behaviour and adiposity, the majority found significant positive associations between television viewing, screen time and self-reported overall sedentary behaviour with markers of adiposity, independent of dietary intake. No significant associations between screen time with blood pressure and cholesterol were reported. Sedentary behaviour appears to be associated with adiposity in adolescents, irrespective of dietary intake. However, the variability of dietary variables between studies suggests further work is needed to understand the role of dietary intake when examining these associations in youth.

Inconsistent associations between sweet drink intake and 2-year change in BMI among Victorian children and adolescents.

OBJECTIVE: The aim of this study was to examine whether baseline (T1) or 2-year change in sweet drink intake in children and adolescents was associated with age- and gender-standardized body mass index (BMIz) at time two (T2), 2 years later. METHODS: Data on 1465 children and adolescents from the comparison groups of two quasi-experimental intervention studies from Victoria, Australia were analysed. At two time points between 2003 and 2008 (mean interval: 2.2 years) height and weight were measured and sweet drink consumption (soft drink and fruit juice/cordial) was assessed. RESULTS: No association was observed between T1 sweet drink intake and BMIz at T2 among children or adolescents. Children from higher socioeconomic status families who reported an increased intake of sweet drinks at T2 compared with T1 had higher mean BMIz at T2 (ß: 0.13, P = 0.05). There was no evidence of a dose-response relationship between sweet drink intake and BMIz. In supplementary analyses, we observed that more frequent usual consumption of fruit juice/cordial was associated with a higher BMIz at T2 among children. CONCLUSION: This study showed limited evidence of an association between sweet drink intake and BMIz. However, the association is complex and may be confounded by both dietary and activity behaviours.

Investigation of the cutaneous response to recall antigen in humans in vivo.

In this paper we provide a detailed description of an experimental method for investigating the induction and resolution of recall immune response to antigen in humans in vivo. This involves the injection of tuberculin purified protein derivative (PPD) into the skin, followed by inducing suction blisters at the site of injection, from which leucocytes and cytokines that are involved in the response can be isolated and characterized. Using this technique we found that although the majority of CD4(+) T cells in the skin that are present early in the response express cutaneous lymphocyte antigen (CLA), the expression of this marker is reduced significantly in later phases. This may enable these cells to leave the skin during immune resolution. Furthermore, interleukin (IL)-2 production can be detected both in CD4(+) T cells and also in the blister fluid at the peak of the response at day 7, indicating that mediators found in the blister fluid are representative of the cytokine microenvironment in vivo. Finally, we found that older humans have defective ability to respond to cutaneous PPD challenge, but this does not reflect a global immune deficit as they have similar numbers of circulating functional PPD-specific CD4(+) T cells as young subjects. The use of the blister technology enables further characterization of the skin specific defect in older humans and also general mechanisms that govern immune regulation in vivo.

Life-skills as a predictor of academic success: an exploratory study.

Traditional predictors of academic performance in college, such as measures of verbal and mathematical abilities [i.e., Scholastic Aptitude Test (SAT)] and academic achievement (i.e., high school GPA), often account for less than 25% of the variance in college performance, thus leaving a considerable amount of variance unexplained. The primary goal of this study was to examine developmental variables that may account for academic achievement beyond the traditional indices mentioned. Specifically, the relationships among four categories of life-skills and cumulative GPA were examined. A hierarchical multiple-regression analysis revealed that the four life-skills categories predicted an additional 9.4% of the variance in cumulative GPA beyond high school GPA and SAT scores. Of the four categories, physical fitness/health maintenance skills made a statistically significant, unique contribution to predicting cumulative GPA. Because of the exploratory nature of the current study, suggestions are made for conducting future research in light of the study's limitations.

The appropriateness of opt-out consent for monitoring childhood obesity in Australia.

Childhood obesity monitoring is a fundamental component of obesity prevention but is poorly done in Australia. Monitoring obesity prevalence in children provides important population health data that can be used to track trends over time, identify areas at greatest risk of obesity, determine the effectiveness of interventions and policies, raise awareness and stimulate action. High participation rates are essential for effective monitoring because these provide more representative data. Passive ('opt-out') consent has been shown to provide high participation rates in international childhood obesity monitoring programs and in a recent Australian federal initiative monitoring early child development. A federal initiative structured like existing child development monitoring programs, but with the authority to collect height and weight measurements using opt-out consent, is recommended to monitor rates of childhood obesity in Australia.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

Association between a common IL10 distal promoter haplotype and IgE production in individuals with atopic dermatitis.

Atopic dermatitis (AD) is a genetically determined inflammatory skin disease characterized by abnormal cytokine production, including increased production of interleukin 10 (IL10). Single nucleotide polymorphisms (SNP) and haplotypes in the IL10 gene promoter region on chromosome 1q31-32 have been implicated in several inflammatory diseases, but generally, only SNPs proximal to the transcription start site (TSS) have been investigated. The aim of this study was to identify proximal, distal and combined haplotype sets in the IL10 promoter region and to study their association with clinical phenotypes in atopic dermatitis. SNPs at positions -3575, -2849, -2779, -2763, -1082, -851, -819 and -592 in the IL10 promoter region were genotyped in individuals with atopic dermatitis (n= 47) and nonatopic control subjects (n= 40) using polymerase chain reaction-based techniques and induced heteroduplex generator (IHG) analysis. Pan-promoter, TSS-proximal and TSS-distal haplotypes were reconstructed using phase analysis. Fifteen haplotypes representing all eight SNPs were identified. Subgrouping identified four 4-locus and three 3-locus TSS-proximal haplotypes; and nine 4-locus TSS-distal haplotypes. No difference was found in haplotype or SNP frequencies between the AD and control groups, or between patients with mild or severe disease. However, a common 4-locus TSS-distal haplotype (TGAC) was significantly increased in patients with IgE levels over 1000 kIU L(-1). This study is the first to analyse the association between haplotype groups in the IL10 promoter region and clinical phenotypes in AD. We have demonstrated a significant association between the TSS-distal haplotype TGAC, and IgE levels in AD patients. It remains to be shown if there is an association between the TGAC haplotype and IL10 production, which might account for the stimulation of IgE production.

Molecular subsets in the gene expression signatures of scleroderma skin.

BACKGROUND: Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production. METHODOLOGY AND FINDINGS: We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc. CONCLUSIONS AND SIGNIFICANCE: Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

Common markers of proliferation.

When normal tissue and tumour samples are compared by microarray analysis, the biggest differences most often occur in the expression levels of genes that control cell proliferation. However, this difference is detected whenever mRNA samples that are taken from two cell populations with different proliferation rates are compared. Although the exact genes that comprise this 'proliferation signature' often differ, they are almost always genes that are involved in the fundamental process of cell proliferation. Can the proliferation signature be used to improve our understanding of the cell cycle and cancer pathogenesis, as well as being used as a biomarker for cancer diagnosis and prognosis?

Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of beta-chemokines.

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.