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Objective Shift work encompasses a broad range of work time arrangements. However, how shift work affects the circadian expression of clock genes remains to be explored. The objective of this study was to evaluate the pattern of clock gene expression in shift workers in the field. Methods We examined clock gene expression in Japanese men who work: (1) one night shift followed by a day off (caregivers: nurses and doctors; the one-night group); (2) three or more consecutive night shifts (factory workers; the consecutive-night group); or (3) daytime only (the daytime group), using beard follicle samples. The expression of Period3, Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), and Nuclear Receptor Subfamily 1 Group D Member 2 (Nr1d2) was examined by real-time polymerase chain reaction. Results Period3 expression in the daytime and one-night groups together with Nr1d2 expression in the one-night group fitted a 24-h-period cosine curve better than in the consecutive-night group (p = 0.004, 0.012, and 0.001, respectively). The level of overall Period3 gene expression, calibrated with that of 18S-rRNA, was decreased in the consecutive-night group compared with that in the daytime group (p = 0.006). The patterns of Period3 and Nr1d2 expression in the daytime and one-night groups were more coherent than those in the consecutive-night group. Conclusions These results suggest that night shift work affects the rhythms and levels of circadian Period3 and Nr1d2 expression dependent on the shift schedule or type of the shift; however, there is substantial variation between individuals.

Background: Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.

Summary The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1ko/H2ko) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1ko/H2eko) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1ko/H2ko phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1ko/H2eko embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency. genesis 52:897–906, 2014. © 2014 Wiley Periodicals, Inc.

Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation.Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling.The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events.

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency.

SummaryWe report on a 25-year-old female heart transplant patient who presented with recurrent episodes of cellular rejection due to decreased adherence to immunosuppressive therapy. She received a heart transplantation in 1994 when she was 10 years old. In order to improve her adherence to immunosuppressive therapy, switching to the once-daily extended-release formulation of tacrolimus was performed in a step-wise fashion. First, the twice-daily formulation of cyclosporin A was replaced with the twice-daily preparation of tacrolimus. When the trough blood levels of tacrolimus reached a plateau in the range of 5.0ng/mL, it was changed to the once-daily extended-release formulation of tacrolimus after confirming the absence of new rejection episodes. There were no significant changes in renal function before and after the switch. After being discharged from the hospital, the patient made significant advancements in adherence to immunosuppressive therapy. Her subsequent clinical course was uneventful, with no adverse events observed. Most patients who undergo solid organ transplantation must receive lifelong immunosuppressive therapy. This case demonstrates that conversion to the extended-release formulation of tacrolimus from other calcineurin inhibitor preparations is a reasonable choice to consider in the management of compromised immunosuppressive therapy adherence in heart transplant patients during the late posttransplant period.