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Budding yeast undergo robust oscillations in oxygen consumption during continuous growth in a nutrient-limited environment. Using liquid chromatography-mass spectrometry and comprehensive 2D gas chromatography-mass spectrometry-based metabolite profiling methods, we have determined that the intracellular concentrations of many metabolites change periodically as a function of these metabolic cycles. These results reveal the logic of cellular metabolism during different phases of the life of a yeast cell. They may further indicate that oscillation in the abundance of key metabolites might help control the temporal regulation of cellular processes and the establishment of a cycle. Such oscillations in metabolic state might occur during the course of other biological cycles.gas chromatography-mass spectrometry ͉ liquid chromatography-mass spectrometry ͉ metabolic cycle ͉ metabolite profiling C ircadian rhythms are driven by biological clocks found in virtually all kingdoms of life (1, 2). Genome-wide expression studies in plants, flies, and mice have shown that many genes are expressed periodically as a function of the circadian cycle (3-6). It is now predictable from studies of oscillating gene expression that the circadian regulatory apparatus should differentially control metabolic state as a function of the circadian cycle (7,8). By contrast, direct and comprehensive measurements of changes in sentinel metabolites have not been performed as a function of the circadian cycle or of biological cycles of other temporal dimensions.The budding yeast Saccharomyces cerevisiae exhibits various modes of oscillatory behavior during continuous growth (9-12). We recently established a continuous culture system that reveals an Ϸ4-to 5-h yeast metabolic cycle (YMC) in which over half of the genome is periodically expressed as a function of robust recurring oscillations in oxygen consumption (12). Expression profiling studies have led to the prediction that a variety of cellular and metabolic processes are temporally orchestrated around three major phases of the YMC [oxidative (Ox), reductive/building (RB), and reductive/ charging (RC)] (12). Cells undergoing the YMC are furthermore synchronized with respect to the cell cycle, because the YMC strictly gates cell division to the RB phase when respiration decreases significantly (12).A prediction of temporal compartmentalization is that the concentrations of critical metabolites might exhibit periodic fluctuation and, in turn, play a reciprocal role in regulating the YMC (8). To determine whether cyclic changes in metabolic state might occur during the YMC, we used both liquid chromatography (LC)/ tandem mass spectrometry (LC-MS/MS) and comprehensive 2D gas chromatography (GC)/time-of-f light mass spectrometry (GCϫGC-TOFMS) to monitor the intracellular concentrations of Ϸ150 common metabolites at regularly spaced intervals throughout the YMC. The combined use of LC-MS-and GC-MS-based methods provides a means to evaluate the consistency of results for metabolites detected by both ...

BRCA1 deficient tumor cells have defects in homologous-recombination repair and in replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1 deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1 deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP inhibitor resistant BRCA1 deficient tumors with acquired replication fork stabilization.

Background: CHD2 is a conserved ATPase and deletions of CHD2 have been linked to developmental and neurological disorders. Results:The regions flanking the ATPase domain of CHD2 confer substrate specificity and couple ATP hydrolysis to remodeling. Conclusion: CHD2 possesses nucleosome assembly activity regulated by its accessory domains. Significance: Understanding the mechanisms of chromatin remodeling is crucial for delineating how remodeling defects contribute to human diseases.

