Leerobinson3601

With these principles and tools, we demonstrate the benefits and versatility of our method, applying it toward the diverse applications of pharmaceutical crystallization, microbatch chemical reactions, and biological assays. Copyright © 2020 the Author(s). Published by PNAS.Epidemiological studies suggest that insulin resistance accelerates progression of age-based cognitive impairment, which neuroimaging has linked to brain glucose hypometabolism. As cellular inputs, ketones increase Gibbs free energy change for ATP by 27% compared to glucose. Here we test whether dietary changes are capable of modulating sustained functional communication between brain regions (network stability) by changing their predominant dietary fuel from glucose to ketones. We first established network stability as a biomarker for brain aging using two large-scale (n = 292, ages 20 to 85 y; n = 636, ages 18 to 88 y) 3 T functional MRI (fMRI) datasets. To determine whether diet can influence brain network stability, we additionally scanned 42 adults, age less then 50 y, using ultrahigh-field (7 T) ultrafast (802 ms) fMRI optimized for single-participant-level detection sensitivity. One cohort was scanned under standard diet, overnight fasting, and ketogenic diet conditions. To isolate the impact of fuel type, an independent overnight fasted cohort was scanned before and after administration of a calorie-matched glucose and exogenous ketone ester (d-β-hydroxybutyrate) bolus. Across the life span, brain network destabilization correlated with decreased brain activity and cognitive acuity. Effects emerged at 47 y, with the most rapid degeneration occurring at 60 y. Networks were destabilized by glucose and stabilized by ketones, irrespective of whether ketosis was achieved with a ketogenic diet or exogenous ketone ester. Together, our results suggest that brain network destabilization may reflect early signs of hypometabolism, associated with dementia. Dietary interventions resulting in ketone utilization increase available energy and thus may show potential in protecting the aging brain. Copyright © 2020 the Author(s). Fluorescein5isothiocyanate Published by PNAS.Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence. Copyright © 2020 the Author(s). Published by PNAS.All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.