Rious effects on the behaviour of cells on the surface or after transplantation. This was demonstrated by the successful loading and delivery of RAFT to an ex vivo porcine eye using a clinical insertion device. Finally, one of the unfavourable aspects of the DMEK procedure is that the isolated membrane is prone to curling, making the tissue difficult to handleeven by experienced surgeons and so often leads to cell loss. RAFT differs significantly in this area, showing no evidence of the spontaneous IQ-1 curling that can lead to cell damage, allowing easy handling during the transplantation 25033180 procedure.ConclusionsHuman corneal endothelial cells cultured on RAFT retain their endothelial characteristics on a biomaterial that has many desirable physical properties allowing easy handling. 1326631 This method provides expanded human corneal endothelial cells with a suitable substrate for transplantation so that one donor cornea could potentially treat multiple patients requiring endothelial replacement.AcknowledgmentsThanks to The Lions Eye Institute for Transplantation and Research, Tampa, FL, US for assistance with procurement of research grade donor corneal tissue.Author ContributionsConceived and designed the experiments: HL GP RD JM JD AS. Performed the experiments: HL GP K-PT RP AS. 
Analyzed the data: HL GP. Contributed reagents/materials/analysis tools: HL GP RD JM JD AS. Wrote the paper: HL GP JM JD K-PT RP AS.
Circadian clocks generate a multitude of circadian rhythms in behavioral, neuronal, physiological, and endocrine functions [1,2]. While these rhythms have endogenous periodicity of circa 24 h, in nature they are entrained by light and temperature cycles associated with solar days. Circadian clocks consist of transcriptional and translational feedback loops working in a cell ML 240 autonomous manner that are largely conserved between Drosophila and humans [3,4]. At the core of the Drosophila circadian clock there are four clock genes: Clock (Clk), cycle (cyc), timeless (tim), and period (per) [5]. They interact in a negative feedback loop, such that loss of function in any of these genes results in disruption of the clock mechanism [6]. The expression levels of per and tim are regulated by transcriptional activators encoded by Clk and cyc. This leads to periodic increase in the levels of PER and TIM proteins. The latter accumulate in cell nuclei, and repress CLK/CYC activators, leading to suppression of per and tim transcription. In addition to per and tim, CLK/ CYC heterodimers activate genes that participate in additional clock feedback loops and a substantial number of clock output genes [7,8]. Clock-controlled output genesmodulate a myriad of metabolic and cellular functions, such as the regulation of energy balance, DNA-damage repair and xenobiotic detoxification in both mammals [9?1] and Drosophila [12?4]. There is emerging evidence that circadian clocks regulate processes that protect an organism from oxidative stress. Previously, we reported that levels of reactive oxygen species (ROS) and protein carbonyls fluctuate in a daily rhythm in heads of young wild type flies, whereas they were non-rhythmic and significantly higher in clock deficient per01 mutants [15]. These mutants also accumulated higher levels of protein carbonyls and peroxidated lipids during aging [16,17], suggesting that antioxidant defenses were compromised by the loss of clock function. In mice, deficiency of the clock protein BMAL1 (homolog of fly CYC protein) leads to incre.Rious effects on the behaviour of cells on the surface or after transplantation. This was demonstrated by the successful loading and delivery of RAFT to an ex vivo porcine eye using a clinical insertion device. Finally, one of the unfavourable aspects of the DMEK procedure is that the isolated membrane is prone to curling, making the tissue difficult to handleeven by experienced surgeons and 
so often leads to cell loss. RAFT differs significantly in this area, showing no evidence of the spontaneous curling that can lead to cell damage, allowing easy handling during the transplantation 25033180 procedure.ConclusionsHuman corneal endothelial cells cultured on RAFT retain their endothelial characteristics on a biomaterial that has many desirable physical properties allowing easy handling. 1326631 This method provides expanded human corneal endothelial cells with a suitable substrate for transplantation so that one donor cornea could potentially treat multiple patients requiring endothelial replacement.AcknowledgmentsThanks to The Lions Eye Institute for Transplantation and Research, Tampa, FL, US for assistance with procurement of research grade donor corneal tissue.Author ContributionsConceived and designed the experiments: HL GP RD JM JD AS. Performed the experiments: HL GP K-PT RP AS. Analyzed the data: HL GP. Contributed reagents/materials/analysis tools: HL GP RD JM JD AS. Wrote the paper: HL GP JM JD K-PT RP AS.
Circadian clocks generate a multitude of circadian rhythms in behavioral, neuronal, physiological, and endocrine functions [1,2]. While these rhythms have endogenous periodicity of circa 24 h, in nature they are entrained by light and temperature cycles associated with solar days. Circadian clocks consist of transcriptional and translational feedback loops working in a cell autonomous manner that are largely conserved between Drosophila and humans [3,4]. At the core of the Drosophila circadian clock there are four clock genes: Clock (Clk), cycle (cyc), timeless (tim), and period (per) [5]. They interact in a negative feedback loop, such that loss of function in any of these genes results in disruption of the clock mechanism [6]. The expression levels of per and tim are regulated by transcriptional activators encoded by Clk and cyc. This leads to periodic increase in the levels of PER and TIM proteins. The latter accumulate in cell nuclei, and repress CLK/CYC activators, leading to suppression of per and tim transcription. In addition to per and tim, CLK/ CYC heterodimers activate genes that participate in additional clock feedback loops and a substantial number of clock output genes [7,8]. Clock-controlled output genesmodulate a myriad of metabolic and cellular functions, such as the regulation of energy balance, DNA-damage repair and xenobiotic detoxification in both mammals [9?1] and Drosophila [12?4]. There is emerging evidence that circadian clocks regulate processes that protect an organism from oxidative stress. Previously, we reported that levels of reactive oxygen species (ROS) and protein carbonyls fluctuate in a daily rhythm in heads of young wild type flies, whereas they were non-rhythmic and significantly higher in clock deficient per01 mutants [15]. These mutants also accumulated higher levels of protein carbonyls and peroxidated lipids during aging [16,17], suggesting that antioxidant defenses were compromised by the loss of clock function. In mice, deficiency of the clock protein BMAL1 (homolog of fly CYC protein) leads to incre.