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Background Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Methods Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Results Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. Conclusion We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. Graphical Abstract A higher resolution version of the Graphical abstract is available as Supplementary information

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. In this work we aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.

Aging is associated with a decline in heart function across the tissue, cellular, and molecular levels. The risk of cardiovascular disease grows significantly over time, and as developed countries continue to see an increase in lifespan, the cost of cardiovascular healthcare for the elderly will undoubtedly rise. The molecular basis for cardiac function deterioration with age is multifaceted and not entirely clear, and there is a limit to what investigations can be performed on human subjects or mammalian models. Drosophila melanogaster has emerged as a useful model organism for studying aging in a short timeframe, benefitting from a suite of molecular and genetic tools and displaying highly conserved traits of cardiac senescence. Here, we discuss recent advances in our understanding of cardiac aging and how the fruit fly has aided in these developments.

Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart. This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin. The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition. The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen., Highlights • The ageing Drosophila heart accumulates collagens (Viking and Pericardin). • This fibrosis-like phenotype accompanied age-dependent cardiac dysfunction. • Reduced expression of the collagen-interacting protein SPARC prevented the age-related cardiac dysfunction. • SPARC overexpression in nephrocytes drives cardiomyopathy and Pericardin deposition. • Reduced SPARC did not prevent collagen accumulation in the ageing heart.

Here we show that a labyrinth channel compartment and slit diaphragms, which are the histological structures\ud enabling insect nephrocytes ultrafiltration, are established during embryogenesis first by the garland nephrocytes (GCNs). The later pericardial nephrocytes, which represent the majority of functional nephrocytes in larvae and adults, lack these characteristic features at the embryonic stage. During larval development, a subpopulation of the pericardial cells survives and matures into functional nephrocytes (PCNs) displaying a fully differentiated slit diaphragm and a labyrinth channel compartment. Likely the embryonic pericardial cells have primary functions other than ultrafiltration (e.g. in production and secretion of ECM constituents). We also show, for the first time, that PCNs in the adult fly undergo dramatic histological degeneration upon ageing. The slit diaphragms disappear, the labyrinth channel system degenerates and the lysosomal compartment becomes highly enriched with electron-dense material. When using nephrocytes as a model for genetic screening purposes or to investigate the specific role of genes involved in endocytosis, histological changes occurring upon ageing need to be taken into account when interpreting structural data.

Vertebrate podocytes are kidney glomerular cells critically required for normal renal filtration. To fulfill their role, podocytes form molecular sieves known as slit diaphragms that contribute to the glomerular filtration barrier. The disruption of podocyte biology or slit diaphragm formation in humans is a precursor to albuminuria, renal failure, and cardiovascular morbidity. Due to genetic and functional similarities, the nephrocytes of Drosophila are increasingly used to model the genetic and metabolic basis of human podocyte biology. They have the advantage that they are a much quicker system to study compared to other murine transgenic models. In this chapter we present methods to modulate and study Drosophila nephrocyte function and diaphragm formation.

Vertebrate podocytes are kidney glomerular cells critically required for normal renal filtration. To fulfill their role, podocytes form molecular sieves known as slit diaphragms that contribute to the glomerular filtration barrier. The disruption of podocyte biology or slit diaphragm formation in humans is a precursor to albuminuria, renal failure, and cardiovascular morbidity. Due to genetic and functional similarities, the nephrocytes of Drosophila are increasingly used to model the genetic and metabolic basis of human podocyte biology. They have the advantage that they are a much quicker system to study compared to other murine transgenic models. In this chapter we present methods to modulate and study Drosophila nephrocyte function and diaphragm formation.

Supplemental Digital Content is available in the text., 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.

