Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

MicroRNAs modulate core-clock gene expression in pancreatic islets during early postnatal life in rats.

Evidence continues to emerge detailing a fine-tuning of the regulation of metabolic processes and energy homeostasis by cell-autonomous circadian clocks. Pancreatic beta cell functional maturation occurs after birth and implies transcriptional changes triggered by a shift in the nutritional supply that occurs at weaning, enabling the adaptation of insulin secretion. So far, the developmental timing and exact mechanisms involved in the initiation of the circadian clock in the growing pancreatic islets have never been addressed. Circadian gene expression was measured by quantitative RT-PCR in islets of rats at different postnatal ages up to 3 months, and by in vitro bioluminescence recording in newborn (10-day-old) and adult (3-month-old) islets. The effect of the microRNAs miR-17-5p and miR-29b-3p on the expression of target circadian genes was assessed in newborn rat islets transfected with microRNA antisense or mimic oligonucleotides, and luciferase reporter assays were performed on the rat insulin-secreting cell line INS832/13 to determine a direct effect. The global regulatory network between microRNAs and circadian genes was computationally predicted. We found up to a sixfold-change in the 24 h transcriptional oscillations and overall expression of Clock, Npas2, Bmal1, Bmal2, Rev-erbα, Per1, Per2, Per3 and Cry2 between newborn and adult rat islets. Synchronisation of the clock machinery in cultured islet cells revealed a delayed cell-autonomous rhythmicity of about 1.5 h in newborn compared with adult rats. Computational predictions unveiled the existence of a complex regulatory network linking over 40 microRNAs displaying modifications in their expression profiles during postnatal beta cell maturation and key core-clock genes. In agreement with these computational predictions, we demonstrated that miR-17-5p and miR-29b-3p directly regulated circadian gene expression in the maturing islet cells of 10-day-old rats. These data show that the circadian clock is not fully operational in newborn islets and that microRNAs potently contribute to its regulation during postnatal beta cell maturation. Defects in this process may have long-term consequences on circadian physiology and pancreatic islet function, favouring the manifestation of metabolic diseases such as diabetes

Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8 <sup>+</sup> T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care

Agricultural Biotechnology's Complementary Intellectual Assets

We formulate and test a hypothesis to explain the dramatic restructuring experienced recently by the plant breeding and seed industry. The reorganization can be explained in part by the desire to exploit complementarities between intellectual assets needed to create genetically modified organisms. This hypothesis is tested using data on agricultural biotechnology patents, notices for field tests of genetically modified organisms, and firm characteristics. The presence of complementarities is identified with a positive covariance in the unexplained variation of asset holdings. Results indicate that coordination of complementary assets have increased under the consolidation of the industry