2004). Phytic acid-phosphorus and other nutritionally important mineral nutrient elements in grains of wild-type and low phytic acid (lpa1-1) rice. AbstractMineral nutrient stores in cereal grains are mainly phytate, a salt of the phosphorus-rich compound phytic acid. Quantitative measures of total phosphorus, phytic acid-phosphorus, potassium, magnesium, calcium, iron, manganese and zinc were obtained for whole grains, embryos and rest-of-grain portions of cv. Kaybonnet rice (wild type) (Oryza sativa L.) and a low phytic acid (lpa1-1) mutant strain with a 45% reduction in phytic acid. P, K and Mg were present in higher amounts than Ca, Mn, Fe and Zn in both grain types. Whole-grain amounts of total P, Ca, Mn and phytic acid-phosphorus were lower in whole lpa1-1 grains than in wild-type grains; K, Mg and Fe amounts were similar, and Zn was higher. Embryos, which comprise 3.5% or less of grain dry weight, were comparatively rich in all measured elements. The lpa1-1 mutation influenced the phytic acid content of the embryo more than that of the aleurone layer. Aleurone-layer cells of wild-type grains had many phosphorus-rich globoids 2 µm or larger in diameter, whereas lpa1-1 grains contained more of the smaller globoids. The reduction in globoid size was consistent with the reduction in phytate. Energy-dispersive X-ray analysis of both aleurone-layer cells and sections of globoids in aleurone-layer cells revealed that P, K and Mg were the main mineral nutrient elements present in both grain types; traces of Ca, Mn, Fe or Zn were present. Starchy endosperm cells contained virtually no P, K or Mg, whereas scutellum cells were rich in these elements. Phytic acid-phosphorus in rice 113 Figure 1. Environmental scanning electron microscope (ESEM) micrographs of the aleurone layer from wild-type (a and c) and lpa1-1 (b and d) mature rice grains. Globoids are the light spheres. Scale bars: (a, b) 10 m; (c, d) 2 m.

Homologous-recombination (HR) deficient tumors with BRCA1 and BRCA2 mutations exhibit replication fork stability defects. To date, PARP inhibitors are the only targeted therapy available in the clinic against HR deficient tumors, and alternative therapies are needed. In this study, we found a deubiquitinase, USP1, to be significantly upregulated in tumors with mutations in BRCA1. SiRNA mediated silencing or small molecule inhibition of USP1 activity resulted in replication fork destabilization and decreased viability of BRCA1 deficient cells, revealing a synthetic lethal relationship between USP1 and BRCA1. The cofactor of USP1, UAF1, had previously been shown to have DNA-binding activity. We observed that USP1 independently binds to and is stimulated by fork DNA. It is therefore the first known deubiquitinase (DUB) to be directly regulated by DNA binding. A truncated form of USP1, lacking its DNA binding region, was not stimulated by DNA and failed to localize and protect the replication fork. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of fork destabilization and cell death in the absence of USP1. Loss of monoubiquitinated PCNA, resulting from RAD18 knockdown, rescued the sensitivity and replication fork instability induced by USP1 inhibition. USP1 therefore is the first DUB enzyme exhibiting DNA-mediated activation at the replication fork, and is required for fork protection in BRCA1 deficient cells. We propose small molecule inhibitors against USP1 as a therapeutic option for BRCA1 deficient cancers. Citation Format: Kah Suan Lim, Heng Li, Emma A. Roberts, Emily F. Gaudiano, Connor Clairmont, Karthikeyan Ponnienselvan, Jessica C. Liu, Kalindi Parmar, Ning Zheng, Alan D'Andrea. USP1 is required for replication fork stability in BRCA1-deficient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 333.

Medical student exposure to oncology is imperative given the prevalence of cancer, growing need for survivorship care, and ever-evolving therapies. Our institution offers a Cancer Care Elective for undergraduate medical students focused on clinical shadowing, but the COVID-19 pandemic necessitated completely redesigning a virtual alternative. In this study, we utilize a post-elective survey to 1) assess whether the novel virtual elective effectively promoted student learning and 2) identify which components were most impactful. We created an entirely virtual, semester-long course with structured mentorship, subspecialty panels, physician-led didactics, and patient exposure. Students attended multidisciplinary tumor boards and presented on oncologic topics. A post-elective survey assessed the course’s impact on students’ knowledge and the perceived value of each elective component. Of the 29 enrolled students, 12 responded to our survey (41%). Most students reported that the elective highly enhanced their understanding of medical (67%), surgical (75%), and pediatric (66%) oncology. The highest rated didactic involved patients discussing their cancer journeys, with 80% of students reporting that this session enhanced their understanding of patient–physician collaboration. Students reported that physician mentorship helped them better understand oncology (90%) and promoted interest in pursuing an oncologic career (100%). This study demonstrates that our virtual Cancer Care Elective was effective at increasing student understanding of oncology in practice. The results also suggest that patient exposure and physician mentorship are particularly educational and encouraging.