The aim of this study was to evaluate the efficacy and tolerability of sitagliptin compared with glimepiride in elderly patients with type 2 diabetes mellitus (T2DM) and inadequate glycemic control with diet and exercise alone. This was a randomized, parallel-group, multinational, non-inferiority clinical trial with an active-controlled, double-blind treatment period in which patients ≥65 and ≤85 years of age with T2DM were screened at 85 sites. Patients were randomized to once-daily sitagliptin (100 or 50 mg, depending on renal function) or glimepiride (in titrated doses) for 30 weeks. The main outcome measures were change from baseline in glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), and body weight; and the incidence of symptomatic hypoglycemia. The mean baseline HbA1c was 7.8 % in both the sitagliptin group (n = 197) and the glimepiride group (n = 191). After 30 weeks, the least squares (LS) mean change in HbA1c baseline was −0.32 % with sitagliptin and −0.51 % with glimepiride, for a between-group difference (95 % CI) of 0.19 % (0.03–0.34). This result met the pre-specified criterion for declaring non-inferiority, which required that the upper 95 % confidence limit lie below 0.4 %. The LS mean change in FPG from baseline was −14.5 mg/dL with sitagliptin and −21.2 mg/dL with glimepiride, for a between-group difference (95 % CI) of 6.7 mg/dL (0.7–12.7). The percentages of patients with adverse events of symptomatic hypoglycemia were 0.8 % in the sitagliptin group and 4.7 % in the glimepiride group (between-treatment difference = −3.9 %, p = 0.009). The LS mean change in body weight from baseline was 0.4 kg with sitagliptin and 1.1 kg with glimepiride, for a between-group difference of −0.7 kg (p = 0.011). In elderly patients with T2DM and inadequate glycemic control with diet and exercise alone, sitagliptin provided non-inferior glycemic control after 30 weeks of treatment compared with glimepiride. Compared with glimepiride, sitagliptin had a lower risk of hypoglycemia. Sitagliptin was weight-neutral; while the between-group difference in change from baseline in body weight was statistically significant, the modest difference may not be clinically meaningful. ClinicalTrials.gov NCT01189890.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Summary. Background: Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep–wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect cell-specific processes. Independent reports support the hypothesis that thrombopoietin (TPO) is under circadian control. Objectives: The current study tested the hypothesis that CLOCK, a circadian transcription factor, may regulate Thpo, the gene encoding TPO. Methods: Circadian gene expression patterns were analyzed in mice and in human cell lines, Small interfering RNA was used to knock down CLOCK expression in cell lines, and gene expression was also examined in ClockΔ19/Δ19 mutant mice. Results: It was found that there was a diurnal rhythm in the expression of Thpoin vivo in mice, and that this was associated with concomitant rhythms of protein abundance. Thpo was rhythmically expressed in human cell lines, consistent with the gene being directly or indirectly regulated by the circadian clock. Silencing of CLOCK in the Huh7 human hepatoma cell line led to a significant reduction in the rhythmicity of Thpo expression. The expression of Mpl in murine marrow also displayed diurnal rhythmicity in vivo. In ClockΔ19/Δ19 mutant mice, Thpo and Mpl expression was disrupted and there was an increase in the number of mature megakaryocytes, but no change in the ploidy distribution within the megakaryocyte population. Conclusions: These findings establish that Clock regulates Thpo and Mpl expression in vivo, and demonstrate an important link between the body’s circadian timing mechanisms and megakaryopoiesis.

SummaryThe 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°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.

The number of circulating platelets in humans exhibits diurnal rhythmicity, with lowest numbers often recorded in the morning. It has been demonstrated that a similar diurnal rhythmicity in the platelet count exists in mice. In this brief communication, it is reported that husbandry conditions affect the diurnal rhythm of platelet abundance in mice. The platelet count in mice, housed one per cage and entrained to a 12 hour : 12 hour light : dark cycle, fluctuated over 24 hours, with peak counts occurring during the animals' rest period. In contrast, this pattern was dramatically altered in mice housed as groups of 4-6 mice per cage. In group-housed mice, there was a transient surge in both platelet and reticulated platelet numbers at the transition from light to dark, corresponding to the time that animals initiate daily locomotor activity. It is speculated that this difference may reflect the circadian regulation of a stress response experienced by group-housed mice, possibly upon sampling. This finding highlights a new component to the mammalian platelet count that has not been reported before. This is an important observation because the surge in platelet and reticulated platelet numbers and the mechanism controlling it, may contribute to the diurnal incidence of cardiovascular events seen in humans.

The regulation of germ cell number in the developing ovary is central to female reproduction. Members of the Bcl-2 family of proapoptotic and antiapoptotic proteins have been implicated in this process in rodents. We investigated the expression of Mcl-1, Bcl-2, Bax, and BAD at 13-21 gestational wk in the human fetal ovary and of Mcl-1 in the adult ovary. mRNA expression of Mcl-1 and its short form Mcl-1s, Bcl-2, Bax, and BAD was demonstrated in fetal ovary by RT-PCR. Hybridization array analysis suggested a selective increase in Mcl-1 expression between 14 and 18 wk gestation, which was confirmed by quantitative PCR. There was a corresponding change in the expression of Mcl-1 protein, detected by immunohistochemistry, from germ cells at the periphery of the ovary at 14-16 wk to the largest germ cells, including oocytes within newly formed primordial follicles, at 21 wk. Mcl-1 was also expressed by oocytes of primordial and preantral follicles in the adult. Bax and BAD immunostaining was detected in both somatic and germ cells in the fetal ovary, whereas Bcl-2 was restricted to somatic cells: no changes in expression were observed. Apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, were observed in all fetal ovaries but were infrequent. These results confirm that Bcl-2 family members are differentially expressed in several cell types within the developing human ovary. Increased mRNA expression and the changing distribution of Mcl-1 in germ cells as they develop into primordial follicles as well as persistence in the growing oocyte in the adult may indicate an important role for this survival/antiapoptotic factor throughout germ cell development and maturation.

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.

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. © 1999 Wiley-Liss, Inc.

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.

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.

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.

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 degrees 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.

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.

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.

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.