Paul S. Hartley

Dietary Modulation of Drosophila Sleep-Wake Behaviour

Background A complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed. Methodology/Principal Findings Using automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep. Conclusions We conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans.

The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets

The ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37 degrees C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4-64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4-64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

Klf15 Is Critical for the Development and Differentiation of Drosophila Nephrocytes

Insect nephrocytes are highly endocytic scavenger cells that represent the only invertebrate model for the study of human kidney podocytes. Despite their importance, nephrocyte development is largely uncharacterised. This work tested whether the insect ortholog of mammalian Kidney Krüppel-Like Factor (Klf15), a transcription factor required for mammalian podocyte differentiation, was required for insect nephrocyte development. It was found that expression of Drosophila Klf15 (dKlf15, previously known as Bteb2) was restricted to the only two nephrocyte populations in Drosophila, the garland cells and pericardial nephrocytes. Loss of dKlf15 function led to attrition of both nephrocyte populations and sensitised larvae to the xenotoxin silver nitrate. Although pericardial nephrocytes in dKlf15 loss of function mutants were specified during embryogenesis, they failed to express the slit diaphragm gene sticks and stones and did not form slit diaphragms. Conditional silencing of dKlf15 in adults led to reduced surface expression of the endocytic receptor Amnionless and loss of in vivo scavenger function. Over-expression of dKlf15 increased nephrocyte numbers and rescued age-dependent decline in nephrocyte function. The data place dKlf15 upstream of sns and Amnionless in a nephrocyte-restricted differentiation pathway and suggest dKlf15 expression is both necessary and sufficient to sustain nephrocyte differentiation. These findings explain the physiological relevance of dKlf15 in Drosophila and imply that the role of KLF15 in human podocytes is evolutionarily conserved.

SPARC-Dependent Cardiomyopathy in Drosophila

Background—The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. Methods and Results—This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte–cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. Conclusions—The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans.

Food-entrained rhythmic expression of PER2 and BMAL1 in murine megakaryocytes does not correlate with circadian rhythms in megakaryopoiesis.

Summary.  Background: Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. Objectives: This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. Methods: Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food‐entrained from light‐entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell‐cycle behavior in megakaryocytes collected from mice over a 24‐h period. Results: Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell‐cycle activity. Conclusions: Normal megakaryopoiesis requires synchrony between food‐ and light‐entrained circadian oscillators.

Fermitins, the Orthologs of Mammalian Kindlins, Regulate the Development of a Functional Cardiac Syncytium in Drosophila melanogaster

The vertebrate Kindlins are an evolutionarily conserved family of proteins critical for integrin signalling and cell adhesion. Kindlin-2 (KIND2) is associated with intercalated discs in mice, suggesting a role in cardiac syncytium development; however, deficiency of Kind2 leads to embryonic lethality. Morpholino knock-down of Kind2 in zebrafish has a pleiotropic effect on development that includes the heart. It therefore remains unclear whether cardiomyocyte Kind2 expression is required for cardiomyocyte junction formation and the development of normal cardiac function. To address this question, the expression of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kind2, was silenced in Drosophila cardiomyocytes. Heart development was assessed in adult flies by immunological methods and videomicroscopy. Silencing both Fit1 and Fit2 led to a severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. A null allele of Fit1 was generated but this had no impact on the heart. Similarly, the silencing of Fit2 failed to affect heart function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1 null flies disrupted syncytium development, leading to severe cardiomyopathy. The data definitively demonstrate a role for Fermitins in the development of a functional cardiac syncytium in Drosophila. The findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. These findings support the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans.

Timed feeding of mice modulates light-entrained circadian rhythms of reticulated platelet abundance and plasma thrombopoietin and affects gene expression in megakaryocytes

Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light‐dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light‐dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light‐dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.

The diurnal tick-tockery of platelet biology

Circadian (∼24 hours) clocks are ubiquitous in nature and are important regulators of behaviour, physiology and metabolism. Circadian clocks can synchronise biological processes with environmental cycles, buffer biological systems to maintain homeostasis and partition mutually antagonistic processes to different temporal spaces within the daily cycle. Clocks act cell-autonomously (intrinsically) and systemically (extrinsically) to coordinate whole organism biology and there is epidemiological evidence indicating that chronic disruption of behavioural rhythms increases the risk of developing cancer and cardiovascular disease. Although the genetic mechanism of the mammalian clock has been largely deciphered, the physiological relevance of clocks often remains elusive. Findings from humans and animal models suggest that the circadian clock and diurnal rhythms have an important role in megakaryopoiesis and the risk of a cardiovascular event. This short review will introduce the mammalian circadian clock and discuss how circadian clocks and diurnal rhythms influence platelet production and function.

Cryopreservation of houbara semen: A pilot study

The potential of producing viable houbara bustard Chlamydotis undulata undulata progeny with cryopreserved semen was investigated during the 1998 breeding season. Houbara semen was diluted and rapidly frozen into pellet form in LN2 using dimethylacetamide (DMA) as a cryoprotectant. Thawed semen exhibited 24.86± 10.61% recovery of fresh sperm motility levels and 36.44±16.17% of fresh viability counts. Two second-year females were inseminated on two and three occasions, respectively, with thawed semen. One female laid four eggs between two clutches and the second female laid two eggs in a single clutch. All eggs were fertile; two developed to 18–21days but failed to pip; one chick died at pip and three chicks hatched. This is the first documented occasion that houbara bustard chicks were conceived with cryopreserved semen and it demonstrates the feasibility of using a rapid, simple, and relatively inexpensive methodology to achieve this result. Zoo Biol 18:147–152, 1999.

Hypoglycaemia predisposes platelets to death by affecting calcium homeostasis and mitochondrial integrity

Factors affecting platelet survival are poorly understood. To explore the hypothesis that platelet lifespan correlates with the lifespan of a key housekeeping process we subjected human platelets to in vitro incubation at 37°C for 24 h to several days under hypoglycaemic conditions. Viability was assessed both by microscopy and flow cytometry using calcein-AM and/or FM4-64. In keeping with previous data we found that, under control conditions platelets died at a linear rate during 120 h of incubation. Hypoglycaemia did not affect the death rate but did lead to an increase in the frequency of platelets unable to accumulate the mitochondrial potentiometric dye 10-Nonyl Acridine Orange (NAO) and promoted platelet death in response to the pro-apoptotic molecule BH3I-2′. Hypoglycaemia led to an increase in intraplatelet calcium that could be prevented 2-aminoethoxydiphenylborate (2-APB), a store operated calcium channel (SOCC) blocker. However, this agent was unable to rescue the platelets’ ability to accumulate NAO. These data suggest that extracellular glucose is utilised by platelets for calcium homeostasis and maintenance of mitochondrial integrity and that hypoglycaemia primes platelets for death.