Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

Understanding hereditary diseases using the dog and human as companion model systems

Animal models are requisite for genetic dissection of, and improved treatment regimens for, human hereditary diseases. While several animals have been used in academic and industrial research, the primary model for dissection of hereditary diseases has been the many strains of the laboratory mouse. However, given its greater (than the mouse) genetic similarity to the human, high number of naturally occurring hereditary diseases, unique population structure, and the availability of the complete genome sequence, the purebred dog has emerged as a powerful model for study of diseases. The major advantage the dog provides is that it is afflicted with approximately 450 hereditary diseases, about half of which have remarkable clinical similarities to corresponding diseases of the human. In addition, humankind has a strong desire to cure diseases of the dog so these two facts make the dog an ideal clinical and genetic model. This review highlights several of these shared hereditary diseases. Specifically, the canine models discussed herein have played important roles in identification of causative genes and/or have been utilized in novel therapeutic approaches of interest to the dog and human

Technologies of sleep research

Sleep is investigated in many different ways, many different species and under many different circumstances. Modern sleep research is a multidisciplinary venture. Therefore, this review cannot give a complete overview of all techniques used in sleep research and sleep medicine. What it will try to do is to give an overview of widely applied techniques and exciting new developments. Electroencephalography has been the backbone of sleep research and sleep medicine since its first application in the 1930s. The electroencephalogram is still used but now combined with many different techniques monitoring body and brain temperature, changes in brain and blood chemistry, or changes in brain functioning. Animal research has been very important for progress in sleep research and sleep medicine. It provides opportunities to investigate the sleeping brain in ways not possible in healthy volunteers. Progress in genomics has brought new insights in sleep regulation, the best example being the discovery of hypocretin/orexin deficiency as the cause of narcolepsy. Gene manipulation holds great promise for the future since it is possible not only to investigate the functions of different genes under normal conditions, but also to mimic human pathology in much greater detail

Activity of the delta-opioid receptor is partially reduced while activity of the K-receptor is maintained in mice lacking the mu-receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between mu-, delta- and kappa-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of delta- and kappa-opioid receptors in mice lacking the mu-opioid receptor (MOR). Measurements of agonist-induced [S-35]GTP gamma S binding and adenylyl cyclase inhibition showed that functional coupling of delta- and kappa-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[D-penicillamine(2), D-penicillamine(5)] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR -/- mice. delta-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail Rick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR -/- mice. Analgesic and respiratory responses produced by the selective kappa-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of delta- and kappa-receptor signaling properties in mice lacking mu-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the kappa-agonist further suggest that the kappa-receptor mainly acts independently from the mu-receptor in vivo. Reduced delta-analgesia and the absence of delta-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between mu-receptors and central delta-receptors in specific neuronal pathways

OPTN/SRTR 2019 Annual Data Report: Heart

The new adult heart allocation policy was approved in 2016 and implemented in October 2018. This year’s Annual Data Report provides early insight into the effects of this policy. In 2019, new listings continued to increase, with 4086 new candidates. Also in 2019, 3597 heart transplants were performed, an increase of 157 (4.6%) from 2018; 509 transplants occurred in children and 3088 in adults. Short‐ and long‐term posttransplant mortality rates improved. Overall, Mortality rates for adult recipients were 6.4% at 6 months and 7.9% at 1 year for transplants in 2018, 14.4% at 3 years for transplants in 2016, and 20.1% at 5 years for transplants in 2014. Mortality rates for pediatric recipients were 6.3% at 6 months and 8.2% at 1 year for transplants in 2018, 10.3% at 3 years for transplants in 2016, and 17.8% at 5 years for transplants in 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166328/1/ajt16492.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166328/2/ajt16492_am.pd