The sirtuins are members of the histone deacetylase family of proteins that participate in a variety of cellular functions and play a role in aging. We identified a potent inhibitor of sirtuin 2 (SIRT2) and found that inhibition of SIRT2 rescued alpha-synuclein toxicity and modified inclusion morphology in a cellular model of Parkinson's disease. Genetic inhibition of SIRT2 via small interfering RNA similarly rescued alpha-synuclein toxicity. Furthermore, the inhibitors protected against dopaminergic cell death both in vitro and in a Drosophila model of Parkinson's disease. The results suggest a link between neurodegeneration and aging.

Plug-flow biofilm reactors colonized by microorganisms in streamwater were used to measure the concentration and composition of biodegradable dissolved organic C (BDOC) in White Clay Creek. During the 4-month study period, DOC ranged from 0.8 to 10.4 mg C liter-' and was, on average, composed of 75% humic substances, 13% carbohydrates, 2% amino acids, and 18% > 100 kDa. The carbohydrates were predominantly polysaccharides, nearly all amino acids were present in the combined form, and most carbohydrates and amino acids were humic bound. BDOC ranged from 0.2 to 2.9 mg C liter-', averaged 25% of the DOC, and was composed of 75% humic substances, 30% carbohydrates, 4% amino acids, and 39% DOC >lOO kDa. The carbohydrate portion of the BDOC was primarily polysaccharide or humic bound. Similarly, the amino acid portion of the BDOC was overwhelmingly present in the combined form and primarily humic bound. Glycine and aspartic acid were the dominant amino acids in White Clay Creek DOC and in the BDOC pool. Our data broaden the perspective on substrates important to microbial metabolism and energy flow in streams and provide the first direct evidence that humic substances, although largely refractory, are an important component of streamwater BDOC.Dissolved organic matter (DOM) comprises most of the reduced carbon in aquatic ecosystems and provides energy and carbon resources for the metabolism of heterotrophic bacteria. Not all DOM is biologically labile or even biodegradable. Although numerous investigations in both freshwater and marine environments have reported on the quantity and composition of DOM, fewer studies have addressed the biodegradable fraction. Identifying biodegradable DOM (BDOM) constituents and quantifying their contribution to heterotrophic metabolism can increase our understanding of ecosystem function and bacterial ecophysiology.From a limited number of studies we know that BDOM in streams and rivers includes both low-molecular-weight (Kaplan and Bott 1983) and high-molecular-weight (Meyer

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis.

Retinoic acid, the active metabolite of vitamin A, plays important roles in various physiological and pathological processes. The two-step production of retinoic acid from vitamin A (retinol) is catalyzed by alcohol dehydrogenases and aldehyde dehydrogenases, which are potential therapeutic targets for numerous diseases, such as obesity, diabetes, and cancer. Currently, the lack of a suitable high-throughput cellular assay hinders efforts to identify therapeutic small molecular inhibitors of aldehyde dehydrogenase, such as ALDH1A1. In this report, we utilized high-content imaging technology and a commercially available cell permeable ALDH substrate to develop a 96-well cellular ALDH1A1 assay. This assay has a robust and sensitive readout and is amenable to automation. With this cellular assay, we identified potent selective ALDH1A1 inhibitors to explore the role of retinoic acid production in various preclinical disease models.

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Basal Insulin Fc (BIF, LY3209590) is an insulin Fc-fusion protein in clinical testing as a once weekly treatment for diabetes mellitus. BIF is comprised of a human single-chain insulin fused to a human IgG2 Fc domain through a peptide linker. The cellular internalization characteristics of BIF compared to native insulin using U2OS cells expressing human insulin receptor (hIR) are reported in this work. Internalization and sub-cellular localization were visualized by immuno-fluorescent confocal microscopy in U2OS cells expressing human insulin receptor. Internalization of BIF was observed, and the insulin and Fc moieties remained co-localized. Co-localization studies using an antibody to the early endosomal marker Early Endosome Antigen 1 (EEA1) showed that human insulin and BIF (insulin and Fc moieties) were rapidly internalized and co-localized with EEA1. During ligand washout, the remaining insulin and Fc moieties of BIF remained largely co-localized with EEA1 and a concomitant loss of immunostaining was observed, similar to human insulin. Studies using an antibody to the lysosomal marker Lysosomal Associated Membrane Protein-1 (LAMP-1) revealed a low level of lysosomal localization for human insulin and BIF (insulin and Fc moieties), indicating a similar intracellular trafficking pattern of human insulin and BIF to the lysosomal degradative pathway. The co-localization of remaining ligand decreased over time. IR internalization and transport to early endosomes was measured using an enzyme complementation assay. The EC50 for BIF was 4.7 ± 2.1 nM (n = 3), and the EC50 for human insulin was 0.06 ± 0.01 nM (n = 3). In summary, BIF stimulated IR internalization to a similar maximum level; however, with decreased potency versus human insulin. The subcellular trafficking pattern of BIF is similar to human insulin. BIF undergoes rapid internalization and transport to early endosomes with limited transport to the lysosomes and undergoes loss of cellular immunostaining during ligand washout. Disclosure C. Volk: None. C. Zhang: Employee; Self; Eli Lilly and Company. J. S. Moyers: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company